Developing Antibody Drug Conjugates (ADCs) with Site-Directed ConjugationMuctarr Sesay, Ph.D. and David Cunningham Goodwin Biotechnology, Inc., Plantation FL 33313

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ADC: Is the Whole Greater than the Sum of its Parts? Understanding the Complexity of the ADC Molecule

Considerations include selecting an appropriate:

• Target: clinically-established vs. novel?

• Monoclonal antibody: innovative vs. biosimilar (biobetter)?

• Linker: cleavable vs. non-cleavable? ❏ Non-cleavable, thioether-based

SteadFastTM Linker Technology

♦ Modular, heterobifunctional linker system which can conjugate a wide range of cytotoxic molecules

♦ Does not require separate modification of cytotoxic payload or antibody to introduce reactive functional groups

❏ Cleavable, disulfide-based and hydrazone-based FlexReleaseTM Linker Technology

• Cytotoxic payload: licensed vs. non-proprietary?

Case Studies: Random vs. Site-Directed Conjugations with Radioisotopes and Cytotoxics

• Two classic examples of site-directed conjugation have been studied in vivo in murine models. In one study1, we first tried a random conjugation strategy, but unfortunately, the increased levels of the chelator resulted in a loss of binding capacity to both the recombinant protein and to tumor cells. We then

utilized our proprietary, site-directed approach and achieved a conjugation efficiency of 93% and the resultant conjugate demonstrated structural intactness and a preserved immunoreactivity toward human aspartyl (asparaginyl) ß-hydroxylase (HAAH).

Also, the biodistribution in 111In-DPTA-PAN622 antibody, the level of cancer cell internalization, and the pathological investigation revealed that the weight of the primary breast cancer tumors was significantly (p=0.04) less than the untreated murine model.

• In the second study, we applied our proprietary site-directed conjugation technology to another client’s IgG1 antibody2, and the resultant maytansine conjugate displayed good binding to the TF-Ag expressing cancer cell lines in vitro and in a murine model, showed good internalization and cell killing ability in triple negative breast cancer tumors.

© Copyright 2017. All rights reserved.

Fig. 7

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PAN622 Antibody (µg/mL)

PAN622 ControlPAN622 CHX-A 10:1 molar ratioPAN622 DTPA-MAL 15 mil

Binding of PAN622 conjugates to HAAH and cancer cells. Binding of Pan-622-DTPA-MAL, 15 minutes, and Pan622-CHX’’ 10:1 conjugates to 4T1, mouse breast cancer cells.

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(A) 4 hours postinjection of 111In-DTPA-PAN622; (B) 24 hours; (C) 48 hours (left panel untreated mice; right panel treated with 213Bi-DTPA-PAN622). The dark blue color corresponds to the highest levels of radioactivity in the organs, the light blue—the lowest. Black arrows are pointing to the liver in all mice, red arrows are pointing to the peritoneal accumulation of radioactivity in untreated tumor-bearing mice.

1. Revskaya E, Jiang Z, Morganstern A, Bruchertseifer F, Sesay, M, et al. A radiolabeled fully human antibody to human aspartyl (asparaginyl) ß-Hydroxylase is a promising agent for imaging and therapy of metastatic breast cancer. Cancer Biotherapy and Radiopharmaceuticals. 2017; 32(2) 57-65.

2. Tati S, Fisk J, Abdullah J, et al. Humanization of JAA-F11, a highly specific anti-Thomsen-Friedenreich pancarcinoma antibody and in vitro efficacy analysis. Neoplasia. (Submitted for publication 2017).

Biodistribution of 111In-DTPA-Pan622 antibody in healthy mice and 4T1 tumor-bearing mice.

Biodistribution of 111In-DTPA-PAN622 antibody in healthy mice and 4T1 tumor-bearing mice.

Conclusions

Engineering ADCs to meet your needs

❏ More homogeneous conjugates

♦ Site-Directed Conjugates produce more homogenous conjugates at a lower average DAR than Random Conjugation.

❏ Improved binding to target cells

♦ Site-Directed Conjugation resulted in improved binding to target cells when compared to Random Conjugation.

❏ Processes created with scale up manufacturing for clinical studies in mind