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A search for synergy in the binding kinetics of Trastuzumab and Pertuzumab whole and F(ab) to Her2

A search for synergy in the binding kinetics of Trastuzumab and Pertuzumab whole and F(ab) to Her2 www.nature.com/npjbcancer All rights reserved 2374-4677/15 BRIEF COMMUNICATION OPEN A search for synergy in the binding kinetics of Trastuzumab and Pertuzumab whole and F(ab) to Her2 1,5 1,2,5 2,4 1,3,4 Wai-Heng Lua , Samuel Ken-En Gan , David Philip Lane and Chandra Shekhar Verma Therapeutic efficacy resulting from combining Trastuzumab and Pertuzumab in the treatment of Her2 overexpressing breast cancer patients has been shown to increase patient survival. This is thought to arise from inhibition of receptor dimerization and the immune tagging of the cancer cells; however, the underlying molecular mechanisms have remained enigmatic. Previously, a molecular modeling study suggested that this resulted from colocalization of the two antibodies on to the extracellular domain of Her2. We report here the experimental characterization of this interaction by measuring the binding kinetics of these two whole antibodies and their F(ab)s to the extracellular domain of Her2 in solution. We found that both antibodies (the whole antibodies and the fragments) colocalized on to Her2, but did not augment the binding of each other. npj Breast Cancer (2015) 1, 15012; doi:10.1038/npjbcancer.2015.12; published online 5 August 2015 CORRESPONDENCE/FINDINGS of Her2 with saturating (200 nM) and non-saturating (50 nM) concentrations of each antibody (shown in plateau sections of Therapeutic efficacy resulting from combining drugs in oncology is Figure 1c–e) followed by measurements of the second antibody increasingly being reported. A significant improvement in survival appears to have little effect on the binding of the latter. An was recently reported in the treatment of Her2 overexpressing observation to note is that the presence of the control IgG does breast cancer patients from combining the antibodies Trastuzumab 1 seem to increase the Kd of both antibodies (compare Figure 1a, c and Pertuzumab. However, the underlying molecular mechanisms and Figure 1b, d) thus making comparisons with binding of have remained enigmatic. A hypothesis was put forward from a antibodies alone, a little complex. molecular modeling study that suggested a partial rationale in the We repeated the experiments to rule out confounding variables form of enhanced affinities arising from colocalization of the two under the following conditions: (1) using only Trastuzumab and antibodies on to the extracellular domain of Her2. We report here Pertuzumab F(ab)s (Figure 1g, h); (2) preloading Her2 with F(ab) the experimental characterization of this interaction by measuring or whole antibody, followed by measurement of the alternative the binding kinetics (using the BLItz biosensor system, ForteBio, Pall, antibody as a whole or F(ab), respectively (not shown); (3) Singapore) of these two antibodies (whole antibodies and F(ab)s; preloading the alternative antibody overnight (performed for both see Figure 1) to the extracellular domain of Her2 in solution. F(ab)s and whole antibodies, data not shown). Under these Briefly, 25 μg/ml of HIS-tagged Her2 (cat no: 10004-H08H, Sino conditions, minimal binding changes of either antibody were Biologicals, China) was bound onto Nickel-nitrilotriacetic acid (Ni- observed. This suggests that the state of the antibodies (F(ab)s NTA) biosensors (ForteBio, Pall). Trastuzumab (Roche, Singapore) or whole Igs, or mixed) were not contributing factors nor was the and Pertuzumab (Roche) to Her2 binding kinetics were measured time of incubation a factor in inducing synergistic binding. using the BLI technology from ForteBio (http://www.fortebio.com/ We have demonstrated, in agreement with the computational bli-technology.html). Whole antibody-Her2 interactions were modeling, that Tratsuzumab binds to the extracellular domain of calculated from five serial 1:2 dilutions. Synergistic binding of Her2 with a higher affinity than does Pertuzumab. We have also the whole antibodies and F(ab)s were measured from successive shown, in agreement with the modeling, that both antibodies can loading of the antibodies. Human