Mechanisms and Investigations into Hematotoxicity Nancy Everds, DVM, Dipl ACVP Seattle Genetics neverds@seagen.com NorCal SOT meeting October 2019
Outline • Introduction and preanalytical effects • Mechanisms of toxicity – Red blood cells – Platelets – Neutrophils
Blood cells as toxicity targets • Blood cells exposed to high drug concentrations • Blood cells are dynamic – New cells constantly being released and exposed – Patrolling function – Rapidly respond to plasma and endothelial mediators – Large numbers of surface receptors – Intimate contact with macrophages • Drugs can affect blood cells as primary or secondary effects – Bind to cell surface and target for removal – Inhibit or activate cells – Cytotoxic to precursors – Downstream from complement activation, immune complexes, etc.
Nonclinical vs clinical hematotoxicity • Concordance of toxicities for 150 test articles (Olsen 2000) – 91% concordance – Lack of concordance for thrombocytopenia • Reported hematotoxicities for biootherapeutics (Everds and Tarrant 2013) RBC NEUT PLT (n=15) (n=17) (n=30) Caveat: Based on literature and EMA/FDA submissions HUMAN Includes drugs that were never tested in humans NON-CLIN BOTH
Nonclinical vs clinical hematotoxicity • General concordance – Conserved targets, pharmacology, or pathways – Target present in health • Poor concordance – Idiosyncratic, immune-mediated, and/or low-incidence effects – Target not present in nonclinical species or in healthy subjects – Off-target binding – Unknown mechanism Everds and Tarrant 2014 Martin and Bugelski 2012
2019 • While lack of efficacy and dose-limiting toxicities are the most common causes of trial failure, the reason(s) why so many new drugs encounter these problems is not well understood. • Using CRISPR-Cas9 mutagenesis, the proteins ostensibly targeted by these drugs are nonessential for cancer cell proliferation. • Efficacy of each drug was unaffected by the loss of its putative target, indicating that these compounds kill cells via off-target effects.
Hematologic effects during infusion reactions (NHPs) • 15/49 biotherapeutic approvals by FDA from 2004 to 2016 had infusion reactions (Mease et al 2017) • No IR-related hematologic changes reported in pharmtox reviews – 4 initial dose reactions (cytokine release, increased CRP) – 12 delayed (ADA-mediated) • Clinical signs, ↓PK, TK, complement activation • Literature differs: hematologic changes reported (Rojko 2014, Heyen 2014, Leach 2014, Chirmule 2012, Everds 2013) Mease et al 2017
Considerations for hematology results • NHP challenges – Low number of animals – High background variability – Intercurrent diseases – Study design confounders (preanalytical effects) • Weight of evidence – Biological plausibility – Dose response and timing – Concordance with other study data – Imprecision/variability of endpoint • Primary vs secondary – Intercurrent diseases – Secondary to pharmacology – Stress • Snapshot, not movie
Preanalytical effects • Can obfuscate true test article-related effects • Control/minimize to increase consistency of data • Primary sources – Husbandry and timing – Intercurrent procedures/restraint/anesthesia – Venipuncture and processing 9
Rhesus monkeys and advanced cognition Top and Bottom Right Collection: Animals in one of four racks of cages sampled Top and Bottom Left once a week for 4 weeks, starting with cage nearest door. Order contributed: Animals farthest from door had longer disturbance time, lower lymphocytes, and higher neutrophils Location mattered: Animals with visual access to anteroom had higher cortisol and lower lymphocyte counts Repeat experiment: Covering window eliminated location effect and confirmed order effect. Cortisol (ug/dL) Lymphocytes (/uL) 20 8000 * * 15 6000 * 10 4000 5 2000 0 0 Bottom Top left Bottom Top right Bottom Top left Bottom 10 Capitanio 1996 right left right left
Understanding decreased blood cell counts • Consider potential mechanisms – Decreased production – Altered trafficking or distribution (increase or decrease counts) – Consumption, destruction, egress, apoptosis, or hemorrhage – More than one of the above • Questions that help identify underlying cause – What cell(s) are affected and when? – Is effect expected based on the pharmacology or known mechanism? – What is the time course of recovery?
