IMMUNOMEDICS, INC.
Advanced Antibody-Based Therapeutics
Oncology
Autoimmune Diseases
Improved Therapeutic Index of IMMU-132 ADC
vs. Irinotecan in Preclinical Studies
David M. Goldenberg
CSO, Immunomedics, Inc.
IMMU – 132
A Trop-2-Targeted ADC
2
hRS-7
(Humanized Antibody
to Trop-2)
3
Trophoblast Cell-surface Antigen (Trop-2, EGP-1)
• Trop-2/EGP1 is a pan-epithelial cancer antigen
– Pancarcinoma antigen – in many solid cancers.
– Related to (49% homology) but distinct from EGP-2 (aka EpCAM).
• Acts like a true oncogene, initiating signaling mechanisms that can
result in increased tumorigenicity, aggressiveness, and metastasis.
• Prognostic marker in several cancer types.
• RS7-3G11: Mab developed against human squamous cell lung cancer.
• Binds human lung, breast, colon, renal, and prostate cancers.
• Expression on corresponding normal tissue weaker and spatially restricted.
• Epitope identified as a 46 kDa glycoprotein (35 kDa protein).
• Rapidly internalized following binding to Trop-2.
4
High Trop-2 Expression in Various Cancers (IHC)
Cancer
N
Focala
Diffuseb
Total
%Pos
Prostate
Pancreas
Colon
Breast
Lung
Adenocarcinoma
Small Cell
Stomach
Adenocarcinoma
Signet
GIST
45
29
44
27
40
18
22
39
19
13
7
1
0
2
2
3
0
3
1
0
1
0
44
29
42
25
34
16
18
28
18
8
2
45
29
44
27
37
16
21
29
18
9
2
100
100
100
100
93
89
95
74
95
69
29
a - Focal : 1%-25% of the cancer tissue labeled with the specific MAb.
b - Diffuse: >25% of the cancer tissue labeled with the specific MAb.
5
Quantitation of Trop-2 on Surface of Cancer Cells by
Quantbrite PE Beads Using Flow Cytometry
6
Cell line
Origin
# Trop-2
BxPC-3
Pancreatic
891,000
NCI-N87
Gastric
383,000
MDA-MB-468
Breast
341,000
Capan-1
Pancreatic
115,000
AGS
Gastric
78,000
Colo 205
Colon
52,000
SK-MES-1
Sq Cell Lung
27,000
Humanized Antibody to Trop-2
• RS7-3G11: mAb against human squamous cell lung
cancer
– Binds human lung, breast, colon, renal, prostate and
other cancers
– Expression on corresponding normal tissue weaker and
spatially restricted
– Epitope identified as a 46 kD glycoprotein (35 kD
protein)
• Rapidly internalized into cancer tumor cells following
binding to Trop-2.
7
Examples of High Trop-2 Expression in Diverse Cancers
Prostate cancer
Pancreatic
cancer
SCLC
TNBC
8
RS7 Binding to Human Prostate Cancer
A-B: PC3 human
prostate cancer
xenograft from nude
mouse.
C-F: Human tumor
specimens with
increasing Gleason
scores.
G-I: LN and liver
metastases.
Normal Prostate
Control IgG
9
RS7 IgG
RS7 Internalization: In vitro studies
Within 60 minutes, RS7 is found in the MDA-MD-468 tumor cells
10
Immunomedics’ ADC Platform
• Designed for targeted delivery of SN-38, a proven,
potent, effective anticancer agent (2-3 logs more potent
than irinotecan).
• Optimized linker chosen to maximize efficacy in vivo.
• Proprietary linker chemistry protects SN-38 lactone ring
from forming less potent carboxylate form.
• Acid-labile linkage for intracellular release.
• 7-8 SN-38 molecules linked per IgG.
• Good stability in vitro and in vivo.
11
Immunomedics’ ADC Platform
Novel linker and
ADC construct
Glucuronidation site
protected while
bound to IgG
• Conjugation of SN38 lactone ring via
PEG-peptide linker
and site-specific IgG
coupling via
maleimide
attachment to interchain SH groups.
