642. CLL: THERAPY, EXCLUDING TRANSPLANTATION: POSTER III |
`
`NOVEMBER 15, 2013
`
`Target Engagement, Pathway Inhibition, and Efficacy
`Of The Bruton’s Tyrosine Kinase (Btk) Inhibitor CC-292
`
`Daniel W Pierce,*,1 Sabine Ponader, PhD,*,2 Kumudha Balakrishnan,*,3
`Varsha Gandhi, PhD,3 William G. Wierda, MD,*,4 Susan O'Brien, MD,5 Carla Heise, PhD,1
`Mariana Nacht, PhD,*,6 Sharon Aslanian,*,6 Xiping Liu,*,1 Sean Hong,*,1 Shijuan Wu,*,1
`Marianna Zavodovskaya, MS,*,1 Jeffrey Marine,*,7 Evelyn Barnett,*,7 Pilar Nava-Parada,*,7
`Jay Mei, MD,7 Rajesh Chopra, MD,7 Jan A. Burger, MD, PhD,4 Juswinder Singh*,6
`1Department of Translational Medicine, Celgene Corporation, San Francisco, CA, USA,
`2University Hospital of Cologne, Cologne, Germany,
`3Department of Experimental Therapeutics, MD Anderson Cancer Center, Houston, TX, USA,
`4Department of Leukemia, MD Anderson Cancer Center, Houston, TX, USA,
`5Department of Leukemia, The University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA,
`6Celgene Avilomics Research, Bedford, MA, USA,
`7Celgene Corporation, Summit, NJ, USA
`
`Blood (2013) 122 (21) : 4169.
`
`http://doi.org/10.1182/blood.V122.21.4169.4169
`
`Abstract
`
`Introduction
`
`The B-cell receptor (BCR) and its downstream effectors have emerged as important therapeutic targets in
`
`B-cell malignancies. CC-292 is a novel, potent, covalent, and highly selective inhibitor of Btk (IC50apparent
`of 0.5 nM, kinact/KI ratio of 7.69 × 104 M-1s-1), that does not appreciably inhibit other kinases involved in
`BCR signaling (eg, IC50 Lyn kinase, 4401 nM) (Evans et al., J Pharmacol Exp Ther. 2013). Here, we
`
`report preclinical characterization and clinical data in CLL from a single-agent phase 1 dose-escalation
`
`trial of CC-292 in B-cell malignancies, with a focus on how target engagement and downstream events
`
`correlate with clinical activity.
`
`Results
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1021, p. 1 of 3
`
`

