Mechanisms and Investigations into Hematotoxicity Nancy Everds, - - PowerPoint PPT Presentation

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)

Caveat: Based on literature and EMA/FDA submissions Includes drugs that were never tested in humans RBC (n=15) PLT (n=30) NEUT (n=17)

HUMAN 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

• 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. 2019

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

10

Top and Bottom Left Top and Bottom Right

Collection: Animals in one of four racks of cages sampled

• nce 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.

Capitanio 1996

5 10 15 20

Bottom right Top left Bottom left

Cortisol (ug/dL)

2000 4000 6000 8000 Top right Bottom right Top left Bottom left

Lymphocytes (/uL)

* * *

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

Hemoglobin synthesis Valent 2018

Human

• erythroblast

Veterinary Pro- Rubriblast Prorubricyte Basophilic- Rubricyte Polychromatic - Polychromatic rubricyte Orthochromatic or Acidophilic metarubricyte Committee for Clarification of the Nomenclature

• f Cells and Diseases of the Blood and Blood-

forming Organs

Redundancy and normal apoptosis allows rapid expansion

Besarab 2010

EPO actions: Decreased apoptosis of erythroid cells (~60%

• f proerythroblasts apoptose in mouse spleen)

Shortened transit time Earlier release of reticulocytes

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

50 100 150 200 250 300

• 8 3 8 1522294357 -8 3 8 1522294357 -5 3 8 15222836435057 -5 3 8 152229364344

10 20 30 40 50 60

• 8 3 8 1522294357 -8 3 8 1522294357 -5 3 8 15222836435057 -5 3 8 152229364344

Hematocrit (%) Reticulocytes 10e3/uL Vehicle Drug A Drug B-low Drug B-high

Reticulocytes and red cell mass must be interpreted together

Drug B: Inappropriately low reticulocyte counts in light of decreased RBC mass parameters

Lack of maturation past rubricyte stage Normal RBC maturation Drug A Drug B-high

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

IL-1 IFN-γ IFN-β TNF ↓ EPO production ↓ RBC precursors ↓ Fe release from macrophages IL-6 Transferrin Hepcidin

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

• 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

development for oncology

• Toxicity: administration to cynos caused

mild-mod decreases in RBC mass

Liu 2015

5 1 0 1 5 2 0 5 1 0 1 5

H G B

g / d L C o n t r o l H u 5 f 9 3 0 m g / k g

SIRPa CD47

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

Liver constitutively produces TPO PLT and MK take up TPO Serum TPO decreases

PLT aging: Desialylation Ashwell-Morris Receptor (AMR) Hepatocyte Increase TPO mRNA transcription and translation IL-6 Megakaryocytes: Take up TPO Young platelets: Take up TPO Hoffmeister 2016

Previous understanding Current view

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 FcγRIIa (CD32) in circulation

– 200,000-600,000 platelets/uL blood in primates and dogs – ~5000 FcγRIIa/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)

Platelets-procedural effects

• Platelets vary up to +/-20% between collection time points in

healthy resting NHPs

• Increased platelets with frequent phlebotomy or inflammation

(increased IL-6)

100 200 300 400 500 600 700

• 8

3 8 15 21 22 29 Platelets

Study Day Platelets (x10e3/uL)

Platelets-decreased production

• Mechanisms

– Effects on precursors, megakaryocytes, or bone marrow microenvironment – Effects on TPO (anti-TPO antibodies) – Phagocytosis

• Correlates in bone marrow megakaryocytes by cytology and/or

histology

– Density, maturity, morphology

Examples of decreased platelet production

100 200 300 400 500 600 700 800

• 14
• 7

1 8 15 22

Example platelet counts

Early stage Later stage

Difficult to tell difference between decreased production and consumption/ sequestration

Platelets-decreased lifespan

• Apoptosis (numerous drugs via mitochondrial depolarization and caspace activation)
• Activation, consumption, sequestration

– Complement, immune complexes, multimers – Vasculitis, DIC – Off-target effect

• Increased phagocytosis

– Interleukins, cytokines – Off-target effect

• Class effects

– Oligonucleotides – Liposomes—transient decrease Characteristics

• Minimal to marked decreased platelets
• Rapid recovery with cessation of treatment
• Increased mean platelet volume

de Silva 2018

Decreased platelets due to off-target activation

• mAb against soluble target not expressed on platelets
• Profound decreases in platelets immediately after dosing*

– Remaining platelets were agranular – In vitro investigative study showed macaque-specific activation – Required both Fab and Fc portion of molecule