• Average of ~ 7.6 SN38 molecules/IgG
– High doses of SN-38
delivered.
12
Conjugation of drug to IgG by
inter-chain disulfide bond
reduction
IgG reduced in this manner clears at
the same rate as native IgG in mice,
suggesting that the coupling method
does not destabilize the IgG.
13
Hydrophobic interaction chromatography (HIC)
Two representative lots of IMMU-132
Figure 5
Figure 7
C/N1301195
0.060
C/N1310040
0.050
0.055
0.050
0.040
7.301
0.045
7.226
0.045
8.886
8.850
0.060
0.055
0.040
0.035
0.030
0.030
AU
AU
0.035
6.495
0.020
0.020
0.015
0.015
0.010
0.010
8
6 7
0.005
6.725
0.025
0.025
0.000
0.000
-0.005
-0.005
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00
Minutes
Minutes
RT
6.498
7.226
8.850
14
8
6 7
0.005
364/
280
corrected
0.6093
0.7087
0.7867
DAR
% of total
6
7
8
7.55
7.0
30.5
62.5
Ave
RT
DAR = 7.6
6.498
7.226
8.850
364/
280
corrected
0.5818
0.7045
0.7831
DAR
% of total
6
7
8
7.59
7.1
27.7
65.3
Ave
IMMU-132: Drug-Antibody Ratio (DAR) Distribution
DAR
LC-MS
HIC
8
69.2
69.0
7
26.1
24.1
6
4.3
6.9
5
0.4
ND
Average
7.64
7.62
LC-MS, liquid chromatography-mass spectroscopy
HIC, hydrophobic interaction chromatography
Average SN-38 per IgG = 7.6
Majority of product has 8 moles of SN-38
15
IMMU-132 Specificity: Detection of dsDNA Breaks
1. Incubate Trop-2 positive or Trop-2
negative TNBC cell line with IMMU132 or irrelevant anti-CD20-SN-38
conjugate for 1 h @ 4oC.
2. Wash and replace media; incubate
overnight 37oC.
3. Stain cells with anti-rH2AX-AF488 to
detect dsDNA breaks
4. Analyze by flow cytometry.
Treatment
Median fluorescence intensity
HCC1806
HCC1395
Cell alone
4.25
5.54
Cell + anti-rH2AX-AF488
168
122
Cell + IMMU-132 + anti-rH2AX-AF488
546
123
Cell + hA20-SN38 + anti-rH2AX-AF488
167
123
HCC139 Trop-2-Negative TNBC
• No difference between no
treatment, irrelevant
conjugate treatment or
specific conjugate
treatment
HCC1806 Trop-2-Positive TNBC
16
•
IMMU-132 shows 3-fold increase in dsDNA break formation.
IMMU-132 ADCC is Reduced Compared to Native IgG
ADCC Against Breast Adenocarcinoma (MDA-MB-468)
4 h incubation; BMC's to Target Cells 50:1 Ratio; 10 min Reaction Time
40
*
2.64)
30
10
2.25)
(8.56
(2.40
2.23)
20
(0.17
Mean % Lysis
SD
(29.78
Ig
S7
G
-C
L2
A
-S
N
-3
8
hR
hR
S7
L2
A
-S
N
-3
8
hL
L2
-C
hL
L2
Ig
G
0
Abs [33.33nM]
17
hLL2 IgG
hLL2-CL2A-SN-38
hRS7 IgG
hRS7-CL2A-SN-38
2.62)
**
In Vitro Stability of IMMU-132: Effect of pH and Serum
• SN-38 is released from the CL2A linker at low
pH with a half-life of ~10 h.
• Release of SN-38 from the CL2A linker is not
affected by cathepsin B.
• SN-38 is released from the CL2A linker in
human serum with a half-life of ~21 h.
• A different linker, CL2E, is highly stable in
serum, but yielded inferior anti-tumor
response in vivo.
