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Rapid Access to Potent Bispecific T Cell Engagers Using Biogenic Tyrosine Click Chemistry
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  • Irene Shajan
    Irene Shajan
    Laboratory of Organic Chemistry, Wageningen University & Research, Stippeneng 4, Wageningen 6807 WE, The Netherlands
    More by Irene Shajan
  • Léa N. C. Rochet
    Léa N. C. Rochet
    Department of Chemistry, University College London, 20 Gordon St, London WC1H 0AJ, U.K.
    More by Léa N. C. Rochet
    Orcidhttps://orcid.org/0009-0003-5594-6290
  • Shannon R. Tracey
    Shannon R. Tracey
    Patrick G Johnston Centre for Cancer Research, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, U.K.
    More by Shannon R. Tracey
  • Bianka Jackowska
    Bianka Jackowska
    Patrick G Johnston Centre for Cancer Research, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, U.K.
    More by Bianka Jackowska
  • Rania Benazza
    Rania Benazza
    Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR 7178, Strasbourg 67000, France
    Infrastructure Nationale de Protéomique ProFI − FR2048, Strasbourg 67087, France
    More by Rania Benazza
  • Oscar Hernandez-Alba
    Oscar Hernandez-Alba
    Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR 7178, Strasbourg 67000, France
    Infrastructure Nationale de Protéomique ProFI − FR2048, Strasbourg 67087, France
    More by Oscar Hernandez-Alba
  • Sarah Cianférani
    Sarah Cianférani
    Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR 7178, Strasbourg 67000, France
    Infrastructure Nationale de Protéomique ProFI − FR2048, Strasbourg 67087, France
    More by Sarah Cianférani
    Orcidhttps://orcid.org/0000-0003-4013-4129
  • Christopher J. Scott
    Christopher J. Scott
    Patrick G Johnston Centre for Cancer Research, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, U.K.
    More by Christopher J. Scott
  • Floris L. van Delft
    Floris L. van Delft
    Laboratory of Organic Chemistry, Wageningen University & Research, Stippeneng 4, Wageningen 6807 WE, The Netherlands
    Synaffix BV − A Lonza Company, Kloosterstraat 9, Oss 5349 AB, The Netherlands
    More by Floris L. van Delft
    Orcidhttps://orcid.org/0000-0003-4455-7727
  • Vijay Chudasama*
    Vijay Chudasama
    Department of Chemistry, University College London, 20 Gordon St, London WC1H 0AJ, U.K.
    *Email: [email protected]
    More by Vijay Chudasama
    Orcidhttps://orcid.org/0000-0002-8876-3285
  • Bauke Albada*
    Bauke Albada
    Laboratory of Organic Chemistry, Wageningen University & Research, Stippeneng 4, Wageningen 6807 WE, The Netherlands
    *Email: [email protected]
    More by Bauke Albada
    Orcidhttps://orcid.org/0000-0003-3659-2434
Open PDFSupporting Information (1)

Bioconjugate Chemistry

Cite this: Bioconjugate Chem. 2023, 34, 12, 2215–2220
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https://doi.org/10.1021/acs.bioconjchem.3c00357
Published November 14, 2023

Copyright © 2023 The Authors. Published by American Chemical Society. This publication is licensed under

CC-BY 4.0 .

Abstract

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Bispecific antibodies as T cell engagers designed to display binding capabilities to both tumor-associated antigens and antigens on T cells are considered promising agents in the fight against cancer. Even though chemical strategies to develop such constructs have emerged, a method that readily converts a therapeutically applied antibody into a bispecific construct by a fully non-genetic process is not yet available. Herein, we report the application of a biogenic, tyrosine-based click reaction utilizing chemoenzymatic modifications of native IgG1 antibodies to generate a synthetic bispecific antibody construct that exhibits tumor-killing capability at picomolar concentrations. Control experiments revealed that a covalent linkage of the different components is required for the observed biological activities. In view of the highly potent nature of the constructs and the modular approach that relies on convenient synthetic methods utilizing therapeutically approved biomolecules, our method expedites the production of potent bispecific antibody constructs with tunable cell killing efficacy with significant impact on therapeutic properties.

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Subjects

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The ability of a monoclonal antibody (mAb) to bind a specific antigen, such as a protein or epitope, on the surface of a cell has led to a variety of therapeutic applications. (1−4) In particular, derivatives like antibody-drug conjugates (ADCs) and bispecific antibodies (bsAbs) in cancer treatment have been developed. (5−11) Whereas ADCs are designed to deliver a toxic payload to malignant tissue, (12,13) bsAbs bind different epitopes such as antigens on separate target cells. (14−17) Currently, T cell or NK cell redirectors and tumor-targeted immunomodulators form the main class of bsAbs. (18) For example, the first FDA-approved bispecific construct, blinatumomab, binds to CD19 on (malignant) B cells and CD3 on T cells, generating a cytolytic synapse that leads to lysis of the targeted cell. (19) This success has led to the evaluation of many different formats of T cell engagers, like diabody, (20) CrossMab, (21) BiTE, (22) dual affinity retargeting antibodies (DART), (23) tandem diabody (TandAb), (24) and more recently synthetic bispecific mAbs (SynAbs). (25)
Most bsAbs are produced via protein engineering of the native mAb framework. (26−28) However, the evolution of bio-orthogonal (click) chemistry has facilitated the generation of novel synthetic antibody conjugates, such as ADCs. (29,30) At the moment, strain-promoted azide alkyne cycloaddition (SPAAC) (31) and the inverse electron-demand Diels–Alder (IEDDA) reactions between strained unsaturated carbon–carbon systems and tetrazine (32,33) or ortho-quinone (34) have been successfully applied for antibody modification. (35)
Herein, we describe the application of biogenic tyrosine-based click chemistry for the synthesis of bsAbs using native mAbs, resulting in constructs that display T-cell activation activity at picomolar concentrations.
In this approach, Fab units of the anti-CD3 antibody OKT3 were rebridged using an appropriate pyridazinedione (PD) construct to provide a single tetrazine handle per Fab, which were then connected to TCO-functionalized HER2-binding trastuzumab prepared via biogenic tyrosine-based click chemistry on the deglycosylated native mAb. As such, the correct abbreviation for this construct is rbFab-dgmAb-rbFab (in which rb = rebridged and dgmAb = deglycosylated monoclonal antibody), but we refer to these as bispecific antibody constructs (bsAcs). Contrary to most current methods, our approach enables the convenient conversion of native IgG1 antibodies into a 2:2 bsAc that combines two antigen-binding sites for each of the two different targets via a few chemical and chemoenzymatic steps.

Results and Discussion

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BsAc Synthesis

To gain synthetic access to bsAcs from native mAbs, we first prepared appropriate BCN-PEG3-TCO (1) and MeTz-PEG3-Br2PD (2) (36) linkers using convenient established procedures (Scheme 1A and Supporting Information). For the preparation of MeTz-functionalized FabCD3 (3a, Scheme 1B), targeted digestion of the hinge region of the anti-CD3 OKT3 mAb by treatment with immobilized papain resulted in two Fab units that could be isolated from the Fc unit using protein A purification. Reduction of the C-terminally positioned intrachain disulfide bond of the obtained FabCD3 fragments with an excess of TCEP enabled rebridging with the dibromopyridazinedione-based tetrazine-functionalized construct, MeTz-PEG3-Br2PD (2), to yield MeTz-rbFabCD3 (3a). Similarly, rebridged HER2-binding Fabs were also prepared to be incorporated in our negative control construct (i.e., MeTz-rbFabHER2 (3b), see insert Scheme 1B). An additional digestion step using immobilized pepsin was necessary to isolate the corresponding HER2-binding Fab fragments this time. (37)

Scheme 1

Scheme 1. Conversion of Native mAbs to Bispecific Constructsa
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a(A) Synthesis of BCN-PEG3-TCO (1) and chemical structure of MeTz-PEG3-Br2PD (2). (B) Installation of MeTz handle on OKT3 Fab by rebridging the light-chain and heavy-chain Fab fragments obtained after digestion and reduction. Structure of MeTz-rbFabHER2 (3b) that is used to construct the negative control is shown in the inset. (C) Chemoenzymatic functionalization of native mAb trastuzumab with a TCO handle using BCN-PEG3-TCO (1), resulting in deglycosylated TCO-functionalized trastuzumab (4). The bispecific antibody construct (bsAc, 5a) was obtained after reaction with MeTz-rbFabCD3 (3a) by TCO-Tz IEDDA.

Benefiting from the 150-fold higher reactivity of BCN with an ortho-quinone when compared to TCO, (38) we were able to functionalize the Fc domain of trastuzumab with two TCO handles by subsequent treatment of the mAb with peptide-N-glycosidase F (PNGase F) and mushroom tyrosinase (mTyr) in the presence of BCN-PEG3-TCO (1) (Scheme 1C). As this IEDDA-compatible click handle is generated from the proteinogenic amino acid residue tyrosine, we refer to this approach as “biogenic click chemistry”. Purification by protein A column chromatography afforded the desired TCO-functionalized mAb (4).
The two MeTz-functionalized Fabs, i.e., MeTz-rbFabCD3 (3a) and MeTz-rbFabHER2 (3b), were subjected to tetrazine-trans-cyclooctene IEDDA conjugation with TCO-functionalized trastuzumab (4) at 4 °C for 2 h in PBS of pH 7.4 (Scheme 1C). Non-reducing SDS-PAGE analysis revealed formation of the desired constructs (see Supporting Information, Figures S10 and S11), which were purified by subsequent filtration over a protein A column and size exclusion chromatography to yield the desired 2:2 HER2xCD3 bsAc (5a) as well as the tetravalent HER2-binding negative control (5b). We found that isolation of the prepared bispecific constructs was challenging (see Supporting Information, S10 and S11). In fact, analysis of the most pure fractions by native SEC-MS, which separates molecules as a function of their hydrodynamic volume and is particularly well adapted to separate, identify, and relatively quantify the different species generated during the formation of both tetrameric constructs, 5a and 5b (Figure 1), revealed that the reaction of TCO-deglycosylated-Trz with the different Fab subunits (either anti-CD3 or anti-HER2) resulted in earlier elution of the main peak in the chromatogram dimension (from 4.5 to 4.0 min). As expected, the hydrodynamic volume increases due to conjugation of the mAb with the Fab domains. Closer inspection showed that the main peaks (at 3.9 min) correspond to the formation of the tetravalent mAb constructs (2:2 bsAc), but that a shoulder centered at 4.1 min corresponding to the trivalent formats of the mAbs (2:1 bsAc) was also observed. Gaussian fitting of the partially coeluting species yielded relative intensities of bsAc 5a and 5b of 54% in both cases. For the trivalent species that are also formed, i.e., the 2:1 HER2xCD3 as a side-product of 5a and the 2:1 HER2xHER2 as a side-product of 5b, relative intensities of 32 and 24% were calculated, respectively. Therefore, even though full purification of these constructs was not possible, the obtained fractions mostly contained the targeted constructs and similar amounts of 2:1 byproduct.

Biological Evaluation

The binding capability of the synthesized constructs, i.e., the isolated fractions of the 2:2 HER2xCD3 bsAc (5a) and the tetravalent HER2-binding negative control (5b), of which the analysis profile is shown in Figure 1, to HCC1954 (HER2+CD3–) and Jurkat (HER2–CD3+) cells was first evaluated by flow cytometry (Figure 2A). Briefly, HCC1954 cells were incubated with the synthesized constructs, and Fc-units of the bound constructs were stained using an FITC-labeled anti-IgG Fc antibody. Those samples stained with the isotype control and FITC-labeled anti-IgG Fc alone did not exhibit an increase in mean fluorescence intensity, while cells preincubated with samples containing the HER2xCD3 bsAc (5a) and the HER2xHER2 control (5b) displayed an increase in FITC mean fluorescence intensity as expected. This indicated that the synthetic constructs containing the HER2-binding element of trastuzumab, including the Fc unit, retained their binding to HER2+ cell lines. The lower staining levels detected for negative control 5b can be attributed to the tetravalent nature of the construct, which saturates more binding sites than the other constructs while not offering additional binding options for the FITC-labeled anti-IgG Fc antibody. Following this, binding to CD3 receptors was assessed using a CD3+-immortalized human T cell lines (Jurkat). As expected only the cells incubated with bsAc 5a, which contained the OKT3-derived fragments, showed an increase in mean fluorescence intensity, while cells preincubated with control construct 5b did not reveal binding to these cells. Therefore, our synthetic constructs retained their expected binding capacity to their targeted cells after the rebridging and biogenic IEDDA click conjugation.

Figure 1

Figure 1. SEC-nMS analysis of trastuzumab samples obtained during assembly of the bispecific constructs via biogenic tyrosine click chemistry. (A) SEC-UV chromatogram of deglycosylated Trz (black), trastuzumab-TCO 4 (blue), HER2xCD3 bsAc 5a (orange), and HER2xHER2 bsAc 5b (pink). Relative quantification of each species is performed upon the integration of chromatographic peak areas. (B) Native MS spectra of the major peak from each sample namely, monomer of deglycosylated Trz (black), monomer of trastuzumab-TCO (4) (blue), 2:2 HER2xCD3 bsAc 5a (orange), and 2:2 HER2xHER2 bsAc 5b (pink). (C) Deconvoluted values of the relevant species are provided with a standard deviation obtained from at least four different charge states; masses of minor species are summarized in Supporting Information (Figure S19).

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Figure 2

Figure 2. Biological activity studies of the bispecific antibody construct and controls. (A) (i) Flow cytometry analysis of binding of the various constructs to HCC1954 (HER2+CD3–) and Jurkat (HER2–CD3+) cells; (ii) binding of the constructs to HCC1954 (HER2+CD3–) cells shown as mean fluorescence intensity (MFI); and (iii) binding of the constructs to Jurkat (HER2–CD3+) cells shown as MFI (n = 3). (B) Induction of IFN-γ production and excretion by the various constructs and controls (5 nM) in T cells or HCC1954 (HER2+CD3–) cells alone or HCC1954/T-cell cocultures (ratio 1:10). Culture supernatant IFN-γ was quantified by ELISA at 48 h following treatment. (C) Cellular metabolism assay as a measure of HCC1954 cell viability affected by the synthetic constructs in the presence of HCC1954 (HER2+CD3–) cells alone or HCC1954/T-cell cocultures (E/T ratio 10:1, 5 nM construct). HCC1954 viability was assessed by Cell Titer-Glo at 48 h following treatment. (D) Cytotoxicity dose–response curve of the bsAc 5a on HCC1954/T-cell cocultures (ratio 1:10) were incubated with varying concentrations (serial dilutions ranging from 0.0128 pM to 5 nM; donors are indicated with D#1, D#9, and D#10). HCC1954 cell viability was assessed by Cell Titer-Glo at 48 h following treatment, where the IC50 value was extrapolated. Statistical analysis was performed in GraphPad Prism (v9.5.1), where the data is presented as mean ± SEM. Statistical significance was established by Two-way ANOVA and Šídák’s multiple comparisons test (ns denotes “no significance” and **** p ≤ 0.0001).

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Next, we determined whether binding was also accompanied by T cell activation (Figure 2B). For this we determined interferon-γ levels as a qualitative indicator of T cell activation using an ELISA performed on the supernatants after 48 h following treatment with the fractions containing the different constructs (T cells were obtained from three healthy blood donors). Specifically, T cell/HCC1954 cocultures (effector:target ratio 10:1), HCC1954 monocultures, or T cell monocultures were incubated with 5 nM of bsAc 5a, or any of the controls (i.e., untreated, tetravalent HER2 binder (5b), trimmed trastuzumab, MeTz-rbFabCD3 (3a), a 1:1 mixture of trimmed trastuzumab and MeTz-rbFabCD3 (3a)). As can be seen, the presence of bsAc 5a in the T cell/HCC1954 coculture was required for significant interferon-γ expression. In fact, the absence of IFN-γ levels in the coculture that was treated with the 1:1 mixture of trimmed trastuzumab and Tz-OKT3 shows that both are required to be present in the same molecular construct in order to elicit T cell activation. Therefore, we conclude that T cell activation is restricted to the tumor microenvironment and not by simultaneous engagement of the antigen-binding site on the surface of the different cells.
After this, the capacity of each construct to activate T cells for the killing of the HER2+ tumor cells was assessed by using HCC1954 cells (Figure 2C). For this, T cells and HCC1954 as monocultures and T cell/HCC1954 cocultures (effector:target ratio 10:1) were incubated with 5 nM of each synthetic construct, and HCC1954 cell viability was measured following 48 h post treatment. To our delight, 90% cell death was observed in cocultures treated with the 2:2 HER2xCD3 bsAc (5a), where no significant cell death was observed for treatment with any of the other constructs. Furthermore, the isolated components separately, or a mixture of the non-linked isolated components, did not trigger T cell-mediated cell killing. This indicated that simultaneous binding of T cells and tumor cells by the same molecular construct was required for proper T cell engagement to the tumor cell. Lastly, IC50 values of the isolated fractions containing our synthetic 2:2 HER2xCD3 bsAc 5a were determined using HCC1954 cells and T cells from three independent donors (effector:target ratio 10:1) (Figure 2D). Exposure of the mixture of cells to increasing concentrations of our bsAc 5a revealed low IC50 values of 1.3–26.7 pM. Despite the variations between the performance of the T cells obtained from the different donors, the IC50 values in the low picomolar range reveal the potency of the constructs generated from approved mAbs by our biogenic tyrosine-based click chemistry approach. Realizing that the fractions contain 54% of the targeted 2:2 constructs and also significant amounts of 2:1 constructs, i.e., 32% for HER2xCD3 and 24% for HER2xHER2, the actual potency of the conjugates is likely higher than was observed in these studies.