Erythropoiesis Human Veterinary -erythroblast Rubriblast Pro- Prorubricyte Basophilic- Rubricyte Polychromatic - Polychromatic rubricyte Orthochromatic or metarubricyte Acidophilic Committee for Clarification of the Nomenclature Hemoglobin of Cells and Diseases of the Blood and Blood- forming Organs synthesis Valent 2018
Redundancy and normal apoptosis allows rapid expansion EPO actions: Decreased apoptosis of erythroid cells (~60% of proerythroblasts apoptose in mouse spleen) Shortened transit time Earlier release of reticulocytes Besarab 2010
RBCs: study design considerations • Excess blood collection: toxicity testing in a challenged animal • Mostly a problem for cynomolgus monkey studies – Rats: satellite animals, sparse sampling, fewer PD markers – Dogs: larger blood volume • Important considerations for interpretation – Volume of blood collected – Concurrent control or data collected with similar study design – Timing and degree of reticulocyte response vs treated animals
Effect of phlebotomy (control animals)
RBCs-decreased production • Mechanisms – Toxicity to precursor cells or bone marrow microenvironment – EPO (anti-EPO antibodies) or other growth factors – Phagocytosis of precursors • Decreased reticulocytes most sensitive marker – Interpreted in context of RBC mass – Appropriate/adequate or inappropriate/inadequate – Downstream effects on RBC mass: depend on duration of effect and maturity stage • Compare to concurrent controls, magnitude of RBC changes, and time course of recovery
Reticulocytes and red cell mass must be interpreted together Vehicle Drug A Drug B-low Drug B-high 60 50 Hematocrit 40 30 (%) 20 10 0 -8 3 8 1522294357 -8 3 8 1522294357 -5 3 8 15222836435057 -5 3 8 152229364344 300 250 Reticulocytes 200 10e3/uL 150 100 50 0 -8 3 8 1522294357 -8 3 8 1522294357 -5 3 8 15222836435057 -5 3 8 152229364344
Drug B: Inappropriately low reticulocyte counts in light of decreased RBC mass parameters Drug B-high Drug A Lack of maturation past Normal RBC maturation rubricyte stage
Mechanisms of decreased RBC lifespan • Most common—due to chronic toxicity • Binding of drug to RBCs – Off-target: Ofatumumab in cynos – Intended target: CD47 mAbs in cynos • Macrophage activation – Interleukins, other cytokines – Off-target activation of macrophages • Mechanical damage – Complement activation, vasculitis, intravascular coagulation • Oxidant/metabolic/membrane alterations
Decreased red cell mass due to toxicity/inflammation • Rapid onset (not really chronic) • Multifactorial process – Decreased EPO, decreased RBC production, increased RBC destruction (eryptosis), iron sequestration • Weight-of-evidence approach – Decreased red cell mass without appropriate reticulocyte response – Clinical signs of weight loss, poor doer – Indicators of inflammation (decreased albumin) – Other toxicities
Anemia of chronic disease TNF IL-1 IL-6 Hepcidin IFN- β IFN- γ Transferrin ↓ EPO ↓ RBC ↓ Fe release production precursors from macrophages Means and Krantz Blood 80 p1639-1647 (1992) Ganz et al Hematology (2006) Am Soc Hematol Educ Program. 29-35, 507 Nairz and Weiss al Wien. Klin. Wochenschr. 2006 Aug;118(15-16):442-62 .
CD47 as an oncology target SIRPa CD47 • CD47: “Don’t eat me” signal on blood cells – CD47 interacts with SIRPa on macrophages to prevent phagocytosis – Overexpressed in cancer, e.g., on AML cells • 5F9 antibody (Hu5F9-G4; Stanford) in H G B development for oncology 1 5 • Toxicity: administration to cynos caused 1 0 g / d L mild-mod decreases in RBC mass 5 C o n t r o l H u 5 f 9 3 0 m g / k g 0 0 5 1 0 1 5 2 0 Liu 2015
Megakaryocytes and platelets • MK maturation over 5 (mice) to 12 (human) days • MKs shed ~4000 platelets prior to apoptosis – Bone marrow and lung vasculature • Platelets circulate for ~5 -10 days • Senescent platelets cleared by hepatocytes and macrophages McArthur 2018
Regulation of platelet production Current view IL-6 Previous understanding Ashwell-Morris Receptor (AMR) Liver constitutively produces TPO Hepatocyte PLT aging: Desialylation Increase TPO mRNA transcription and translation Serum TPO PLT and MK decreases take up TPO Megakaryocytes: Take up TPO Young platelets: Take up TPO Hoffmeister 2016
Biology of platelets • Primary hemostasis (platelet plug) • Large number and variety of surface receptors – Binding / crosslinking of receptors leads to activation • Express largest pool of Fc γ RIIa (CD32) in circulation – 200,000-600,000 platelets/uL blood in primates and dogs – ~5000 Fcγ RIIa/platelet • Activated by large molecules, especially Abs – Binding of mAb via Fab and Fc portions to same or different platelets – ADAs against a drug that associates with platelets (heparin) – Multimeric IgGs (aggregates, ADA/drug immune complexes)
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