18
IMMU-132: Nonclinical Studies
Pharmacokinetics
Efficacy in Mice
Safety Studies
19
IMMU-132 Nonclinical Studies: Summary
• Pharmacotoxicology studies in nude mice with human tumor
xenografts exhibit a significantly higher tumor uptake of SN-38
when IMMU-132 is administered compared to irinotecan (as much
as 135-fold).
• GLP Toxicology studies in Cynomolgus monkeys completed.
• Nonclinical studies in many solid tumors (e.g., breast cancer
[including TNBC], SCLC and NSCLC, esophageal cancer, colorectal
cancer, pancreatic cancer, prostate cancer, renal cell cancer) show:
– high expression of Trop-2 on cancer cells and no/low expression
on normal tissues by immunohistochemistry
– broad binding and internalization of IMMU-132 to tumor cells
– therapeutic efficacy in xenograft studies.
20
IMMU-132: Biodistribution in Tumor-Bearing Mice
Group
21
(N)
1
30
2
30
Amount Injected
(IMMU-132)
20 µCi, 250 µg IV
111In-hRS7 IgG
20 µCi, 250 µg IV
111In-hRS7-SN38
Necropsy Schedule
5 min, 4, 24, 48, 72, and 168 h
5 min, 4, 24, 48, 72, and 169 h
•
IMMU-132 clears faster from blood than hRS7 IgG alone
•
Tumor uptake of IMMU-132 not appreciably affected by hRS7 IgG
IMMU-132 vs. Irinotecan
SN-38 and SN-38G Concentrations in Serum
Serum (ng/mL)
In irinotecan-treated animals:
• 53% of total SN-38 is SN-38G.
In IMMU-132-treated animals:
• Only 3.1% of the SN-38 [TOTAL] is
in an unbound form over 72 h.
• Only 4.0% of the Unbound SN-38 is
in the form of SN-38G.
*
*
*
*Undetected after 6
h
There is NO SN-38G bound to IMMU-132
IMMU-132-treated animals
22
Irinotecan-treated animals
SN-38 [TOTAL] AUC1-72 h
148,752
SN-38 AUC0.083-6 h
2520
Free SN-38 AUC1-48 h
4468
SN-38G AUC0.083-6 h
2843
Free SN-38G AUC1-6 h
184
IMMU-132 vs. Irinotecan SN-38 Concentrations in Tissues
• Nude mice bearing Capan-1 human pancreatic cancer xenografts
(~0.06-0.27 g) were injected IV with
– Irinotecan
• 40 mg/kg (773 µg; SN-38 equivalents = 448 µg )
– This dose is MTD in mice.
– Human dose equivalent = 3.25 mg/kg or ~126 mg/m2
– IMMU-132
• 1.0 mg (DAR= 7.6; SN-38 equivalents = 20 µg )
– Well below the MTD in mice.
– Human equivalent dose ~4 mg/kg.
• Necropsy (3 animals per interval)
– Irinotecan: 5 min, 1, 2, 6 and 24 h.
– IMMU-132: 1, 6, 24, 48 and 72 h.
23
SN-38 (ng/g)
Irinotecan-treated animals
SN-38 (ng/g)
IMMU-132-treated animals
IMMU-132 vs. Irinotecan SN-38 Concentrations in Capan-1
AUClast (µg/g·h)
Irinotecan-treated
IMMU-132-treated
0.40
54.25
IMMU-132 delivers up to 135-fold more SN38 to Capan-1 xenografts than irinotecan
Keep in mind these facts:
• Mice convert irinotecan to SN-38 more efficiently than humans
• Mice were given 22-fold more SN-38 equivalents with the
irinotecan dose than the IMMU-132 dose, yet IMMU-132 delivers
135-fold more SN-38
24
IMMU-132: Preclinical Studies Summary
• Efficacious at doses well below the MTD in 5 wellestablished human cancer cell lines grown
subcutaneously in nude mice, including colorectal
(N=1), pancreatic (N=2), lung (N=2), and breast (TNBC)
(N=1) cancers.