Conclusions

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We developed a convenient and modular approach using both biogenic and artificial IEDDA click reactions to convert native antibodies into potent bispecific T cell-engaging bioconjugates. As such, our method provides convenient access to antibody constructs that display two different paratopes by using therapeutically approved monoclonal antibodies. While purification of the 2:2 construct from the 2:1 constructs, which are also formed, requires optimization, the synthesized 2:2 HER2xCD3 constructs displayed enhanced binding to cells that expressed either the HER2 or CD3 receptor. In fact, we found that T cell activation was restricted to a bioconjugate that contained both antigen-binding sites in one molecular construct. Clearly, our synthetic method that uses native antibodies does not hamper the biological activity of the parent mAbs, and the generated bispecific constructs retain the activity of both the mAbs, activating the T lymphocytes against HER2+ cells, inducing tumor cell death even in the very low pM range. Current studies are focused on dissecting the potency of the 2:2 constructs in relation to the 2:1 constructs and improving our synthetic method toward the preparation of different and more diverse bsAcs.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.3c00357.

  • Synthetic chemistry experimental details, conjugation experiments and in vitro assays, 1H and 13C spectra, and full LC-MS spectra including TIC trace and deconvoluted spectra (PDF)

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Author Information

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  • Corresponding Authors
  • Authors
    • Irene Shajan - Laboratory of Organic Chemistry, Wageningen University & Research, Stippeneng 4, Wageningen 6807 WE, The Netherlands
    • Léa N. C. Rochet - Department of Chemistry, University College London, 20 Gordon St, London WC1H 0AJ, U.K.;  Orcidhttps://orcid.org/0009-0003-5594-6290
    • Shannon R. Tracey - Patrick G Johnston Centre for Cancer Research, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, U.K.
    • Bianka Jackowska - Patrick G Johnston Centre for Cancer Research, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, U.K.
    • Rania Benazza - Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR 7178, Strasbourg 67000, France;  Infrastructure Nationale de Protéomique ProFI − FR2048, Strasbourg 67087, France
    • Oscar Hernandez-Alba - Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR 7178, Strasbourg 67000, France;  Infrastructure Nationale de Protéomique ProFI − FR2048, Strasbourg 67087, France
    • Sarah Cianférani - Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR 7178, Strasbourg 67000, France;  Infrastructure Nationale de Protéomique ProFI − FR2048, Strasbourg 67087, France;  Orcidhttps://orcid.org/0000-0003-4013-4129
    • Christopher J. Scott - Patrick G Johnston Centre for Cancer Research, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, U.K.
    • Floris L. van Delft - Laboratory of Organic Chemistry, Wageningen University & Research, Stippeneng 4, Wageningen 6807 WE, The Netherlands;  Synaffix BV − A Lonza Company, Kloosterstraat 9, Oss 5349 AB, The Netherlands;  Orcidhttps://orcid.org/0000-0003-4455-7727
  • Author Contributions

    I.S. synthesized the bifunctional linker and the antibody conjugates. L.N.C.R. synthesized the MeTz-Br2PD linker and rebridged Fab fragments. I.S. generated and purified the bispecific antibody constructs and performed the biology experiments. S.R.T. performed ELISA for IL-2. C.J.S. designed the biology experiments. R.B. performed native SEC-nMS. S.C. and O.H.A. developed the SEC-nMS method. B.A., F.L.V.D., and V.C. shaped the project. I.S. and B.A. cowrote the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work is funded by the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 859458. This study was further supported by the CNRS, the University of Strasbourg, the Agence Nationale de la Recherche, and the French Proteomic Infrastructure (ProFI; ANR-10-INBS-08-03).

