Ulf Grawunder - Academia.edu (original) (raw)
Papers by Ulf Grawunder
Springer eBooks, 1994
The molecular steps and cellular stages which define the development of B lymphocytes in the mous... more The molecular steps and cellular stages which define the development of B lymphocytes in the mouse have been reviewed by us in several publications (Rolink and Melchers 1991, 1993a, b; Melchers 1993 and references therein). This presentation, given as an introduction to normal B cell development in relation to immunodeficiencies and autoimmune diseases, summarizes the major conclusions for normal B cell development reached in these reviews. It gives one example of a B lineage-expressed immunodeficiency induced by targeted disruption by a defective copy of the gene for λ5, one component of the surrogate L chain (Kitamura et al. 1992; Rolink et al. 1993), and describes the example of the New Zealand black (NZB) × New Zealand white (NZW) mouse strain, where recent experimental progress in the analysis of normal B cell development in vitro and in vivo has helped to define defects expressed in the B lineage which lead to lupus-like autoimmune disease (Reininger et al. 1992).
PLOS ONE, 2015
<p>The tag sequences are shown on top, with the sortase A recognition motif in bold print, ... more <p>The tag sequences are shown on top, with the sortase A recognition motif in bold print, and the Strep II tag underlined. (A) Spacer-free design; (B) Design using a 5 amino acid (GGGGS) spacer on the IgL chain. Antibody was incubated with 2-fold serial dilutions of sortase A in the presence of a 20-fold excess of Gly<sub>5</sub>-modified FITC. The crude reaction products were then separated by SDS-PAGE, and the FITC conjugates visualized by placing the gel on a UV box. IgH, heavy chain; IgL, light chain; GF, Gly<sub>5</sub>-FITC.</p
Symposium in Immunology III, 1994
The molecular steps and cellular stages which define the development of B lymphocytes in the mous... more The molecular steps and cellular stages which define the development of B lymphocytes in the mouse have been reviewed by us in several publications (Rolink and Melchers 1991, 1993a, b; Melchers 1993 and references therein). This presentation, given as an introduction to normal B cell development in relation to immunodeficiencies and autoimmune diseases, summarizes the major conclusions for normal B cell development reached in these reviews. It gives one example of a B lineage-expressed immunodeficiency induced by targeted disruption by a defective copy of the gene for λ5, one component of the surrogate L chain (Kitamura et al. 1992; Rolink et al. 1993), and describes the example of the New Zealand black (NZB) × New Zealand white (NZW) mouse strain, where recent experimental progress in the analysis of normal B cell development in vitro and in vivo has helped to define defects expressed in the B lineage which lead to lupus-like autoimmune disease (Reininger et al. 1992).
Journal of Clinical Oncology, May 20, 2021
TPS1108 Background: The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is highly expresse... more TPS1108 Background: The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is highly expressed during embryonic development, but is minimally present or absent on post-partum healthy tissues. ROR1 is expressed in a variety of hematological and solid tumors and is associated with aggressive cancer phenotype and poor clinical outcomes. NBE-002 is an ADC targeting ROR1, obtained by site-specific, enzymatic conjugation of the anthracycline-derivative PNU-159682, modified with a non-cleavable linker to a humanized recombinant IgG1 monoclonal antibody, based on a novel anti-human ROR1 monoclonal antibody XBR1-402 (Peng et al. (2017) J. Mol. Biol. 429: 2954-73). Direct anti-tumor activity of NBE-002 was evaluated in immunodeficient, ROR1 expression-low/-intermediate/-high PDX models of several carcinoma and sarcoma subtypes. The most pronounced anti-tumor effect was achieved in triple-negative breast cancer (TNBC), at doses as low as 0.033 mg/kg, suggesting a best-in-class therapeutic index in light of the high tolerability in preclinical toxicology models. Administration in a fully immune competent setting (EMT6/ROR1 orthotopic breast cancer model) led to a strong anti-tumor response and a long-lasting anti-tumor immune protection dependent on CD8 T cells. Methods: NBE-002-01 (NCT04441099) is a first-in-human, open-label, multi-center, phase (Ph) 1/2 study of NBE-002 in adult patients with advanced solid tumors. Ph 1 of the study consists of a Dose Escalation Cohort (DEC), utilizing an accelerated titration design, followed by a traditional 3+3 design, and an optional Safety Expansion Cohort (SEC). Ph 2 will include two parallel Expansion Cohorts (EC), enrolling patients with advanced TNBC (EC1) or other solid tumors (EC2), with Simon’s two-stage design. Key eligibility criteria include Eastern Cooperative Oncology Group performance status of 0-2 (Ph 1) or 0-1 (Ph 2), adequate organ function defined as: hemoglobin ≥9.0 g/dL, neutrophils ≥1500 /µL, platelets ≥100000/µL, aspartate and alanine aminotransferases ≤2.5x the upper limit of normal (ULN), total bilirubin ≤1.5x ULN, creatinine ≤1.5x ULN, left ventricular ejection fraction ≥50%. The primary objectives are to assess safety and tolerability and to establish the recommended dose for further development (Ph 1), and to evaluate anti-tumor activity of (Ph 2). Secondary and exploratory objectives include characterization of immunogenicity as well as pharmacokinetic and pharmacodynamic profiles. NBE-002 is given intravenously once every three weeks until disease progression, unacceptable toxicity, withdrawal of consent, or other protocol-specific criteria are met. Ph 1 dose escalation was initiated on 17 JULY 2020 and is still recruiting in the US. Ph 2 is planned to be initiated in 2022. Clinical trial information: NCT04441099 .
