University of Iowa
Iowa Research Online
Theses and Dissertations
Summer 2012
Next generation monoclonal antibodies and their
mechanisms of action against B-cell lymphomas
Delila Peri
University of Iowa
Copyright 2012 Delila Peri
This thesis is available at Iowa Research Online: http://ir.uiowa.edu/etd/3367
Recommended Citation
Peri, Delila. "Next generation monoclonal antibodies and their mechanisms of action against B-cell lymphomas." MS (Master of
Science) thesis, University of Iowa, 2012.
http://ir.uiowa.edu/etd/3367.
Follow this and additional works at: http://ir.uiowa.edu/etd
Part of the Immunology of Infectious Disease Commons
NEXT GENERATION ANTI-CD20 MONOCLONAL
ANTIBODIES AND THEIR MECHANISMS OF ACTION AGAINST
B-CELL LYMPHOMAS
by
Delila Peri
A thesis submitted in partial fulfillment
of the requirements for the Interdisciplinary Studies-Master of
Science degree in Immunology
in the Graduate College of
The University of Iowa
July 2012
Thesis Supervisor: Professor George Weiner
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
MASTER’S THESIS
_______________
This is to certify that the Master’s thesis of
Delila Peri
has been approved by the Examining Committee for the thesis
requirement for the Interdisciplinary Studies-Master of Science degree
in Immunology at the July 2012 graduation.
Thesis Committee: ______________________________
George Weiner, Thesis Supervisor
______________________________
David Lubaroff
______________________________
Siegfried Janz
ACKNOWLEDGMENTS
First I would like to thank my darling children Jonathan and Elizabeth, who
have brought light into my life. I would also like to thank my beloved husband
Moshe for his support throughout my time in the program, as well as my parents
Jerry and DeAnna Kern for their endless support of me.
Additionally I would like to thank my advisor Dr. George Weiner for his
support and patience, as well as my thesis committee and the University of Iowa
Immunology Program.
ii
ABSTRACT
Next generation monoclonal antibodies (mAbs) are unique in that they are
specifically designed to enhance their mechanisms of action, primarily
complement fixation and antibody-dependent cellular cytotoxicity (ADCC).
Recent studies suggest that complement-fixing properties of a mAb can counter
its ability to activate NK cells and mediate ADCC. GA101, a third generation
(type II anti-CD20) mAb, and rituximab-MAGE (glyco-engineered type I mAb)
show enhanced ADCC and direct cell killing; while ofatumumab, a second
generation anti-CD20 mAb, shows enhanced complement-mediated cytotoxicity
(CMC). These studies set out to determine the primary mechanisms of actions of
these various mAbs, and compare the effect of complement on their ability to
activate NK cells and mediate ADCC or CMC. We also studied the efficiency of
rituximab vs. rituximab-MAGE to deplete B-cells in vivo in mice expressing
human transgenic CD20. In vitro, rituximab and ofatumumab fixed more
complement and mediated a greater degree of CMC, than GA101 and rituximabMAGE. Additionally, complement inhibited the ability of both rituximab and
ofatumumab to bind to and activate NK cells, whereas, addition of complement to
GA101 or rituximab-MAGE did not affect their NK cell activating ability.
Complement also blocked rituximab-induced NK-cell mediated ADCC, but not
GA101-induced NK-cell mediated ADCC. Finally, GA101 and rituximab-MAGE
depleted a higher percentage of B cells in whole blood compared to rituximab
and ofatumumab, whereas rituximab-MAGE depleted fewer B cells, in vivo, in a
complement-dependent fashion. We conclude from these studies that there are
iii
significant differences among these antibodies and that the ability of a given
antibody to mediate CMC and complement fixation correlates with the ability of
complement to block the interaction between the antibody and NK cells.
iv
TABLE OF CONTENTS
LIST OF TABLES ................................................................................................ vii
LIST OF FIGURES .............................................................................................. viii
LIST OF ABBREVIATIONS .................................................................................. ix
CHAPTER
I.
INTRODUCTION .................................................................................. 1
B cell lymphomas and current therapies ....................................... 1
Antibody structure and anti-CD20 monoclonal antibodies ............ 4
Third generation monoclonal antibodies ....................................... 7
Mechanisms of action for antibody therapy ................................... 9
II.
MATERIALS AND METHODS ........................................................... 13
Cell lines, antibodies and serum ................................................. 13
C3b deposition assay .................................................................. 13
Complement mediated cytotoxicity 51Cr release assay ............... 14
Complement mediated propidium iodide assay .......................... 15
NK cell adhesion assay ............................................................... 15
NK cell activation ......................................................................... 16
Antibody dependent cellular cytotoxicity 51Cr release assay ....... 16
B cell depletion assay .................................................................. 17
Mouse studies ............................................................................. 18
Statistical analysis ....................................................................... 18
III.
THE COMPARISON OF RITUXIMAB TO VARIOUS SECOND
AND THIRD GENERATION MONOCLONAL ANTIBODIES AND
THEIR MECHANISMS OF ACTION ................................................... 19
Introduction .................................................................................. 19
Differences in complement concentrations are highly
variable between various compartments ..................................... 19
Rituximab and ofatumumab have superior complement
fixation properties as compared to GA101 and rituximabMAGE .......................................................................................... 21
Rituximab and mediates CMC of Raji cells while GA101
does not mediate CMC ................................................................ 22
Serum inhibitis binding of NK cells to rituximab but not to
GA101 ......................................................................................... 22
GA101 is superior to rituximab and ofatumumab in activating
NK cells in the presence of complement ..................................... 23
C5-depleted serum blocks ADCC mediated by rituximab, but
not mediated by GA101............................................................... 24
GA101 and rituximab-MAGE have superior B cell depleting
properties as compared to ofatumumab and rituximab ............... 25
v
Rituximab depleted more CD20+ B cells than rituximabMAGE in an in vivo model ........................................................... 26
IV.
DISCUSSION AND FUTURE DIRECTIONS ...................................... 28
REFERENCES .................................................................................................... 53
vi
LIST OF TABLES
Table 1. Description of anti-CD20 monoclonal antibody therapies used in these
studies…………………………………………………………………………………..36
Table 2. Complement fixation properties of type I and type II mAbs used in these
studies…………………………………………………………………………………..37
vii
LIST OF FIGURES
Figure
1.
Immunoglobulin structure depicting sugar residues .................................... 34
2.
Schematic representation of the interaction between complement,
mAbs, the FcγRIIIa, and the primary mechanism of action used ................ 35
3.
Complement levels vary between various compartments ........................... 38
4.
Rituximab and ofatumumab have increased complement fixation
capabilities compared to GA101 ................................................................. 39
5.
Rituximab and ofatumumab have increased complement fixation
capabilities compared to GA101 at various concentrations ........................ 40
6.
GA101 and rituximab-MAGE have decreased abilities to fix
complement ................................................................................................. 41
7.
Rituximab-MAGE and GA101 have decreased complement fixation
properties in comparison to rituximab alone ................................................ 42
8.
GA101 induces lower complement mediated cytotoxicity in the
presence of serum using a 51Cr release assay ........................................... 43
9.