• GLP Tox: Cynomolgus monkeys express Trop-2.
• No evidence of clinically significant toxicity in any
Trop-2-expressing normal tissue.
25
Therapeutic Efficacy of IMMU-132 with Different DARs
26
IMMU-132: Efficacy Well-Characterized in
Nonclinical Studies
• Breast cancer
– MDA-MB-231 (TNBC Adenocarcinoma; ATCC# HTB-26)
– MDA-MB-468 (TNBC Adenocarcinoma; ATCC# HTB-132)
• Colon cancer
– LoVo (Colonic Adenocarcinoma; ATCC# CCL-229)
– COLO 205 (Colonic Adenocarcinoma; ATCC# CCL-222)
• Gastric cancer
– NCI-N87 (Gastric Carcinoma; ATCC# CRL-5822)
– Hs 746T (Gastric Carcinoma; ATCC# HTB-135)
• Lung cancer
– Calu-3 (NSCLC; ATCC# HTB-55)
– SK-MES-1 (Squamous Cell Lung Carcinoma; ATCC# HTB-58)
27
IMMU-132: Efficacy Well-Characterized in
Nonclinical Studies
• Ovarian cancer
– SK-OV-3 (Ovarian Adneocarcinoma; ATCC# HTB-77)
• Pancreatic cancer
– BxPC-3 (Pancreatic Adenocarcinoma; ATCC# CRL-1687)
– Capan-1 (Pancreatic Adenocarcinoma; ATCC# HTB-79)
• Prostate cancer
– PC-3 (Prostate Adenocarcinoma; ATCC# CRL- 1435)
– DU 145 (Prostate Adenocarcinoma; ATCC# HTB-81)
– MDA-PCa 2b (Prostate Adenocarcinoma; ATCC# CRL-2422)
28
IMMU-132: In Vivo Efficacy
Various Human Xenograft Models Having Different Trop-2 Levels
Lung Cancer Cell Lines
Calu-3
Pancreatic Cancer Cell Lines
SK-MES-1
Capan-1
Moderate Trop-2
Low Trop-2
(128,201 ± 50,708)
(29,488 ± 5,824)
BxPC-3
Moderate Trop-2
HighTrop-2
(157,376 ± 36,976)
(493,773 ± 97,779)
1.50
1.25
Saline Control
1.00
50 mg conjugate =
0.8 mg SN-38 equivalents
0.75
0.50
0.25
0.00
0
14
28
Injection
Schedule
42 56 70 84
Days from
tumor implantation
98
112
1.50
Irinotecan
1.25
anti-CD20-SN38 (500 mg)
1.00
Irinotecan (40 mg/kg q2dx5, i.v.)
0.75
hRS7 IgG (500 mg i.p.)
Saline Control
0.50
500 mg conjugate = 8 mg SN-38
40 mg/kg irinotecan = 23.2 mg/kg SN-38
or 464 mg SN-38 equivalents/dose
0.25
0.00
0
7
14
21 28 35 42
Days from
tumor implantation
Colon Cancer Cell Line
COLO 205
Low Trop-2
Mean Tumor Volume (cm 3 ± SD)
(58,179 ± 6,909)
29
Dosing
Conjugated and
unconjugated IgG
3.0
hRS7-CL2-SN38 (500 mg)
anti-CD20-SN-38 (500 mg)
Unconjugated hRS7 IgG (1 mg)
2.5
2.0
1.5
Irinotecan (40 mg/kg q2dx5)
Saline
1.0
500 mg conjugte = 8 mg SN-38
40 mg/kg irinotecan = 464 mg SN-38 per dose
0.5
0.0
0
7 14 21 28 35 42 49 56 63
Days from
tumor implantation
hRS7-SN38 (500 mg)
49
2.50 Dosing - all agents
2.25
hRS7-SN38 (500 m g)
2.00
anti-CD20-SN38 (500 m g)
1.75
Irinotecan (130 m g) i.v.
