Intracellular delivery and trafficking dynamics of a lymphoma-targeting antibody-polymer conjugate.
Ratiometric fluorescence and cellular fractionation studies were employed to characterize the intracellular trafficking dynamics of antibody-poly(propylacrylic acid) (PPAA) conjugates in CD22+ RAMOS-AW cells. The HD39 monoclonal antibody (mAb) directs CD22-dependent, receptor-mediated uptake in human B-cell lymphoma cells, where it is rapidly trafficked to the lysosomal compartment. To characterize the intracellular-release dynamics of the polymer-mAb conjugates, HD39-streptavidin (HD39/SA) was dual-labeled with pH-insensitive Alexa Fluor 488 and pH-sensitive pHrodo fluorophores. The subcellular pH distribution of the HD39/SA-polymer conjugates was quantified as a function of time by live-cell fluorescence microscopy, and the average intracellular pH value experienced by the conjugates was also characterized as a function of time by flow cytometry. PPAA was shown to alter the intracellular trafficking kinetics strongly relative to HD39/SA alone or HD39/SA conjugates with a control polymer, poly(methacryclic acid) (PMAA). Subcellular trafficking studies revealed that after 6 h, only 11% of the HD39/SA-PPAA conjugates had been trafficked to acidic lysosomal compartments with values at or below pH 5.6. In contrast, the average intracellular pH of HD39/SA alone dropped from 6.7 ± 0.2 at 1 h to 5.6 ± 0.5 after 3 h and 4.7 ± 0.6 after 6 h. Conjugation of the control polymer PMAA to HD39/SA showed an average pH drop similar to that of HD39/SA. Subcellular fractionation studies with tritium-labeled HD39/SA demonstrated that after 6 h, 89% of HD39/SA was associated with endosomes (Rab5+) and lysosomes (Lamp2+), while 45% of HD39/SA-PPAA was translocated to the cytosol (lactate dehydrogenase+). These results demonstrate the endosomal-releasing properties of PPAA with antibody-polymer conjugates and detail their intracellular trafficking dynamics and subcellular compartmental distributions over time.
Department of Bioengineering, University of Washington , Seattle, Washington 98195-5061, United States.
Geoffrey Y
GY
Berguig
Anthony J
AJ
Convertine
Julie
J
Shi
Maria Corinna
MC
Palanca-Wessels
Craig L
CL
Duvall
Suzie H
SH
Pun
Oliver W
OW
Press
Patrick S
PS
Stayton
D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D002051:Burkitt Lymphoma / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism; D016503:Drug Delivery Systems; D005434:Flow Cytometry; D006801:Humans; D008856:Microscopy, Fluorescence; D011108:Polymers / Q000737:chemistry / Q000494:pharmacology*; D021381:Protein Transport; D051918:Sialic Acid Binding Ig-like Lectin 2 / Q000378:metabolism; D013347:Subcellular Fractions; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; C497867:CD22 protein, human; D011108:Polymers; D051918:Sialic Acid Binding Ig-like Lectin 2