GA101 induces lower complement mediated cytotoxicity in the
presence of serum ....................................................................................... 44
10. NK cells adhere to GA101 but not rituximab in the presence of serum ....... 45
11. Complement blocks ability of rituximab coated target cells to activate
NK cells, but not GA101coated Raji cells to activate NK cells .................... 46
12
Complement blocks the ability of rituximab and ofatumumab coated
target cells to activate NK cells, but not GA101 coated cells to activate
NK cells ....................................................................................................... 47
13. GA101 has enhanced ADCC in the presence of C5-depleted serum ......... 48
14. GA101 has superior B cell depleting capabilities compared to
rituximab and ofatumumab .......................................................................... 49
15. GA101 has superior B cell depleting capabilities compared to
rituximab and ofatumumab at various concentrations ................................. 50
16. Rituximab-MAGE shows superior ability to deplete B cells compared to
rituximab alone ............................................................................................ 51
17. Rituximab depletes more circulating B cells in vivo than rituximabMAGE .......................................................................................................... 52
viii
LIST OF ABBREVIATIONS
Ab
ADCC
Ag
C3b
C5
CD20
CDR
CH
CL
CMC
51
Cr
Fab
Fc
FcγR
Fuc
GlcNAc
HI
IgG
IP
MAb
MAGE
Man
NHL
NK
PBS
PCD
RPMI
S
Tg
VH
VL
Antibody
Antibody-dependent cellular cytotoxicity
Antigen
Complement protein 3b
Complement protein 5
Cluster of differentiation 20
Complementarity determining regions
Constant domain, heavy chain
Constant domain, light chain
Complement mediated cytotoxicity
Chromium isotope 51
Fragment, antigen binding
Fragment, crystallizable
Fragment crystallized gamma receptor
Fucose
N-Acetylglucosamine
heat inactivated
Immunoglobulin G
Intraperitoneally
Monoclonal antibody
Monoclonal antibody glyco-engineered
Mannose
Non-Hodgkin’s lymphoma
Natural killer
Phosphate buffered saline
Programmed cell death
Roswell Park Memorial Institute medium
Serum
Transgenic
Variable domain, heavy chain
Variable domain, light chain
ix
1
CHAPTER I: INTRODUCTION
B cell lymphomas and current therapies
Lymphomas constitute a large portion of malignancies diagnosed each
year representing nearly 19% of all cancers diagnosed in 2011 according to data
from the American Cancer Society. Lymphomas can be derived from B, T, or NK
cell origin, with B cell lymphoma being the most common (Kuppers 2005). There
are two major types of lymphomas: Non-Hodgkin’s Lymphoma (NHL) and
Hodgkin’s lymphoma (HL). Currently, the average survival rate of patients
diagnosed and treated for NHL is around 60% for patients deemed with favorable
prognostic factors based on age, stage, response to treatment, and location of
the cancer (Moore and Cabanillas 1998). Lymphoma, by definition, is a
malignancy of lymphocytes, which are cells that comprise the immune system
(Kuppers 2005). In the case of B cell lymphoma, malignant B cells result from
the acquisition of genetic abnormalities during differentiation (Lenz and Staudt
2010) with most being clonal descendants of distinct populations of B-cell
precursors (Maloney 2012). Lymphoma typically presents as a solid tumor and is
characterized by its clinical-pathological features such as its immune phenotype
and the presence or absence of surface markers.
Currently, treatment for B cell lymphoma consists of chemotherapeutic
agents, a corticosteroid, and a monoclonal antibody (mAb) specific for cluster of
differentiation 20 (CD20), followed by a consolidation phase of radiation therapy
in some cases. For example, R-CHOP (rituximab, cyclophosphamide,
2
doxorubicin hydrochloride, vincristine sulfate, and prednisone) is one regimen
used to treat diffuse large B cell lymphoma, one of the nearly 30 types of NHL
(Hainsworth, Litchy et al. 2005).
The expression of CD20 on most B cell lymphomas allows for the specific
targeting of these cells by monoclonal antibodies specific for CD20. CD20 is an
activated-glycosylated phosphoprotein expressed on the surface of all B-cells
during the pro-B phase of B cell development to the mature and activated
phases, thus known as a B cell marker. Its main function is to enable a B-cell
immune response against T-independent antigens. In addition, CD20 has been
shown to regulate calcium channel transport (Maloney 2012). Other cell surface
markers are also used to distinguish the subtype of lymphoma, such as CD15,
CD19, and CD30, to name a few (Bertrand, Billips et al. 1997).
The discovery of CD20 expression on B lymphocytes by Lee Nadler
(Stashenko, Nadler et al. 1980), has allowed for the development of
pharmaceutical drugs that target CD20. The first anti-CD20 monoclonal antibody
approved by the FDA for the treatment of B cell lymphomas was rituximab in
1997. Rituximab is used as either a single agent or in combination with
chemotherapy for treating B cell lymphomas. Since the development of
rituximab, anti-CD20 mAbs have revolutionized the way B cell lymphomas are
treated. Patients incur fewer side effects and morbidities with mAb therapy alone
compared to chemotherapy alone, and survival rates have improved with the
addition of mAbs to treatment regimens (Maloney, Grillo-Lopez et al. 1997;
McLaughlin, Grillo-Lopez et al. 1998).
3
Although the use of rituximab as a first line therapy has increased the
overall survival rates for some patients, there is still the need to improve
treatment efficacy for those patients who are refractory or relapsed, or for
patients who may have developed a resistance to rituximab therapy (Michallet,
Lebras et al. 2012). Thus, there is a great need for additional anti-CD20 mAbs
that may be used as alternatives to rituximab that may be further engineered into
more efficient mAbs with enhanced cytotoxicity. Based on the success of
rituximab against B cell tumors, second and third generation mAbs have been
developed. Second generation antibodies are humanized or fully human with
unmodified Fc domain, with the aim of reducing immunogenicity. Third
generation antibodies are modified to include engineered Fc domains with the
aim of improving the therapeutic activity in all patients, particularly in genetically
defined subpopulations that express a low affinity version of the Fc receptor on
their immune effector cells (Oflazoglu and Audoly 2010). Ofatumumab, a second
generation mAb and obinutuzumab (GA101), a third generation mAb have also
been approved for treatment of other B cell diseases such as rheumatoid arthritis
(Weiner, Surana et al. 2010).
Accordingly, there is still much we do not know about the mechanisms
responsible for the clinical efficacy of mAbs. There is considerable evidence that
both antibody-dependent cellular cytotoxicity (ADCC) and complement fixation
contribute to the efficacy of rituximab therapy, in vitro and in animal models as
reviewed in (Wang and Weiner 2008). Thus, a better understanding of the
relative roles and interactions between these two mechanisms is vital as the next
4
generation of mAb for cancer therapy are developed and tested. Here, we set
out to determine the primary mechanisms of action of a variety of mAbs (first,
second, and third generation) and the effect of complement on their ability to
activate NK cells, fix complement, and mediate either ADCC, CMC, or direct cell
killing.
Antibody structure and anti-CD20 monoclonal
antibodies
Antibodies, also known as immunoglobulins (Ig) are composed of two
identical light chains (Lκ) and two identical heavy chains covalently linked
through inter-chain disulfide bonds (Jefferis 1993; Wright and Morrison 1997). A
portion of the heavy chains, comprise the Fc region (Fragment, crystallizable)
and the Fab (Fragment, antigen binding) regions of the antibody. Each light
chain consists of two domains, a variable domain (VL) and a constant domain
(CL). The heavy chains consist of four domains, three constant domains (CH1,
CH2 and CH3 domains) and a variable domain (VH) and the complementarity
determining regions (CDRs) of IgG molecules are located in the VL and VH
domains as depicted in Figure 1 (Beale and Feinstein 1976; Davies and Metzger
1983; Jefferis 1993; Wright and Morrison 1997). The VL, VH, CL and CH1
domains together constitute the Fab portion of IgGs whereas CH2 and CH3
domains constitute the Fc portion. The Fab and Fc portions of IgGs are linked
through a highly flexible hinge region, which in turn covalently connects the two
heavy chains through inter-chain disulfide bonds (Wright and Morrison 1997).