References

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    A review. Monoclonal antibodies constitute a promising class of targeted anticancer agents that enhance natural immune system functions to suppress cancer cell activity and eliminate cancer cells. The successful application of IgG monoclonal antibodies has inspired the development of various types of therapeutic antibodies, such as antibody fragments, bispecific antibodies, and antibody derivs. (e.g., antibody-drug conjugates and immunocytokines). The miniaturization and multifunctionalization of antibodies are flexible and viable strategies for diagnosing or treating malignant tumors in a complex tumor environment. In this review, we summarize antibodies of various mol. types, antibody applications in cancer therapy, and details of clin. study advances. We also discuss the rationale and mechanism of action of various antibody formats, including antibody-drug conjugates, antibody-oligonucleotide conjugates, bispecific/multispecific antibodies, immunocytokines, antibody fragments, and scaffold proteins. With advances in modern biotechnol., well-designed novel antibodies are finally paving the way for successful treatments of various cancers, including precise tumor immunotherapy, in the clinic.
  5. 5
    Ackerman, S. E.; Pearson, C. I.; Gregorio, J. D.; Gonzalez, J. C.; Kenkel, J. A.; Hartmann, F. J.; Luo, A.; Ho, P. Y.; LeBlanc, H.; Blum, L. K. Immune-Stimulating Antibody Conjugates Elicit Robust Myeloid Activation and Durable Antitumor Immunity. Nat. Cancer 2021, 2, 18– 33,  DOI: 10.1038/s43018-020-00136-x
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    Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity
    Ackerman, Shelley E.; Pearson, Cecelia I.; Gregorio, Joshua D.; Gonzalez, Joseph C.; Kenkel, Justin A.; Hartmann, Felix J.; Luo, Angela; Ho, Po Y.; LeBlanc, Heidi; Blum, Lisa K.; Kimmey, Samuel C.; Luo, Andrew; Nguyen, Murray L.; Paik, Jason C.; Sheu, Lauren Y.; Ackerman, Benjamin; Lee, Arthur; Li, Hai; Melrose, Jennifer; Laura, Richard P.; Ramani, Vishnu C.; Henning, Karla A.; Jackson, David Y.; Safina, Brian S.; Yonehiro, Grant; Devens, Bruce H.; Carmi, Yaron; Chapin, Steven J.; Bendall, Sean C.; Kowanetz, Marcin; Dornan, David; Engleman, Edgar G.; Alonso, Michael N.
    Nature Cancer (2021), 2 (1), 18-33CODEN: NCAADQ; ISSN:2662-1347. (Springer International Publishing AG)
    Innate pattern recognition receptor agonists, including Toll-like receptors (TLRs), alter the tumor microenvironment and prime adaptive antitumor immunity. However, TLR agonists present toxicities assocd. with widespread immune activation after systemic administration. To design a TLR-based therapeutic suitable for systemic delivery and capable of safely eliciting tumor-targeted responses, we developed immune-stimulating antibody conjugates (ISACs) comprising a TLR7/8 dual agonist conjugated to tumor-targeting antibodies. Systemically administered human epidermal growth factor receptor 2 (HER2)-targeted ISACs were well tolerated and triggered a localized immune response in the tumor microenvironment that resulted in tumor clearance and immunol. memory. Mechanistically, ISACs required tumor antigen recognition, Fcγ-receptor-dependent phagocytosis and TLR-mediated activation to drive tumor killing by myeloid cells and subsequent T-cell-mediated antitumor immunity. ISAC-mediated immunol. memory was not limited to the HER2 ISAC target antigen since ISAC-treated mice were protected from rechallenge with the HER2- parental tumor. These results provide a strong rationale for the clin. development of ISACs.
  6. 6
    He, L.; Wang, L.; Wang, Z.; Li, T.; Chen, H.; Zhang, Y.; Hu, Z.; Dimitrov, D. S.; Du, J.; Liao, X. Immune Modulating Antibody-Drug Conjugate (IM-ADC) for Cancer Immunotherapy. J. Med. Chem. 2021, 64, 15716– 15726,  DOI: 10.1021/acs.jmedchem.1c00961
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    Immune Modulating Antibody-Drug Conjugate (IM-ADC) for Cancer Immunotherapy
    He, Lei; Wang, Liangliang; Wang, Zhisong; Li, Tiantian; Chen, Hui; Zhang, Yaning; Hu, Zeping; Dimitrov, Dimiter S.; Du, Juanjuan; Liao, Xuebin
    Journal of Medicinal Chemistry (2021), 64 (21), 15716-15726CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Antibody-drug conjugate (ADC) and immune checkpoint blockade (ICB) offer promising approaches for cancer treatment. Here, we describe an ADC constructed by conjugating anti-PD-L1 THIOMAB with a bifunctional immunomodulator D18 via a redox-cleavable linker. The resulting ADC HE-S2 not only triggers a potent antitumor immune response by blocking the PD-1/PD-L1 interaction and activating the Toll-like receptor 7/8 (TLR7/8) signaling pathway but also upregulates its targeted PD-L1 expression via epigenetic regulation and/or IFN-γ induction, thus conferring more sensitivity to the PD-1/PD-L1 blockade. We identify that ADC HE-S2 treatment could lead to more pronounced tumor suppression than the treatment of D18 in combination with the anti-PD-L1 antibody. Accordingly, this study provides a novel ADC strategy to enhance the antitumor immune response to ICB therapy.
  7. 7
    Fu, Z.; Li, S.; Han, S.; Shi, C.; Zhang, Y. Antibody Drug Conjugate: The “Biological Missile” for Targeted Cancer Therapy. Signal Transduct. Targeted Ther. 2022, 7, 93,  DOI: 10.1038/s41392-022-00947-7
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    Antibody drug conjugate: the "biological missile" for targeted cancer therapy
    Fu, Zhiwen; Li, Shijun; Han, Sifei; Shi, Chen; Zhang, Yu
    Signal Transduction and Targeted Therapy (2022), 7 (1), 93CODEN: STTTCB; ISSN:2059-3635. (Nature Portfolio)
    A review. Antibody-drug conjugate (ADC) is typically composed of a monoclonal antibody (mAbs) covalently attached to a cytotoxic drug via a chem. linker. It combines both the advantages of highly specific targeting ability and highly potent killing effect to achieve accurate and efficient elimination of cancer cells, which has become one of the hotspots for the research and development of anticancer drugs. Since the first ADC, Mylotarg (gemtuzumab ozogamicin), was approved in 2000 by the US Food and Drug Administration (FDA), there have been 14 ADCs received market approval so far worldwide. Moreover, over 100 ADC candidates have been investigated in clin. stages at present. This kind of new anti-cancer drugs, known as "biol. missile", is leading a new era of targeted cancer therapy. Herein, we conducted a review of the history and general mechanism of action of ADCs, and then briefly discussed the mol. aspects of key components of ADCs and the mechanisms by which these key factors influence the activities of ADCs. Moreover, we also reviewed the approved ADCs and other promising candidates in phase-3 clin. trials and discuss the current challenges and future perspectives for the development of next generations, which provide insights for the research and development of novel cancer therapeutics using ADCs.
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    Brinkmann, U.; Kontermann, R. E. The Making of Bispecific Antibodies. MAbs 2017, 9, 182– 212,  DOI: 10.1080/19420862.2016.1268307
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    The making of bispecific antibodies
    Brinkmann, Ulrich; Kontermann, Roland E.
    mAbs (2017), 9 (2), 182-212CODEN: MABSCP; ISSN:1942-0870. (Taylor & Francis, Inc.)
    During the past two decades we have seen a phenomenal evolution of bispecific antibodies for therapeutic applications. The 'zoo' of bispecific antibodies is populated by many different species, comprising around 100 different formats, including small mols. composed solely of the antigen-binding sites of two antibodies, mols. with an IgG structure, and large complex mols. composed of different antigen-binding moieties often combined with dimerization modules. The application of sophisticated mol. design and genetic engineering has solved many of the tech. problems assocd. with the formation of bispecific antibodies such as stability, soly. and other parameters that confer drug properties. These parameters may be summarized under the term 'developability'. In addn., different 'target product profiles', i.e., desired features of the bispecific antibody to be generated, mandates the need for access to a diverse panel of formats. These may vary in size, arrangement, valencies, flexibility and geometry of their binding modules, as well as in their distribution and pharmacokinetic properties. There is not 'one best format' for generating bispecific antibodies, and no single format is suitable for all, or even most of, the desired applications. Instead, the bispecific formats collectively serve as a valuable source of diversity that can be applied to the development of therapeutics for various indications. Here, a comprehensive overview of the different bispecific antibody formats is provided.
  9. 9
    Tissot, A. C.; Brinkmann, U. Bispecific Antibodies. In Next-generation therapies and technologies for immune-mediated inflammatory diseases, 1st edition; Mina-Osorio, P., Ed.; Springer International Publishing: New York, 2017; p 75– 97.
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    Brinkmann, U.; Kontermann, R. E. Bispecific Antibodies. Science 1979, 2021 (372), 916– 917,  DOI: 10.1126/science.abg1209
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    Wang, Q.; Chen, Y.; Park, J.; Liu, X.; Hu, Y.; Wang, T.; McFarland, K.; Betenbaugh, M. J. Design and Production of Bispecific Antibodies. Antibodies 2019, 8, 43,  DOI: 10.3390/antib8030043
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    11
    Design and production of bispecific antibodies
    Wang, Qiong; Chen, Yiqun; Park, Jaeyoung; Liu, Xiao; Hu, Yifeng; Wang, Tiexin; McFarland, Kevin; Betenbaugh, Michael J.
    Antibodies (2019), 8 (3), 43CODEN: ANTICA; ISSN:2073-4468. (MDPI AG)
    With the current biotherapeutic market dominated by antibody mols., bispecific antibodies represent a key component of the next-generation of antibody therapy. Bispecific antibodies can target two different antigens at the same time, such as simultaneously binding tumor cell receptors and recruiting cytotoxic immune cells. Structural diversity has been fast-growing in the bispecific antibody field, creating a plethora of novel bispecific antibody scaffolds, which provide great functional variety. Two common formats of bispecific antibodies on the market are the single-chain variable fragment (scFv)-based (no Fc fragment) antibody and the full-length IgG-like asym. antibody. Unlike the conventional monoclonal antibodies, great prodn. challenges with respect to the quantity, quality, and stability of bispecific antibodies have hampered their wider clin. application and acceptance. In this review, we focus on these two major bispecific types and describe recent advances in the design, prodn., and quality of these mols., which will enable this important class of biologics to reach their therapeutic potential.
  12. 12
    Marei, H. E.; Cenciarelli, C.; Hasan, A. Potential of Antibody–Drug Conjugates (ADCs) for Cancer Therapy. Cancer Cell Int. 2022, 22, 255,  DOI: 10.1186/s12935-022-02679-8
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    12
    Potential of antibody-drug conjugates (ADCs) for cancer therapy
    Marei, Hany E.; Cenciarelli, Carlo; Hasan, Anwarul
    Cancer Cell International (2022), 22 (1), 255CODEN: CCIACC; ISSN:1475-2867. (BioMed Central Ltd.)
    A review. The primary purpose of ADCs is to increase the efficacy of anticancer medications by minimizing systemic drug distribution and targeting specific cells. Antibody conjugates (ADCs) have changed the way cancer is treated. However, because only a tiny fraction of patients experienced long-term advantages, current cancer preclin. and clin. research has been focused on combination trials. The complex interaction of ADCs with the tumor and its microenvironment appear to be reliant on the efficacy of a certain ADC, all of which have significant therapeutic consequences. Several clin. trials in various tumor types are now underway to examine the potential ADC therapy, based on encouraging preclin. results. This review tackles the potential use of ADCs in cancer therapy, emphasizing the essential processes underlying their pos. therapeutic impacts on solid and hematol. malignancies. Addnl., opportunities are explored to understand the mechanisms of ADCs action, the mechanism of resistance against ADCs, and how to overcome potential resistance following ADCs administration. Recent clin. findings have aroused interest, leading to a large increase in the no. of ADCs in clin. trials. The rationale behind ADCs, as well as their primary features and recent research breakthroughs, will be discussed. We then offer an approach for maximizing the potential value that ADCs can bring to cancer patients by highlighting key ideas and distinct strategies.
  13. 13
    Dumontet, C.; Reichert, J. M.; Senter, P. D.; Lambert, J. M.; Beck, A. Antibody-Drug Conjugates Come of Age in Oncology. Nat. Rev. Drug Discovery 2023, 22, 641– 661,  DOI: 10.1038/s41573-023-00709-2
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    Antibody-drug conjugates come of age in oncology
    Dumontet, Charles; Reichert, Janice M.; Senter, Peter D.; Lambert, John M.; Beck, Alain
    Nature Reviews Drug Discovery (2023), 22 (8), 641-661CODEN: NRDDAG; ISSN:1474-1776. (Nature Portfolio)
    Abstr.: Antibody-drug conjugates (ADCs) combine the specificity of monoclonal antibodies with the potency of highly cytotoxic agents, potentially reducing the severity of side effects by preferentially targeting their payload to the tumor site. ADCs are being increasingly used in combination with other agents, including as first-line cancer therapies. As the technol. to produce these complex therapeutics has matured, many more ADCs have been approved or are in late-phase clin. trials. The diversification of antigenic targets as well as bioactive payloads is rapidly broadening the scope of tumor indications for ADCs. Moreover, novel vector protein formats as well as warheads targeting the tumor microenvironment are expected to improve the intratumor distribution or activation of ADCs, and consequently their anticancer activity for difficult-to-treat tumor types. However, toxicity remains a key issue in the development of these agents, and better understanding and management of ADC-related toxicities will be essential for further optimization. This Review provides a broad overview of the recent advances and challenges in ADC development for cancer treatment.
  14. 14
    Beishenaliev, A.; Loke, Y. L.; Goh, S. J.; Geo, H. N.; Mugila, M.; Misran, M.; Chung, L. Y.; Kiew, L. V.; Roffler, S.; Teo, Y. Y. Bispecific Antibodies for Targeted Delivery of Anti-Cancer Therapeutic Agents: A Review. J. Controlled Release 2023, 359, 268– 286,  DOI: 10.1016/j.jconrel.2023.05.032
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    14
    Bispecific antibodies for targeted delivery of anti-cancer therapeutic agents: A review
    Beishenaliev, Adilet; Loke, Yean Leng; Goh, Sook Jing; Geo, Hui Nee; Mugila, Malar; Misran, Misni; Chung, Lip Yong; Kiew, Lik Voon; Roffler, Steve; Teo, Yin Yin
    Journal of Controlled Release (2023), 359 (), 268-286CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
    A review. Monospecific antibodies have been utilized increasingly for anti-cancer drug targeting owing to their ability to minimise off-target toxicity by binding specifically to a tumor epitope, hence selectively delivering drugs to the tumor cells. Nevertheless, the monospecific antibodies only engage a single cell surface epitope to deliver their drug payload. Hence, their performance is often unsatisfactory in cancers where multiple epitopes need to be engaged for optimal cellular internalization. In this context, bispecific antibodies (bsAbs) that simultaneously target two distinct antigens or two distinct epitopes of the same antigen offer a promising alternative in antibody-based drug delivery. This review describes the recent advances in developing bsAb-based drug delivery strategies, encompassing the direct conjugation of drug to bsAbs to form bispecific antibody-drug conjugates (bsADCs) and the surface functionalization of nanoconstructs with bsAbs to form bsAb-coupled nanoconstructs. The article first details the roles of bsAbs in enhancing the internalisation and intracellular trafficking of bsADCs with subsequent release of chemotherapeutic drugs for an augmented therapeutic efficacy, particularly among heterogeneous tumor cell populations. Then, the article discusses the roles of bsAbs in facilitating the delivery of drug-encapsulating nanoconstructs, including org./inorg. nanoparticles and large bacteria-derived minicells, that provide a larger drug loading capacity and better stability in blood circulation than bsADCs. The limitations of each type of bsAb-based drug delivery strategy and the future prospects of more versatile strategies (e.g., trispecific antibodies, autonomous drug delivery systems, theranostics) are also elaborated.
  15. 15
    Shin, H. G.; Yang, H. R.; Yoon, A.; Lee, S. Bispecific Antibody-Based Immune-Cell Engagers and Their Emerging Therapeutic Targets in Cancer Immunotherapy. Int. J. Mol. Sci. 2022, 23, 5686,  DOI: 10.3390/ijms23105686
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    15
    Bispecific Antibody-Based Immune-Cell Engagers and Their Emerging Therapeutic Targets in Cancer Immunotherapy
    Shin, Ha Gyeong; Yang, Ha Rim; Yoon, Aerin; Lee, Sukmook
    International Journal of Molecular Sciences (2022), 23 (10), 5686CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    A review. Cancer is the second leading cause of death worldwide after cardiovascular diseases. Harnessing the power of immune cells is a promising strategy to improve the antitumor effect of cancer immunotherapy. Recent progress in recombinant DNA technol. and antibody engineering has ushered in a new era of bispecific antibody (bsAb)-based immune-cell engagers (ICEs), including T- and natural-killer-cell engagers. Since the first approval of blinatumomab by the United States Food and Drug Administration (US FDA), various bsAb-based ICEs have been developed for the effective treatment of patients with cancer. Simultaneously, several potential therapeutic targets of bsAb-based ICEs have been identified in various cancers. Therefore, this review focused on not only highlighting the action mechanism, design and structure, and status of bsAb-based ICEs in clin. development and their approval by the US FDA for human malignancy treatment, but also on summarizing the currently known and emerging therapeutic targets in cancer. This review provides insights into practical considerations for developing next-generation ICEs.
  16. 16
    Chen, R. P.; Shinoda, K.; Rampuria, P.; Jin, F.; Bartholomew, T.; Zhao, C.; Yang, F.; Chaparro-Riggers, J. Bispecific Antibodies for Immune Cell Retargeting against Cancer. Expert Opin. Biol. Ther. 2022, 22, 965– 982,  DOI: 10.1080/14712598.2022.2072209
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    Bispecific antibodies for immune cell retargeting against cancer
    Chen, Rebecca P.; Shinoda, Kenta; Rampuria, Pragya; Jin, Fang; Bartholomew, Tin; Zhao, Chunxia; Yang, Fan; Chaparro-Riggers, Javier
    Expert Opinion on Biological Therapy (2022), 22 (8), 965-982CODEN: EOBTA2; ISSN:1471-2598. (Taylor & Francis Ltd.)
    A review. Following the approval of the T cell engaging bispecific antibody blinatumomab, immune cell retargeting with bispecific or multispecific antibodies has emerged as a promising cancer immunotherapy strategy, offering alternative mechanisms compared to immune checkpoint blockade. As we gain more understanding of the complex tumor microenvironment, rules and design principles have started to take shape on how to best harness the immune system to achieve optimal anti-tumor activities. In the present review, we aim to summarize the most recent advances and challenges in using bispecific antibodies for immune cell retargeting and to provide insights into various aspects of antibody engineering. Discussed herein are studies that highlight the importance of considering antibody engineering parameters, such as binding epitope, affinity, valency, and geometry to maximize the potency and mitigate the toxicity of T cell engagers. Beyond T cell engaging bispecifics, other bispecifics designed to recruit the innate immune system are also covered. Diverse and innovative mol. designs of bispecific/multispecific antibodies have the potential to enhance the efficacy and safety of immune cell retargeting for the treatment of cancer. Whether or not clin. data support these different hypotheses, esp. in solid tumor settings, remains to be seen.
  17. 17
    Ordóñez-Reyes, C.; Garcia-Robledo, J. E.; Chamorro, D. F.; Mosquera, A.; Sussmann, L.; Ruiz-Patiño, A.; Arrieta, O.; Zatarain-Barrón, L.; Rojas, L.; Russo, A. Bispecific Antibodies in Cancer Immunotherapy: A Novel Response to an Old Question. Pharmaceutics 2022, 14, 1243,  DOI: 10.3390/pharmaceutics14061243
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    17
    Bispecific Antibodies in Cancer Immunotherapy: A Novel Response to an Old Question
    Ordonez-Reyes, Camila; Garcia-Robledo, Juan Esteban; Chamorro, Diego F.; Mosquera, Andres; Sussmann, Liliana; Ruiz-Patino, Alejandro; Arrieta, Oscar; Zatarain-Barron, Lucia; Rojas, Leonardo; Russo, Alessandro; de Miguel-Perez, Diego; Rolfo, Christian; Cardona, Andres F.
    Pharmaceutics (2022), 14 (6), 1243CODEN: PHARK5; ISSN:1999-4923. (MDPI AG)
    Immunotherapy has redefined the treatment of cancer patients and it is constantly generating new advances and approaches. Among the multiple options of immunotherapy, bispecific antibodies (bsAbs) represent a novel thoughtful approach. These drugs integrate the action of the immune system in a strategy to redirect the activation of innate and adaptive immunity toward specific antigens and specific tumor locations. Here we discussed some basic aspects of the design and function of bsAbs, their main challenges and the state-of-the-art of these mols. in the treatment of hematol. and solid malignancies and future perspectives.
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    Dahlén, E.; Veitonmäki, N.; Norlén, P. Bispecific Antibodies in Cancer Immunotherapy. Ther. Adv. Vaccines Immunother. 2018, 6, 3– 17,  DOI: 10.1177/2515135518763280
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    Bispecific antibodies in cancer immunotherapy
    Dahlen, Eva; Veitonmaeki, Niina; Norlen, Per
    Therapeutic Advances in Vaccines and Immunotherapy (2018), 6 (1), 3-17CODEN: TAVIAY; ISSN:2515-1363. (Sage Publications Ltd.)
    A review. Following the clin. success of immune checkpoint antibodies targeting CTLA-4, PD-1 or PD-L1 in cancer treatment, bispecific antibodies are now emerging as a growing class of immunotherapies with potential to further improve clin. efficacy and safety. We describe three classes of immunotherapeutic bispecific antibodies: (a) cytotoxic effector cell redirectors; (b) tumor-targeted immunomodulators; and (c) dual immunomodulators. Cytotoxic effector cell redirectors are dominated by T-cell redirecting compds., bispecific compds. engaging a tumor-assocd. antigen and the T-cell receptor/CD3 complex, thereby redirecting T-cell cytotoxicity to malignant cells. This is the most established class of bispecific immunotherapies, with two compds. having reached the market and numerous compds. in clin. development. Tumor-targeted immunomodulators are bispecific compds. binding to a tumor-assocd. antigen and an immunomodulating receptor, such as CD40 or 4-1BB. Such compds. are usually designed to be inactive until binding the tumor antigen, thereby localizing immune stimulation to the tumor environment, while minimizing immune activation elsewhere. This is expected to induce powerful activation of tumor-specific T cells with reduced risk of immune-related adverse events. Finally, dual immunomodulators are bispecific compds. that bind two distinct immunomodulating targets, often combining targeting of PD-1 or PD-L1 with that of LAG-3 or TIM-3. The rationale is to induce superior tumor immunity compared to monospecific antibodies to the same targets. In this review, we describe each of these classes of bispecific antibodies, and present examples of compds. in development.
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    Huo, Y.; Sheng, Z.; Lu, D. R.; Ellwanger, D. C.; Li, C. M.; Homann, O.; Wang, S.; Yin, H.; Ren, R. Blinatumomab-Induced T Cell Activation at Single Cell Transcriptome Resolution. BMC Genom. 2021, 22, 145,  DOI: 10.1186/s12864-021-07435-2
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    Kipriyanov, S. M.; Moldenhauer, G.; Strauss, G.; Little, M. Bispecific CD3 X CD19 Diabody for T Cell-Mediated Lysis of Malignant Human B Cells. Int. J. Cancer 1998, 77, 763– 772,  DOI: 10.1002/(SICI)1097-0215(19980831)77:5<763::AID-IJC16>3.0.CO;2-2
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    Bispecific CD3 x CD19 diabody for T cell-mediated lysis of malignant human B cells
    Kipriyanov, Sergey M.; Moldenhauer, Gerhard; Strauss, Gudrun; Little, Melvyn
    International Journal of Cancer (1998), 77 (5), 763-772CODEN: IJCNAW; ISSN:0020-7136. (Wiley-Liss, Inc.)
    For the treatment of minimal residual disease in patients with leukemias and malignant lymphomas, we constructed a heterodimeric diabody specific for human CD19 on B cells and CD3E chain of the T cell receptor complex. The bispecific diabody was expressed in Escherichia coli using a vector contg. a dicistronic operon for co-secretion of VH3-VL19 and VH19-VL3 single-chain Fv fragments (scFv). It was purified in one step by immobilized metal affinity chromatog. (IMAC) from the periplasmic ext. and culture medium. Flow cytometry expts. revealed specific interactions of the diabody with both CD3 and CD19 pos. cells, to which it bound with affinities close to those of the parental scFvs. It was less stable than anti-CD3 scFv but more stable than anti-CD19 scFv when incubated in human serum at 37°C. In cytotoxicity tests, the diabody proved to be a potent agent for retargeting peripheral blood lymphocytes to lyse tumor cells expressing the CD19 antigen. The efficiency of cell lysis compared favorably with that obtained with a bispecific antibody (BsAb) of the same dual specificity that was prepd. by the quadroma technique.
  21. 21
    Klein, C.; Schaefer, W.; Regula, J. T.; Dumontet, C.; Brinkmann, U.; Bacac, M.; Umaña, P. Engineering Therapeutic Bispecific Antibodies Using CrossMab Technology. Methods 2019, 154, 21– 31,  DOI: 10.1016/j.ymeth.2018.11.008
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    Engineering therapeutic bispecific antibodies using CrossMab technology
    Klein, Christian; Schaefer, Wolfgang; Regula, Joerg T.; Dumontet, Charles; Brinkmann, Ulrich; Bacac, Marina; Umana, Pablo
    Methods (Amsterdam, Netherlands) (2019), 154 (), 21-31CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)
