Nonclinical safety evaluation of immunoconjuates

Melissa M Schutten, D

Antibody-Drug Conjugates:Modes of Toxicity

Melissa M. Schutten, DVM, PhD, DACVPSafety Assessment Pathology

NorCal Society of Toxicology MeetingSeptember 27, 2012

Overview

• Introduction to Antibody Drug Conjugates (ADCs)

• Modes of ADC toxicity

• Challenges associated with nonclinical safety evaluation of ADCs

• Summary

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Genentech Confidential—Internal Use Only

Anatomy of an Antibody-Drug Conjugate (ADC)

Antibody targeted to tumor

Very potentchemotherapeutic drug

• Tubulin polymerization inhibitors• Maytansines (DM1, DM4)• Auristatins (MMAE, MMAF)

• DNA damaging agents• Calicheamicins• Duocarmycins• Anthracyclines (doxorubicin)

• Humanized monoclonal Ab (IgG1)

• mAb with Fc modifications (modulate ADCC, CDC activity)

• Other mAb fragments

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Linker stable in circulation

• Linker biochemistry• Acid labile (hydrazone)• Enzyme dipeptides (cleavable) • Thioether (uncleavable)• Hindered disulfide (uncleavable)

• Site of conjugation• Fc, HC, LC

Improving the Therapeutic WindowD

RU

G D

OS

E

TOXIC DOSE (MTD)

EFFICACIOUS DOSE (MED)Therapeutic Window

Therapeutic Window

TOXIC DOSE (MTD)

EFFICACIOUS DOSE (MED)

Chemotherapy ADC

MTD: Maximum tolerated dose; MED: Minimum Efficacious Dose

• ADCs can selectively deliver a potent cytotoxic drug to tumor cells via tumor-specific and/or over-expressed antigens

• Increase drug delivery to tumor

• Reduce normal tissue drug exposure

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ADC More Efficacious than Free Cytotoxin in Mice

Parsons et al, AACR (2007); Modified from S. Spencer

T-DM1 (ADC)

Free DM1 (cytotoxin)

DM1

ADC Better Tolerated than Free Cytotoxin in Rats

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Free DM1 (2400 µg DM1/m²)Early mortality (100%)

T-DM1 (2040 µg DM1/m²)

Bod

y W

eigh

t(%

cha

nge

from

bas

elin

e)

Time (Day)

Single IV dose; rats

T-DM1: Trastuzumab emtansine

ADC Better Tolerated than Free Cytotoxin in Monkeys

A. Kim, D. Danilenko, N. Dybdal, K. Flagella, K. Achilles-Poon

• No neutrophil decreases when cytotoxic drug delivered linked to an antibody

• ~2-3 times more cytotoxic drug can be given as an ADC

Wh

ite

Blo

od

Cel

ls

6 mg/kg ADC (~750 μg MMAE/m2)0.063 mg/kg MMAE (~750 μg MMAE/m2)

Genentech Confidential—Internal Use Only

Modes of Anti-tumor Activity of ADCs

Tumor cytotoxicity is target-directed

ADC-Ag binding → internalization in lysosomes → ADC degradation → release of toxin intracellularly → tumor cell death

Tumor Cell Tumor

Cells

Tumor cytotoxicity is target-enhanced (bystander effect)ADC-Ag binding → extracellular cleavage of toxin → release of toxin in local tumor environment → diffusion of toxin intracellularly to neighboring tumor cells → tumor cell death

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Tissue Antigen Characteristics Are Key in ADCs

Careful selection of target antigens are an important criterion for both the safety and efficacy of an ADC

•The ‘ideal’ tissue antigen should have:– High level of target expression in cancer cells

– Little to no expression in normal cells

– Expressed on the cell surface

– Readily internalized

– No shedding into the blood by cleavage of the antigen from cancer cell surface

•The number of antigen molecules and antibody binding affinity for the antigen may affect the potency of the ADC

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Genentech Confidential—Internal Use Only

Unwanted ADC-mediated cytotoxicity• Targeted binding to normal tissues

expressing antigen• Off-target (cross reactive) binding to normal

tissues • Non-antigen-mediated ADC uptake (e.g., Fc-

mediated uptake, pinocytosis)

Systemic release of toxin • Instability of linker• Catabolism of ADC

+

Modes of Toxicity of ADCs

Normal Cell

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Genentech Confidential—Internal Use Only

Unwanted ADC-mediated cytotoxicity• Targeted binding to normal tissues

expressing antigen• Off-target (cross reactive) binding to normal

tissues • Non-antigen-mediated ADC uptake (e.g., Fc-

mediated uptake, pinocytosis)

Systemic release of toxin • Instability of linker• Catabolism of ADC

+

Modes of Toxicity of ADCs

Normal Cell

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Slower Drug Deconjugation With Uncleavable Linker

12Polson, et al., Cancer Res., 69(6), 2009

Days post dose

Co

nc

entr

ati

on

(µg/

ml)

Single IV dose 20 mg/kg ADC

Uncleavablelinker

Cleavable linker

Total Ab

More Stable Linker Reduces Systemic Toxicity of ADC in Rats

13Polson, et al., Cancer Res., 69(6), 2009

Ch

ang

e in

bo

dyw

eig

ht

(gra

ms)

Days post dose

Single IV dose given on Day 1 :

CD22-DM1 with cleavable linker

More Stable Linker Reduces Systemic Toxicity of ADC in Rats

14Polson, et al., Cancer Res., 69(6), 2009

Single IV dose given on Day 1 :

Genentech Confidential—Internal Use Only

Unwanted ADC-mediated cytotoxicity• Targeted binding to normal tissues

expressing antigen• Off-target (cross reactive) binding to normal

tissues • Non-antigen-mediated ADC uptake (e.g., Fc-

mediated uptake, pinocytosis)

