Development and Validation of an Immuno-PET Tracer as a Companion Diagnostic Agent for Antibody-Drug Conjugate Therapy to Target the CA6 Epitope.
OBJECTIVE
To develop and compare three copper 64 ((64)Cu)-labeled antibody fragments derived from a CA6-targeting antibody (huDS6) as immuno-positron emission tomography (immuno-PET)-based companion diagnostic agents for an antibody-drug conjugate by using huDS6.


METHODS
Three antibody fragments derived from huDS6 were produced, purified, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and evaluated in the following ways: (a) the affinity of the fragments and the DOTA conjugates was measured via flow cytometry, (b) the stability of the labeled fragments was determined ex vivo in human serum over 24 hours, and (c) comparison of the in vivo imaging potential of the fragments was evaluated in mice bearing subcutaneous CA6-positive and CA6-negative xenografts by using serial PET imaging and biodistribution. Isotype controls with antilysozyme and anti-DM4 B-Fabs and blocking experiments with an excess of either B-Fab or huDS6 were used to determine the extent of the antibody fragment (64)Cu-DOTA-B-Fab binding specificity. Immunoreactivity and tracer kinetics were evaluated by using cellular uptake and 48-hour imaging experiments, respectively. Statistical analyses were performed by using t tests, one-way analysis of variance, and Wilcoxon and Mann-Whitney tests.


RESULTS
The antibody fragment (64)Cu-DOTA-B-Fab was more than 95% stable after 24 hours in human serum, had an immunoreactivity of more than 70%, and allowed differentiation between CA6-positive and CA6-negative tumors in vivo as early as 6 hours after injection, with a 1.7-fold uptake ratio between tumors. Isotype and blocking studies experiments showed tracer-specific uptake in antigen-positive tumors, despite some nonspecific uptake in both tumor models.