    A review. Bispecific antibodies have recently gained major interest as they allow novel mechanisms-of-action and/or therapeutic applications that cannot be achieved using conventional IgG-based antibodies. A major issue in engineering IgG-based bispecific antibodies has been to enable the correct assocn. of heavy and light chains resulting in correct assembly of the desired bispecific antibody in sufficient yield. Various approaches have been described during recent years to tackle this challenge. We have developed the so-called CrossMab technol. that enforces correct light chain assocn. based on the domain crossover of Ig domains in the Fab region of the bispecific antibody. This versatile technol. allows the generation of different bispecific antibody formats including asym. heterodimeric monovalent 1 + 1 bispecific antibodies and asym. heterodimeric bispecific antibodies with 2 + 1 valency in combination with approaches enabling Fc-hetermodimerization like knob-into-hole technol. as well as the generation of tetravalent sym. bispecific antibodies with 2 + 2 valency, also known as Tandem-Fab based IgG antibodies, using processes suitable for the large scale prodn. of therapeutic bispecific antibodies. Notably, as of now, at least eight different bispecific antibodies using CrossMab technol. entered clin. development, and addnl. CrossMabs are in late preclin. development. This review provides a summary of the status and progress with the engineering and generation of CrossMab technol. based bispecific antibodies as well as their therapeutic application.
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    Ellerman, D. Bispecific T-Cell Engagers: Towards Understanding Variables Influencing the in Vitro Potency and Tumor Selectivity and Their Modulation to Enhance Their Efficacy and Safety. Methods 2019, 154, 102– 117,  DOI: 10.1016/j.ymeth.2018.10.026
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    Bispecific T-cell engagers: Towards understanding variables influencing the in vitro potency and tumor selectivity and their modulation to enhance their efficacy and safety
    Ellerman, Diego
    Methods (Amsterdam, Netherlands) (2019), 154 (), 102-117CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)
    A review. Bispecific mols. redirecting the cytotoxicity of T-cells are a growing class of therapeutics with numerous mols. being tested in clin. trials. However, it has been a long way since the proof of concept studies in the mid 1980's. In the process we have learnt about the impact of different variables related to the bispecific mol. and the target antigen on the potency of this type of drugs. This work reviews the insights gained and how that knowledge has been used to design more potent bispecific T-cell engagers. The more recent advancement of antibodies with this modality into safety studies in non-human primates and as well as in clin. studies has revealed potential toxicity liabilities for the mode of action. Modifications in existing antibody formats and new exptl. mols. designed to mitigate these problems are discussed.
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    Moore, P. A.; Zhang, W.; Rainey, G. J.; Burke, S.; Li, H.; Huang, L.; Gorlatov, S.; Veri, M. C.; Aggarwal, S.; Yang, Y.; Shah, K. Application of Dual Affinity Retargeting Molecules to Achieve Optimal Redirected T-Cell Killing of B-Cell Lymphoma. Blood 2011, 117, 4542– 4551,  DOI: 10.1182/blood-2010-09-306449
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    Application of dual affinity retargeting molecules to achieve optimal redirected T-cell killing of B-cell lymphoma
    Moore, Paul A.; Zhang, Wenjun; Rainey, G. Jonah; Burke, Steve; Li, Hua; Huang, Ling; Gorlatov, Sergey; Veri, Maria Concetta; Aggarwal, Sudeepta; Yang, Yinhua; Shah, Kalpana; Jin, Linda; Zhang, Sunan; He, Leilei; Zhang, Tengfei; Ciccarone, Valentina; Koenig, Scott; Bonvini, Ezio; Johnson, Syd
    Blood (2011), 117 (17), 4542-4551CODEN: BLOOAW; ISSN:0006-4971. (American Society of Hematology)
    We describe the application of a novel, bispecific antibody platform termed dual affinity retargeting (DART) to eradicate B-cell lymphoma through coengagement of the B cell-specific antigen CD19 and the TCR/CD3 complex on effector T cells. Comparison with a single-chain, bispecific antibody bearing identical CD19 and CD3 antibody Fv sequences revealed DART mols. to be more potent in directing B-cell lysis. The enhanced activity with the CD19 × CD3 DART mols. was obsd. on all CD19-expressing target B cells evaluated using resting and prestimulated human PBMCs or purified effector T-cell populations. Characterization of a CD19 × TCR bispecific DART mol. revealed equiv. potency with the CD19 × CD3 DART mol., demonstrating flexibility of the DART structure to support T-cell/B-cell assocns. for redirected T cell-killing applications. The enhanced level of killing mediated by DART mols. was not accompanied by any increase in nonspecific T-cell activation or lysis of CD19- cells. Cell-assocn. studies indicated that the DART architecture is well suited for maintaining cell-to-cell contact, apparently contributing to the high level of target cell killing. Finally, the ability of the CD19 × TCR DART to inhibit B-cell lymphoma in NOD/SCID mice when coadministered with human PBMCs supports further evaluation of DART mols. for the treatment of B-cell malignancies.
  24. 24
    Reusch, U.; Harrington, K. H.; Gudgeon, C. J.; Fucek, I.; Ellwanger, K.; Weichel, M.; Knackmuss, S. H. J.; Zhukovsky, E. A.; Fox, J. A.; Kunkel, L. A. Characterization of CD33/CD3 Tetravalent Bispecific Tandem Diabodies (TandAbs) for the Treatment of Acute Myeloid Leukemia. Clin. Cancer Res. 2016, 22, 5829– 5838,  DOI: 10.1158/1078-0432.CCR-16-0350
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    24
    Characterization of CD33/CD3 Tetravalent Bispecific Tandem Diabodies (TandAbs) for the Treatment of Acute Myeloid Leukemia
    Reusch, Uwe; Harrington, Kimberly H.; Gudgeon, Chelsea J.; Fucek, Ivica; Ellwanger, Kristina; Weichel, Michael; Knackmuss, Stefan H. J.; Zhukovsky, Eugene A.; Fox, Judith A.; Kunkel, Lori A.; Guenot, Jeanmarie; Walter, Roland B.
    Clinical Cancer Research (2016), 22 (23), 5829-5838CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
    Purpose: Randomized studies with gemtuzumab ozogamicin have validated CD33 as a target for antigen-specific immunotherapy of acute myelogenous leukemia (AML). Here, we investigated the potential of CD33/CD3-directed tandem diabodies (TandAbs) as novel treatment approach for AML. These tetravalent bispecific antibodies provide two binding sites for each antigen to maintain the avidity of a bivalent antibody and have a mol. wt. exceeding the renal clearance threshold, thus offering a longer half-life compared to smaller antibody constructs. Exptl. Design: We constructed a series of TandAbs composed of anti-CD33 and anti-CD3 variable domains of diverse binding affinities and profiled their functional properties in CD33+ human leukemia cell lines, xenograft models, and AML patient samples. Results: Our studies demonstrated that several CD33/CD3 TandAbs could induce potent, dose-dependent cytolysis of CD33+ AML cell lines. This effect was modulated by the effector-to-target cell ratio and strictly required the presence of T cells. Activation and proliferation of T cells and maximal AML cell cytolysis correlated with high avidity to both CD33 and CD3. High-avidity TandAbs were broadly active in primary specimens from patients with newly diagnosed or relapsed/refractory AML in vitro, with cytotoxic properties independent of CD33 receptor d. and cytogenetic risk. Tumor growth delay and inhibition were obsd. in both prophylactic and established HL-60 xenograft models in immunodeficient mice. Conclusions: Our data show high efficacy of CD33/CD3 TandAbs in various preclin. models of human AML. Together, these findings support further study of CD33/CD3 TandAbs as novel immunotherapeutics for patients with AML. Clin Cancer Res; 22(23); 5829-38. ©2016 AACR.
  25. 25
    Thoreau, F.; Szijj, P. A.; Greene, M. K.; Rochet, L. N. C.; Thanasi, I. A.; Blayney, J. K.; Maruani, A.; Baker, J. R.; Scott, C. J.; Chudasama, V. Modular Chemical Construction of IgG-like Mono- and Bispecific Synthetic Antibodies (SynAbs). ACS Cent. Sci. 2023, 9, 476– 487,  DOI: 10.1021/acscentsci.2c01437
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    25
    Modular Chemical Construction of IgG-like Mono- and Bispecific Synthetic Antibodies (SynAbs)
    Thoreau, Fabien; Szijj, Peter A.; Greene, Michelle K.; Rochet, Lea N. C.; Thanasi, Ioanna A.; Blayney, Jaine K.; Maruani, Antoine; Baker, James R.; Scott, Christopher J.; Chudasama, Vijay
    ACS Central Science (2023), 9 (3), 476-487CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
    In recent years there has been rising interest in the field of protein-protein conjugation, esp. related to bispecific antibodies (bsAbs) and their therapeutic applications. These constructs contain two paratopes capable of binding two distinct epitopes on target mols. and are thus able to perform complex biol. functions (mechanisms of action) not available to monospecific mAbs. Traditionally these bsAbs have been constructed through protein engineering, but recently chem. methods for their construction have started to (re)emerge. While these have been shown to offer increased modularity, speed, and for some methods even the inherent capacity for further functionalization (e.g., with small mol. cargo), most of these approaches lacked the ability to include a fragment crystallizable (Fc) modality. The Fc component of IgG antibodies offers effector function and increased half-life. Here we report a first-in-class disulfide rebridging and click-chem.-based method for the generation of Fc-contg., IgG-like mono- and bispecific antibodies. These are in the FcZ-(FabX)-FabY format, i.e., two distinct Fabs and an Fc, potentially all from different antibodies, attached in a homogeneous and covalent manner. We have dubbed these mols. synthetic antibodies (SynAbs). We have constructed a T cell-engager (TCE) SynAb, FcCD20-(FabHER2)-FabCD3, and have confirmed that it exhibits the expected biol. functions, including the ability to kill HER2+ target cells in a coculture assay with T cells.
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    Schaefer, W.; Regula, J. T.; Bähner, M.; Schanzer, J.; Croasdale, R.; Dürr, H.; Gassner, C.; Georges, G.; Kettenberger, H.; Imhof-Jung, S. Immunoglobulin Domain Crossover as a Generic Approach for the Production of Bispecific IgG Antibodies. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 11187– 11192,  DOI: 10.1073/pnas.1019002108
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    Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies
    Schaefer, Wolfgang; Regula, Jorg T.; Bahner, Monika; Schanzer, Jurgen; Croasdale, Rebecca; Durr, Harald; Gassner, Christian; Georges, Guy; Kettenberger, Hubert; Imhof-Jung, Sabine; Schwaiger, Manfred; Stubenrauch, Kay G.; Sustmann, Claudio; Thomas, Markus; Scheuer, Werner; Klein, Christian
    Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (27), 11187-11192, S11187/1-S11187/10CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technol. that enables heterodimerization of the heavy chains, correct assocn. of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This "crossover" retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible "CrossMab" formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by std. techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMabCH1-CL was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.
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    Madsen, A. V.; Kristensen, P.; Buell, A. K.; Goletz, S. Generation of Robust Bispecific Antibodies through Fusion of Single-Domain Antibodies on IgG Scaffolds: A Comprehensive Comparison of Formats. MAbs 2023, 15, 1,  DOI: 10.1080/19420862.2023.2189432
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    Kühl, L.; Aschmoneit, N.; Kontermann, R. E.; Seifert, O. The EIg Technology to Generate Ig-like Bispecific Antibodies. MAbs 2022, 14, 1,  DOI: 10.1080/19420862.2022.2063043
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    de Bever, L.; Popal, S.; van Schaik, J.; Rubahamya, B.; van Delft, F. L.; Thurber, G. M.; van Berkel, S. S. Generation of DAR1 Antibody-Drug Conjugates for Ultrapotent Payloads Using Tailored GlycoConnect Technology. Bioconjugate Chem. 2023, 34, 538– 548,  DOI: 10.1021/acs.bioconjchem.2c00611
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    29
    Generation of DAR1 Antibody-Drug Conjugates for Ultrapotent Payloads Using Tailored GlycoConnect Technology
    de Bever, Laureen; Popal, Sorraya; van Schaik, Jord; Rubahamya, Baron; van Delft, Floris L.; Thurber, Greg M.; van Berkel, Sander S.
    Bioconjugate Chemistry (2023), 34 (3), 538-548CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    GlycoConnect technol. can be readily adapted to provide different drug-to-antibody ratios (DARs) and is currently also evaluated in various clin. programs, including ADCT-601 (DAR2), MRG004a (DAR4), and XMT-1660 (DAR6). While antibody-drug conjugates (ADCs) typically feature a DAR2-8, it has become clear that ADCs with ultrapotent payloads (e.g., PBD dimers and calicheamicin) can only be administered to patients at low doses (<0.5 mg/kg), which may compromise effective biodistribution and may be insufficient to reach target receptor satn. in the tumor. Here, we show that GlycoConnect technol. can be readily extended to DAR1 ADCs without the need of antibody re-engineering. We demonstrate that various ultrapotent, cytotoxic payloads are amenable to this methodol. In a follow-up expt., HCC-1954 tumor spheroids were treated with either an AlexaFluor647-labeled DAR1 or DAR2 PBD-based ADC to study the effect on tumor penetration. Significant improvement of tumor spheroid penetration was obsd. for the DAR1 ADC compared to the DAR2 ADC at an equal payload dose, underlining the potential of a lower DAR for ADCs bearing ultrapotent payloads.
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    Van Berkel, S. S.; van Delft, F. L. Enzymatic Strategies for (near) Clinical Development of Antibody-Drug Conjugates. Drug Discovery Today: Technol. 2018, 30, 3– 10,  DOI: 10.1016/j.ddtec.2018.09.005
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    Enzymatic strategies for (near) clinical development of antibody-drug conjugates
    van Berkel Sander S; van Delft Floris L
    Drug discovery today. Technologies (2018), 30 (), 3-10 ISSN:.
    Target-specific killing of tumor cells with antibody-drug conjugates (ADCs) is an elegant concept in the continued fight against cancer. However, despite more than 20 years of clinical development, only four ADC have reached market approval, while at least 50 clinical programs were terminated early. The high attrition rate of ADCs may, at least in part, be attributed to heterogeneity and instability of conventional technologies. At present, various (chemo)enzymatic approaches for site-specific and stable conjugation of toxic payloads are making their way to the clinic, thereby potentially providing ADCs with increased therapeutic window.
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    Van Geel, R.; Wijdeven, M. A.; Heesbeen, R.; Verkade, J. M. M.; Wasiel, A. A.; Van Berkel, S. S.; Van Delft, F. L. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native MAbs Provides Homogeneous and Highly Efficacious Antibody-Drug Conjugates. Bioconjugate Chem. 2015, 26, 2233– 2242,  DOI: 10.1021/acs.bioconjchem.5b00224
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    31
    Chemoenzymatic conjugation of toxic payloads to the globally conserved N-glycan of native mAbs provides homogeneous and highly efficacious antibody-drug conjugates
    van Geel, Remon; Wijdeven, Marloes A.; Heesbeen, Ryan; Verkade, Jorge M. M.; Wasiel, Anna A.; van Berkel, Sander S.; van Delft, Floris L.
    Bioconjugate Chemistry (2015), 26 (11), 2233-2242CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    A robust, generally applicable, nongenetic technol. is presented to convert monoclonal antibodies into stable and homogeneous ADCs. Starting from a native (nonengineered) mAb, a chemoenzymic protocol allows for the highly controlled attachment of any given payload to the N-glycan residing at asparagine-297, based on a two-stage process: first, enzymic remodeling (trimming and tagging with azide), followed by ligation of the payload based on copper-free click chem. The technol., termed GlycoConnect, is applicable to any IgG isotype irresp. of glycosylation profile. Application to trastuzumab and maytansine, both components of the marketed ADC Kadcyla, demonstrate a favorable in vitro and in vivo efficacy for GlycoConnect ADC. Moreover, the superiority of the native glycan as attachment site was demonstrated by in vivo comparison to a range of trastuzumab-based glycosylation mutants. A side-by-side comparison of the copper-free click probes bicyclononyne (BCN) and a dibenzoannulated cyclooctyne (DBCO) showed a surprising difference in conjugation efficiency in favor of BCN, which could be even further enhanced by introduction of electron-withdrawing fluoride substitutions onto the azide. The resulting mAb-conjugates were in all cases found to be highly stable, which in combination with the demonstrated efficacy warrants ADCs with a superior therapeutic index.
  32. 32
    Evans, H. L.; Nguyen, Q. D.; Carroll, L. S.; Kaliszczak, M.; Twyman, F. J.; Spivey, A. C.; Aboagye, E. O. A Bioorthogonal 68Ga-Labelling Strategy for Rapid in Vivo Imaging. Chem. Commun. 2014, 50, 9557– 9560,  DOI: 10.1039/C4CC03903C
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    A bioorthogonal 68Ga-labelling strategy for rapid in vivo imaging
    Evans, Helen L.; Nguyen, Quang-De; Carroll, Laurence S.; Kaliszczak, Maciej; Twyman, Frazer J.; Spivey, Alan C.; Aboagye, Eric O.
    Chemical Communications (Cambridge, United Kingdom) (2014), 50 (67), 9557-9560CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    Herein, we describe a fast and robust method for achieving 68Ga-labeling of the EGFR-selective monoclonal antibody (mAb) Cetuximab using the bioorthogonal Inverse-electron-Demand Diels-Alder (IeDDA) reaction. The in vivo imaging of EGFR is demonstrated, as well as the translation of the method within a two-step pretargeting strategy.
  33. 33
    Taylor, R. J.; Geeson, M. B.; Journeaux, T.; Bernardes, G. J. L. Chemical and Enzymatic Methods for Post-Translational Protein-Protein Conjugation. J. Am. Chem. Soc. 2022, 144, 14404– 14419,  DOI: 10.1021/jacs.2c00129
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    Chemical and Enzymatic Methods for Post-Translational Protein-Protein Conjugation
    Taylor, Ross J.; Geeson, Michael B.; Journeaux, Toby; Bernardes, Goncalo J. L.
    Journal of the American Chemical Society (2022), 144 (32), 14404-14419CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A review. Fusion proteins play an essential role in the biosciences but suffer from several key limitations, including the requirement for N-to-C terminal ligation, incompatibility of constituent domains, incorrect folding, and loss of biol. activity. This perspective focuses on chem. and enzymic approaches for the post-translational generation of well-defined protein-protein conjugates, which overcome some of the limitations faced by traditional fusion techniques. Methods discussed range from chem. modification of nucleophilic canonical amino acid residues to incorporation of unnatural amino acid residues and a range of enzymic methods, including sortase-mediated ligation. Through summarizing the progress in this rapidly growing field, the key successes and challenges assocd. with using chem. and enzymic approaches are highlighted and areas requiring further development are discussed.
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    Bruins, J. J.; Damen, J. A. M.; Wijdeven, M. A.; Lelieveldt, L. P. W. M.; Van Delft, F. L.; Albada, B. Non-Genetic Generation of Antibody Conjugates Based on Chemoenzymatic Tyrosine Click Chemistry. Bioconjugate Chem. 2021, 32, 2167– 2172,  DOI: 10.1021/acs.bioconjchem.1c00351
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    34
    Non-Genetic Generation of Antibody Conjugates Based on Chemo enzymatic Tyrosine Click Chemistry
    Bruins, Jorick J.; Damen, Johannes A. M.; Wijdeven, Marloes A.; Lelieveldt, Lianne P. W. M.; van Delft, Floris L.; Albada, Bauke
    Bioconjugate Chemistry (2021), 32 (10), 2167-2172CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    The availability of tools to generate homogeneous and stable antibody conjugates without recombinant DNA technol. is a valuable asset in fields spanning from in vitro diagnostics to in vivo imaging and therapeutics. We present here a general approach for the conjugation to human IgG1 antibodies, by employing a straightforward two-stage protocol based on antibody deglycosylation followed by tyrosinase-mediated ortho-quinone strain-promoted click chem. The technol. is validated by the efficient and clean generation of highly potent DAR2 and DAR4 antibody-drug conjugates (ADCs) with cytotoxic payloads MMAE or PBD dimer, and their in vitro evaluation.
  35. 35
    Oliveira, B. L.; Guo, Z.; Bernardes, G. J. L. Inverse Electron Demand Diels-Alder Reactions in Chemical Biology. Chem. Soc. Rev. 2017, 46, 4895– 4950,  DOI: 10.1039/C7CS00184C
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    Inverse electron demand Diels-Alder reactions in chemical biology
    Oliveira, B. L.; Guo, Z.; Bernardes, G. J. L.
    Chemical Society Reviews (2017), 46 (16), 4895-4950CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    The emerging inverse electron demand Diels-Alder (IEDDA) reaction stands out from other bioorthogonal reactions by virtue of its unmatchable kinetics, excellent orthogonality and biocompatibility. With the recent discovery of novel dienophiles and optimal tetrazine coupling partners, attention has now been turned to the use of IEDDA approaches in basic biol., imaging and therapeutics. Here we review this bioorthogonal reaction and its promising applications for live cell and animal studies. We first discuss the key factors that contribute to the fast IEDDA kinetics and describe the most recent advances in the synthesis of tetrazine and dienophile coupling partners. Both coupling partners have been incorporated into proteins for tracking and imaging by use of fluorogenic tetrazines that become strongly fluorescent upon reaction. Selected notable examples of such applications are presented. The exceptional fast kinetics of this catalyst-free reaction, even using low concns. of coupling partners, make it amenable for in vivo radiolabelling using pretargeting methodologies, which are also discussed. Finally, IEDDA reactions have recently found use in bioorthogonal decaging to activate proteins or drugs in gain-of-function strategies. We conclude by showing applications of the IEDDA reaction in the construction of biomaterials that are used for drug delivery and multimodal imaging, among others. The use and utility of the IEDDA reaction is interdisciplinary and promises to revolutionize chem. biol., radiochem. and materials science.
  36. 36
    Maruani, A.; Szijj, P. A.; Bahou, C.; Nogueira, J. C. F.; Caddick, S.; Baker, J. R.; Chudasama, V. A Plug-and-Play Approach for the de Novo Generation of Dually Functionalized Bispecifics. Bioconjugate Chem. 2020, 31, 520– 529,  DOI: 10.1021/acs.bioconjchem.0c00002
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    36
    A Plug-and-Play Approach for the De Novo Generation of Dually Functionalized Bispecifics
    Maruani, Antoine; Szijj, Peter A.; Bahou, Calise; Nogueira, Joao C. F.; Caddick, Stephen; Baker, James R.; Chudasama, Vijay
    Bioconjugate Chemistry (2020), 31 (3), 520-529CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    Diseases are multifactorial, with redundancies and synergies between various pathways. However, most of the antibody-based therapeutics on the market interact with only one target, thus limiting their efficacy. The targeting of multiple epitopes could improve the therapeutic index of treatment and counteract mechanisms of resistance. To this effect, a new class of therapeutics has emerged: bispecific antibodies. Bispecific formation using chem. methods is rare and low-yielding and/or requires a large excess of one of the two proteins to avoid homodimerization and heterogeneity. In order for chem. prepd. bispecifics to deliver their full potential, high-yielding, modular, and reliable crosslinking technologies are required. Herein, we describe a novel approach not only for the rapid and high-yielding chem. generation of bispecific antibodies from native antibody fragments, but also for the site-specific dual functionalization of the resulting bioconjugates. Based on orthogonal clickable functional groups, this strategy enables the assembly of functionalized bispecifics with controlled loading in a modular and convergent manner.
  37. 37
    Maruani, A.; Smith, M. E. B.; Miranda, E.; Chester, K. A.; Chudasama, V.; Caddick, S. A Plug-and-Play Approach to Antibody-Based Therapeutics via a Chemoselective Dual Click Strategy. Nat. Commun. 2015, 6, 2– 10,  DOI: 10.1038/ncomms7645
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    Damen, J. A. M.; Escorihuela, J.; Zuilhof, H.; van Delft, F.; Albada, B. High Rates of Quinone-Alkyne Cycloaddition Reactions Are Dictated by Entropic Factors. Chem.─Eur. J. 2023, 29, e202300231  DOI: 10.1002/chem.202300231
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    38
    High Rates of Quinone-Alkyne Cycloaddition Reactions are Dictated by Entropic Factors
    Damen, Johannes A. M.; Escorihuela, Jorge; Zuilhof, Han; van Delft, Floris L.; Albada, Bauke
    Chemistry - A European Journal (2023), 29 (39), e202300231CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Reaction rates of strained cycloalkynes and cycloalkenes with 1,2-quinone were quantified by stopped flow UV-Vis spectroscopy and computational anal. We found that the strained alkyne BCN-OH 3 (ks 1824 M-1 s-1) reacts >150 times faster than the strained alkene TCO-OH 5 (k2 11.56 M-1 s-1), and that derivatization with a carbamate can lead to a redn. of the rate const. with almost half. Also, the 8-membered strained alkyne BCN-OH 3 reacts 16 times faster than the more strained 7-membered THS 2 (k2 110.6 M-1 s-1). Using the linearized Eyring equation we detd. the thermodn. activation parameters of these two strained alkynes, revealing that the SPOCQ reaction of quinone 1 with THS 2 is assocd. with ΔH≠ of 0.80 kcal/mol, ΔS≠=-46.8 cal/K·mol, and ΔG≠=14.8 kcal/mol (at 25°C), whereas the same reaction with BCN-OH 3 is assocd. with, ΔH≠=2.25 kcal/mol, ΔS≠=-36.3 cal/K·mol, and ΔG≠=13.1 kcal/mol (at 25°C). Computational anal. supported the values obtained by the stopped-flow measurements, with calcd. ΔG≠ of 15.6 kcal/mol (in H2O) for the SPOCQ reaction with THS 2, and with ΔG≠ of 14.7 kcal/mol (in H2O) for the SPOCQ reaction with BCN-OH 3. With these empirically detd. thermodn. parameters, we set an important step towards a more fundamental understanding of this set of rapid click reactions.