5
Human immunoglobulins are divided into four isotypes; IgG1, IgG2, IgG3
and IgG4 (Raju 2008). The number of disulfide bonds linking the two heavy
chains, as well as the primary amino acid sequences between isotypes
determines differences between these four isotypes. In addition, the flexibility
and length of the hinge region also affects the mobility of the Fc and Fab regions.
These biological and physicochemical properties of IgG isotypes, as well as their
ability to induce complement mediated cytotoxicity (CMC) and antibody
dependent cellular cytotoxicity (ADCC) allows them to significantly differ from
each other. IgG1 is the preferred isotype for designing mAbs that rely on active
Fc effector functions, such as rituximab (Jefferis 2007; Brezski, Vafa et al. 2009).
The Fab portion of mAbs are important for antibody-antigen interactions while the
Fc portion is crucial for defining the antibody effector functions and
pharmacokinetic properties of IgG molecules (Jefferis 2007). The Fc region of the
antibody plays a role in modulating immune response through the binding of
specific proteins, such as Fc receptors (FcγRIIIa) (Nimmerjahn and Ravetch
2008) as well as complement proteins such as C1q, the first component of the
complement cascade (Putnam, Liu et al. 1979; Huber 1980).
Thus, the Fc region of mAbs is the most important region for ADCC and CMC
mechanisms.
In humans and animals, naturally occurring antibodies are secreted from B
cells when the B cell receptor (BCR) is bound by crosslinking antigen (Meffre,
Casellas et al. 2000). MAbs, on the other hand, can be developed by using a
variety of techniques and can differ in their structure from antibodies produced by
6
human plasma cells (Birch and Racher 2006). For example, mAbs may be
murine, chimeric, humanized, or fully humanized. One way to produce
monoclonal antibodies is to immunize mice repeatedly with relevant antigen. The
resulting B cells are then fused in vitro with myeloma cells lines to form
immortalized B cells that secrete monoclonal IgGs. Other ways to produce mAbs
include phage or yeast display methods, using human antibodies and antigens
and that can be developed completely in vitro (Birch and Racher 2006). These
antibodies are then screened to derive mAbs that are highly specific to the target
antigen, such as CD20 expressed on B cell lymphomas.
Similarly to naturally occurring antibodies, pharmaceutical anti-CD20
mAbs also consist of Fc and Fab portions, and in the case of anti-CD20 mAbs,
the Fab variable regions are specific for human CD20. Since a large portion of
mAbs are produced by using mouse B cells, the chimerization or humanization of
murine monoclonal antibodies to reduce immunogenicity is an essential step
required to develop newly engineered mAbs that may be administered to patients
repeatedly (Wu, Tan et al. 2001).
Rituximab is a chimeric mAb, in which a human Fc portion replaces the
murine Fc portion. GA101, on the contrary, is a fully humanized antibody
(Fishwild, O'Donnell et al. 1996). Anti-CD20 mAbs can be further distinguished
not only by the way they are engineered, but by their abilities to fix complement
and their binding to CD20 (Cragg, Morgan et al. 2003). These mAbs can be
referred to as type I or type II. MAbs, such as rituximab and ofatumumab are
type I. They translocate CD20 into membrane rafts, and are effective at fixing
7
complement. MAbs such as ofatumumab (humanized) also binds to the CD20
molecule at positions 159 to 166 on the large loop and directly to the small loop.
This unique binding pattern of ofatumumab, which results in increased proximity
of the antibody to the cell membrane, accounts for the ability of this mAb to
induce complement-mediated cytotoxicity. This is in direct comparison to other
mAbs like rituximab, which bind to the larger of two extracellular loops within the
CD20 molecule; including the alanine-N-proline (ANP) residues at positions 170
to 172. In contrast, type II anti-CD20 mAb such as B1 (Ivanov, Beers et al. 2009)
or GA101 (Mossner, Brunker et al. 2010) bind to CD20 in a different manner.
These mAbs are unable to translocate CD20 into lipid rafts and do not fix
complement effectively. One unique mAb is Rituximab-MAGE which is a type I
antibody, but has been glycoengineered to be poor at complement fixation.
Table 1 and Table 2 depict the different characteristics between these different
anti-CD20 monoclonal antibodies as well as their complement fixation properties.
Third generation monoclonal antibodies
Modifying mAbs holds promise for future clinical therapies against
malignancies. GA101 is a third generation type II anti-CD20 antibody that that
was derived by humanization of the parental B-Ly1 mouse antibody with
subsequent glycoengineering using GlycoMab® technology (Roche) (Robak
2009). The Fc-region of GA101 was glycoengineered to contain bisected,
afucosylated carbohydrates. As a result GA101 has increased affinity for the
FcγRIIIa. These modifications in the Fc glycosylation pattern can alter the ability
8
of mAbs to fix complement. Previous studies performed on GA101 have
demonstrated other biological differences from rituximab. First, GA101 induced
significantly larger amounts of direct programmed cell death (PCD) than
rituximab and ofatumumab in both cell lines and primary B-cell tumors. Second,
actin reorganization is critical for GA101-induced cell death. Third, cell death
evoked by GA101 is independent of BCL-2 and caspases, and could circumvent
resistance to apoptosis (Alduaij, Ivanov et al. 2011). Finally, GA101-induced
apoptosis is mediated by lysosomes undergoing lysosomal membrane
permeabilization (LMP), confirming that this mode of death is a defining feature
of type II anti-CD20 mAbs (Ivanov, Beers et al. 2009).
The process of glycoengineering mAbs is important for modifying IgG
effector functions. Modifications primarily take place at glycans N-linked to the
Fc region of IgGs (T. Shantha Raju 2010). Typically, mAbs are N-glycosylated in
the CH2 domain of the Fc region at asparagine (Asn) residue 297. In human
IgGs, the majority of the Fc glycans are complex biantennary structures with a
high degree of heterogeneity due to the presence or absence of different terminal
sugar residues, such as fucose (Raju, Briggs et al. 2000). It has been shown
that Fc glycosylation is required for mAbs to bind various Fc receptors such as
the FcγRIIIa receptor (CD16a) found on Natural Killer (NK) cells (Mimura,
Ghirlando et al. 2001) as well as binding to C1q protein. The effector functions of
IgG antibodies such as ADCC and CDC are therefore dependent on Fc
glycosylation (Raju and Scallon 2006).
9
Several strategies have been described by a number of groups to reduce
fucosylation of mAbs in the core region (Scallon, McCarthy et al. 2007). These
strategies include the development of alternative cell lines that either lack the
expression of α1,6-fucosyltransferase or express the enzyme at reduced levels
(Shields, Lai et al. 2002; Shinkawa, Nakamura et al. 2003). Alternative strategies
include, silencing of the gene that encodes the transferase using an RNAi
method (Scallon, McCarthy et al. 2007). Therefore, mAbs that are either
completely non-fucosylated or contain significantly reduced amounts of core Fuc
residues are currently in human clinical trials for development as human
therapeutics, such as GA101 (Scallon, McCarthy et al. 2007). The use of newly
developed glyco-engineered antibodies that are afucosylated may show
promising results for use in future clinical therapies for the treatment of
lymphomas through inducing more efficient ADCC.
Mechanisms of action for antibody therapies
There are various mechanisms of cytotoxicity that can be induced by
monoclonal antibodies. These include cell signaling agonism/antagonism,
complement activation, and ADCC, with the latter thought to be playing a
predominant role in the efficacy of some mAbs (Kohrt, Houot et al. 2012).