`

`Pharmacodynamic effects of Btk inhibition by CC-292 can be monitored by occupancy of the Btk
`
`catalytic site, Btk autophosphorylation on Y223, and downstream phosphorylation of Plc-γ2 and Erk.
`We developed a sensitive (10 pg/mL lower limit of quantification) and quantitative assay to measure
`
`covalent binding of CC-292 to Btk (Evans et al., J Pharmacol Exp Ther. 2013), as well as Western and
`
`novel phos-flow assays to probe downstream signal transduction. These methods showed that CC-292
`
`treatment blocks Btk autophosphorylation and downstream pathway activation in both tumor cells and
`
`human peripheral blood mononuclear cells (PBMCs). The extent of CC-292 binding to Btk correlated
`
`with its in vitro and in vivo effects. The occupancy assay demonstrated that CC-292 effectively targets
`
`Btk in tumor cell lines, PBMCs, spleen, and lymph nodes (LNs) in animal models, and in PBMC and
`
`lymph node samples from clinical trial subjects. In rats and non-human primates treated with CC-292,
`
`Btk occupancy in spleen and LNs was dose-dependent. Measured occupancy in rat spleen and axillary,
`
`mesenteric, and superficial cervical LNs was 94%, 92%, 90%, and 76% respectively, 4 hours (hrs) after
`
`a single 30-mg/kg dose. Interim data from the phase 1 CLL trial showed that PBMC Btk was completely
`
`occupied in the majority of subjects 4 hrs post-dose with both QD and BID dosing. Twenty-four hrs post-
`
`dose at 750 and 1000 mg QD, CC-292 exhibited 83% ± 17% Btk occupancy, whereas with BID dosing
`
`at 375 and 500 mg, occupancy was 94% ± 16% at the corresponding time point (12 hrs after the second
`
`dose). Thus, while both schedules achieved extensive and sustained Btk occupancy, residual free Btk
`
`levels were lower with the BID schedule, offering a rationale for an early trend towards more rapid nodal
`
`responses, lymphocytosis, and partial responses on the BID schedule observed to date in the phase 1
`
`study. In the 10 clinical LN biopsies tested to date, no measurable levels of unoccupied Btk have been
`
`detected, although Btk protein was present as determined by Western blotting, showing that CC-292 was
`
`able to penetrate LNs and inhibit Btk in human subjects as it did in preclinical models. For monitoring
`
`downstream signal transduction, we developed reagents and assays including a phos-flow assay based
`
`on a novel rabbit monoclonal antibody to detect Btk pY223 levels in PBMC subsets. CC-292 effectively
`
`inhibited constitutive and induced phosphorylation of Btk and Plc-γ2 at low nanomolar concentrations.
`CC-292 also inhibited BCR activation and nurse-like cell–supported survival of CLL cells. Furthermore,
`
`CC-292 reduced CLL cell migration and actin polymerization in response to chemokines (CXCL12,
`
`CXCL13) and inhibited secretion of the chemokines CCL3 and CCL4 by CLL cells. These chemokines
`
`are essential for migration and retention of normal and neoplastic B cells in the marrow and secondary
`
`lymphatic tissues. Consistent with this preclinical data, CC-292 treatment resulted in rapid reductions in
`
`circulating CCL3 and CCL4 levels. In subjects treated at the 750 mg QD, 1000 mg QD, 375 mg BID, and
`
`500 mg BID dose levels, plasma CCL3 was reduced from 99 ± 16 pg/ml before treatment to 28 ± 5 pg/ml
`
`(N = 48, mean ± SEM) at 24 hrs after the first dose, while CCL4 was reduced from 235 ± 59 pg/ml to 74 ±
`
`16 pg/ml (N = 51).
`
`Conclusions
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1021, p. 2 of 3
`
`

`

`These data demonstrate that CC-292 achieves significant and durable occupancy of Btk in vitro
`
`and in vivo, inhibits Btk-mediated downstream signaling events and chemokine production, and that
`
`these preclinical activities have translated into the clinic. Taken together, these results argue that Btk
`
`inhibition is necessary and sufficient for clinical activity in CLL. These emerging data support continued
`
`development of CC-292 for the treatment of B-cell malignancies.
`
`Disclosures:
`
`Pierce: Celgene: Employment, Equity Ownership. O'Brien: Genentech: Consultancy, Research
`
`Funding; Emergent: Consultancy, Research Funding; CLL Global Research Foundation: Membership
`
`on an entity’s Board of Directors or advisory committees; Celgene: Consultancy; Gilead Sciences:
`
`Consultancy, Research Funding; Infinity: Consultancy, Research Funding; MorphoSys: Research
`
`Funding; Pharmacyclics: Consultancy, Research Funding; Talon: Consultancy, Research Funding;
`
`Teva/Cephalon: Consultancy. Heise: Celgene: Employment, Equity Ownership. Nacht: Celgene:
`
`Employment, Equity Ownership. Aslanian: Celgene: Employment, Equity Ownership. Liu: Celgene:
`
`Employment, Equity Ownership. Hong: Celgene: Employment, Equity Ownership. Wu: Celgene:
`
`Employment, Equity Ownership. Zavodovskaya: Celgene: Employment, Equity Ownership. Marine:
`
`Celgene: Employment, Equity Ownership. Barnett: Celgene: Employment, Equity Ownership. Nava-
`
`Parada: Celgene: Employment, Equity Ownership. Mei: Celgene: Employment, Equity Ownership.
`
`Chopra: Celgene: Employment, Equity Ownership. Burger: Pharmacyclics: Research Funding; Gilead:
`
`Research Funding. Singh: Celgene: Employment, Equity Ownership.
`
`Author notes
`
`* Asterisk with author names denotes non-ASH members.
`
`© 2013 by the American Society of Hematology
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1021, p. 3 of 3
`
`