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  • Abstract

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    Scheme 1

    Scheme 1. Conversion of Native mAbs to Bispecific Constructsa
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    a(A) Synthesis of BCN-PEG3-TCO (1) and chemical structure of MeTz-PEG3-Br2PD (2). (B) Installation of MeTz handle on OKT3 Fab by rebridging the light-chain and heavy-chain Fab fragments obtained after digestion and reduction. Structure of MeTz-rbFabHER2 (3b) that is used to construct the negative control is shown in the inset. (C) Chemoenzymatic functionalization of native mAb trastuzumab with a TCO handle using BCN-PEG3-TCO (1), resulting in deglycosylated TCO-functionalized trastuzumab (4). The bispecific antibody construct (bsAc, 5a) was obtained after reaction with MeTz-rbFabCD3 (3a) by TCO-Tz IEDDA.

    Figure 1

    Figure 1. SEC-nMS analysis of trastuzumab samples obtained during assembly of the bispecific constructs via biogenic tyrosine click chemistry. (A) SEC-UV chromatogram of deglycosylated Trz (black), trastuzumab-TCO 4 (blue), HER2xCD3 bsAc 5a (orange), and HER2xHER2 bsAc 5b (pink). Relative quantification of each species is performed upon the integration of chromatographic peak areas. (B) Native MS spectra of the major peak from each sample namely, monomer of deglycosylated Trz (black), monomer of trastuzumab-TCO (4) (blue), 2:2 HER2xCD3 bsAc 5a (orange), and 2:2 HER2xHER2 bsAc 5b (pink). (C) Deconvoluted values of the relevant species are provided with a standard deviation obtained from at least four different charge states; masses of minor species are summarized in Supporting Information (Figure S19).

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    Figure 2

    Figure 2. Biological activity studies of the bispecific antibody construct and controls. (A) (i) Flow cytometry analysis of binding of the various constructs to HCC1954 (HER2+CD3–) and Jurkat (HER2–CD3+) cells; (ii) binding of the constructs to HCC1954 (HER2+CD3–) cells shown as mean fluorescence intensity (MFI); and (iii) binding of the constructs to Jurkat (HER2–CD3+) cells shown as MFI (n = 3). (B) Induction of IFN-γ production and excretion by the various constructs and controls (5 nM) in T cells or HCC1954 (HER2+CD3–) cells alone or HCC1954/T-cell cocultures (ratio 1:10). Culture supernatant IFN-γ was quantified by ELISA at 48 h following treatment. (C) Cellular metabolism assay as a measure of HCC1954 cell viability affected by the synthetic constructs in the presence of HCC1954 (HER2+CD3–) cells alone or HCC1954/T-cell cocultures (E/T ratio 10:1, 5 nM construct). HCC1954 viability was assessed by Cell Titer-Glo at 48 h following treatment. (D) Cytotoxicity dose–response curve of the bsAc 5a on HCC1954/T-cell cocultures (ratio 1:10) were incubated with varying concentrations (serial dilutions ranging from 0.0128 pM to 5 nM; donors are indicated with D#1, D#9, and D#10). HCC1954 cell viability was assessed by Cell Titer-Glo at 48 h following treatment, where the IC50 value was extrapolated. Statistical analysis was performed in GraphPad Prism (v9.5.1), where the data is presented as mean ± SEM. Statistical significance was established by Two-way ANOVA and Šídák’s multiple comparisons test (ns denotes “no significance” and **** p ≤ 0.0001).

