Cambridge Healthtech Institute’s Kent Simmons recently spoke with Dr. Thomas Mikita, Director of Pfizer’s Centers for Therapeutic Innovation, about his upcoming presentation “High Affinity Factor XIa-specific IgGs and Reversal Agent as Potential Treatment for Thrombotic Disease”, to be delivered in the Emerging Indications for Therapeutic Antibodies meeting at the 2018 PEGS event. PEGS is scheduled for April 30 - May 4, 2018 in Boston, with the Emerging Indications program set for April 30-May 1.
Why did you target FXIa?
The coagulation cascade is structured such that two pathways, intrinsic and extrinsic, funnel into a common pathway that drives blood coagulation. All proteases on the cascade exist in zymogen form until activated by a triggering event. The extrinsic and common pathways are essential for maintaining hemostasis. Current anticoagulants like warfarin, which broadly inhibit multiple proteases on the coagulation cascade, or the new oral FXa and Thrombin inhibitors, which target a specific protease, carry a bleeding risk as these drugs target the extrinsic and common pathway. FXI resides on the intrinsic pathway, and considerable experimental data suggests that inhibiting FXIa (the active form of FXI) will reduce thrombosis risk without an appreciable increase in bleeding risk. This is why we chose to develop an antibody to FXIa. The goal was to make a potentially safer anti-thrombotic drug candidate.
What was one of the more challenging aspects to achieving potency?
FXI is present in the blood stream at 30 nM, and only a small percentage is activated during a coagulation event. This means that before FXI is activated to FXIa, our active-site-targeting IgG has no target to bind to. Once FXI is cleaved (by FXIIa) to generate FXIa, the active site is exposed, and now FIX (the next protease activated on the cascade) is in a race with our IgG to bind to the FXIa active site. FIX is present in the circulation at 90 nM and has a very fast on-rate for FXIa. To generate a potent enough inhibitor of FXIa we had to make a highly selective and high affinity (Kd~ 30 pM) IgG that would not bind to any of the other proteases in either active or zymogen form, and which also had an on-rate for FXIa that was faster than FIX. This required considerable affinity maturation. But we got it.
Were there any surprises when you obtained the co-crystal of the mAb with FXIa?
Yes, prior to having the crystal structure, we suspected that the heavy chain of CDR2 played an important role in binding the FXIa active site, as substitutions that increased affinity and potency in this IgG series involved substitutions of negatively charged residues in this loop. However, the crystal structure revealed that the important active site contacts were made by light chain CDR loops which didn’t change during affinity maturation. Rather, we believe that the negative charges introduced in CDR2 of the heavy chain placed negative charge across from a positively charged patch outside of the active site on FXIa. We hypothesize that this charge-based interaction likely increases docking such that the overall affinity and on-rate for the antibody was increased further.
Why did you also make two different reversal agents for the antibody?
Our team discussed both options, a highly specific antibody reversal agent that binds only our lead IgG (called DEF), and the design of an inactive protease domain as a reversal agent that works more generally for both IgG and small molecule active site inhibitors of FXIa. In the end we did both, in the spirit of healthy internal competition. Reversal agents are a focus for both old and newer anti-coagulants as they allow you to reverse a dangerous bleeding event that might arise during the course of a treatment. Because of this, we always knew we would need to develop one for our lead anti-FXIa antibody.
This work was done as collaboration between your team at Pfizer’s Centers for Therapeutic Innovation (CTI) and Dr Shaun Coughlin’s lab at UCSF, can you tell us a little more about CTI?
Pfizer’s Centers for Therapeutic Innovation carry out collaborative drug development projects with academic researchers who come to us with an idea for drug development. All proposed projects undergo rigorous review and a Statement of Work outlining the project is entered into before the program starts. From the start of lab work, both CTI scientists and our academic partner scientists work collaboratively. When it works well, as it did with Dr Coughlin’s lab, I think it can be a fantastic, fast-moving, and productive experience for all involved.
Speaker Biography:
Thomas Mikita, Ph.D., Director, Centers for Therapeutic Innovation, Pfizer, Inc.
I am currently a Director and Research Project Leader at Pfizer's Centers for Therapeutic Innovation. I lead or have led projects in Thrombosis, Oncology and Fibrosis. Prior to Pfizer I worked at CV Therapeutics (now Gilead) where my research focused on target discovery with a focus on atherosclerosis. My postdoc at Tularik (now Amgen) focused on IL-4/Stat6 signaling from receptor to nucleus. I have a PhD in Molecular Biophysics and Biochemistry from Yale University where I focused on the study of DNA replication inhibitors involved in cancer chemotherapy.