TM
XmAb 2513
PRECLINICAL PHARMACOLOGY OF
AN FC ENGINEERED HUMANIZED ANTI-CD30 MAB
Phil Hammond, Greg Lazar, Sher Karki, David Carmichael, Seung Chu
Xencor, Inc. 111 W. Lemon Ave., Monrovia, CA 91016
XmAb™2513

Humanized Version Of Murine Mab AC10
XmAb Fc Region



Antiproliferative Against HL And ALCL Cell Lines
Blocks CD30 Ligand Binding
Has Improved Effector Functions
–
–




0.75
0.5
0.25
0.1
1
10
100
CD30+
Karpas299
Cell
1000
Antibody concentration (ng/ml)
2.2
2.0
0 .8 0
0 .7 5
0 .7 0
1.6
XmAb2513
cAC10-IgG1
xAC10-IgG1
5F11
BSA
hIgG
1.2
1.0
0.1
1
10
100
1000
Antibody concentration (ng/ml)
h u m a n ize d
m u rin e
•
•
•
•
2000
1000
Percent Survival
Percent phagocytosis
10000
100000
19
FcRIIIA(158F)
23
599
26
FcRIIB
120
2190
18
Increases cytotoxicity for FcRIIIa allotypes
Efficacy Improved 4.7 ± 1.4 - Fold
Potency Improved 2.4 ± 1.4 - Fold
40
35
20
5F11
15
cAC10
10
XmAb2513
20
5F11
cAC10
15
10
30
25
20
15
10
5
5
0
0
1
10
100
Ab Concentration (ng/ml)
0.01
1000
0.1
1
10
100
1000
F/F
F/V
10000
F/V
60
PBS
0.3 mg/kg
1 mg/kg
3 mg/kg
50
40
30
70
60
50
PBS
3 mg/kg
10 mg/kg
30 mg/kg
40
30
0
10
20
30
40
50
60
70
0
0
10
20
30
40
50
60
70
Day
Tumor
regressions
PBS
0/9
3 mg/kg 0/9
10 mg/kg 4/9
30 mg/kg 5/9
9
8
7
6
5
4
3
2
1
0
For animal 3503 tumor began
to regrow on day 43. On day
45 animal 3508 no longer had
measurable tumor
0
• Female mice from the ICR-SCID strain were injected subcutaneously with 5x107 cells of the L540 HL line.
• Animals were randomized to treatment groups when established tumors of 50-100 mm3 were measured by microcaliper.
cultured from donor PBMCs isolated from Leukopaks.
• Dosing was performed by intraperitoneal injection every 4 days for 10 doses (Q4Dx10).
• The target for these data was the HL L540 cell line.
• Animals were sacrificed when tumors reached 1500
• Donor allotype was determined by sequencing after PCR from genomic DNA.
day 70.
mm 3,
for humane considerations, or at the completion of the study on
10
20
30
F/V
V/V
V/V
FcRIIIa 158 Allotype
Ab Concentration (ng/ml)
Tumor clearance
• Antibody dependent cell-mediated phagocytosis (ADCP) assays were performed with monocyte-derived macrophages
• No H/H donors were identified in the donor populations used for these studies.
173
Lazar et al. PNAS 2006
Caused Tumor Regression in L540 Xenograft
70
Donor Duplicates
XENP2477
9
Cell Counting Assay
XmAb2513
High Range
Time (days)
XmAb2513
FcRIIIA(158V)
Log10 scale
• Binding affinities were determined using a biacore method.
• Antibodies were immobilized on a Protein A chip and receptors included in the mobile phase.
-7
Antibody dependent cell-mediated cytotoxicity was measured by both lactate dehydrogenase (LDH) release and cell counting
(Guava). Human PBMC effector cells were purified from a Leukopack using a ficoll gradient. L540 Hodgkin Lymphoma target
cells were seeded into 96-well plates at 10,000 (LDH) or 15,000 (Sorting) cells/well and opsonized using antibody at the indicated
concentration. Effector cells were added at 25x E:T and the plate incubated at 37C for 4 hrs prior to assay. For LDH, data were
normalized to maximal (Triton X100 lysis of target cells alone) and minimal (PBMCs alone) lysis. Reported cytotoxicity was
derived from LDH data.