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  • References

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      A Novel Carcinoembryonic Antigen T-Cell Bispecific Antibody (CEA TCB) for the Treatment of Solid Tumors
      Bacac, Marina; Fauti, Tanja; Sam, Johannes; Colombetti, Sara; Weinzierl, Tina; Ouaret, Djamila; Bodmer, Walter; Lehmann, Steffi; Hofer, Thomas; Hosse, Ralf J.; Moessner, Ekkehard; Ast, Oliver; Bruenker, Peter; Grau-Richards, Sandra; Schaller, Teilo; Seidl, Annette; Gerdes, Christian; Perro, Mario; Nicolini, Valeria; Steinhoff, Nathalie; Dudal, Sherri; Neumann, Sebastian; von Hirschheydt, Thomas; Jaeger, Christiane; Saro, Jose; Karanikas, Vaios; Klein, Christian; Umana, Pablo
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      Purpose: CEA TCB is a novel IgG-based T-cell bispecific (TCB) antibody for the treatment of CEA-expressing solid tumors currently in phase I clin. trials (NCT02324257). Its format incorporates bivalent binding to CEA, a head-to-tail fusion of CEA- and CD3e-binding Fab domains and an engineered Fc region with completely abolished binding to FcγRs and C1q. The study provides novel mechanistic insights into the activity and mode of action of CEA TCB. Exptl. Design: CEA TCB activity was characterized on 110 cell lines in vitro and in xenograft tumor models in vivo using NOG mice engrafted with human peripheral blood mononuclear cells. Results: Simultaneous binding of CEA TCB to tumor and T cells leads to formation of immunol. synapses, T-cell activation, secretion of cytotoxic granules, and tumor cell lysis. CEA TCB activity strongly correlates with CEA expression, with higher potency obsd. in highly CEA-expressing tumor cells and a threshold of approx. 10,000 CEA-binding sites/cell, which allows distinguishing between high- and low-CEA-expressing tumor and primary epithelial cells, resp. Genetic factors do not affect CEA TCB activity confirming that CEA expression level is the strongest predictor of CEA TCB activity. In vivo, CEA TCB induces regression of CEA-expressing xenograft tumors with variable amts. of immune cell infiltrate, leads to increased frequency of activated T cells, and converts PD-L1 neg. into PD-L1-pos. tumors. Conclusions: CEA TCB is a novel generation TCB displaying potent antitumor activity; it is efficacious in poorly infiltrated tumors where it increases T-cell infiltration and generates a highly inflamed tumor microenvironment. Clin Cancer Res; 22(13); 3286-97. ©2016 AACR.
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      A review. It has been more than three decades since the first monoclonal antibody was approved by the United States Food and Drug Administration (US FDA) in 1986, and during this time, antibody engineering has dramatically evolved. Current antibody drugs have increasingly fewer adverse effects due to their high specificity. As a result, therapeutic antibodies have become the predominant class of new drugs developed in recent years. Over the past five years, antibodies have become the best-selling drugs in the pharmaceutical market, and in 2018, eight of the top ten bestselling drugs worldwide were biologics. The global therapeutic monoclonal antibody market was valued at approx. US$115.2 billion in 2018 and is expected to generate revenue of $150 billion by the end of 2019 and $300 billion by 2025. Thus, the market for therapeutic antibody drugs has experienced explosive growth as new drugs have been approved for treating various human diseases, including many cancers, autoimmune, metabolic and infectious diseases. As of Dec. 2019, 79 therapeutic mAbs have been approved by the US FDA, but there is still significant growth potential. This review summarizes the latest market trends and outlines the preeminent antibody engineering technologies used in the development of therapeutic antibody drugs, such as humanization of monoclonal antibodies, phage display, the human antibody mouse, single B cell antibody technol., and affinity maturation. Finally, future applications and perspectives are also discussed.
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      Jin, S.; Sun, Y.; Liang, X.; Gu, X.; Ning, J.; Xu, Y.; Chen, S.; Pan, L. Emerging New Therapeutic Antibody Derivatives for Cancer Treatment. Signal Transduct. Targeted Ther. 2022, 7, 39,  DOI: 10.1038/s41392-021-00868-x
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      Emerging new therapeutic antibody derivatives for cancer treatment
      Jin, Shijie; Sun, Yanping; Liang, Xiao; Gu, Xinyu; Ning, Jiangtao; Xu, Yingchun; Chen, Shuqing; Pan, Liqiang
      Signal Transduction and Targeted Therapy (2022), 7 (1), 39CODEN: STTTCB; ISSN:2059-3635. (Nature Portfolio)
      A review. Monoclonal antibodies constitute a promising class of targeted anticancer agents that enhance natural immune system functions to suppress cancer cell activity and eliminate cancer cells. The successful application of IgG monoclonal antibodies has inspired the development of various types of therapeutic antibodies, such as antibody fragments, bispecific antibodies, and antibody derivs. (e.g., antibody-drug conjugates and immunocytokines). The miniaturization and multifunctionalization of antibodies are flexible and viable strategies for diagnosing or treating malignant tumors in a complex tumor environment. In this review, we summarize antibodies of various mol. types, antibody applications in cancer therapy, and details of clin. study advances. We also discuss the rationale and mechanism of action of various antibody formats, including antibody-drug conjugates, antibody-oligonucleotide conjugates, bispecific/multispecific antibodies, immunocytokines, antibody fragments, and scaffold proteins. With advances in modern biotechnol., well-designed novel antibodies are finally paving the way for successful treatments of various cancers, including precise tumor immunotherapy, in the clinic.
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      Ackerman, S. E.; Pearson, C. I.; Gregorio, J. D.; Gonzalez, J. C.; Kenkel, J. A.; Hartmann, F. J.; Luo, A.; Ho, P. Y.; LeBlanc, H.; Blum, L. K. Immune-Stimulating Antibody Conjugates Elicit Robust Myeloid Activation and Durable Antitumor Immunity. Nat. Cancer 2021, 2, 18– 33,  DOI: 10.1038/s43018-020-00136-x
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      Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity
      Ackerman, Shelley E.; Pearson, Cecelia I.; Gregorio, Joshua D.; Gonzalez, Joseph C.; Kenkel, Justin A.; Hartmann, Felix J.; Luo, Angela; Ho, Po Y.; LeBlanc, Heidi; Blum, Lisa K.; Kimmey, Samuel C.; Luo, Andrew; Nguyen, Murray L.; Paik, Jason C.; Sheu, Lauren Y.; Ackerman, Benjamin; Lee, Arthur; Li, Hai; Melrose, Jennifer; Laura, Richard P.; Ramani, Vishnu C.; Henning, Karla A.; Jackson, David Y.; Safina, Brian S.; Yonehiro, Grant; Devens, Bruce H.; Carmi, Yaron; Chapin, Steven J.; Bendall, Sean C.; Kowanetz, Marcin; Dornan, David; Engleman, Edgar G.; Alonso, Michael N.
      Nature Cancer (2021), 2 (1), 18-33CODEN: NCAADQ; ISSN:2662-1347. (Springer International Publishing AG)
      Innate pattern recognition receptor agonists, including Toll-like receptors (TLRs), alter the tumor microenvironment and prime adaptive antitumor immunity. However, TLR agonists present toxicities assocd. with widespread immune activation after systemic administration. To design a TLR-based therapeutic suitable for systemic delivery and capable of safely eliciting tumor-targeted responses, we developed immune-stimulating antibody conjugates (ISACs) comprising a TLR7/8 dual agonist conjugated to tumor-targeting antibodies. Systemically administered human epidermal growth factor receptor 2 (HER2)-targeted ISACs were well tolerated and triggered a localized immune response in the tumor microenvironment that resulted in tumor clearance and immunol. memory. Mechanistically, ISACs required tumor antigen recognition, Fcγ-receptor-dependent phagocytosis and TLR-mediated activation to drive tumor killing by myeloid cells and subsequent T-cell-mediated antitumor immunity. ISAC-mediated immunol. memory was not limited to the HER2 ISAC target antigen since ISAC-treated mice were protected from rechallenge with the HER2- parental tumor. These results provide a strong rationale for the clin. development of ISACs.
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      He, L.; Wang, L.; Wang, Z.; Li, T.; Chen, H.; Zhang, Y.; Hu, Z.; Dimitrov, D. S.; Du, J.; Liao, X. Immune Modulating Antibody-Drug Conjugate (IM-ADC) for Cancer Immunotherapy. J. Med. Chem. 2021, 64, 15716– 15726,  DOI: 10.1021/acs.jmedchem.1c00961
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      Immune Modulating Antibody-Drug Conjugate (IM-ADC) for Cancer Immunotherapy
      He, Lei; Wang, Liangliang; Wang, Zhisong; Li, Tiantian; Chen, Hui; Zhang, Yaning; Hu, Zeping; Dimitrov, Dimiter S.; Du, Juanjuan; Liao, Xuebin
      Journal of Medicinal Chemistry (2021), 64 (21), 15716-15726CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Antibody-drug conjugate (ADC) and immune checkpoint blockade (ICB) offer promising approaches for cancer treatment. Here, we describe an ADC constructed by conjugating anti-PD-L1 THIOMAB with a bifunctional immunomodulator D18 via a redox-cleavable linker. The resulting ADC HE-S2 not only triggers a potent antitumor immune response by blocking the PD-1/PD-L1 interaction and activating the Toll-like receptor 7/8 (TLR7/8) signaling pathway but also upregulates its targeted PD-L1 expression via epigenetic regulation and/or IFN-γ induction, thus conferring more sensitivity to the PD-1/PD-L1 blockade. We identify that ADC HE-S2 treatment could lead to more pronounced tumor suppression than the treatment of D18 in combination with the anti-PD-L1 antibody. Accordingly, this study provides a novel ADC strategy to enhance the antitumor immune response to ICB therapy.
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      Fu, Z.; Li, S.; Han, S.; Shi, C.; Zhang, Y. Antibody Drug Conjugate: The “Biological Missile” for Targeted Cancer Therapy. Signal Transduct. Targeted Ther. 2022, 7, 93,  DOI: 10.1038/s41392-022-00947-7
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      Antibody drug conjugate: the "biological missile" for targeted cancer therapy
      Fu, Zhiwen; Li, Shijun; Han, Sifei; Shi, Chen; Zhang, Yu
      Signal Transduction and Targeted Therapy (2022), 7 (1), 93CODEN: STTTCB; ISSN:2059-3635. (Nature Portfolio)
      A review. Antibody-drug conjugate (ADC) is typically composed of a monoclonal antibody (mAbs) covalently attached to a cytotoxic drug via a chem. linker. It combines both the advantages of highly specific targeting ability and highly potent killing effect to achieve accurate and efficient elimination of cancer cells, which has become one of the hotspots for the research and development of anticancer drugs. Since the first ADC, Mylotarg (gemtuzumab ozogamicin), was approved in 2000 by the US Food and Drug Administration (FDA), there have been 14 ADCs received market approval so far worldwide. Moreover, over 100 ADC candidates have been investigated in clin. stages at present. This kind of new anti-cancer drugs, known as "biol. missile", is leading a new era of targeted cancer therapy. Herein, we conducted a review of the history and general mechanism of action of ADCs, and then briefly discussed the mol. aspects of key components of ADCs and the mechanisms by which these key factors influence the activities of ADCs. Moreover, we also reviewed the approved ADCs and other promising candidates in phase-3 clin. trials and discuss the current challenges and future perspectives for the development of next generations, which provide insights for the research and development of novel cancer therapeutics using ADCs.
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      The making of bispecific antibodies
      Brinkmann, Ulrich; Kontermann, Roland E.
      mAbs (2017), 9 (2), 182-212CODEN: MABSCP; ISSN:1942-0870. (Taylor & Francis, Inc.)
      During the past two decades we have seen a phenomenal evolution of bispecific antibodies for therapeutic applications. The 'zoo' of bispecific antibodies is populated by many different species, comprising around 100 different formats, including small mols. composed solely of the antigen-binding sites of two antibodies, mols. with an IgG structure, and large complex mols. composed of different antigen-binding moieties often combined with dimerization modules. The application of sophisticated mol. design and genetic engineering has solved many of the tech. problems assocd. with the formation of bispecific antibodies such as stability, soly. and other parameters that confer drug properties. These parameters may be summarized under the term 'developability'. In addn., different 'target product profiles', i.e., desired features of the bispecific antibody to be generated, mandates the need for access to a diverse panel of formats. These may vary in size, arrangement, valencies, flexibility and geometry of their binding modules, as well as in their distribution and pharmacokinetic properties. There is not 'one best format' for generating bispecific antibodies, and no single format is suitable for all, or even most of, the desired applications. Instead, the bispecific formats collectively serve as a valuable source of diversity that can be applied to the development of therapeutics for various indications. Here, a comprehensive overview of the different bispecific antibody formats is provided.
    9. 9
      Tissot, A. C.; Brinkmann, U. Bispecific Antibodies. In Next-generation therapies and technologies for immune-mediated inflammatory diseases, 1st edition; Mina-Osorio, P., Ed.; Springer International Publishing: New York, 2017; p 75– 97.
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      Brinkmann, U.; Kontermann, R. E. Bispecific Antibodies. Science 1979, 2021 (372), 916– 917,  DOI: 10.1126/science.abg1209
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      Wang, Q.; Chen, Y.; Park, J.; Liu, X.; Hu, Y.; Wang, T.; McFarland, K.; Betenbaugh, M. J. Design and Production of Bispecific Antibodies. Antibodies 2019, 8, 43,  DOI: 10.3390/antib8030043
      11
      Design and production of bispecific antibodies
      Wang, Qiong; Chen, Yiqun; Park, Jaeyoung; Liu, Xiao; Hu, Yifeng; Wang, Tiexin; McFarland, Kevin; Betenbaugh, Michael J.
      Antibodies (2019), 8 (3), 43CODEN: ANTICA; ISSN:2073-4468. (MDPI AG)
      With the current biotherapeutic market dominated by antibody mols., bispecific antibodies represent a key component of the next-generation of antibody therapy. Bispecific antibodies can target two different antigens at the same time, such as simultaneously binding tumor cell receptors and recruiting cytotoxic immune cells. Structural diversity has been fast-growing in the bispecific antibody field, creating a plethora of novel bispecific antibody scaffolds, which provide great functional variety. Two common formats of bispecific antibodies on the market are the single-chain variable fragment (scFv)-based (no Fc fragment) antibody and the full-length IgG-like asym. antibody. Unlike the conventional monoclonal antibodies, great prodn. challenges with respect to the quantity, quality, and stability of bispecific antibodies have hampered their wider clin. application and acceptance. In this review, we focus on these two major bispecific types and describe recent advances in the design, prodn., and quality of these mols., which will enable this important class of biologics to reach their therapeutic potential.
    12. 12
      Marei, H. E.; Cenciarelli, C.; Hasan, A. Potential of Antibody–Drug Conjugates (ADCs) for Cancer Therapy. Cancer Cell Int. 2022, 22, 255,  DOI: 10.1186/s12935-022-02679-8
      12
      Potential of antibody-drug conjugates (ADCs) for cancer therapy
      Marei, Hany E.; Cenciarelli, Carlo; Hasan, Anwarul
      Cancer Cell International (2022), 22 (1), 255CODEN: CCIACC; ISSN:1475-2867. (BioMed Central Ltd.)
      A review. The primary purpose of ADCs is to increase the efficacy of anticancer medications by minimizing systemic drug distribution and targeting specific cells. Antibody conjugates (ADCs) have changed the way cancer is treated. However, because only a tiny fraction of patients experienced long-term advantages, current cancer preclin. and clin. research has been focused on combination trials. The complex interaction of ADCs with the tumor and its microenvironment appear to be reliant on the efficacy of a certain ADC, all of which have significant therapeutic consequences. Several clin. trials in various tumor types are now underway to examine the potential ADC therapy, based on encouraging preclin. results. This review tackles the potential use of ADCs in cancer therapy, emphasizing the essential processes underlying their pos. therapeutic impacts on solid and hematol. malignancies. Addnl., opportunities are explored to understand the mechanisms of ADCs action, the mechanism of resistance against ADCs, and how to overcome potential resistance following ADCs administration. Recent clin. findings have aroused interest, leading to a large increase in the no. of ADCs in clin. trials. The rationale behind ADCs, as well as their primary features and recent research breakthroughs, will be discussed. We then offer an approach for maximizing the potential value that ADCs can bring to cancer patients by highlighting key ideas and distinct strategies.
    13. 13
      Dumontet, C.; Reichert, J. M.; Senter, P. D.; Lambert, J. M.; Beck, A. Antibody-Drug Conjugates Come of Age in Oncology. Nat. Rev. Drug Discovery 2023, 22, 641– 661,  DOI: 10.1038/s41573-023-00709-2
      13
      Antibody-drug conjugates come of age in oncology
      Dumontet, Charles; Reichert, Janice M.; Senter, Peter D.; Lambert, John M.; Beck, Alain
      Nature Reviews Drug Discovery (2023), 22 (8), 641-661CODEN: NRDDAG; ISSN:1474-1776. (Nature Portfolio)
      Abstr.: Antibody-drug conjugates (ADCs) combine the specificity of monoclonal antibodies with the potency of highly cytotoxic agents, potentially reducing the severity of side effects by preferentially targeting their payload to the tumor site. ADCs are being increasingly used in combination with other agents, including as first-line cancer therapies. As the technol. to produce these complex therapeutics has matured, many more ADCs have been approved or are in late-phase clin. trials. The diversification of antigenic targets as well as bioactive payloads is rapidly broadening the scope of tumor indications for ADCs. Moreover, novel vector protein formats as well as warheads targeting the tumor microenvironment are expected to improve the intratumor distribution or activation of ADCs, and consequently their anticancer activity for difficult-to-treat tumor types. However, toxicity remains a key issue in the development of these agents, and better understanding and management of ADC-related toxicities will be essential for further optimization. This Review provides a broad overview of the recent advances and challenges in ADC development for cancer treatment.
    14. 14
      Beishenaliev, A.; Loke, Y. L.; Goh, S. J.; Geo, H. N.; Mugila, M.; Misran, M.; Chung, L. Y.; Kiew, L. V.; Roffler, S.; Teo, Y. Y. Bispecific Antibodies for Targeted Delivery of Anti-Cancer Therapeutic Agents: A Review. J. Controlled Release 2023, 359, 268– 286,  DOI: 10.1016/j.jconrel.2023.05.032