Comparison of in vitro cell killing activity of sortase A-conjugated ADCs with cleavable and non-... more Comparison of in vitro cell killing activity of sortase A-conjugated ADCs with cleavable and non-cleavable linkers.
Zenodo (CERN European Organization for Nuclear Research), Dec 13, 2021
We report the first method of enzyme protection enabling the production of partially shielded enz... more We report the first method of enzyme protection enabling the production of partially shielded enzymes capable of processing substrates as large as proteins. We show that partially shielded sortase retains its transpeptidase activity and can perform bioconjugation reactions on antibodies. Moreover, a partially shielded trypsin is shown to outperform its soluble counterpart in terms of proteolytic kinetics. Remarkably, partial enzyme shielding results in a drastic increase in temporal stability of the enzyme.
Quantification of HER2 expression levels on T47D and SKBR3 breast cancer cells.
Legends to Supplementary Figures S1-S6
In vivo evaluation of CD30-specific ADCs in mouse xenograft model.
In vitro cytotoxicity assays with HER2-positive and -negative cell lines using sortase A-conjugat... more In vitro cytotoxicity assays with HER2-positive and -negative cell lines using sortase A-conjugated, anthracycline-based anti-HER2 ADCs.
Annals of Oncology, Nov 1, 2016
In vivo evaluation of HER2- and CD30-specific ADCs in mouse xenograft models.
Legends to Supplementary Figures S1-S6
Comparison of in vitro cell killing activity of sortase A-conjugated ADCs with cleavable and non-... more Comparison of in vitro cell killing activity of sortase A-conjugated ADCs with cleavable and non-cleavable linkers.
Biochemical Journal, 2019
Sortase enzymes play an important role in Gram-positive bacteria. They are responsible for the co... more Sortase enzymes play an important role in Gram-positive bacteria. They are responsible for the covalent attachment of proteins to the surface of the bacteria and perform this task via a highly sequence-specific transpeptidation reaction. Since these immobilized proteins are often involved in pathogenicity of Gram-positive bacteria, characterization of this type of enzyme is also of medical relevance. Different classes of sortases (A–F) have been found, which recognize characteristic recognition sequences present in substrate proteins. Up to date, sortase A from Staphylococcus aureus, a housekeeping class A sortase, is the most thoroughly studied representative of the sortase family of enzymes. Here we report the in-depth characterization of the class F sortase from Propionibacterium acnes, a class of sortases that has not been investigated before. As Sortase F is the only transpeptidase found in the P. acnes genome, it is the housekeeping sortase of this organism. Sortase F from P. ...
Antibody–drug conjugates (ADC) are highly potent and specific antitumor drugs, combining the spec... more Antibody–drug conjugates (ADC) are highly potent and specific antitumor drugs, combining the specific targeting of mAbs with the potency of small-molecule toxic payloads. ADCs generated by conventional chemical conjugation yield heterogeneous mixtures with variable pharmacokinetics, stability, safety, and efficacy profiles. To address these issues, numerous site-specific conjugation technologies are currently being developed allowing the manufacturing of homogeneous ADCs with predetermined drug-to-antibody ratios. Here, we used sortase-mediated antibody conjugation (SMAC) technology to generate homogeneous ADCs based on a derivative of the highly potent anthracycline toxin PNU-159682 and a noncleavable peptide linker, using the anti-HER2 antibody trastuzumab (part of Kadcyla) and the anti-CD30 antibody cAC10 (part of Adcetris). Characterization of the resulting ADCs in vitro and in vivo showed that they were highly stable and exhibited potencies exceeding those of ADCs based on conv...