IgG control (cat no: PN 18-1073, colocalize onto the extracellular domain of Her2. However, in lot no: 3060036, ForteBio, Pall) was used as the control IgG. The contrast to the modeling, we did not observe significant synergistic F(ab)s of both Trastuzumab and Pertuzumab were prepared by binding effects through prior Trastuzumab or Pertuzumab loading TM papain digestion using the Pierce Fab Preparation Kit (cat no: (note that only the extracellular portion of Her2 was used in our 44985, Life Technologies, Singapore). experiments). The discrepancy between these findings and the Our measurements found the kinetics of whole Trastuzumab hypotheses generated from the modelling may arise from the fact to be comparable to previous surface plasmon resonance that the modeling may not have captured the complex nature of measurements. We see that Trastuzumab (in both its IgG and F the interactions which, owing to the much larger system size of the (ab) states) (see Figure 1a,b,f, respectively) binds tighter than the ternary complexes of Her2–Trastuzumab–Pertuzumab, would likely corresponding Pertuzumab states, an observation in support of the require longer simulation times than that have been used. However, earlier computational model based on the F(ab) states. Preloading the complex nature of these interactions and the vast differences 1 2 Bioinformatics Institute, Agency for Science, Technology, and Research (A*STAR), Singapore, Singapore; p53 Laboratory, Agency for Science, Technology, and Research 3 4 (A*STAR), Singapore, Singapore; Department of Biological Sciences, National University of Singapore (NUS), Singapore, Singapore and School of Biological Sciences, Nanyang Technological University (NTU), Singapore, Singapore. Correspondence: SK-E Gan or CS Verma (samuelg@bii.a-star.edu.sg or chandra@bii.a-star.edu.sg) These authors contributed equally to the work. Received 21 December 2014; revised 23 May 2015; accepted 8 July 2015 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited Synergistic binding kinetics of Trastuzumab and Pertuzumab to Her2 W-H Lua et al npj Breast Cancer (2015) 15012 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited Synergistic binding kinetics of Trastuzumab and Pertuzumab to Her2 W-H Lua et al Figure 1. Binding assays of Trastuzumab and Pertuzumab to Her2 on a BLItz system. (a) Whole Trastuzumab binding to Her2. Binding of 12.5– 200 nM of Trastuzumab to 25 μg/ml of Ni-NTA probe bound Her2-His-tagged on the BLItz system. The binding kinetics of Trastuzumab was titratable to 25 nM. (b) Whole Pertuzumab binding to Her2. Binding of 12.5–200 nM of Pertuzumab to 25 μg/ml of Ni-NTA probe bound Her2- His-tagged on the BLItz system. The binding kinetics of Pertuzumab was titratable to 25 nM. (c) Whole Trastuzumab binding with Pert preloaded. Binding of 200 nM Trastuzumab to (i) Her2-His-tagged (ii) 50 and 200 nM Pertuzumab or 200 nM human IgG. Unbound sensor was used as negative control. (d) Whole Pertuzumab binding with Trast preloaded. Binding of 200 nM Pertuzumab to (i) Her2-His-tagged (ii) 50 and 200 nM Trastuzumab or 200 nM human IgG. Unbound sensor was used as negative control. (e) Whole Trastuzumab binding with half Pert preloaded. Binding of 200 nM Trastuzumab to (i) Her2-His-tagged (ii) 50 nM Pertuzumab or 200 nM human IgG at 1 min loading time. One minute loading time was used to prevent saturation of Pertuzumab, which may interfere with Trastuzumab binding. (f) F(ab) binding to Her2. Binding assay showing 200 nM of Trastuzumab or Pertuzumab F(ab) to Her2-His-tagged. Decreased maximum binding kinetics reflect the decreased bound protein size. (top left) SDS-PAGE showing purified Trastuzumab and Pertuzumab F(ab) prepared using the Fab preparation kit. (g) Trastuzumab F(ab) binding with Pert F(ab) preloaded. Binding of 200 nM Trastuzumab F(ab) to (i) Her2-His-tagged (ii) 200 nM of Pertuzumab F(ab) or human IgG. (h) Pertuzumab F(ab) binding with Trast F(ab) preloaded. Binding of 200 nM Pertuzumab F(ab) to (i) Her2-His- tagged (ii) 200 nM of Trastuzumab F(ab) or human IgG. Bl, baseline, as measured using phosphate-buffered saline; hIgG, human IgG control; Ni-NTA, Nickel-nitrilotriacetic acid; Pert, Pertuzumab; Trast, Trastuzumab. 