      14
      Bispecific antibodies for targeted delivery of anti-cancer therapeutic agents: A review
      Beishenaliev, Adilet; Loke, Yean Leng; Goh, Sook Jing; Geo, Hui Nee; Mugila, Malar; Misran, Misni; Chung, Lip Yong; Kiew, Lik Voon; Roffler, Steve; Teo, Yin Yin
      Journal of Controlled Release (2023), 359 (), 268-286CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
      A review. Monospecific antibodies have been utilized increasingly for anti-cancer drug targeting owing to their ability to minimise off-target toxicity by binding specifically to a tumor epitope, hence selectively delivering drugs to the tumor cells. Nevertheless, the monospecific antibodies only engage a single cell surface epitope to deliver their drug payload. Hence, their performance is often unsatisfactory in cancers where multiple epitopes need to be engaged for optimal cellular internalization. In this context, bispecific antibodies (bsAbs) that simultaneously target two distinct antigens or two distinct epitopes of the same antigen offer a promising alternative in antibody-based drug delivery. This review describes the recent advances in developing bsAb-based drug delivery strategies, encompassing the direct conjugation of drug to bsAbs to form bispecific antibody-drug conjugates (bsADCs) and the surface functionalization of nanoconstructs with bsAbs to form bsAb-coupled nanoconstructs. The article first details the roles of bsAbs in enhancing the internalisation and intracellular trafficking of bsADCs with subsequent release of chemotherapeutic drugs for an augmented therapeutic efficacy, particularly among heterogeneous tumor cell populations. Then, the article discusses the roles of bsAbs in facilitating the delivery of drug-encapsulating nanoconstructs, including org./inorg. nanoparticles and large bacteria-derived minicells, that provide a larger drug loading capacity and better stability in blood circulation than bsADCs. The limitations of each type of bsAb-based drug delivery strategy and the future prospects of more versatile strategies (e.g., trispecific antibodies, autonomous drug delivery systems, theranostics) are also elaborated.
    15. 15
      Shin, H. G.; Yang, H. R.; Yoon, A.; Lee, S. Bispecific Antibody-Based Immune-Cell Engagers and Their Emerging Therapeutic Targets in Cancer Immunotherapy. Int. J. Mol. Sci. 2022, 23, 5686,  DOI: 10.3390/ijms23105686
      15
      Bispecific Antibody-Based Immune-Cell Engagers and Their Emerging Therapeutic Targets in Cancer Immunotherapy
      Shin, Ha Gyeong; Yang, Ha Rim; Yoon, Aerin; Lee, Sukmook
      International Journal of Molecular Sciences (2022), 23 (10), 5686CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
      A review. Cancer is the second leading cause of death worldwide after cardiovascular diseases. Harnessing the power of immune cells is a promising strategy to improve the antitumor effect of cancer immunotherapy. Recent progress in recombinant DNA technol. and antibody engineering has ushered in a new era of bispecific antibody (bsAb)-based immune-cell engagers (ICEs), including T- and natural-killer-cell engagers. Since the first approval of blinatumomab by the United States Food and Drug Administration (US FDA), various bsAb-based ICEs have been developed for the effective treatment of patients with cancer. Simultaneously, several potential therapeutic targets of bsAb-based ICEs have been identified in various cancers. Therefore, this review focused on not only highlighting the action mechanism, design and structure, and status of bsAb-based ICEs in clin. development and their approval by the US FDA for human malignancy treatment, but also on summarizing the currently known and emerging therapeutic targets in cancer. This review provides insights into practical considerations for developing next-generation ICEs.
    16. 16
      Chen, R. P.; Shinoda, K.; Rampuria, P.; Jin, F.; Bartholomew, T.; Zhao, C.; Yang, F.; Chaparro-Riggers, J. Bispecific Antibodies for Immune Cell Retargeting against Cancer. Expert Opin. Biol. Ther. 2022, 22, 965– 982,  DOI: 10.1080/14712598.2022.2072209
      16
      Bispecific antibodies for immune cell retargeting against cancer
      Chen, Rebecca P.; Shinoda, Kenta; Rampuria, Pragya; Jin, Fang; Bartholomew, Tin; Zhao, Chunxia; Yang, Fan; Chaparro-Riggers, Javier
      Expert Opinion on Biological Therapy (2022), 22 (8), 965-982CODEN: EOBTA2; ISSN:1471-2598. (Taylor & Francis Ltd.)
      A review. Following the approval of the T cell engaging bispecific antibody blinatumomab, immune cell retargeting with bispecific or multispecific antibodies has emerged as a promising cancer immunotherapy strategy, offering alternative mechanisms compared to immune checkpoint blockade. As we gain more understanding of the complex tumor microenvironment, rules and design principles have started to take shape on how to best harness the immune system to achieve optimal anti-tumor activities. In the present review, we aim to summarize the most recent advances and challenges in using bispecific antibodies for immune cell retargeting and to provide insights into various aspects of antibody engineering. Discussed herein are studies that highlight the importance of considering antibody engineering parameters, such as binding epitope, affinity, valency, and geometry to maximize the potency and mitigate the toxicity of T cell engagers. Beyond T cell engaging bispecifics, other bispecifics designed to recruit the innate immune system are also covered. Diverse and innovative mol. designs of bispecific/multispecific antibodies have the potential to enhance the efficacy and safety of immune cell retargeting for the treatment of cancer. Whether or not clin. data support these different hypotheses, esp. in solid tumor settings, remains to be seen.
    17. 17
      Ordóñez-Reyes, C.; Garcia-Robledo, J. E.; Chamorro, D. F.; Mosquera, A.; Sussmann, L.; Ruiz-Patiño, A.; Arrieta, O.; Zatarain-Barrón, L.; Rojas, L.; Russo, A. Bispecific Antibodies in Cancer Immunotherapy: A Novel Response to an Old Question. Pharmaceutics 2022, 14, 1243,  DOI: 10.3390/pharmaceutics14061243
      17
      Bispecific Antibodies in Cancer Immunotherapy: A Novel Response to an Old Question
      Ordonez-Reyes, Camila; Garcia-Robledo, Juan Esteban; Chamorro, Diego F.; Mosquera, Andres; Sussmann, Liliana; Ruiz-Patino, Alejandro; Arrieta, Oscar; Zatarain-Barron, Lucia; Rojas, Leonardo; Russo, Alessandro; de Miguel-Perez, Diego; Rolfo, Christian; Cardona, Andres F.
      Pharmaceutics (2022), 14 (6), 1243CODEN: PHARK5; ISSN:1999-4923. (MDPI AG)
      Immunotherapy has redefined the treatment of cancer patients and it is constantly generating new advances and approaches. Among the multiple options of immunotherapy, bispecific antibodies (bsAbs) represent a novel thoughtful approach. These drugs integrate the action of the immune system in a strategy to redirect the activation of innate and adaptive immunity toward specific antigens and specific tumor locations. Here we discussed some basic aspects of the design and function of bsAbs, their main challenges and the state-of-the-art of these mols. in the treatment of hematol. and solid malignancies and future perspectives.
    18. 18
      Dahlén, E.; Veitonmäki, N.; Norlén, P. Bispecific Antibodies in Cancer Immunotherapy. Ther. Adv. Vaccines Immunother. 2018, 6, 3– 17,  DOI: 10.1177/2515135518763280
      18
      Bispecific antibodies in cancer immunotherapy
      Dahlen, Eva; Veitonmaeki, Niina; Norlen, Per
      Therapeutic Advances in Vaccines and Immunotherapy (2018), 6 (1), 3-17CODEN: TAVIAY; ISSN:2515-1363. (Sage Publications Ltd.)
      A review. Following the clin. success of immune checkpoint antibodies targeting CTLA-4, PD-1 or PD-L1 in cancer treatment, bispecific antibodies are now emerging as a growing class of immunotherapies with potential to further improve clin. efficacy and safety. We describe three classes of immunotherapeutic bispecific antibodies: (a) cytotoxic effector cell redirectors; (b) tumor-targeted immunomodulators; and (c) dual immunomodulators. Cytotoxic effector cell redirectors are dominated by T-cell redirecting compds., bispecific compds. engaging a tumor-assocd. antigen and the T-cell receptor/CD3 complex, thereby redirecting T-cell cytotoxicity to malignant cells. This is the most established class of bispecific immunotherapies, with two compds. having reached the market and numerous compds. in clin. development. Tumor-targeted immunomodulators are bispecific compds. binding to a tumor-assocd. antigen and an immunomodulating receptor, such as CD40 or 4-1BB. Such compds. are usually designed to be inactive until binding the tumor antigen, thereby localizing immune stimulation to the tumor environment, while minimizing immune activation elsewhere. This is expected to induce powerful activation of tumor-specific T cells with reduced risk of immune-related adverse events. Finally, dual immunomodulators are bispecific compds. that bind two distinct immunomodulating targets, often combining targeting of PD-1 or PD-L1 with that of LAG-3 or TIM-3. The rationale is to induce superior tumor immunity compared to monospecific antibodies to the same targets. In this review, we describe each of these classes of bispecific antibodies, and present examples of compds. in development.
    19. 19
      Huo, Y.; Sheng, Z.; Lu, D. R.; Ellwanger, D. C.; Li, C. M.; Homann, O.; Wang, S.; Yin, H.; Ren, R. Blinatumomab-Induced T Cell Activation at Single Cell Transcriptome Resolution. BMC Genom. 2021, 22, 145,  DOI: 10.1186/s12864-021-07435-2
      There is no corresponding record for this reference.
    20. 20
      Kipriyanov, S. M.; Moldenhauer, G.; Strauss, G.; Little, M. Bispecific CD3 X CD19 Diabody for T Cell-Mediated Lysis of Malignant Human B Cells. Int. J. Cancer 1998, 77, 763– 772,  DOI: 10.1002/(SICI)1097-0215(19980831)77:5<763::AID-IJC16>3.0.CO;2-2
      20
      Bispecific CD3 x CD19 diabody for T cell-mediated lysis of malignant human B cells
      Kipriyanov, Sergey M.; Moldenhauer, Gerhard; Strauss, Gudrun; Little, Melvyn
      International Journal of Cancer (1998), 77 (5), 763-772CODEN: IJCNAW; ISSN:0020-7136. (Wiley-Liss, Inc.)
      For the treatment of minimal residual disease in patients with leukemias and malignant lymphomas, we constructed a heterodimeric diabody specific for human CD19 on B cells and CD3E chain of the T cell receptor complex. The bispecific diabody was expressed in Escherichia coli using a vector contg. a dicistronic operon for co-secretion of VH3-VL19 and VH19-VL3 single-chain Fv fragments (scFv). It was purified in one step by immobilized metal affinity chromatog. (IMAC) from the periplasmic ext. and culture medium. Flow cytometry expts. revealed specific interactions of the diabody with both CD3 and CD19 pos. cells, to which it bound with affinities close to those of the parental scFvs. It was less stable than anti-CD3 scFv but more stable than anti-CD19 scFv when incubated in human serum at 37°C. In cytotoxicity tests, the diabody proved to be a potent agent for retargeting peripheral blood lymphocytes to lyse tumor cells expressing the CD19 antigen. The efficiency of cell lysis compared favorably with that obtained with a bispecific antibody (BsAb) of the same dual specificity that was prepd. by the quadroma technique.
    21. 21
      Klein, C.; Schaefer, W.; Regula, J. T.; Dumontet, C.; Brinkmann, U.; Bacac, M.; Umaña, P. Engineering Therapeutic Bispecific Antibodies Using CrossMab Technology. Methods 2019, 154, 21– 31,  DOI: 10.1016/j.ymeth.2018.11.008
      21
      Engineering therapeutic bispecific antibodies using CrossMab technology
      Klein, Christian; Schaefer, Wolfgang; Regula, Joerg T.; Dumontet, Charles; Brinkmann, Ulrich; Bacac, Marina; Umana, Pablo
      Methods (Amsterdam, Netherlands) (2019), 154 (), 21-31CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)
      A review. Bispecific antibodies have recently gained major interest as they allow novel mechanisms-of-action and/or therapeutic applications that cannot be achieved using conventional IgG-based antibodies. A major issue in engineering IgG-based bispecific antibodies has been to enable the correct assocn. of heavy and light chains resulting in correct assembly of the desired bispecific antibody in sufficient yield. Various approaches have been described during recent years to tackle this challenge. We have developed the so-called CrossMab technol. that enforces correct light chain assocn. based on the domain crossover of Ig domains in the Fab region of the bispecific antibody. This versatile technol. allows the generation of different bispecific antibody formats including asym. heterodimeric monovalent 1 + 1 bispecific antibodies and asym. heterodimeric bispecific antibodies with 2 + 1 valency in combination with approaches enabling Fc-hetermodimerization like knob-into-hole technol. as well as the generation of tetravalent sym. bispecific antibodies with 2 + 2 valency, also known as Tandem-Fab based IgG antibodies, using processes suitable for the large scale prodn. of therapeutic bispecific antibodies. Notably, as of now, at least eight different bispecific antibodies using CrossMab technol. entered clin. development, and addnl. CrossMabs are in late preclin. development. This review provides a summary of the status and progress with the engineering and generation of CrossMab technol. based bispecific antibodies as well as their therapeutic application.
    22. 22
      Ellerman, D. Bispecific T-Cell Engagers: Towards Understanding Variables Influencing the in Vitro Potency and Tumor Selectivity and Their Modulation to Enhance Their Efficacy and Safety. Methods 2019, 154, 102– 117,  DOI: 10.1016/j.ymeth.2018.10.026
      22
      Bispecific T-cell engagers: Towards understanding variables influencing the in vitro potency and tumor selectivity and their modulation to enhance their efficacy and safety
      Ellerman, Diego
      Methods (Amsterdam, Netherlands) (2019), 154 (), 102-117CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)
      A review. Bispecific mols. redirecting the cytotoxicity of T-cells are a growing class of therapeutics with numerous mols. being tested in clin. trials. However, it has been a long way since the proof of concept studies in the mid 1980's. In the process we have learnt about the impact of different variables related to the bispecific mol. and the target antigen on the potency of this type of drugs. This work reviews the insights gained and how that knowledge has been used to design more potent bispecific T-cell engagers. The more recent advancement of antibodies with this modality into safety studies in non-human primates and as well as in clin. studies has revealed potential toxicity liabilities for the mode of action. Modifications in existing antibody formats and new exptl. mols. designed to mitigate these problems are discussed.
    23. 23
      Moore, P. A.; Zhang, W.; Rainey, G. J.; Burke, S.; Li, H.; Huang, L.; Gorlatov, S.; Veri, M. C.; Aggarwal, S.; Yang, Y.; Shah, K. Application of Dual Affinity Retargeting Molecules to Achieve Optimal Redirected T-Cell Killing of B-Cell Lymphoma. Blood 2011, 117, 4542– 4551,  DOI: 10.1182/blood-2010-09-306449
      23
      Application of dual affinity retargeting molecules to achieve optimal redirected T-cell killing of B-cell lymphoma
      Moore, Paul A.; Zhang, Wenjun; Rainey, G. Jonah; Burke, Steve; Li, Hua; Huang, Ling; Gorlatov, Sergey; Veri, Maria Concetta; Aggarwal, Sudeepta; Yang, Yinhua; Shah, Kalpana; Jin, Linda; Zhang, Sunan; He, Leilei; Zhang, Tengfei; Ciccarone, Valentina; Koenig, Scott; Bonvini, Ezio; Johnson, Syd
      Blood (2011), 117 (17), 4542-4551CODEN: BLOOAW; ISSN:0006-4971. (American Society of Hematology)
      We describe the application of a novel, bispecific antibody platform termed dual affinity retargeting (DART) to eradicate B-cell lymphoma through coengagement of the B cell-specific antigen CD19 and the TCR/CD3 complex on effector T cells. Comparison with a single-chain, bispecific antibody bearing identical CD19 and CD3 antibody Fv sequences revealed DART mols. to be more potent in directing B-cell lysis. The enhanced activity with the CD19 × CD3 DART mols. was obsd. on all CD19-expressing target B cells evaluated using resting and prestimulated human PBMCs or purified effector T-cell populations. Characterization of a CD19 × TCR bispecific DART mol. revealed equiv. potency with the CD19 × CD3 DART mol., demonstrating flexibility of the DART structure to support T-cell/B-cell assocns. for redirected T cell-killing applications. The enhanced level of killing mediated by DART mols. was not accompanied by any increase in nonspecific T-cell activation or lysis of CD19- cells. Cell-assocn. studies indicated that the DART architecture is well suited for maintaining cell-to-cell contact, apparently contributing to the high level of target cell killing. Finally, the ability of the CD19 × TCR DART to inhibit B-cell lymphoma in NOD/SCID mice when coadministered with human PBMCs supports further evaluation of DART mols. for the treatment of B-cell malignancies.
    24. 24
      Reusch, U.; Harrington, K. H.; Gudgeon, C. J.; Fucek, I.; Ellwanger, K.; Weichel, M.; Knackmuss, S. H. J.; Zhukovsky, E. A.; Fox, J. A.; Kunkel, L. A. Characterization of CD33/CD3 Tetravalent Bispecific Tandem Diabodies (TandAbs) for the Treatment of Acute Myeloid Leukemia. Clin. Cancer Res. 2016, 22, 5829– 5838,  DOI: 10.1158/1078-0432.CCR-16-0350
      24
      Characterization of CD33/CD3 Tetravalent Bispecific Tandem Diabodies (TandAbs) for the Treatment of Acute Myeloid Leukemia
      Reusch, Uwe; Harrington, Kimberly H.; Gudgeon, Chelsea J.; Fucek, Ivica; Ellwanger, Kristina; Weichel, Michael; Knackmuss, Stefan H. J.; Zhukovsky, Eugene A.; Fox, Judith A.; Kunkel, Lori A.; Guenot, Jeanmarie; Walter, Roland B.
      Clinical Cancer Research (2016), 22 (23), 5829-5838CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
      Purpose: Randomized studies with gemtuzumab ozogamicin have validated CD33 as a target for antigen-specific immunotherapy of acute myelogenous leukemia (AML). Here, we investigated the potential of CD33/CD3-directed tandem diabodies (TandAbs) as novel treatment approach for AML. These tetravalent bispecific antibodies provide two binding sites for each antigen to maintain the avidity of a bivalent antibody and have a mol. wt. exceeding the renal clearance threshold, thus offering a longer half-life compared to smaller antibody constructs. Exptl. Design: We constructed a series of TandAbs composed of anti-CD33 and anti-CD3 variable domains of diverse binding affinities and profiled their functional properties in CD33+ human leukemia cell lines, xenograft models, and AML patient samples. Results: Our studies demonstrated that several CD33/CD3 TandAbs could induce potent, dose-dependent cytolysis of CD33+ AML cell lines. This effect was modulated by the effector-to-target cell ratio and strictly required the presence of T cells. Activation and proliferation of T cells and maximal AML cell cytolysis correlated with high avidity to both CD33 and CD3. High-avidity TandAbs were broadly active in primary specimens from patients with newly diagnosed or relapsed/refractory AML in vitro, with cytotoxic properties independent of CD33 receptor d. and cytogenetic risk. Tumor growth delay and inhibition were obsd. in both prophylactic and established HL-60 xenograft models in immunodeficient mice. Conclusions: Our data show high efficacy of CD33/CD3 TandAbs in various preclin. models of human AML. Together, these findings support further study of CD33/CD3 TandAbs as novel immunotherapeutics for patients with AML. Clin Cancer Res; 22(23); 5829-38. ©2016 AACR.
