Antibody Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies. Michael J Flynn 1,2, Patrick van Berkel 3, Francesca Zammarchi 3, ...
Pre-Clinical Activity of ADCT-301, a Novel Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies Michael J Flynn , Patrick van Berkel , Francesca Zammarchi , Jean-Noel Levy , Arnaud Tiberghien , Luke A Masterson , 2 2 2 2 2,3 1,2,3 and John A Hartley François D’Hooge , Peter C Tyrer , Lauren Adams , David G Williams , Philip W Howard 1,2
3
2
2
2
University College London, London, United Kingdom; 2 Medimmune, Cambridge, United Kingdom; 3 ADC Therapeutics Sarl, Epalinges, Switzerland
ADCT-301 Non-binding ADC
CD25
α P
-6
The relationship between increased CD25 expression and poor prognosis 5 is well established and raises the possibility of using an anti-CD25 antibody to deliver a cytotoxin to these cells in patients. Clinical proof of concept for treatment of CD25-positive malignancies has previously been established using radio-immunoconjugates 6 and immunotoxins 7 utilizing antibodies basiliximab and daclizumab. ADCT-301 is an antibody drug conjugate (ADC) composed of HuMax®-TAC, a human IgG1 anti-CD25 antibody, stochastically conjugated via a cathepsin-cleavable valine-alanine (val-ala) linker to a pyrrolobenzodiazepine (PBD) warhead with a resultant drug antibody ratio (DAR) of 2.3 ± 0.3. O
O
O
O
O
O
O
4
O
O
O
O
H N
O
N H
O
3
4
5
6
20.5
20.5
20.7
17.9
21.2
24.4
HuMax-TAC 14.6
16.0
19.1
14.8
14.4
N
H N
O
O
O
O
The number of CD25 and CD30 molecules expressed on the cell surface was determined on various lymphoma cell lines by flow cytometric Qifikit® assay. Cytotoxicity of ADCT-301 on these cell lines was determined by the 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay.
0.2
The single cell gel electrophoresis (Comet) assay was carried out on cells treated with ADCT-
In vivo, ADCT-301 was administered as a single dose in different Karpas 299 xenograft models and compared to AdcetrisTM. The Maximal Tolerated Dose (MTD) was evaluated in non-tumor bearing SCID mice.
10
1
2
L540 HuT 78 Daudi
-3
-2
-1
4
ADCT-301 Karpas 299 warhead Karpas 299 ADCT-301 SU-DHL-1 warhead SU-DHL-1
40
log10 [ADCT-301] (nM)
B CD25-expressing cell line
CD25-negative cell line
SU-DHL-1
HDLM-2
Karpas 299
L540
HuT 78
Daudi
tumor type*
ALCL
HL
ALCL
HL
CTCL
BL
Mean GI50 ng/ml (pM)
2.48 (16.50)
7.67 (51.14)
5.47 (36.53)
3.25 (21.63)
>> 150 ( >> 10, 000)
>>150 (>>10, 000)
Mean CD25 molecules/cell (x 1000)
341
175
112
91
0
0
*: ALCL, Anaplastic large cell lymphoma; HL, Hodgkin’s lymphoma; CTCL, Cutaneous T cell lymphoma; BL, Burkitt’s lymphoma.
A. In vitro cytotoxicity of ADCT-301 after a 96-hour exposure on six lymphoma cell lines, as measured by the MTS cell viability assay. B. Although CD25 expression is required for sensitivity to ADCT-301, the drug concentration resulting in a 50% reduction in the net tetrazolium reduction product increase in untreated control cells (GI50) did not correlate with the number of CD25 molecules expressed per cell (Pearson’s correlation coefficient r=-0.37).
1200 800 400 0 0
5
10
15
20
25
30
35
40
45
3000
50
55
60
payload amount
Vehicle, qd x 1
0
4
8
12
16
20
24
28
32
For ADCT-301, the peak of DNA cross-linking occurred between 4 and 8 hours and persisted for 36 hours.
C
XL50 (mean ± SD) (pM) GI50 (mean ± SD) (pM)
80 ng
Adcetris qd x 1
200 ng
Adcetris q4d x 4
800 ng
Adcetris, 0.5 mg/kg, qd x 1 2000
Adcetris, 0.5 mg/kg, q4d x 4 ADCT-301, 0.5 mg/kg, qd x 1
1500 1000 500 0
10
0
20
30
40
50
60
70
4× 10 5
A
ADCT-301 ([warhead]) 42.7±20.04 (98.21) 57.11±15.86 (123.35)
B
110 100 90 80 70 60 50 40 30 20 10 0
1× 10 5
0
CD25
110 100 90 80 70 60 50 40 30 20 10 0
342.95±132.20
The concentration of drug which resulted in 50% of the maximum level of DNA cross-linking (XL50) correlated with the GI50 values (Pearson’s correlation coefficient r=0.91), which supports cross-linking as the mode of action of ADCT-301.