The role of complement has also been of great debate as to its role in
mAb therapy. The complement system is part of the immune system and is
composed of a number of proteins found in the blood. When stimulated by one
of several triggers, proteases in the system cleave specific proteins to release
10
cytokines and initiate an amplifying cascade of further cleavages. The end-result
of this activation cascade is massive amplification of the response and activation
of the cell-killing membrane attack complex. In relation to anti-CD20 mediated
mAb therapy, complement mediated cytotoxicity consists of the binding of
antibody to CD20 which activates the complement cascade through C1q, leading
to cell death or deposition of complement proteins on the cell membrane, which
ultimately punctures the cell wall of the tumor cell causing lysis (Maloney 2012).
ADCC works via a different mechanism. During ADCC, the mAb binds to
the tumor antigen and then interacts with the Fcγ receptors on the surface of
effector cells such as NK cells, DC’s or macrophages (Kohrt, Houot et al. 2012).
In NK cells antibody-dependent cellular cytotoxicity (ADCC) is mediated by the
FcγR type IIIa (CD16a) (Freundlich, Trinchieri et al. 1984) a trans membrane
glycoprotein of M, 50-70 kDa (Stahls, Heiskala et al. 1992). Triggering of the
FcγRIIIa (CD16a) on natural killer (NK) cells by monoclonal antibodies or
antibody-coated target cells stimulates a rapid phospholipase C (PLC)-mediated
hydrolysis of inositol phospholipids and results in subsequent delivery of the lytic
hit (Stahls, Heiskala et al. 1992). Previous studies have demonstrated that
genetic polymorphisms in the FcγRIIIa can determine the efficacy of mAb therapy
(Weng and Levy 2003). It should also be noted that DCs are not considered to
be primary ADCC cells, but can take up mAb-bound tumor cells for antigen
processing (Kohrt, Houot et al. 2012).
Finally, mAbs have the ability to induce programmed cell death (PCD).
During direct cytotoxicity the anti-CD20 antibody induces internal signaling within
11
the tumor cell, causing anti-proliferative effects or cell death, which may involve
apoptosis or other cell-death pathways. Thus, different mAbs have the ability to
induce different mechanisms of actions based on their structure.
The relationship between ADCC and complement fixation is complex. A
number of investigators have found complement mediated cytotoxicity (CMC)
contributes to rituximab-induced lysis of malignant B cells (Bellosillo, Villamor et
al. 2001; Weng and Levy 2001; Di Gaetano, Cittera et al. 2003; Golay, Cittera et
al. 2006). In contrast, our laboratory recently demonstrated in vitro that the C3b
component of complement can inhibit mAb-induced NK cell activation and ADCC
by interfering with the interaction between rituximab Fc and CD16 on the NK cell
(Wang, Racila et al. 2008). Additional studies have also demonstrated that
depletion of complement can enhance the efficacy of mAb therapy in a murine
model (Wang, Veeramani et al. 2009). These studies raise the possibility that, if
ADCC is a prime mechanism of action, the ability of a mAb to fix complement
may actually decrease mAb efficacy, as illustrated in Figure 2.
Given that complement fixation can inhibit the ability of rituximab-coated
target cells to activate NK cells and mediate ADCC, we assessed the effect of
complement on the ability of GA101 or rituximab-MAGE to bind to NK cells,
activate NK cells, mediate ADCC, as well as their abilities to deplete B cells both
in vitro and in vivo. Due to the inability of GA101 and rituximab-MAGE to
effectively bind complement, and the fact that GA101 is a type II mAb and
rituximab-MAGE is modified in the Fc portion of the antibody, we hypothesized
that GA101 and rituximab-MAGE would be able to more effectively bind to the
12
FcγRIIIa of NK cells (CD16a) and induce NK activation, in the presence of
complement.
13
CHAPTER II: MATERIALS AND METHODS
Cell lines, antibodies and serum
Raji and Daudi cells (Burkitt’s Lymphoma B-cells) were obtained from
American Type Culture Collection (ATCC, Manassas, VA). All cell lines were
tested for their expression of CD20 using antibodies specific for CD20 and
analyzed on the LSR flow cytometry machine. The GJW cell line was constructed
in the lab and was EBV transformed. Rituximab was obtained commercially
(South San Francisco, CA). GA101 was provided by Genentech (South San
Francisco, CA), ofatumumab was obtained commercially (Middlesex, UK), and
rituximab-MAGE was provided as a gift from Dr. Hong Liu (Eureka Therapeutics,
Inc. Emeryville, CA). Normal human serum was obtained from normal donors
after obtaining informed consent in agreement with the University of Iowa human
subjects protocol. Heat inactivated serum was produced by heating normal
human serum to 56 oC for 30 minutes. Serum and NK cells from the same donor
were used in each individual experiment. C5-depleted serum obtained
commercially from Complement Technology (Tyler, TX).
C3b deposition assay
5X105 Raji, Daudi, or GJW cells were incubated with 5,1,0.2, 0.04, 0 µg/ml
of mAbs with 25% serum or 25% heat-inactivated serum for 30 min at 36 oC.
Cells were then washed with 1mL PBS, spun for 10min at 1250rpm, supernatant
removed, washed again with 1mL PBS, spun for 10min at 1250rpm and then the
14
pellet was resuspended in 100µl of FACS buffer. 1µg of primary anti-C3b (3E7)
mAb (obtained from Dr. Ron Taylor, University of Virginia) was added and cells
were incubated for 30min at 4 oC. Cells were rinsed and spun, and then 1µg of a
secondary IgG1 mAb conjugated to FITC was added (Sigma-Aldrich F0767).
Samples were rinsed, spun, and resuspended in 300µl FACS buffer. Controls
used were cells plus no Ab+ serum, and cells plus no mAb + Heat inactivated
serum. Additional flow cytometry controls used were isotype controls. Samples
were then immediately analyzed using a flow cytometer and median fluorescence
intensity was calculated and plotted. For studies comparing levels of
complement with different patients, ascites or serum was incubated with 5µg/ml
rituximab and 5X105 Raji cells, and followed the protocol according as described
above.
Complement-mediated cytotoxicity 51Cr release
assay
Serum and either rituximab or GA101 at various concentrations were
added to104 51Cr labeled Raji cells in a 96-well V bottomed plate and incubated
for 2 hours at 37oC. All samples were evaluated in triplicate. After centrifugation,
supernatant was collected and counts determined. Percent specific lysis was
calculated as (experimental value - spontaneous lysis) / (maximum lysis spontaneous lysis) x 100.
15
Complement-mediated cytotoxicity propidium
iodide assay
5X105 Raji or Daudi cells were incubated with 5, 2.5, 1.25, 0.625 or 0
µg/ml of monoclonal antibodies, with 25% serum or pleural fluid for 30 min at 36
o
C. Cells were then spun at 1250 rpm for 10 min, resuspended with 0.5ml PBS,
and 50 µl PI was added at a concentration of 50µg. Tubes were then analyzed
via flow cytometry and calculated for % PI positive cells. Controls received no
Ab, no serum, or were unstained.
NK cell adhesion assay
96 wells EIA/RIA U-bottomed microtiter plates (Corning, Corning, NY)
were coated overnight at 4oC with 10µg/ml rituximab or GA101. After coating,
plates were washed x3 times with PBS. Wells were then coated with neat heatinactivated fetal calf serum and again washed x3 with PBS. Human serum or
heat-inactivated serum was added to individual wells at concentrations of 0 to
25% at 37o C for 1 hour. Plates were washed x3 with PBS. NK cells from
healthy donors were obtained from leukocyte reduction filters and isolated using
the MACS NK-cell isolation kit from Milteny Biotec (Auburn, CA). NK cells (5x104
cells/well) were added to wells of the microtiter plates and allowed to adhere for 1
hour at room temperature. Plates were spun gently at 100g for 1 min.