Systemic release of toxin • Instability of linker• Catabolism of ADC

• DAR

+

Modes of Toxicity of ADCs

Normal Cell

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Early Observation: Highly Drugged ADCs More Toxic

DAR 2

DAR 4

DAR 6

DAR: Drug-to-Antibody Ratio

-100

100

300

500

700

900

1100

1300

1500

Vehicle Control 840 ug/m2Herceptin-MC-vc-PAB-MMAF (2P)

840 ug/m2Herceptin-MC-vc-PAB-MMAF (4P)

840 ug/m2Herceptin-MC-vc-PAB-MMAF (6P)

AST

(U/L

)

Study Day 0

Study Day 3

Study Day 5

DAR 2

DAR 4

DAR 6

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•Engineered ThioMAb backbone allows more homogenous drug load (MMAE)

•Efficacy of TDC ADC (mg/kg basis) and 2 x ADC (ug MMAE/m2 basis)

ThioMAb Technology: Controlling Heterogeneity

DAR

DAR

ADC

TDC

Junutula, et al., Nat. Biotech., 26(8), 2008 17

Catabolism and Deconjugation of TDC is Slower than ADC in Rats

Junutula, et al., Nat. Biotech., 26(8), 2008

Catabolism of the Antibody Deconjugation of the Antibody

Single dose I.V. PK study: ADC or TDC with matched cytotoxin (MMAE) doses

MMAE TDC is Better Tolerated Than ADC in Monkeys

Junutula, et al., Nat. Biotech., 26(8), 2008

• No neutrophil decreases with TDC compared to equivalent ug/m2 dose of ADC

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Repeat IV doses of ADC or TDC, Days 1 and 23:

Genentech Confidential—Internal Use Only

Unwanted ADC-mediated cytotoxicity• Targeted binding to normal tissues

expressing antigen• Off-target (cross reactive) binding to normal

tissues • Non-antigen-mediated ADC uptake (e.g., Fc-

mediated uptake, pinocytosis)

Systemic release of toxin • Instability of linker• Catabolism of ADC

• DAR• Site of conjugation

+

Modes of Toxicity of ADCs

Normal Cell

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Unwanted ADC-mediated cytotoxicity• Targeted binding to normal tissues

expressing antigen• Off-target (cross reactive) binding to normal

tissues • Non-antigen-mediated ADC uptake (e.g., Fc-

mediated uptake, pinocytosis)

Systemic release of toxin • Instability of linker• Catabolism of ADC

+

Modes of Toxicity of ADCs

Normal Cell

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Target Antigen Binding Causes “On-Target” Lymphoid Depletion

Anti-cyCD79b Anti-cyCD79b MCC DM1

Ki-67

Vehicle

CD20

Polson, et. al, Mol Cancer Ther 8(10), 2009

B-cell target depletion in splenic follicles: An example of “exaggerated pharmacology”

Target Toxicity to Normal Tissues

Results: Seven patients received a total of 23 weekly doses of bivatuzumab mertansine. One patient at the 100 mg/m2 and one at the 120 mg/m2 level experienced stable disease during treatment phase but also developed grade 1 skin toxicity (desquamation). One of them received a second treatment course. At the highest dose level achieved in this study (140 mg/m2), one patient developed toxic epidermal necrolysis after two infusions and died. Massive apoptosis of skin keratinocytes had occurred, whereas only symptomatic therapy for skin toxicity was available. The risk-benefit assessment of all patients treated in the total phase I program (4 clinical trials, 70 patients) turned out to be negative after consideration of this case of a toxic epidermal necrolysis and the skin-related adverse events observed in the other trials. Therefore, development of the conjugate was discontinued.

Unwanted ADC-mediated cytotoxicity• Targeted binding to normal tissues

expressing antigen• Off-target (cross reactive) binding to

normal tissues • Non-antigen-mediated ADC uptake (e.g.,

Fc-mediated uptake, pinocytosis)

Systemic release of toxin • Instability of linker• Catabolism of ADC

+

Modes of Toxicity of ADCs

Normal Cell

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Summary

• An ADC is both a “large molecule” and a “small molecule”.

• ADCs hold great promise for improving current oncology therapies.

– Highly potent cytotoxic agents are delivered directly to cancer cells, sparing normal tissues.

– ADCs tend to be better tolerated than standard chemotherapy.

– Increased therapeutic window allows for better balance between safety/efficacy.

• There is a fine balance between efficacy and toxicity.

─ Choice of linker, cytotoxic drug and mAb are all important determinants of safety, PK, and efficacy.

─ Toxicity is usually antigen-independent, ADC/drug-dependent.

─ Linker stability, DAR, and site of drug conjugation impacts toxicity.

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Acknowledgements

• Reina Fuji• Kelly Flagella• Willy Solis• Kirsten Achilles-Poon• Jacqueline Tarrant• Rama Pai• Ning Ma• Joe Beyer• Trung Nguyen• Nghi La• Fiona Zhong• Michelle McDowell• Noel Dybdal• Donna Dambach• Theresa Reynolds

• Susan Spencer• Paul Polakis• Bonnee Rubinfeld• Jagath Junutula• Shang-Fan Yu• David Kan• Ivan Inigo• Wai Lee Wong• Kathy Kozak• Elaine Mai• Jeff Gorrell• Michael Mamounas• Andrew Polson• Seattle Genetics

• Angela Hendricks• Amy Oldendorp• Surinder Kaur• Ben Shen• Jay Tibbitts• Joo-Hee Yi• Kedan Lin• Doug Leipold• Ola Saad• Montserrat Carrasco-Triguero• Keyang Xu

• Luna Liu• Andy Boswell• Helen Davis• Margaret Kenrick

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