-10 -20 -30 -40
0
5
10
15
Days
ADCT-301 was administered i.v. as a single dose to non-tumor bearing CB.17 SCID mice. The lower dashed line indicates 20% of body weight loss. The MTD of ADCT301 in non-tumor bearing SCID mice was 9 mg/kg.
ADCT-301 has picomolar affinity for the IL-2R-α, comparable to that of the naked antibody, HuMax-TAC.
ADCT-301, 0.2 mg, qd x 1 ADCT-301, 0.4 mg, qd x 1 ADCT-301, 0.6 mg, qd x 1
10 15 20 25 30 35 40 45 50 55 60 65
Days
vehicle, qd x 1 ADCT-301, 0.6 mg/kg, qd x1 Adcetris, 0.6 mg/kg, qd x 1 Adcetris, 0.5 mg/kg, q4d x 4
ADCT-301 shows potent and highly targeted cytotoxicity in CD25expressing cell lines. Bystander killing can be demonstrated on CD25-negative cells. DNA cross-linking is the likely mode of cytotoxic action of ADCT-301. These cross-links persist for at least 36 hours in vitro. In vivo, single-dose ADCT-301 administration shows excellent efficacy, superior to a single dose of Adcetris in Karpas 299 xenografts. Together, these data clearly demonstrate the potent anti-tumor activity of ADCT-301 against CD25-expressing hematological tumors and warrants the rapid development of this agent into the clinic.
Acknowledgments In vivo experiments: Charles River Laboratories. In vitro experimental setup - flow cytometric affinity: Maria Mellinas-Gomez (Medimmune) and Karin Havenith (ADC Therapeutics); cytotoxicity assays: Simon Corbett (Medimmune); comet assay: Janet Hartley and Victoria Spanswick (UCL).
References 1. Burchill MA et al. Interleukin-2 receptor signaling in regulatory T cell development and homeostasis. Immunol Lett. 2007 Nov 30; 114(1): 1-8.
2. Srivastava MD et al. Soluble interleukin-2 receptor, soluble CD8 and soluble intercellular adhesion molecule-1 levels in hematologic malignancies. Leuk Lymphoma. 1994 Jan; 12(3-4): 0
5
10 15 20 25 30 35 40 45 50 55 60 65
Days
Warhead 272.72±145.50
0
CD30
ADCT-301, 0.1 mg, qd x 1
5
10
Conclusions
2× 10 5
Vehicle, qd x 1 non-binding ADC, 0.6 mg/kg, qd x 1
0
Vehicle, qd x 1 ADCT-301, 1 mg/kg, qd x 1 ADCT-301, 3 mg/kg, qd x 1 ADCT-301, 6 mg/kg, qd x 1 ADCT-301, 9 mg/kg, qd x 1 ADCT-301, 12 mg/kg, qd x 1
3× 10 5
Figure 7- In vivo antitumor efficacy in disseminated model
36
B. Percentage reduction in OTM relative to untreated control in Karpas 299 and SUDHL-1 cells treated with either 133 pM ADCT-301 or with an equimolar concentration of free warhead.
2hr pulse treatment with:
ADCT-301 qd x 1
Non-binding ADC, 0.5 mg/kg, qd x 1
2500
20
A. Cells were incubated in medium or treated with 1.67 nM of ADCT-301 for 2 hours, washed and incubated in fresh medium for a further 24 hours. Different panels show comet assay carried out on Karpas 299 cells that were: 1. Untreated and unirradiated 2. Untreated and irradiated 3. Treated and unirradiated 4. Treated and irradiated.
1
0
1600
Duration of study (days)
Time post exposure (hrs) 0 -4
ADCT-301, 0.6 mg/kg, qdx1
A. ADCT-301 was administered i.v. at a mean tumor volume of 160 mm3. Time to endpoint (TTE) survival analyses compared to vehicle control were superior at 0.1 mg/kg (log-rank test p ≤ 0.05) and at 0.2, 0.4 and 0.6 mg/kg (p ≤ 0.001). B. ADCT-301 (DAR 2.2), Adcetris (DAR ~4) and non-binding control ADC (DAR 2.1) were administered i.v. at a mean tumor volume of 130 mm3. ADCT-301 activity was superior to Adcetris for single dose administration (p ≤ 0.01). Table indicates equivalent payload amount (ng) administered in each treatment.
60
Karpas 299
301 and free warhead. The mean reduction in the product of the tail length and the fraction of total DNA in the tail i.e. the Olive Tail Moment (OTM) was measured.