Absorbance at 405 nm was then read using a plate reader. Low absorbance
indicated binding of NK cells to the walls of the well with little pelleting. High
16
absorbance indicated little binding of NK cells to the walls of the plate with
extensive pelleting.
NK cell activation
Peripheral blood mononuclear cells (PBMC) were obtained from normal
donors. Freshly isolated PBMCs were combined at a 1:1 ratio with Raji cells at a
final concentration of 2.5x106 PBMCs and 2.5x106 Raji per mL. MAb at
concentrations of 0µg/mL to 5µg/mL were added along with media, autologous
serum or autologous heat inactivated serum at various concentrations. Cultures
were incubated for 16-20 hrs at 37°C before flow cytometry analysis. For
analysis, cells were washed and stained with directly conjugated antibodies
including anti-human CD56 AlexaFluor 647, CD54 PE (BD Pharmigen), CD16
FITC (Serotec, Raleigh, NC), CD3 PE-Cy7 (Caltag Laboratories, Burlingame,
CA), and CD19 APC-Cy7, per the manufacture’s protocol. Flow cytometry
analysis of NK cell phenotype was performed on an LSR (BD Immunocytometric
Systems, San Jose, CA). NK cells were identified as CD19-, CD3-, CD56+ cells
in the lymphocyte gate. Specific measurements included assessment of %NK
cells that were CD54bright.
Antibody dependent cellular cytotoxicity 51Cr
release assay
Raji cells served as target cells and were radiolabeled with 51Cr. NK cells
were isolated using the MACS NK isolation kit per the manufacturer’s protocol as
17
outlined above. NK cells were added to Raji cells at a concentration of 1x104
cells/well at effector target ratios of 50:1, 25:1. 12.5:1, 1:1, and 0:1. Rituximab or
GA101 was then added at a concentration of 5mg/ml. In select samples, C5
depleted serum or Heat Inactivated C5 depleted serum was added at a final
concentration of 25%. Samples were incubated for 4 hours at 37oC, centrifuged
at 400g for 5 min and supernatant removed. Percent specific lysis was
calculated as (experimental value - spontaneous lysis) / (maximum lysis spontaneous lysis) x 100.
B cell depletion assay
Whole blood from healthy donors was used. Triplicates of each test
antibody concentration + 3x blood without antibody were placed in a 96-well deep
plate (96-well Polypropylene Deep-Well Plates, 2 ml total volume, conicalbottom, BD #353966 or 96-well Polypropylene Deep-Well Plates, 1.2 ml total
volume, round-bottom, (TreffLab # 96.9118.9.01). 280 µl blood + 20 µl (diluted)
test antibody was used for each well. Blood was incubate at 37°C for 24h (cell
incubator). After 24 hours, antibody mix was added to each well. 7 µl of antiCD3-FITC (BD #555332), 7 µl of anti-CD19-PE (BD #555413), 7 µl of anti-CD45PE-Cy5 (BD #555484). Plate was shaken and 35 µl of blood was removed and
put into the U-bottom 96 well plate with the staining abs. Cells were incubated
for 15 min at room temperature in the dark, then 200 µl BD FACS Lysis Solution
(BD #349202, working solution = 1:10 diluted with Aqua ad injection) was added,
and incubated again for 15 min at RT in the dark. Ratio between numbers of
18
CD19+ cells (B-cells) and number of CD3+ cells (T-cells) were calculated, and
samples without antibody were set as 0% killing.
Mouse Studies
Three transgenic C57Bl/6 mice expressing human CD20 protein on their B
cells were age/sex matched and 6 months old. Mice received either 1mg of
rituximab or 1mg of rituximab-MAGE intraperitoneally. Mice were analyzed after
7 days for B cell depletion. 100µl of blood was isolated from mice and stained
with mouse specific anti-CD19 mAb-FITC 1µg/ml. Samples were read until
10,000 events on a flow cytometer. Percent (%) B cells depleted are
represented in the graphs. All experiments were performed under the guidelines
in accordance with the IACUC Animal Care and Use Protocols from the
University of Iowa.
Statistical analysis
All statistical analyses were performed on experiments using a two-tailed
unpaired Student’s t test for all in vitro studies. Values with a p-value <0.05 or
<0.001 were considered to be statistically significant, and values denoted ns
were non significant.
19
CHAPTER III: THE COMPARISON OF RITUXIMAB TO VARIOUS
SECOND AND THIRD GENERATION ANTIBODIES AND THEIR
MECHANISMS OF ACTIONS
Introduction
We performed the studies presented in order to compare and contrast the
different mechanisms of action between various mAbs including rituximab,
ofatumumab, GA101 and rituximab-MAGE. Previous studies performed in our
laboratory determined that complement inhibited the binding of the anti-CD20
mAb rituximab to the FcgRIIIa on NK cells. We therefore studied other antiCD20 mAbs, that had either changes in their glycosylation pattern of the Fc
region, had different epitopes, or different binding affinities to CD20 (type II
mAbs), and wanted to determine if complement inhibited activation of NK cells
and ADCC. We therefore studied the complement fixation properties of each
mAb, their ability to mediate CMC, their ability to bind to NK cells, and thus
mediate ADCC and deplete B cells through in vitro and in vivo analyses. It
should be noted that due to limited availability of these mAbs, not all mAbs were
used in each assay at the same time.
Differences in complement concentrations are
highly variable between various compartments
Antibody-mediated complement effects may vary based on complement
concentrations in various compartments. Little is known about complement
20
levels in the extravascular compartment. We therefore used ascites fluid from
patients without cancer or known active infection as a surrogate for extravascular
fluid, and evaluated complement levels and activity. We obtained samples of
ascites fluid from five different patients. All samples used in the study were
obtained with consent as per HIPAA protocols during various medical
procedures. We then incubated these samples at a 25% concentration with Raji
cells and 5µg/ml of rituximab. Raji cells were used because they highly express
CD20 and were readily available. Samples were then washed and stained for
C3b deposition. As the results show in Figure 3, ascites fluid in most subjects
contained much lower levels of complement compared to the levels found in
serum, however patient #1411 had increased C3b deposition compared to the
other individuals, which may be due to unknown non-specific inflammation. Due
to privacy regulations any medical issues that may have affected complement
levels were not disclosed. Blood is full of complement proteins, however it is
hypothesized that complement terminal fragments are lacking in extravascular
tissues. These studies confirm that complement levels vary by compartments,
and are typically found at lower levels in the extravascular fluid. Thus one might
consider than CMC may play a less pivotal role in the extravascular compartment
compared to the intravascular compartment where complement levels are higher.
Future studies may be carried out to confirm complement varies by compartment
using ascites and serum from the same donor.
21
Rituximab and ofatumumab have superior
complement fixation properties as compared
to GA101 and rituximab-MAGE
We next compared the complement fixation capabilities of all four mAbs in
different scenarios. In figures 4 and 5 we compared rituximab vs. GA101 vs.
ofatumumab, in the presence of serum using Daudi cells. We wanted to utilize a
variety of different B cell lymphoma lines to ensure that these differences were
not attributed to the physical properties of one cell line alone. We found that
indeed that GA101 did have inferior complement binding capabilities, which
would coincide with the fact that GA101 is a type II mAb that has been
afucosylated. Ofatumumab had similar complement fixation capabilities to
rituximab. When comparing rituximab, a type I mAb, to a glycoengineered type I
mAb using a Raji cell line, we found that rituximab-MAGE did have lower
deposits of C3b on its surface, but it was not completely reduced to undetectable
levels, shown in Figure 6. There still maintains some complement fixation, albeit
very low and it was comparable to the third generation mAb GA101.