Springer eBooks, 1994
The molecular steps and cellular stages which define the development of B lymphocytes in the mous... more The molecular steps and cellular stages which define the development of B lymphocytes in the mouse have been reviewed by us in several publications (Rolink and Melchers 1991, 1993a, b; Melchers 1993 and references therein). This presentation, given as an introduction to normal B cell development in relation to immunodeficiencies and autoimmune diseases, summarizes the major conclusions for normal B cell development reached in these reviews. It gives one example of a B lineage-expressed immunodeficiency induced by targeted disruption by a defective copy of the gene for λ5, one component of the surrogate L chain (Kitamura et al. 1992; Rolink et al. 1993), and describes the example of the New Zealand black (NZB) × New Zealand white (NZW) mouse strain, where recent experimental progress in the analysis of normal B cell development in vitro and in vivo has helped to define defects expressed in the B lineage which lead to lupus-like autoimmune disease (Reininger et al. 1992).
PLOS ONE, 2015
<p>The tag sequences are shown on top, with the sortase A recognition motif in bold print, ... more <p>The tag sequences are shown on top, with the sortase A recognition motif in bold print, and the Strep II tag underlined. (A) Spacer-free design; (B) Design using a 5 amino acid (GGGGS) spacer on the IgL chain. Antibody was incubated with 2-fold serial dilutions of sortase A in the presence of a 20-fold excess of Gly<sub>5</sub>-modified FITC. The crude reaction products were then separated by SDS-PAGE, and the FITC conjugates visualized by placing the gel on a UV box. IgH, heavy chain; IgL, light chain; GF, Gly<sub>5</sub>-FITC.</p
Symposium in Immunology III, 1994
The molecular steps and cellular stages which define the development of B lymphocytes in the mous... more The molecular steps and cellular stages which define the development of B lymphocytes in the mouse have been reviewed by us in several publications (Rolink and Melchers 1991, 1993a, b; Melchers 1993 and references therein). This presentation, given as an introduction to normal B cell development in relation to immunodeficiencies and autoimmune diseases, summarizes the major conclusions for normal B cell development reached in these reviews. It gives one example of a B lineage-expressed immunodeficiency induced by targeted disruption by a defective copy of the gene for λ5, one component of the surrogate L chain (Kitamura et al. 1992; Rolink et al. 1993), and describes the example of the New Zealand black (NZB) × New Zealand white (NZW) mouse strain, where recent experimental progress in the analysis of normal B cell development in vitro and in vivo has helped to define defects expressed in the B lineage which lead to lupus-like autoimmune disease (Reininger et al. 1992).
Journal of Clinical Oncology, May 20, 2021
TPS1108 Background: The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is highly expresse... more TPS1108 Background: The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is highly expressed during embryonic development, but is minimally present or absent on post-partum healthy tissues. ROR1 is expressed in a variety of hematological and solid tumors and is associated with aggressive cancer phenotype and poor clinical outcomes. NBE-002 is an ADC targeting ROR1, obtained by site-specific, enzymatic conjugation of the anthracycline-derivative PNU-159682, modified with a non-cleavable linker to a humanized recombinant IgG1 monoclonal antibody, based on a novel anti-human ROR1 monoclonal antibody XBR1-402 (Peng et al. (2017) J. Mol. Biol. 429: 2954-73). Direct anti-tumor activity of NBE-002 was evaluated in immunodeficient, ROR1 expression-low/-intermediate/-high PDX models of several carcinoma and sarcoma subtypes. The most pronounced anti-tumor effect was achieved in triple-negative breast cancer (TNBC), at doses as low as 0.033 mg/kg, suggesting a best-in-class therapeutic index in light of the high tolerability in preclinical toxicology models. Administration in a fully immune competent setting (EMT6/ROR1 orthotopic breast cancer model) led to a strong anti-tumor response and a long-lasting anti-tumor immune protection dependent on CD8 T cells. Methods: NBE-002-01 (NCT04441099) is a first-in-human, open-label, multi-center, phase (Ph) 1/2 study of NBE-002 in adult patients with advanced solid tumors. Ph 1 of the study consists of a Dose Escalation Cohort (DEC), utilizing an accelerated titration design, followed by a traditional 3+3 design, and an optional Safety Expansion Cohort (SEC). Ph 2 will include two parallel Expansion Cohorts (EC), enrolling patients with advanced TNBC (EC1) or other solid tumors (EC2), with Simon’s two-stage design. Key eligibility criteria include Eastern Cooperative Oncology Group performance status of 0-2 (Ph 1) or 0-1 (Ph 2), adequate organ function defined as: hemoglobin ≥9.0 g/dL, neutrophils ≥1500 /µL, platelets ≥100000/µL, aspartate and alanine aminotransferases ≤2.5x the upper limit of normal (ULN), total bilirubin ≤1.5x ULN, creatinine ≤1.5x ULN, left ventricular ejection fraction ≥50%. The primary objectives are to assess safety and tolerability and to establish the recommended dose for further development (Ph 1), and to evaluate anti-tumor activity of (Ph 2). Secondary and exploratory objectives include characterization of immunogenicity as well as pharmacokinetic and pharmacodynamic profiles. NBE-002 is given intravenously once every three weeks until disease progression, unacceptable toxicity, withdrawal of consent, or other protocol-specific criteria are met. Ph 1 dose escalation was initiated on 17 JULY 2020 and is still recruiting in the US. Ph 2 is planned to be initiated in 2022. Clinical trial information: NCT04441099 .