1.50
Irinotecan (13 m g) i.v.
Saline
1.25
1.00
Mean Tumor Volume ± SD (cm 3)
anti-CD22-SN38 (50 m g)
hRS7-IgG + Free SN-38
(8 m g or 0.4 mg/kg)
500 mg conjugate = 8 mg SN-38
13 mg irinotecan = 7.5 mg SN-38
0.75
0.50
0.25
0.00
0
7 14 21 28 35 42 49 56 63 70
Days from
tumor implantation
TNBC Cell Line
Dosing - all agents
3.0
2.5
hRS7-SN38 (500 m g)
2.0
anti-CD20-SN38 (500 m g)
1.5
Irinotecan (130 mg)
Saline
1.0
500 mg conjugate = 8 mg SN-38
130 mg irinotecan = 75 mg SN-38 equivalents
0.5
0.0
0
7
14 21 28 35 42 49
Days from
tumor implantation
Gastric Cancer Cell Line
NCI-N87
MDA-MB-468
High Trop-2
High Trop-2
(246,857 ± 64,651)
(301,603 ± 29,470)
Therapy
Mean Tumor Vol ± SD(cm 3)
hRS7-SN38 (50 m g)
1.75
Mean Tumor Volumes ± SD (cm3 )
2.00
Mean Tumor Volume ± SD (cm3)
Mean Tumor Volume (cm 3) ± SD
Conjugate
1.50
hRS7-SN38 (350 mg)
anti-CD20-SN38 (350 mg)
Saline
1.25
1.00
0.75
0.50
0.25
0.00
0
7 14 21 28 35 42 49 56 63 70
Days from
tumor implantation
Therapeutic Efficacy of IMMU-132 in TNBC
Xenograft Models
A
Individual animal data for this group
Tumor volume (cm3)
B
% change in tumor volume
C
30
Nonclinical Safety Studies
• GLP Toxicology studies in Cynomolgus monkeys; they
express Trop-2.
– 60 mg/kg administered on days 1 and 4 are safe .
– 120 mg/kg administered on days 1 and 4 (total 240
mg/kg) exceeded the MTD; toxicity similar to
irinotecan (neutropenia, GI toxicity).
31
IMMU-132 Nonclinical Studies: Summary
• Pharmacotoxicology studies in nude mice with human tumor
xenografts exhibit a significantly higher tumor uptake of SN-38
when IMMU-132 administered than for irinotecan (as much as
135-fold)
• GLP Toxicology studies in Cynomolgus monkeys completed
• Nonclinical studies in many solid tumors (e.g., breast cancer
including TNBC, SCLC and NSCLC, esophageal cancer, colorectal
cancer, pancreatic cancer, prostate cancer, renal cell cancer) show:
– high expression of Trop-2 on cancer cells and no/low expression
on normal tissues by immunohistochemistry
– broad binding and internalization of IMMU-132 to tumor cells
– therapeutic efficacy in xenograft studies
32
IMMU-132:
Mild, Predictable and Manageable Toxicity
Adverse events (Grades 3 and 4): Starting dose of 8 or 10 mg/kg (n=109)
Grade 3
Grade 4
17 (16 %)
6 (6%)
Anemia
6 (6 %)
0
Fatigue
6 (6 %)
0
Diarrhea
4 (4 %)
0
Febrile neutropenia
3 (3 %)
2 (2 %)
Neutropenia
Note: Grade 2 Alopecia
n= 20 (18%)
• Grade 3 Lymphopenia and vomiting - 2 patients each
• Grade 3 Asthenia, dizziness, UTI, leucopenia, WBC count decreased – 1
patient each
• No anti-antibody responses detected to-date, even after repeated
dosing.
33
IMMU-132 has Higher Therapeutic Index Compared
to Irinotecan
• Clinical results, to be presented tomorrow
afternoon, confirm that doses showing modest
toxicities (lower than irinotecan) induce therapeutic
responses in multiple Trop-2 expressing, metastatic,
heavily-pretreated solid cancers.
34