Furthermore when tested in an autologous system as shown in Figure 7 we were
also able to confirm that GA101 and rituximab-MAGE had decreased
complement fixation further supporting the evidence that third generation type II,
and glyco-engineered mAbs may work preferentially through ADCC mechanisms
and less through CMC due to their inability to fix complement.
22
Rituximab mediates CMC of Raji cells while
GA101 does not mediate CMC
To coincide with the ability of these mAbs at fixing complement, we
assessed their ability to mediate CMC through inducing lysis of opsonized Raji
cells in the presence of complement. As illustrated in Figure 8, rituximab was
effective at mediating CMC while GA101 was ineffective at concentrations of
mAb up to 5 µg/ml in a 51Cr release assay. We performed an additional
experiment staining the samples with Propidium iodide to analyze cell death in
Figure 9 and also found that rituximab-MAGE and GA101 mediated lower CMC
compared to rituximab. This experiment did not use annexin V to analyze
apoptosis, so we cannot determine to what extent the mAbs contributed to cell
death via apoptosis, but the trend of lower CMC is similar to that seen in Figure 8
where we can exclude apoptosis as a mechanism of action in this particular
assay. Furthermore, rituximab-MAGE induced neither the highest nor lowest
CMC as seen in Figure 9, which may be due to unknown effects from the glycomodifications of the Fc effector region of rituximab-MAGE. We are thus able to
conclude that the ability of these mAbs to fix complement, coincided with their
abilities to induce CMC.
Serum inhibits binding of NK cells to rituximab
but not to GA101
Prior studies performed in our laboratory demonstrated that serum
complement blocks NK cell binding to rituximab (Wang, Racila et al. 2008). More
23
specifically, C3b binds to rituximab Fc and blocks binding of the Fc to CD16.
Given that GA101 fixes complement poorly, we assessed whether complement in
serum is able to block binding of NK cells to GA101. We used an adherence
assay to assess interactions between NK cells and plastic bound rituximab or
GA101. As reported previously, NK cells bound to rituximab in the absence of
serum. This interaction was blocked by even low concentrations of serum
(3.125%). NK cells also bound to GA101. However, in contrast to rituximab,
serum complement had little effect on NK binding to GA101 (Figure 10). The
mAb in this assay are bound directly to plastic, thus this assay reflects
differences that are independent of the epitope specificity of the mAb and
whether it is a type I or a type II anti-CD20 mAb. The differences in the ability of
NK cells to adhere to GA101 and rituximab are therefore due, at least in part, to
the Fc structure of the mAbs. Most importantly, these results demonstrate
complement interferes with the interaction between rituximab and NK cells, but
not GA101 and NK cells.
GA101 is superior to rituximab and ofatumumab in
activating NK cells in the presence of complement
Previously, our laboratory demonstrated that the addition of rituximab to a
mixture of target B cells and PBMCs induces activation of NK cells as measured
by a number of phenotypic changes including up-regulation of CD54.
Furthermore, we found that serum complement, more specifically C3b, blocks NK
activation induced by rituximab-coated target cells. In the current studies we
24
compared the ability of rituximab, GA101, and ofatumumab to induce NK
activation and assessed the effect of complement on this activation. In the
absence of serum, Raji cells coated with either rituximab or GA101 are able to
induce upregulation of CD54 (results not shown). Similar results are seen when
heat inactivated autologous serum was added (Figure 11b). In the presence of
unmodified serum, rituximab-coated Raji cells were unable to induce CD54
upregulation (Figure 11a). In contrast, GA101-coated Raji cells induced
upregulation of NK cell CD54 even in the presence of serum Figure 12. This
suggests the ability of rituximab to fix complement limits its ability to activate NK
cells in the presence of complement, while the inability of GA101 to fix
complement allows GA101 to activate NK cells even in the presence of
complement. Additionally, we also can see in Figure 12 that using another cell
line, Daudi cells, we were still able to find a statistical significance between the
various groups, with GA101 having the highest NK cell activation in either the
presence or absence of complement, further supporting our hypothesis that
complement binding may actually reduce efficacy of certain mAb therapies such
as rituximab, which has a higher affinity to fix complement, since this binding can
inhibit engagement to NK cells.
C5-depleted serum blocks ADCC mediated by
rituximab, but not ADCC mediated by GA101
We next evaluated whether there is a difference between rituximab and
GA101 in their ability to mediate ADCC in the presence of the early components
25
of complement. These studies were done using C5-depleted serum because
such serum allows for activation of the earlier steps in the complement cascade,
including C3b deposition, without development of the membrane attack complex.
As expected, C5-depleted serum was unable to induce CMC of rituximab or
GA101-coated Raji cells (data not shown). In the absence of complement,
purified NK cells induced ADCC of both rituximab-coated Raji cells and GA101coated Raji cells. ADCC mediated by rituximab was inhibited by C5-depleted
serum. In contrast, C5-depleted serum had no effect on ADCC mediated by
GA101 Figure 13. These studies demonstrate that complement fixation
upstream of C5 activation impairs the ADCC of rituximab-coated target cells by
NK cells, but has no effect on ADCC mediated by GA101.
GA101 and rituximab-MAGE have superior B cell
depleting properties as compared to ofatumumab
and rituximab
Coinciding with GA101’s ability to induce higher amounts of ADCC, we
wanted to determine which mAb might be efficacious for depleting B cells in
whole blood, and if ADCC was the primary mechanism of action. These studies
were done using normal blood obtained from healthy donors to determine their B
cell depletion properties. We found that GA101 induced more B cell depletion
than rituximab or ofatumumab as shown in Figure 14 and 15. We observed this
same effect when we compared both type I mAbs (rituximab vs. rituximabMAGE) in Figure 16. These studies demonstrate that mAbs that are
26
glycoengineered or type II produce superior B cell depleting assay results. This
effect could be due to ADCC, and ADCC may be the preferred mechanism of
action, because mAbs like rituximab and ofatumumab mediate higher CMC and
lower ADCC as previously described.
Rituximab depleted more CD20+ B cells than
rituximab-MAGE in an in vivo model
In addition to comparing GA101 to rituximab, we found it valuable to
compare a newly glyco-engineered rituximab (Rituximab-MAGE) in order to
directly compare 2 similar type I mAbs. Differences that were seen could be
attributed to differences in functionality of the mAb and their effects on lipid rafts,
but here we have 2 fully functional, identical type I mAbs that only differ in their
capabilities to fix complement and induce ADCC. We performed in vivo
experiments using mice that were huCD20+ transgenic.
In this mouse model, we injected three huCD20+ Tg mice on a C57Bl/6
background with either rituximab, rituximab-MAGE, or PBS. Mice were analyzed
for B cell depletion one week later. We found that in blood, B cells were depleted
more effectively using rituximab rather than its glyco-engineered twin rituximabMAGE (8.15% vs. 39.7%) as seen in Figure 17. When compared to control PBS,
circulating B cells were reduced from 50.9% to 39.7% with rituximab-MAGE
indicating that rituximab-MAGE did have some depleting properties. It is a
possible that although fewer B cells were depleted in a week, that perhaps
rituximab-MAGE works at a different optimal concentration or at a slower kinetics
27
compared to its counterpart. Since these mice were treated with chimeric
antibodies that have their Fc portion replaced with a human Fc portion, rituximabMAGE was unable to induce ADCC mediated death. Murine NK cells in this
model do not express human CD16, and thus would not be able to engage the
Fc receptor and become activated. Therefore, further studies are needed to
elucidate the optimal dosage and time course needed to obtain similar depletion
and to determine if ADCC is a preferred mechanism of action in this mouse
model.