4,5 between in silico, in vitro, and in vivo measurements rule out any REFERENCES unambiguous conclusions. Nevertheless, the simple fact that both 1 Baselga J, Cortés J, Kim SB, Im SA, Hegg R, Im YH et al. Pertuzumab plus trastu- zumab plus docetaxel for metastatic breast cancer. N Engl J Med 2012; 366: antibodies can bind simultaneously and without significant 109–119. interference, may be sufficient to further impede Her2 dimerization 2 Fuentes G, Scaltriti M, Baselga J, Verma CS. Synergy between trastuzumab and and enforce the engagement of downstream immune effectors to pertuzumab for human epidermal growth factor 2 (Her2) from colocalization: an in explain the clinical observations. silico based mechanism. Breast Cancer Res 2011; 13:R54. 3 Karagiannis P, Singer J, Hunt J, Gan SKE, Rudman SM, Mechtcheriakova D et al. Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells. Cancer Immunol Immun- ACKNOWLEDGMENTS other 2009; 58: 915–930. This work was supported by JCO1334i00050 from the Joint Council Office, Agency for 4 Cass RM, Anderson BR. The disappearance rate of skin sensitizing antibody activity Science, Technology, and Research, Singapore. after intradermal administration. J Allergy 1968; 42:29–35. 5 Wan T, Beavil RL, Fabiane SM, Beavil AJ, Sohi MK, Keown M et al. The crystal structure of IgE Fc reveals an asymmetrically bent conformation. Nat Immunol 2002; 3:681–686. CONTRIBUTIONS WHL prepared the figures, manuscript and did the binding assays. SKEG conceived the design of the project and supervised all aspects of the project and writing. DPL This work is licensed under a Creative Commons Attribution 4.0 and CSV edited and approved the manuscript. International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the COMPETING INTERESTS material. To view a copy of this license, visit http://creativecommons.org/licenses/ The authors declare no conflict of interest. by/4.0/ © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15012 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png npj Breast Cancer Springer Journals

A search for synergy in the binding kinetics of Trastuzumab and Pertuzumab whole and F(ab) to Her2

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Springer Journals
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Copyright © 2015 by The Author(s)
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Biomedicine; Biomedicine, general; Cancer Research; Oncology; Human Genetics; Cell Biology
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10.1038/npjbcancer.2015.12
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Abstract

www.nature.com/npjbcancer All rights reserved 2374-4677/15 BRIEF COMMUNICATION OPEN A search for synergy in the binding kinetics of Trastuzumab and Pertuzumab whole and F(ab) to Her2 1,5 1,2,5 2,4 1,3,4 Wai-Heng Lua , Samuel Ken-En Gan , David Philip Lane and Chandra Shekhar Verma Therapeutic efficacy resulting from combining Trastuzumab and Pertuzumab in the treatment of Her2 overexpressing breast cancer patients has been shown to increase patient survival. This is thought to arise from inhibition of receptor dimerization and the immune tagging of the cancer cells; however, the underlying molecular mechanisms have remained enigmatic. Previously, a molecular modeling study suggested that this resulted from colocalization of the two antibodies on to the extracellular domain of Her2. We report here the experimental characterization of this interaction by measuring the binding kinetics of these two whole antibodies and their F(ab)s to the extracellular domain of Her2 in solution. We found that both antibodies (the whole antibodies and the fragments) colocalized on to Her2, but did not augment the binding of each other. npj Breast Cancer (2015) 1, 15012; doi:10.1038/npjbcancer.2015.12; published online 5 August 2015 CORRESPONDENCE/FINDINGS of Her2 with saturating (200 nM) and non-saturating (50 nM) concentrations of each antibody (shown in plateau sections of Therapeutic efficacy resulting from combining drugs in oncology is Figure 1c–e) followed by measurements of the second antibody increasingly being reported. A significant improvement in survival appears to have little effect on the binding of the latter. An was recently reported in the treatment of Her2 overexpressing observation to note is that the presence of the control IgG does breast