0
1
A
HDLM-2
50
ADCT-301, 0.4 mg/kg, qdx1
2000
Figure 5- Cross-linking as mechanism of action
SU-DHL-1
Bystander kill of non-CD25 expressing cells was determined with CD25-positive Karpas 299 cells co-cultured with PKH26-labeled CD25-negative Ramos cells.
B
Histograms depicting percentage viable cells in monoculture and co-cultures wells of CD25-negative Burkitt lymphoma Ramos cells and CD25-positive Karpas 299 cells. A. Cells treated with 1 or 10 ng/ml ADCT-301. B. Cells treated with 1 or 10 ng/ml non-binding ADC.
B
Aim of this study
binding affinity and binding kinetics of ADCT-301.
15.8
ADCT-301, 0.2 mg/kg, qdx1
50
3
100
N
Flow cytometry and Surface Plasmon Resonance (SPR, Biacore) were used to measure
0.2
A
O
Materials & Methods
20.9
ADCT-301, 0.1 mg/kg, qdx1
2400
Duration of study (days)
Figure 3- Targeted cytotoxicity
OH H
Characterization of the in vitro mechanism of action and in vivo efficacy and tolerability of ADCT-301.
SD
ADCT-301 showed high (picomolar) affinity for CD25.
O N
[ADCT-301] ng/ml
10
B. A dilution series of ADCT-301 and HuMax®-TAC were run several times across a Biacore® CM3 chip which had soluble CD25 ectodomain immobilized onto its surface.
O
O
mean
A. Affinity of ADCT-301 (FITC-labelled) for human CD25, as determined by flow cytometry on concanavalin-A activated CD25-positive human PBMCs (peripheral blood mononuclear cells).
H N
O
1
[non-binding ADC] ng/ml
2
% cell viability ± SD
Figure 1. CD25 immunohistochemical staining of tissue microarrays of lymph node tissue from a patient with A. Diffuse large B cell lymphoma and B. Classical Hodgkin’s lymphoma. Black arrows indicate Reed-Sternberg cells.
=
2
1
ADCT-301
H N
0
log10 concentration ( µg/ml)
B
Run
N
-2
Equilibrium dissociation constant (KD) in pM on human sCD25 ectodomain
B
S
-4
0
100
1000
CD25 is expressed in many hematological malignancies 2 including B and T cell lymphomas some of which show near 100% expression e.g. hairy cell leukemia 3 and adult T-cell lymphoma 4.
A
B
50
Vehicle, qdx1
Percent survival
P
2000
% cell viability per well ± SD
CD132
% reduction in OTM ± SD
IL-2R complex
CD122
3000
MFI (FL1H) arbitrary units
IL-2
A
Mean ± SEM
4000
100
Figure 8- MTD determination
% Body Weight Change
A
Karpas mono-culture Karpas co-culture Ramos mono-culture Ramos co-culture
Molecules per cell surface
A
Figure 2- Affinity of ADCT-301 for human CD25
Figure 6- In vivo antitumor efficacy in subcutaneously implanted model
Percent survival
The Interleukin-2 receptor-α (IL-2R-α, CD25) is one of a heterotrimer that makes up the IL-2R . It plays a key role in signal transduction pathways involved in the pathogenesis of autoimmunity and graft rejection 1.
Figure 4- Bystander killing
Mean Tumor Volume (mm3) ± SEM
Results
Mean Tumor Volume (mm3) ± SEM
Introduction
% cell viability per well ± SD
1
3
241-51.
3. Shao H et al. Distinguishing hairy cell leukemia variant from hairy cell leukemia: development and validation of diagnostic criteria. Leuk Res. 2013 Apr; 37(4): 401-9.
4. Dasanu CA. Newer developments in adult T-cell leukemia/lymphoma therapeutics. Expert Data represent Kaplan-Meier survival curves for each group of 10 mice that were treated 12 days after injection of 107 Karpas 299 cells: A. Dose-dependent extensions of survival compared to vehicle control at all four doses of ADCT-301 tested were shown (p ≤ 0.001). B. ADCT-301 activity was superior to Adcetris for single dose administration (p ≤ 0.05).
Opin Pharmacother. 2011 Aug; 12(11): 1709-17.
5. Yoshida N et al. Clinical significance of sIL-2R levels in B-cell lymphomas. PLoS One. 2013 Nov 13; 8(11).
6. Dancey G et al. A Phase I Clinical Trial of CHT-25 a 131I-Labeled Chimeric Anti-CD25 Antibody Showing Efficacy in Patients with Refractory Lymphoma. Clin Cancer Res. 2009 Dec 15; 15(24): 7701-7710.
ADCT-301 showed superior efficacy despite Adcetris having a higher DAR and the relative increased level of CD30 target cell surface expression (Figure 6B).
7. Kreitman RJ et al. Phase I trial of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) in patients with hematologic malignancies. J Clin Oncol. 2000 Apr; 18(8): 1622-36.