28
CHAPTER IV: DISCUSSION AND FUTURE DIRECTIONS
The anti-CD20 mAb rituximab has changed our approach to the treatment
of B cell malignancies. Nevertheless, rituximab alone does not result in complete
regression in all cases, and many patients relapse or develop resistance
following combination therapy with rituximab and chemotherapy. Thus,
understanding mechanisms of action and development of resistance to antiCD20 mAb therapy is key to the rational design and evaluation of the next
generation therapy (Weiner 2010).
Indirect, but convincing evidence suggests ADCC is central to the efficacy
of anti-CD20 antibody therapy. Previous work in our laboratory demonstrated
that complement fixation can block ADCC by interfering with the interaction
between the anti-CD20 Fc and CD16a on NK cells (Wang, Veeramani et al.
2009). Studies have shown that the therapeutic effect of mAbs is lost in Fcγreceptor knock-out mice (Clynes, Takechi et al. 1998). In addition, three
independent studies demonstrated that single agent rituximab was more effective
in patients with Fcγ receptor III (CD16) polymorphisms associated with higher
affinities for human IgG suggesting that Fc receptors on effector cells may play a
key role in the therapeutic effect of rituximab (Cartron, Dacheux et al. 2002;
Weng and Levy 2003; Treon, Hansen et al. 2005). This evidence points towards
ADCC and CD16 as being vital to the clinical activity of rituximab and has led to
the generation and evaluation of a number of next-generation anti-CD20 mAbs
with an enhanced ability to bind to CD16 (Bowles, Wang et al. 2006). We also
29
found that rituximab-MAGE, which is inefficient in fixing complement, was able to
deplete circulating B cells but did not deplete cells as much as rituximab using a
human CD20+ transgenic mouse model. Further studies are needed in the
future to optimize the dose and validate the results. These mAbs look promising
in vitro, but whether they are more effective clinically remains to be determined.
There is also evidence the complement-mediated lysis can contribute to
efficacy in some situations. The role of complement in mediating the anti-tumor
activity of anti-CD20 mAb has received considerable attention, and is proving to
be quite complex. There is solid evidence that complement can induce CMC of
rituximab coated target cells in vitro (Bellosillo, Villamor et al. 2001; Golay,
Lazzari et al. 2001) and can contribute to the anti-tumor activity of mAb in animal
models (Golay, Cittera et al. 2006). Initial clinical studies indicate ofatumumab,
which has an enhanced ability to fix complement is effective, particularly in CLL
(Coiffier, Lepretre et al. 2008) where malignant cells are exposed to the mAb in
the blood where complement in high. Therefore one might consider utilizing
mAbs that induce CMC to treat circulating tumor cells.
Though complement proteins are mostly studied in the serum, much of the
anti-tumor activity of anti-CD20 mAbs takes place in the extravascular
compartment. Here we have shown that the levels of complement are not
abundant in extravascular fluid, however, previous studies done by our laboratory
have shown that it was present in sufficient concentration to inhibit activation of
NK cells by rituximab-coated target cells (Wang, Veeramani et al. 2009). Thus,
it is unclear whether complement fixation is "friend" or "foe" when it comes to
30
anti-CD20 antibody therapy. Complement may be a “friend” for anti-CD20 mAbcoated target cells in the circulation where complement levels are high and CMC
is important, but a “foe” in the tissues where complement levels are lower and
ADCC may play a more significant role.
A number of new anti-CD20 antibodies have been developed in the past
several years that have varying ability to mediated ADCC and fix complement.
Modifications that enhance effector function, for example incorporation of a
bisecting GlcNAc into the N-glycan, offer a way of enhancing the therapeutic
efficacy of mAbs, which act via ADCC and/or CMC. GA101 is unique among
these anti-CD20 mAbs. It is a type II anti-CD20 mAb, in contrast to most of the
other anti-CD20 mAbs used in this study that are type I anti-CD20 mAb
(Mossner, Brunker et al. 2010). This is thought to be a potential advantage in
that cross-linking by type II anti-CD20 mAb in vitro induces a greater degree of B
cell death when compared to type I anti-CD20 mAb (Beers, Chan et al. 2008).
Type II anti-CD20 mAbs are less effective at fixing complement and mediating
CMC than type I anti-CD20 mAbs.
The differences between rituximab and GA101 with respect to
complement fixation are not due solely to rituximab being a type I anti-CD20, and
GA101 being a type II anti-CD20. The in vitro adherence assay measured NK
cell binding to mAb utilized rituximab and GA101 was done on plastic wells and
there was no involvement of target cells or target antigen. The difference in their
epitope specificity of the mAb and/or movement of the mAb-antigen complex in
the target cell membrane, were not factors as there was no movement of plastic-
31
bound mAb. NK cells bound to either rituximab or GA101 on the plastic in the
absence of complement. However, these two mAbs behaved very differently in
the presence of complement. Complement blocked the ability of NK cells to bind
to rituximab, but had little effect on the ability of NK cells to bind to GA101.
Whether this difference in complement fixation is completely related to
glycoengineering or if other unknown differences play a role remains to be
determined. Since we also had the availability of a glycoengineered version of
rituximab (rituximab-MAGE), we were able to compare the behavior between two
type I mAbs and found that rituximab-MAGE fixed less complement and induced
lower CMC than rituximab alone, due to its glyco-engineered Fc portion.
Therefore, at least in the case of rituximab-MAGE, glyco-engineering played a
significant role.
In these studies we evaluated GA101, ofatumumab, and rituximab-MAGE
in relation to rituximab on their ability to fix complement and mediate CMC. We
also assessed the impact complement has on the ability of these antibodies to
bind to NK cells, activate NK cells, and mediate ADCC. We conclude from these
studies that there are significant differences among these antibodies. The ability
of a given antibody to mediate CMC and complement fixation correlates with the
ability of complement to block the interaction between the antibody and NK cells.
In the presence of complement, GA101 was very efficient in binding to and
activating NK cells and in mediating ADCC on mAb-coated target cells, when
compared to rituximab. Similarly, rituximab-MAGE was very efficient in depleting
B cells in the presence of complement, at least in vitro, when compared to
32
Rituximab. This provides further evidence that ADCC and complement fixation
may be antagonist mechanisms. Modification of the ability of a "next generation"
anti-CD20 antibody to either by increase or decrease complement fixation will
likely also impact on the ability of that antibody to mediate ADCC when
complement is present.
Clinical evaluation of GA101 is ongoing. The results of such clinical trials,
and ongoing performance of translational studies evaluating the relationship
between activating complement fixation and ADCC and anti-CD20 mAb efficacy,
will be crucial as we work to understand how we can take advantage of the
complex interactions between complement and ADCC with the goal of optimizing
the efficacy of this valuable therapeutic modality. Therapies targeting NK cells,
γδ T cells, macrophages and DCs may ultimately be used in combination to
further augment ADCC. Encouraging preclinical studies have led to a number of
promising therapeutics, and the results of proof-of-concept clinical trials are
eagerly awaited (Kohrt, Houot et al. 2012).
Additionally, the individual complement levels of patients may also
contribute to effectiveness of mAb therapy. Patients with lower complement
levels may benefit from a mAb that works via ADCC rather than CMC. Finally,
other factors that may play a role in mAb therapies should be evaluated. These
include: CD20 expression levels on malignant B cells, levels of circulating soluble
CD20 which may limit binding of mAb to target cells, the presence and
abundance of effector cells locally, CD20 binding epitope and kinetics, tissue
distribution and tumor burden (Oflazoglu and Audoly 2010). Hence, many
33
different variables need to be studied and considered in order to optimize mAb
therapy in patients.