CONCLUSIONS
Three antibody fragments were produced and examined as potential companion diagnostic agents. (64)Cu-DOTA-B-Fab is a stable and effective immuno-PET tracer for CA6 imaging in vivo.
From the Department of Radiology (O.I., A.N., S.H., A. Sathirachinda, R.K., A. Srinivasan, S.S.G.) and Departments of Bioengineering and Materials Science & Engineering (S.S.G.), Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305; Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (M.G., J.K., I.F., C.L.); Sanofi Oncology, Vitry, France (C.C., I.S., V.B.); and Sanofi Oncology, Cambridge, Mass (S.K.S.).
Ohad
O
Ilovich
From the Department of Radiology (O.I., A.N., S.H., A. Sathirachinda, R.K., A. Srinivasan, S.S.G.) and Departments of Bioengineering and Materials Science & Engineering (S.S.G.), Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305; Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (M.G., J.K., I.F., C.L.); Sanofi Oncology, Vitry, France (C.C., I.S., V.B.); and Sanofi Oncology, Cambridge, Mass (S.K.S.).
Arutselvan
A
Natarajan
From the Department of Radiology (O.I., A.N., S.H., A. Sathirachinda, R.K., A. Srinivasan, S.S.G.) and Departments of Bioengineering and Materials Science & Engineering (S.S.G.), Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305; Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (M.G., J.K., I.F., C.L.); Sanofi Oncology, Vitry, France (C.C., I.S., V.B.); and Sanofi Oncology, Cambridge, Mass (S.K.S.).
Sharon
S
Hori
From the Department of Radiology (O.I., A.N., S.H., A. Sathirachinda, R.K., A. Srinivasan, S.S.G.) and Departments of Bioengineering and Materials Science & Engineering (S.S.G.), Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305; Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (M.G., J.K., I.F., C.L.); Sanofi Oncology, Vitry, France (C.C., I.S., V.B.); and Sanofi Oncology, Cambridge, Mass (S.K.S.).
Ataya
A
Sathirachinda
From the Department of Radiology (O.I., A.N., S.H., A. Sathirachinda, R.K., A. Srinivasan, S.S.G.) and Departments of Bioengineering and Materials Science & Engineering (S.S.G.), Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305; Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (M.G., J.K., I.F., C.L.); Sanofi Oncology, Vitry, France (C.C., I.S., V.B.); and Sanofi Oncology, Cambridge, Mass (S.K.S.).
Richard
R
Kimura
From the Department of Radiology (O.I., A.N., S.H., A. Sathirachinda, R.K., A. Srinivasan, S.S.G.) and Departments of Bioengineering and Materials Science & Engineering (S.S.G.), Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305; Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (M.G., J.K., I.F., C.L.); Sanofi Oncology, Vitry, France (C.C., I.S., V.B.); and Sanofi Oncology, Cambridge, Mass (S.K.S.).
Ananth
A
Srinivasan
From the Department of Radiology (O.I., A.N., S.H., A. Sathirachinda, R.K., A. Srinivasan, S.S.G.) and Departments of Bioengineering and Materials Science & Engineering (S.S.G.), Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305; Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (M.G., J.K., I.F., C.L.); Sanofi Oncology, Vitry, France (C.C., I.S., V.B.); and Sanofi Oncology, Cambridge, Mass (S.K.S.).
Mathias
M
Gebauer
From the Department of Radiology (O.I., A.N., S.H., A. Sathirachinda, R.K., A. Srinivasan, S.S.G.) and Departments of Bioengineering and Materials Science & Engineering (S.S.G.), Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305; Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (M.G., J.K., I.F., C.L.); Sanofi Oncology, Vitry, France (C.C., I.S., V.B.); and Sanofi Oncology, Cambridge, Mass (S.K.S.).
Jochen
J
Kruip
From the Department of Radiology (O.I., A.N., S.H., A. Sathirachinda, R.K., A. Srinivasan, S.S.G.) and Departments of Bioengineering and Materials Science & Engineering (S.S.G.), Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305; Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (M.G., J.K., I.F., C.L.); Sanofi Oncology, Vitry, France (C.C., I.S., V.B.); and Sanofi Oncology, Cambridge, Mass (S.K.S.).
Ingo
I
Focken
From the Department of Radiology (O.I., A.N., S.H., A. Sathirachinda, R.K., A. Srinivasan, S.S.G.) and Departments of Bioengineering and Materials Science & Engineering (S.S.G.), Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305; Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (M.G., J.K., I.F., C.L.); Sanofi Oncology, Vitry, France (C.C., I.S., V.B.); and Sanofi Oncology, Cambridge, Mass (S.K.S.).
Christian
C
Lange
From the Department of Radiology (O.I., A.N., S.H., A. Sathirachinda, R.K., A. Srinivasan, S.S.G.) and Departments of Bioengineering and Materials Science & Engineering (S.S.G.), Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305; Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (M.G., J.K., I.F., C.L.); Sanofi Oncology, Vitry, France (C.C., I.S., V.B.); and Sanofi Oncology, Cambridge, Mass (S.K.S.).
Chantal
C
Carrez
From the Department of Radiology (O.I., A.N., S.H., A. Sathirachinda, R.K., A. Srinivasan, S.S.G.) and Departments of Bioengineering and Materials Science & Engineering (S.S.G.), Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305; Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (M.G., J.K., I.F., C.L.); Sanofi Oncology, Vitry, France (C.C., I.S., V.B.); and Sanofi Oncology, Cambridge, Mass (S.K.S.).
Ingrid
I
Sassoon
From the Department of Radiology (O.I., A.N., S.H., A. Sathirachinda, R.K., A. Srinivasan, S.S.G.) and Departments of Bioengineering and Materials Science & Engineering (S.S.G.), Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305; Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (M.G., J.K., I.F., C.L.); Sanofi Oncology, Vitry, France (C.C., I.S., V.B.); and Sanofi Oncology, Cambridge, Mass (S.K.S.).
Veronique
V
Blanc
From the Department of Radiology (O.I., A.N., S.H., A. Sathirachinda, R.K., A. Srinivasan, S.S.G.) and Departments of Bioengineering and Materials Science & Engineering (S.S.G.), Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305; Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (M.G., J.K., I.F., C.L.); Sanofi Oncology, Vitry, France (C.C., I.S., V.B.); and Sanofi Oncology, Cambridge, Mass (S.K.S.).
Susanta K
SK
Sarkar
From the Department of Radiology (O.I., A.N., S.H., A. Sathirachinda, R.K., A. Srinivasan, S.S.G.) and Departments of Bioengineering and Materials Science & Engineering (S.S.G.), Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305; Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (M.G., J.K., I.F., C.L.); Sanofi Oncology, Vitry, France (C.C., I.S., V.B.); and Sanofi Oncology, Cambridge, Mass (S.K.S.).
Sanjiv S
SS
Gambhir
D000818:Animals; D002478:Cells, Cultured; D003301:Copper Radioisotopes; D004358:Drug Therapy; D000939:Epitopes; D006801:Humans; D007128:Immunoglobulin Fragments; D007159:Immunologic Tests; D051379:Mice; D049268:Positron-Emission Tomography / Q000379:methods*; D011849:Radioactive Tracers
D003301:Copper Radioisotopes; D000939:Epitopes; D007128:Immunoglobulin Fragments; D011849:Radioactive Tracers