cancer patients from combining the antibodies Trastuzumab 1 seem to increase the Kd of both antibodies (compare Figure 1a, c and Pertuzumab. However, the underlying molecular mechanisms and Figure 1b, d) thus making comparisons with binding of have remained enigmatic. A hypothesis was put forward from a antibodies alone, a little complex. molecular modeling study that suggested a partial rationale in the We repeated the experiments to rule out confounding variables form of enhanced affinities arising from colocalization of the two under the following conditions: (1) using only Trastuzumab and antibodies on to the extracellular domain of Her2. We report here Pertuzumab F(ab)s (Figure 1g, h); (2) preloading Her2 with F(ab) the experimental characterization of this interaction by measuring or whole antibody, followed by measurement of the alternative the binding kinetics (using the BLItz biosensor system, ForteBio, Pall, antibody as a whole or F(ab), respectively (not shown); (3) Singapore) of these two antibodies (whole antibodies and F(ab)s; preloading the alternative antibody overnight (performed for both see Figure 1) to the extracellular domain of Her2 in solution. F(ab)s and whole antibodies, data not shown). Under these Briefly, 25 μg/ml of HIS-tagged Her2 (cat no: 10004-H08H, Sino conditions, minimal binding changes of either antibody were Biologicals, China) was bound onto Nickel-nitrilotriacetic acid (Ni- observed. This suggests that the state of the antibodies (F(ab)s NTA) biosensors (ForteBio, Pall). Trastuzumab (Roche, Singapore) or whole Igs, or mixed) were not contributing factors nor was the and Pertuzumab (Roche) to Her2 binding kinetics were measured time of incubation a factor in inducing synergistic binding. using the BLI technology from ForteBio (http://www.fortebio.com/ We have demonstrated, in agreement with the computational bli-technology.html). Whole antibody-Her2 interactions were modeling, that Tratsuzumab binds to the extracellular domain of calculated from five serial 1:2 dilutions. Synergistic binding of Her2 with a higher affinity than does Pertuzumab. We have also the whole antibodies and F(ab)s were measured from successive shown, in agreement with the modeling, that both antibodies can loading of the antibodies. Human IgG control (cat no: PN 18-1073, colocalize onto the extracellular domain of Her2. However, in lot no: 3060036, ForteBio, Pall) was used as the control IgG. The contrast to the modeling, we did not observe significant synergistic F(ab)s of both Trastuzumab and Pertuzumab were prepared by binding effects through prior Trastuzumab or Pertuzumab loading TM papain digestion using the Pierce Fab Preparation Kit (cat no: (note that only the extracellular portion of Her2 was used in our 44985, Life Technologies, Singapore). experiments). The discrepancy between these findings and the Our measurements found the kinetics of whole Trastuzumab hypotheses generated from the modelling may arise from the fact to be comparable to previous surface plasmon resonance that the modeling may not have captured the complex nature of measurements. We see that Trastuzumab (in both its IgG and F the interactions which, owing to the much larger system size of the (ab) states) (see Figure 1a,b,f, respectively) binds tighter than the ternary complexes of Her2–Trastuzumab–Pertuzumab, would likely corresponding Pertuzumab states, an observation in support of the require longer simulation times than that have been used. However, earlier computational model based on the F(ab) states. Preloading the complex nature of these interactions and the vast differences 1 2 Bioinformatics Institute, Agency for Science, Technology, and Research (A*STAR), Singapore, Singapore; p53 Laboratory, Agency for Science, Technology, and Research 3 4 (A*STAR), Singapore, Singapore; Department of Biological Sciences, National University of Singapore (NUS), Singapore, Singapore and School of Biological Sciences, Nanyang Technological University (NTU), Singapore, Singapore. Correspondence: SK-E Gan or CS Verma (samuelg@bii.a-star.edu.sg or chandra@bii.a-star.edu.sg) These authors contributed equally to the work. Received 21 December 2014; revised 23 May 2015; accepted 8 July 2015 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited Synergistic