    25. 25
      Thoreau, F.; Szijj, P. A.; Greene, M. K.; Rochet, L. N. C.; Thanasi, I. A.; Blayney, J. K.; Maruani, A.; Baker, J. R.; Scott, C. J.; Chudasama, V. Modular Chemical Construction of IgG-like Mono- and Bispecific Synthetic Antibodies (SynAbs). ACS Cent. Sci. 2023, 9, 476– 487,  DOI: 10.1021/acscentsci.2c01437
      25
      Modular Chemical Construction of IgG-like Mono- and Bispecific Synthetic Antibodies (SynAbs)
      Thoreau, Fabien; Szijj, Peter A.; Greene, Michelle K.; Rochet, Lea N. C.; Thanasi, Ioanna A.; Blayney, Jaine K.; Maruani, Antoine; Baker, James R.; Scott, Christopher J.; Chudasama, Vijay
      ACS Central Science (2023), 9 (3), 476-487CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      In recent years there has been rising interest in the field of protein-protein conjugation, esp. related to bispecific antibodies (bsAbs) and their therapeutic applications. These constructs contain two paratopes capable of binding two distinct epitopes on target mols. and are thus able to perform complex biol. functions (mechanisms of action) not available to monospecific mAbs. Traditionally these bsAbs have been constructed through protein engineering, but recently chem. methods for their construction have started to (re)emerge. While these have been shown to offer increased modularity, speed, and for some methods even the inherent capacity for further functionalization (e.g., with small mol. cargo), most of these approaches lacked the ability to include a fragment crystallizable (Fc) modality. The Fc component of IgG antibodies offers effector function and increased half-life. Here we report a first-in-class disulfide rebridging and click-chem.-based method for the generation of Fc-contg., IgG-like mono- and bispecific antibodies. These are in the FcZ-(FabX)-FabY format, i.e., two distinct Fabs and an Fc, potentially all from different antibodies, attached in a homogeneous and covalent manner. We have dubbed these mols. synthetic antibodies (SynAbs). We have constructed a T cell-engager (TCE) SynAb, FcCD20-(FabHER2)-FabCD3, and have confirmed that it exhibits the expected biol. functions, including the ability to kill HER2+ target cells in a coculture assay with T cells.
    26. 26
      Schaefer, W.; Regula, J. T.; Bähner, M.; Schanzer, J.; Croasdale, R.; Dürr, H.; Gassner, C.; Georges, G.; Kettenberger, H.; Imhof-Jung, S. Immunoglobulin Domain Crossover as a Generic Approach for the Production of Bispecific IgG Antibodies. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 11187– 11192,  DOI: 10.1073/pnas.1019002108
      26
      Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies
      Schaefer, Wolfgang; Regula, Jorg T.; Bahner, Monika; Schanzer, Jurgen; Croasdale, Rebecca; Durr, Harald; Gassner, Christian; Georges, Guy; Kettenberger, Hubert; Imhof-Jung, Sabine; Schwaiger, Manfred; Stubenrauch, Kay G.; Sustmann, Claudio; Thomas, Markus; Scheuer, Werner; Klein, Christian
      Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (27), 11187-11192, S11187/1-S11187/10CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technol. that enables heterodimerization of the heavy chains, correct assocn. of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This "crossover" retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible "CrossMab" formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by std. techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMabCH1-CL was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.
    27. 27
      Madsen, A. V.; Kristensen, P.; Buell, A. K.; Goletz, S. Generation of Robust Bispecific Antibodies through Fusion of Single-Domain Antibodies on IgG Scaffolds: A Comprehensive Comparison of Formats. MAbs 2023, 15, 1,  DOI: 10.1080/19420862.2023.2189432
      There is no corresponding record for this reference.
    28. 28
      Kühl, L.; Aschmoneit, N.; Kontermann, R. E.; Seifert, O. The EIg Technology to Generate Ig-like Bispecific Antibodies. MAbs 2022, 14, 1,  DOI: 10.1080/19420862.2022.2063043
      There is no corresponding record for this reference.
    29. 29
      de Bever, L.; Popal, S.; van Schaik, J.; Rubahamya, B.; van Delft, F. L.; Thurber, G. M.; van Berkel, S. S. Generation of DAR1 Antibody-Drug Conjugates for Ultrapotent Payloads Using Tailored GlycoConnect Technology. Bioconjugate Chem. 2023, 34, 538– 548,  DOI: 10.1021/acs.bioconjchem.2c00611
      29
      Generation of DAR1 Antibody-Drug Conjugates for Ultrapotent Payloads Using Tailored GlycoConnect Technology
      de Bever, Laureen; Popal, Sorraya; van Schaik, Jord; Rubahamya, Baron; van Delft, Floris L.; Thurber, Greg M.; van Berkel, Sander S.
      Bioconjugate Chemistry (2023), 34 (3), 538-548CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      GlycoConnect technol. can be readily adapted to provide different drug-to-antibody ratios (DARs) and is currently also evaluated in various clin. programs, including ADCT-601 (DAR2), MRG004a (DAR4), and XMT-1660 (DAR6). While antibody-drug conjugates (ADCs) typically feature a DAR2-8, it has become clear that ADCs with ultrapotent payloads (e.g., PBD dimers and calicheamicin) can only be administered to patients at low doses (<0.5 mg/kg), which may compromise effective biodistribution and may be insufficient to reach target receptor satn. in the tumor. Here, we show that GlycoConnect technol. can be readily extended to DAR1 ADCs without the need of antibody re-engineering. We demonstrate that various ultrapotent, cytotoxic payloads are amenable to this methodol. In a follow-up expt., HCC-1954 tumor spheroids were treated with either an AlexaFluor647-labeled DAR1 or DAR2 PBD-based ADC to study the effect on tumor penetration. Significant improvement of tumor spheroid penetration was obsd. for the DAR1 ADC compared to the DAR2 ADC at an equal payload dose, underlining the potential of a lower DAR for ADCs bearing ultrapotent payloads.
    30. 30
      Van Berkel, S. S.; van Delft, F. L. Enzymatic Strategies for (near) Clinical Development of Antibody-Drug Conjugates. Drug Discovery Today: Technol. 2018, 30, 3– 10,  DOI: 10.1016/j.ddtec.2018.09.005
      30
      Enzymatic strategies for (near) clinical development of antibody-drug conjugates
      van Berkel Sander S; van Delft Floris L
      Drug discovery today. Technologies (2018), 30 (), 3-10 ISSN:.
      Target-specific killing of tumor cells with antibody-drug conjugates (ADCs) is an elegant concept in the continued fight against cancer. However, despite more than 20 years of clinical development, only four ADC have reached market approval, while at least 50 clinical programs were terminated early. The high attrition rate of ADCs may, at least in part, be attributed to heterogeneity and instability of conventional technologies. At present, various (chemo)enzymatic approaches for site-specific and stable conjugation of toxic payloads are making their way to the clinic, thereby potentially providing ADCs with increased therapeutic window.
    31. 31
      Van Geel, R.; Wijdeven, M. A.; Heesbeen, R.; Verkade, J. M. M.; Wasiel, A. A.; Van Berkel, S. S.; Van Delft, F. L. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native MAbs Provides Homogeneous and Highly Efficacious Antibody-Drug Conjugates. Bioconjugate Chem. 2015, 26, 2233– 2242,  DOI: 10.1021/acs.bioconjchem.5b00224
      31
      Chemoenzymatic conjugation of toxic payloads to the globally conserved N-glycan of native mAbs provides homogeneous and highly efficacious antibody-drug conjugates
      van Geel, Remon; Wijdeven, Marloes A.; Heesbeen, Ryan; Verkade, Jorge M. M.; Wasiel, Anna A.; van Berkel, Sander S.; van Delft, Floris L.
      Bioconjugate Chemistry (2015), 26 (11), 2233-2242CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      A robust, generally applicable, nongenetic technol. is presented to convert monoclonal antibodies into stable and homogeneous ADCs. Starting from a native (nonengineered) mAb, a chemoenzymic protocol allows for the highly controlled attachment of any given payload to the N-glycan residing at asparagine-297, based on a two-stage process: first, enzymic remodeling (trimming and tagging with azide), followed by ligation of the payload based on copper-free click chem. The technol., termed GlycoConnect, is applicable to any IgG isotype irresp. of glycosylation profile. Application to trastuzumab and maytansine, both components of the marketed ADC Kadcyla, demonstrate a favorable in vitro and in vivo efficacy for GlycoConnect ADC. Moreover, the superiority of the native glycan as attachment site was demonstrated by in vivo comparison to a range of trastuzumab-based glycosylation mutants. A side-by-side comparison of the copper-free click probes bicyclononyne (BCN) and a dibenzoannulated cyclooctyne (DBCO) showed a surprising difference in conjugation efficiency in favor of BCN, which could be even further enhanced by introduction of electron-withdrawing fluoride substitutions onto the azide. The resulting mAb-conjugates were in all cases found to be highly stable, which in combination with the demonstrated efficacy warrants ADCs with a superior therapeutic index.
    32. 32
      Evans, H. L.; Nguyen, Q. D.; Carroll, L. S.; Kaliszczak, M.; Twyman, F. J.; Spivey, A. C.; Aboagye, E. O. A Bioorthogonal 68Ga-Labelling Strategy for Rapid in Vivo Imaging. Chem. Commun. 2014, 50, 9557– 9560,  DOI: 10.1039/C4CC03903C
      32
      A bioorthogonal 68Ga-labelling strategy for rapid in vivo imaging
      Evans, Helen L.; Nguyen, Quang-De; Carroll, Laurence S.; Kaliszczak, Maciej; Twyman, Frazer J.; Spivey, Alan C.; Aboagye, Eric O.
      Chemical Communications (Cambridge, United Kingdom) (2014), 50 (67), 9557-9560CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      Herein, we describe a fast and robust method for achieving 68Ga-labeling of the EGFR-selective monoclonal antibody (mAb) Cetuximab using the bioorthogonal Inverse-electron-Demand Diels-Alder (IeDDA) reaction. The in vivo imaging of EGFR is demonstrated, as well as the translation of the method within a two-step pretargeting strategy.
    33. 33
      Taylor, R. J.; Geeson, M. B.; Journeaux, T.; Bernardes, G. J. L. Chemical and Enzymatic Methods for Post-Translational Protein-Protein Conjugation. J. Am. Chem. Soc. 2022, 144, 14404– 14419,  DOI: 10.1021/jacs.2c00129
      33
      Chemical and Enzymatic Methods for Post-Translational Protein-Protein Conjugation
      Taylor, Ross J.; Geeson, Michael B.; Journeaux, Toby; Bernardes, Goncalo J. L.
      Journal of the American Chemical Society (2022), 144 (32), 14404-14419CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A review. Fusion proteins play an essential role in the biosciences but suffer from several key limitations, including the requirement for N-to-C terminal ligation, incompatibility of constituent domains, incorrect folding, and loss of biol. activity. This perspective focuses on chem. and enzymic approaches for the post-translational generation of well-defined protein-protein conjugates, which overcome some of the limitations faced by traditional fusion techniques. Methods discussed range from chem. modification of nucleophilic canonical amino acid residues to incorporation of unnatural amino acid residues and a range of enzymic methods, including sortase-mediated ligation. Through summarizing the progress in this rapidly growing field, the key successes and challenges assocd. with using chem. and enzymic approaches are highlighted and areas requiring further development are discussed.
    34. 34
      Bruins, J. J.; Damen, J. A. M.; Wijdeven, M. A.; Lelieveldt, L. P. W. M.; Van Delft, F. L.; Albada, B. Non-Genetic Generation of Antibody Conjugates Based on Chemoenzymatic Tyrosine Click Chemistry. Bioconjugate Chem. 2021, 32, 2167– 2172,  DOI: 10.1021/acs.bioconjchem.1c00351
      34
      Non-Genetic Generation of Antibody Conjugates Based on Chemo enzymatic Tyrosine Click Chemistry
      Bruins, Jorick J.; Damen, Johannes A. M.; Wijdeven, Marloes A.; Lelieveldt, Lianne P. W. M.; van Delft, Floris L.; Albada, Bauke
      Bioconjugate Chemistry (2021), 32 (10), 2167-2172CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      The availability of tools to generate homogeneous and stable antibody conjugates without recombinant DNA technol. is a valuable asset in fields spanning from in vitro diagnostics to in vivo imaging and therapeutics. We present here a general approach for the conjugation to human IgG1 antibodies, by employing a straightforward two-stage protocol based on antibody deglycosylation followed by tyrosinase-mediated ortho-quinone strain-promoted click chem. The technol. is validated by the efficient and clean generation of highly potent DAR2 and DAR4 antibody-drug conjugates (ADCs) with cytotoxic payloads MMAE or PBD dimer, and their in vitro evaluation.
    35. 35
      Oliveira, B. L.; Guo, Z.; Bernardes, G. J. L. Inverse Electron Demand Diels-Alder Reactions in Chemical Biology. Chem. Soc. Rev. 2017, 46, 4895– 4950,  DOI: 10.1039/C7CS00184C
      35
      Inverse electron demand Diels-Alder reactions in chemical biology
      Oliveira, B. L.; Guo, Z.; Bernardes, G. J. L.
      Chemical Society Reviews (2017), 46 (16), 4895-4950CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      The emerging inverse electron demand Diels-Alder (IEDDA) reaction stands out from other bioorthogonal reactions by virtue of its unmatchable kinetics, excellent orthogonality and biocompatibility. With the recent discovery of novel dienophiles and optimal tetrazine coupling partners, attention has now been turned to the use of IEDDA approaches in basic biol., imaging and therapeutics. Here we review this bioorthogonal reaction and its promising applications for live cell and animal studies. We first discuss the key factors that contribute to the fast IEDDA kinetics and describe the most recent advances in the synthesis of tetrazine and dienophile coupling partners. Both coupling partners have been incorporated into proteins for tracking and imaging by use of fluorogenic tetrazines that become strongly fluorescent upon reaction. Selected notable examples of such applications are presented. The exceptional fast kinetics of this catalyst-free reaction, even using low concns. of coupling partners, make it amenable for in vivo radiolabelling using pretargeting methodologies, which are also discussed. Finally, IEDDA reactions have recently found use in bioorthogonal decaging to activate proteins or drugs in gain-of-function strategies. We conclude by showing applications of the IEDDA reaction in the construction of biomaterials that are used for drug delivery and multimodal imaging, among others. The use and utility of the IEDDA reaction is interdisciplinary and promises to revolutionize chem. biol., radiochem. and materials science.
    36. 36
      Maruani, A.; Szijj, P. A.; Bahou, C.; Nogueira, J. C. F.; Caddick, S.; Baker, J. R.; Chudasama, V. A Plug-and-Play Approach for the de Novo Generation of Dually Functionalized Bispecifics. Bioconjugate Chem. 2020, 31, 520– 529,  DOI: 10.1021/acs.bioconjchem.0c00002
      36
      A Plug-and-Play Approach for the De Novo Generation of Dually Functionalized Bispecifics
      Maruani, Antoine; Szijj, Peter A.; Bahou, Calise; Nogueira, Joao C. F.; Caddick, Stephen; Baker, James R.; Chudasama, Vijay
      Bioconjugate Chemistry (2020), 31 (3), 520-529CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      Diseases are multifactorial, with redundancies and synergies between various pathways. However, most of the antibody-based therapeutics on the market interact with only one target, thus limiting their efficacy. The targeting of multiple epitopes could improve the therapeutic index of treatment and counteract mechanisms of resistance. To this effect, a new class of therapeutics has emerged: bispecific antibodies. Bispecific formation using chem. methods is rare and low-yielding and/or requires a large excess of one of the two proteins to avoid homodimerization and heterogeneity. In order for chem. prepd. bispecifics to deliver their full potential, high-yielding, modular, and reliable crosslinking technologies are required. Herein, we describe a novel approach not only for the rapid and high-yielding chem. generation of bispecific antibodies from native antibody fragments, but also for the site-specific dual functionalization of the resulting bioconjugates. Based on orthogonal clickable functional groups, this strategy enables the assembly of functionalized bispecifics with controlled loading in a modular and convergent manner.
    37. 37
      Maruani, A.; Smith, M. E. B.; Miranda, E.; Chester, K. A.; Chudasama, V.; Caddick, S. A Plug-and-Play Approach to Antibody-Based Therapeutics via a Chemoselective Dual Click Strategy. Nat. Commun. 2015, 6, 2– 10,  DOI: 10.1038/ncomms7645
      There is no corresponding record for this reference.
    38. 38
      Damen, J. A. M.; Escorihuela, J.; Zuilhof, H.; van Delft, F.; Albada, B. High Rates of Quinone-Alkyne Cycloaddition Reactions Are Dictated by Entropic Factors. Chem.─Eur. J. 2023, 29, e202300231  DOI: 10.1002/chem.202300231
      38
      High Rates of Quinone-Alkyne Cycloaddition Reactions are Dictated by Entropic Factors
      Damen, Johannes A. M.; Escorihuela, Jorge; Zuilhof, Han; van Delft, Floris L.; Albada, Bauke
      Chemistry - A European Journal (2023), 29 (39), e202300231CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Reaction rates of strained cycloalkynes and cycloalkenes with 1,2-quinone were quantified by stopped flow UV-Vis spectroscopy and computational anal. We found that the strained alkyne BCN-OH 3 (ks 1824 M-1 s-1) reacts >150 times faster than the strained alkene TCO-OH 5 (k2 11.56 M-1 s-1), and that derivatization with a carbamate can lead to a redn. of the rate const. with almost half. Also, the 8-membered strained alkyne BCN-OH 3 reacts 16 times faster than the more strained 7-membered THS 2 (k2 110.6 M-1 s-1). Using the linearized Eyring equation we detd. the thermodn. activation parameters of these two strained alkynes, revealing that the SPOCQ reaction of quinone 1 with THS 2 is assocd. with ΔH≠ of 0.80 kcal/mol, ΔS≠=-46.8 cal/K·mol, and ΔG≠=14.8 kcal/mol (at 25°C), whereas the same reaction with BCN-OH 3 is assocd. with, ΔH≠=2.25 kcal/mol, ΔS≠=-36.3 cal/K·mol, and ΔG≠=13.1 kcal/mol (at 25°C). Computational anal. supported the values obtained by the stopped-flow measurements, with calcd. ΔG≠ of 15.6 kcal/mol (in H2O) for the SPOCQ reaction with THS 2, and with ΔG≠ of 14.7 kcal/mol (in H2O) for the SPOCQ reaction with BCN-OH 3. With these empirically detd. thermodn. parameters, we set an important step towards a more fundamental understanding of this set of rapid click reactions.

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