Santostefano 2012

100 200 300 400 500 600 700 Prestudy 0.25 hr 24 hr 72 hr 168 hr 0 mg/kg 100 mg/kg 15 mg/kg 300 mg/kg

Platelets (K/uL 3 other molecules against same target had no effect on macaque platelets *clinical signs of red face and loss of consciousness after dosing

Decreased platelets due to vasculitis

• Clinical trials with 2 calicheamicin ADCs targeting unrelated antigens

– Thrombocytopenia and increases in serum markers of liver injury in patients

• Investigative studies

– Decreased platelets at day 3 in cynomolgus monkeys (nadir ~ 125K/uL) – Inconsistent minimal increases in ALT, AST, PT, APTT, and/or FIB – No changes in platelet activation state or bone marrow

• No in vitro effect on platelets

– Day 3: liver-specific endothelial injury associated with platelet sequestration in sinusoids (detected via IHC, EM)

• Sinusoidal obstruction syndrome (aka venoocclusive disease; VOD)

Guffroy 2017

Hemophagocytic syndromes

CD4+

IL-2

Activated MΦ

IL-1, IL-6, IFNγ TNFα

Defective NK cell activity: no cytotoxic control

• ver T cells or activated macrophages

IFNγ, M-CSF sCD163

Uncontrolled T cell activity

CD8+ NK

Decreased platelets (and RBCs) due to hemophagocytosis

• rhIL-21

– Cyclical decreases in RBC, platelets after each dose

• Max decrease to ~60-70% of controls

– Striking rebound increases in RETIC and platelets between doses (e.g., up to 800-1000 x 10e3/uL) – Monocyte erythrophagocytosis on blood smears – Similar syndromes observed with other interleukins

• mAbY.1

– mAb against cellular target not expressed on blood cells – At 40 hrs post dose

• Decreased red cell mass as low as ~50% of prestudy
• Decreased platelets as low as ~5% of prestudy

– Enlarged spleen with prominent macrophages and hemophagocytosis – Interference with CD47/SIRPa pathway?

Waggie 2012; Everds 2013

Splenic cytology: macrophage activation / proliferation, and erythrophagocytosis

Cyno: 72 hours post-mAbY.1 Cyno: Stock animal

Neutrophil production

• G-CSF is primary modulator

– Myeloid differentiation, proliferation of precursors, mobilization

• Time from committed precursor to release ~12 days

Hong 2017

Neutrophil life cycle

• Bone marrow

– Stromal CXCR12 tethers young neutrophils via CXCL4 – Increased G-CSF decreases CXCL4 expression allowing neutrophils to exit marrow – Old neutrophils express more CXCL4 and CD62L and return to bone marrow – Macrophages phagocytose old neutrophils causing release of G-CSF

• Aged neutrophils are removed in gut, spleen, liver, and bone marrow
• T1/2 ~6 hrs, lifespan ~11 hours

– Marginal and sequestered neutrophils ≈ circulating neutrophils – Circulating neutrophils ~1-2% of total body mature neutrophils (mouse)

Neutrophils-procedural effects

• Mostly with monkeys at first few time points
• Increased neutrophils most prominent
• Somewhat unpredictable

2 4 6 8 10 12 14 Prestudy 3 8 15 29 Prestudy 3 8 15 22 29

Group A Group B Neutrophil counts (K/uL)

Stress-related increases in neutrophil counts

• Leukocyte cytoskeleton softens in response to catecholamines or

dexamethasone

Fay 2016 Softened marginated leukocytes experience lift force and move to vessel center Softened leukocytes leave capillary bed for larger vessels

Neutrophils-decreased production

• Effects on precursors or bone marrow microenvironment
• Time course

– Depends on level of maturation targeted – Depends on duration of pharmacology

• Clinical sequelae primarily in dogs and NHPs
• Correlates in bone marrow

– Decreased cellularity – Altered maturation patterns – Necrosis

• In vitro colony assays and microphysiologic systems

Summary

• Nonclinical studies may predict human toxicities
• Differences due to
• Impact of procedures and intercurrent

processes

• Different biology
• CBC is window into perturbations
• Regulation of circulating blood cell counts
• Kinetics of blood cell production, lifespan, and

removal

QUESTIONS?

Sequelae of acute platelet activation

• Post-dosing clinical signs

– Intravascular platelet aggregates – Release of vasoactive mediators

• Activation of complement and clotting cascades

– Thrombosis, thromboemboli

• Decreased platelet counts and functionality

Resting (inactive GPIIb/IIIa) Adhesion via GP1b-V-IX Activation (GP IIb/IIIa)