binding kinetics of Trastuzumab and Pertuzumab to Her2 W-H Lua et al npj Breast Cancer (2015) 15012 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited Synergistic binding kinetics of Trastuzumab and Pertuzumab to Her2 W-H Lua et al Figure 1. Binding assays of Trastuzumab and Pertuzumab to Her2 on a BLItz system. (a) Whole Trastuzumab binding to Her2. Binding of 12.5– 200 nM of Trastuzumab to 25 μg/ml of Ni-NTA probe bound Her2-His-tagged on the BLItz system. The binding kinetics of Trastuzumab was titratable to 25 nM. (b) Whole Pertuzumab binding to Her2. Binding of 12.5–200 nM of Pertuzumab to 25 μg/ml of Ni-NTA probe bound Her2- His-tagged on the BLItz system. The binding kinetics of Pertuzumab was titratable to 25 nM. (c) Whole Trastuzumab binding with Pert preloaded. Binding of 200 nM Trastuzumab to (i) Her2-His-tagged (ii) 50 and 200 nM Pertuzumab or 200 nM human IgG. Unbound sensor was used as negative control. (d) Whole Pertuzumab binding with Trast preloaded. Binding of 200 nM Pertuzumab to (i) Her2-His-tagged (ii) 50 and 200 nM Trastuzumab or 200 nM human IgG. Unbound sensor was used as negative control. (e) Whole Trastuzumab binding with half Pert preloaded. Binding of 200 nM Trastuzumab to (i) Her2-His-tagged (ii) 50 nM Pertuzumab or 200 nM human IgG at 1 min loading time. One minute loading time was used to prevent saturation of Pertuzumab, which may interfere with Trastuzumab binding. (f) F(ab) binding to Her2. Binding assay showing 200 nM of Trastuzumab or Pertuzumab F(ab) to Her2-His-tagged. Decreased maximum binding kinetics reflect the decreased bound protein size. (top left) SDS-PAGE showing purified Trastuzumab and Pertuzumab F(ab) prepared using the Fab preparation kit. (g) Trastuzumab F(ab) binding with Pert F(ab) preloaded. Binding of 200 nM Trastuzumab F(ab) to (i) Her2-His-tagged (ii) 200 nM of Pertuzumab F(ab) or human IgG. (h) Pertuzumab F(ab) binding with Trast F(ab) preloaded. Binding of 200 nM Pertuzumab F(ab) to (i) Her2-His- tagged (ii) 200 nM of Trastuzumab F(ab) or human IgG. Bl, baseline, as measured using phosphate-buffered saline; hIgG, human IgG control; Ni-NTA, Nickel-nitrilotriacetic acid; Pert, Pertuzumab; Trast, Trastuzumab. 4,5 between in silico, in vitro, and in vivo measurements rule out any REFERENCES unambiguous conclusions. Nevertheless, the simple fact that both 1 Baselga J, Cortés J, Kim SB, Im SA, Hegg R, Im YH et al. Pertuzumab plus trastu- zumab plus docetaxel for metastatic breast cancer. N Engl J Med 2012; 366: antibodies can bind simultaneously and without significant 109–119. interference, may be sufficient to further impede Her2 dimerization 2 Fuentes G, Scaltriti M, Baselga J, Verma CS. Synergy between trastuzumab and and enforce the engagement of downstream immune effectors to pertuzumab for human epidermal growth factor 2 (Her2) from colocalization: an in explain the clinical observations. silico based mechanism. Breast Cancer Res 2011; 13:R54. 3 Karagiannis P, Singer J, Hunt J, Gan SKE, Rudman SM, Mechtcheriakova D et al. Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells. Cancer Immunol Immun- ACKNOWLEDGMENTS other 2009; 58: 915–930. This work was supported by JCO1334i00050 from the Joint Council Office, Agency for 4 Cass RM, Anderson BR. The disappearance rate of skin sensitizing antibody activity Science, Technology, and Research, Singapore. after intradermal administration. J Allergy 1968; 42:29–35. 5 Wan T, Beavil RL, Fabiane SM, Beavil AJ, Sohi MK, Keown M et al. The crystal structure of IgE Fc reveals an asymmetrically bent conformation. Nat Immunol 2002; 3:681–686. CONTRIBUTIONS WHL prepared the figures, manuscript and did the binding assays. SKEG conceived the design of the project and supervised all aspects of the project and writing. DPL This work is licensed under a Creative Commons Attribution 4.0 and CSV edited and approved the manuscript. International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the COMPETING INTERESTS material. To view a copy of this license, visit http://creativecommons.org/licenses/ The authors declare no conflict of interest. by/4.0/ © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15012

Journal

npj Breast CancerSpringer Journals

Published: Aug 5, 2015

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