10
FcRIIa Allotype
6
1.00E-10
45
0.1
0
Donor Duplicates
511
F/F
1000
20
R/R
92
1.00E-09
• Dissociation constants were determined from Langmuir curve fit.
5
10
R/R
FcRIIA(131R)
25
CD30L binding assay was performed using L540 cells expressing CD30.
Fixed concentrations of antibody-crosslinked his-CD30L were added as indicated in the legend.
A dose response curve of XmAb2513 was also added as indicated on the X axis.
Binding of CD30L to cells was detected using flow cytometry and reagents for the cross-linking antibody.
20
R/R
7
25
80
H/R
977
30
80
35
H/R
147
30
CD30L 1000ng/mL
CD30L 300ng/mL
CD30L 100ng/mL
90
H/R
-11 -10
-9
-8
Log [Variant], 2M
Mediates enhanced cytotoxicity
90
40
0
-12
X e n co r
100
5
FcRIIA(131H)
1.00E-08
Lazar et al, Mol Immunol. 2007
Increased Survival in an L540 Xenograft
45
10
3
1.00E-07
30
(o p tim ize d )
VH HSC Comparison
100
15
40
0
-13
hum an
4000
Low Range
20
3
1.00E-06
50
LDH Release Assay
3000
Dissociation Constant (KD)
1.00E-05
XmAb2513
% ADCP Improved 2.2 ± 0.7 - Fold
25
1
XENP2477a
60
10
XmAb2513 concentration (ng/mL)
Increases phagocytosis for FcRIIa allotypes
30
70
0 .6 0
Animals with
measurable tumor
1.4
80
XmAb2513
Fold
Improvement
FcRI
NK cell dependent
0
100
Differences in anti-proliferative effects between antiCD30 antibodies have been attributed to different epitope
clusters (Horn-Lohrens et al); AC10 is a cluster C
antibody while 5F11 is a cluster A antibody to CD30. To
determine antibody effects on cell proliferation, either
Karpas299 or L540 cells were grown for 4 days in the
presence of antibody at varying concentrations with 10x
molar access of cross linking antibody. Cell growth was
measured using an ATP dependent luminescence assay.
hAC10_variant
hAC10_variant
hAC10_variant
hAC10_variant
hAC10_variant
90
IgG1
XENP
2477
XmAb
2513
20
0 .6 5
0
1.8
AC10_control
hAC10_STD
100
0 .8 5
5000
Percent Survival
Proliferation (RLU x107)
XmAb2513
cAC10
xAC10-IgG1
5F11
BSA
hIgG
Measured by competitive AlphaScreen assay
110
XmAb
IgG1
1.00E-04
4x increase in antigen binding affinity
0 .9 0
% Cytotoxicity
Anti-CD30 (100 ng/ml)
1.0
0
L540 (HD)
Mean Fluorescence Intensity (a.u.)
Proliferation (RLU x107)
Karpas 299
(ALCL)
Cytotoxicity (ADCC)
Phagocytosis (ADCP)
Blocks CD30 Ligand Binding
Anti-human IgG (10x excess)
Humanized template
Increased HSC
Increased affinity
Human String Content
VH = 0.84%, VL = 0.93%
Exhibits No Complement Dependent Cytotoxicity
(CDC) Activity
Is Active In Subcutaneous Xenograft Models Of HL
Well Tolerated In Toxicology Studies
PK Suggests at Least Every Other Week Dosing
Is anti-proliferative against HL and ALCL
1.3
Engineered for high affinity Fc receptor binding
Receptor
(Allotype)
Percent ADCC
–
Has Increased Fc Receptor binding affinity
Dissociation
Constant (KD)
VH, 27 mutations
VL, 15 mutations
Murine = mAC10
Chimeric = cAC10
Humanized = xAC10
% Cytotoxicity

Cluster C epitope
% Maximal Signal
–
XmAb2513 is a new humanized monoclonal antibody (mAb), to the human cell surface antigen CD30, with an engineered
Fc region to enhance recruitment of effector cells and potentiate anti-tumor efficacy. It is being developed for CD30-positive
(CD30+) diseases such as Hodgkin Lymphoma (HL) and anaplastic large cell lymphoma (ALCL). XmAb2513 was derived
from the murine mAb AC10 by humanizing the variable domain using the method of human sequence content optimization
while retaining high binding affinity for CD30. In addition, the Fc region was engineered to increase the binding affinity for all
Fc receptors (FcRs).