Comparison of in vitro cell killing activity of sortase A-conjugated ADCs with cleavable and non-... more Comparison of in vitro cell killing activity of sortase A-conjugated ADCs with cleavable and non-cleavable linkers.
Zenodo (CERN European Organization for Nuclear Research), Dec 13, 2021
We report the first method of enzyme protection enabling the production of partially shielded enz... more We report the first method of enzyme protection enabling the production of partially shielded enzymes capable of processing substrates as large as proteins. We show that partially shielded sortase retains its transpeptidase activity and can perform bioconjugation reactions on antibodies. Moreover, a partially shielded trypsin is shown to outperform its soluble counterpart in terms of proteolytic kinetics. Remarkably, partial enzyme shielding results in a drastic increase in temporal stability of the enzyme.
Quantification of HER2 expression levels on T47D and SKBR3 breast cancer cells.
Legends to Supplementary Figures S1-S6
In vivo evaluation of CD30-specific ADCs in mouse xenograft model.
In vitro cytotoxicity assays with HER2-positive and -negative cell lines using sortase A-conjugat... more In vitro cytotoxicity assays with HER2-positive and -negative cell lines using sortase A-conjugated, anthracycline-based anti-HER2 ADCs.
Annals of Oncology, Nov 1, 2016
In vivo evaluation of HER2- and CD30-specific ADCs in mouse xenograft models.
Legends to Supplementary Figures S1-S6
Comparison of in vitro cell killing activity of sortase A-conjugated ADCs with cleavable and non-... more Comparison of in vitro cell killing activity of sortase A-conjugated ADCs with cleavable and non-cleavable linkers.
Biochemical Journal, 2019
Sortase enzymes play an important role in Gram-positive bacteria. They are responsible for the co... more Sortase enzymes play an important role in Gram-positive bacteria. They are responsible for the covalent attachment of proteins to the surface of the bacteria and perform this task via a highly sequence-specific transpeptidation reaction. Since these immobilized proteins are often involved in pathogenicity of Gram-positive bacteria, characterization of this type of enzyme is also of medical relevance. Different classes of sortases (A–F) have been found, which recognize characteristic recognition sequences present in substrate proteins. Up to date, sortase A from Staphylococcus aureus, a housekeeping class A sortase, is the most thoroughly studied representative of the sortase family of enzymes. Here we report the in-depth characterization of the class F sortase from Propionibacterium acnes, a class of sortases that has not been investigated before. As Sortase F is the only transpeptidase found in the P. acnes genome, it is the housekeeping sortase of this organism. Sortase F from P. ...
Antibody–drug conjugates (ADC) are highly potent and specific antitumor drugs, combining the spec... more Antibody–drug conjugates (ADC) are highly potent and specific antitumor drugs, combining the specific targeting of mAbs with the potency of small-molecule toxic payloads. ADCs generated by conventional chemical conjugation yield heterogeneous mixtures with variable pharmacokinetics, stability, safety, and efficacy profiles. To address these issues, numerous site-specific conjugation technologies are currently being developed allowing the manufacturing of homogeneous ADCs with predetermined drug-to-antibody ratios. Here, we used sortase-mediated antibody conjugation (SMAC) technology to generate homogeneous ADCs based on a derivative of the highly potent anthracycline toxin PNU-159682 and a noncleavable peptide linker, using the anti-HER2 antibody trastuzumab (part of Kadcyla) and the anti-CD30 antibody cAC10 (part of Adcetris). Characterization of the resulting ADCs in vitro and in vivo showed that they were highly stable and exhibited potencies exceeding those of ADCs based on conv...