Using biacore measurements, XmAb2513 was determined to have a binding affinity of 465 pM for CD30. The Fc
engineering increased the binding affinity of XmAb2513 for FcRI, FcRIIa. FcRIIb, and FcRIIIa by between 3- and 26-fold
when compared to the binding affinity of an unengineered comparator mAb.
XmAb2513 retains the potent anti-proliferative activity exhibited by the parental antibody against HL and ALCL cell lines. In
addition, as a result of the Fc engineering, XmAb2513 exhibited superior antibody-dependent cell-mediated cytotoxicity
(ADCC), mediated by NK cells that primarily express FcγRIIIa, when compared to the unengineered mAb. The mean
efficacy (percentage of cells specifically lysed) improvement was 4.7–fold and the mean potency (concentration giving 50%
of maximal lysis) improvement was 2.4-fold over the unengineered mAb. XmAb2513 was also 2.1-fold more efficacious
than the unengineered mAb in antibody-dependent cell-mediated phagocytosis (ADCP) assays using FcγRIIa/b and
FcγRIIIa expressing macrophages.
The in vivo anti-tumor activity of XmAb2513 was evaluated using subcutaneous xenograft models in SCID mice.
Statistically significant reductions in tumor growth, together with enhanced survival, were observed at 3 mg/kg while at 10
and 30 mg/kg XmAb2513 was even able to eliminate established tumors.
XmAb2513 has been successfully engineered to possess multiple mechanisms of action, including ADCC and ADCP, with
significant improvement over those of an unengineered IgG1 mAb comparator. Additionally, XmAb2513 has potent antiproliferative effects and was efficacious against HL xenografts. These in vitro and in vivo pharmacology data provide a
rationale for the clinical testing of XmAb2513 in patients with CD30+ hematologic malignancies.
Was humanized with an increase in affinity
HSC
Abstract
40
50
60
70
Day
• During the study, the tumors of several animals regressed to the point of being unmeasurable or in fact undetectable.
• For all except one of the animals, indicated in the 10 mg/kg group, the effect was durable.
XENP2477
• Antibody dependent cell-mediated cytotoxicity (ADCC) assays were performed with donor PBMCs isolated from Leukopaks.
• The target for these data was the HL L540 cell line.
• Donor allotype was determined by sequencing after PCR from genomic DNA.
Was well tolerated in pharmacokinetic and
toxicology studies
• Was well tolerated on a Q5D X6 dosing regimen in cynomolgus monkeys.
• Terminal T½ was between 12.6 And 17.1 days.
• Exposure to XmAb2513 was proportional to dose (after 1st dose).
Conclusions
The XmAb™2513 engineered antibody has been shown to have enhanced potency and efficacy as compared to IgG1
antibodies. This was observed both in ADCC assays as well as in antiproliferation assays where the antibody crosslinking
required for an antiproliferative effect was mediated by Fc receptor binding. Published results from clinical trials with
bispecific antibodies directed to CD30 provide clinical evidence in Hodgkin Lymphoma that enhanced recruitment of
effector function is a successful means of generating a cytotoxic antibody (Hartmann, 2001: Borchmann, 2002). However,
manufacturing limitations prevent bispecifics from being practical in widespread use. Preclinical results with XmAb™2513
support further testing in the clinic to validate the role of enhanced Fc effector function.
References
Lazar et al. A molecular immunology approach to antibody humanization and functional optimization. Mol Immunol. 2007 Mar;44(8):1986-98
Lazar et al. Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4005-10
Borchmann et al. Phase 1 trial of the novel bispecific molecule H22xKi-4 in patients with refractory Hodgkin lymphoma. Blood. 2002 Nov
1;100(9):3101-7.
Hartmann et al. Anti-CD16/CD30 bispecific antibody treatment for Hodgkin's disease: role of infusion schedule and costimulation with cytokines. Clin
Cancer Res. 2001 Jul;7(7):1873-81
Acknowledgement
We wish to thank the many Xencor employees who supported this work.