34
Fv
VH
Fab
VL
CH1
CL
hinge
Asn
297
Fc
C
H
2
Man GlcNAcGal
GlcNAc
GlcNAc
Asn297
Man GlcNAc
Fuc
Man GlcNAc Gal
N-­‐linked glycan
C
H
3
Adapted from (Raju, Briggs et al. 2000)
Figure 1. Immunoglobulin structure depicting sugar residues. The basic
structure of an IgG immunoglobulin with biantennary N-glycan containing
bisecting GlcNAc residue and a fucose. Asparagine (Asn), Mannose (Man),
Galactose (Gal), Fucose (Fuc), Fragment, variable domain (Fv).
35
CMC
mechanism
of action
mAbs that fix complement
ADCC primary
Mechanism Of
Action
mAbs that do not fix complement
Figure 2. Schematic representation of the interaction between
complement, mAbs, the FcγRIIIa and the primary mechanism of action
used. MAbs that are able to fix complement are unable to bind to the FcγRIIIa
and activate NK cells, thus using CMC as a primary mechanism of action.
MAbs that are unable to fix complement are able to bind to CD16 on NK cells
and mediate ADCC.
36
Monoclonal
Major
Antibody
Mechanism
Rituximab
Rituximab-MAGE
Approval
Type
Generation
Specificity
CMC/ADCC
1997
I
First
Chimeric
ADCC
N/A
I
First-
Chimeric
modified
GA101
Ofatumumab
ADCC, PCD
Phase 2
II
Third
Humanized
CMC
2009
I
Second
Fully human
Table 1. Description of anti-CD20 monoclonal antibody therapies used in these
studies.
37
Type I mAb
Type II mAb
Complement fixation
Ofatumumab
properties
Rituximab
---
Rituximab-MAGE
GA101
Complement fixation
properties
Table 2. Complement fixation properties of type I and type II mAbs used in these
studies.
38
20000
MFI
15000
*
10000
5000
U
ns
t
Is ain
e
ot
yp d
e
C
tl
R
it
R +S
it
+H
Pt I S
.1
6
Pt 4 4
.1
6
Pt 3 9
.1
6
Pt 0 3
.1
3
Pt 7 9
.1
41
1
0
Unstained
Isotype Ctl
Rit+S
Rit +HIS
Pt. 1644
Pt. 1639
Pt. 1603
Pt. 1379
Pt. 1411
Ascites Fluid
Figure 3. Complement levels vary between various compartments.
Raji
Cells were incubated with mAb, donor serum (S), heat inactivated serum (HI S),
or donor ascites fluid for 1hr at 37C. Cells were washed, stained w/Anti-C3b Ab
and analyzed by flow cytometry.
represents 3-5 experiments.
Samples were run in duplicate and data
Patient #1411 had statistically higher levels
compared to all other patients with a p-value <0.05.
39
25000
Median Fluorescence
C3b Deposition using Daudi Cells
Rituximab+S
GA101+S
Ofatumumab+S
Rituximab+HI S
GA101+HI S
Ofatumumab+HI S
20000
15000
10000
5000
IS
ab
+H
O
fa
tu
m
um
10
1+
H
IS
A
G
IS
ab
+H
itu
x
R
tu
m
fa
im
um
ab
+S
10
1+
S
A
G
O
R
itu
x
im
ab
+
S
0
mAb Conc (ug/ml)
Figure 4. Rituximab and ofatumumab have increased complement fixation
capabilities compared to GA101. Daudi cells were incubated with allogenic
serum (S) or heat inactivated serum (HI S) at 25% with various mAbs at 2.5
µg/ml for 30min at 360C. Cells were washed and stained using an anti-C3b
antibody and analyzed via flow cytometry. Median fluorescence intensity was
calculated and averaged between groups. Data represents at least 3 different
experiments.
40
C3b Deposition using Daudi Cells
Rituximab+S
GA101+S
Ofatumumab+S
Rituximab+HI S
GA101+HI S
Ofatumumab+HI S
60000
40000
5
1
0.2
0.04
0
5
1
0.2
0.04
0
5
1
0.2
0.04
0
5
1
0.2
0.04
0
0
5
1
0.2
0.04
0
20000
5
1
0.2
0.04
0
Median Fluorescence
80000
mAb Conc (ug/ml)
Figure 5. Rituximab and ofatumumab have increased complement fixation
capabilities compared to GA101 at various concentrations. Daudi cells were
incubated with allogenic serum (S) or heat inactivated serum (HI S) at 25% with
mAbs at 5,1,0.2,0.04 and 0 µg/ml for 30min at 360C. Cells were washed and
stained using an anti-C3b antibody and analyzed via flow cytometry. Median
fluorescence intensity was calculated and plotted. Samples were run in triplicate.
Isotype and unstained controls not shown.
41
60000
5
1.25
0.3125
0
MFI
40000
20000
10
1+
H
IS
R
itM
A
G
R
E+
itM
S
A
G
E+
H
IS
G
A
10
1+
S
G
A
+H
IS
itu
x
R
R
itu
x
+S
0
% Serum
Figure 6. GA101 and Rituximab-MAGE have decreased abilities to fix
complement. Raji cells were incubated With 25% serum (S) or heat inactivated
serum (HI S) and different Concentrations of mAb for 30 min. Cells were then
washed and Stained with anti-C3b Ab (3E7), And a 2ndary IgG1-PE was used.
Median fluorescence intensity was calculated and plotted. Data represented are
of 3 experiments. Isotype controls and unstained controls not shown.
42
C3b Deposition
Median Fluorescence
80000
Rituximab+S
GA101+S
RitMAGE+S
Rituximab+HI S
GA101+HI S
RitMAGE+HI S
60000
40000
20000
5
1
0.2
0
5
1
0.2
0
5
1
0.2
0
5
1
0.2
0
5
1
0.2
0
5
1
0.2
0
0
mAb conc (ug/ml)
Figure 7. Rituximab-MAGE and GA101 have decreased complement
fixation properties in comparison to rituximab alone. EBV transformed B
cells were incubated with autologous serum (S) or autologous heat inactivated
serum (HI S) at 25% with mAbs at 5,1,0.2,0.04 and 0 µg/ml for 30min at 360C.
Cells were washed and stained using an anti-C3b antibody and analyzed via flow
cytometry. Median fluorescence intensity was calculated and plotted.
% Specific Lysis
43
80
Rituximab
70
GA101
60
50
40
30
20
10
0
0 ug/ml
.04 ug/ml
.2 ug/ml
1 ug/ml
5 ug/ml
mAb concentration
Figure 8. GA101 induces lower complement mediated cytotoxicity in the
presence of serum using 51Cr release assay. Raji Cells were labeled with 51Cr,
incubated with mAb and serum (S) or heat inactivated serum (HI S) for 1hr at
37oC, analysis using a gamma reader and % specific lysis was calculated. Data
is representative of 3 different experiments, and samples were run in triplicate.
Controls using heat-inactivated serum were used, but not shown).
44
% Cell Death
100
Ritux+S
RituxMAGE+S
GA101+S
Ritux+HIS
RitMAGE+HIS
GA101+HIS
80
60
40
20
0
0.
62
5
1.
25
2.
5
5
0
Serum (µg/ml)
mAb%
concentration
Figure 9. GA101 Induces lower complement mediated cytotoxicity in the
presence of serum. Raji Cells were incubated with mAb, serum (S) or heat
inactivated serum (HI S) for 1hr at 37oC. Propidium Iodide at a 50µg
concentration was added to determine cell death, and samples were analyzed by
flow cytometry and percent cells that were positive for PI were plotted.
Rituximab+S was significantly higher to rituximab+HI S with a p-value <0.05.
Samples were run in duplicates and experiments were performed 3 times.
Isotype and unstained controls not shown.
45
NKC Adherence
Absorbance (405nm)
0.3
Rituximab+S
Rituximab+HI S
GA101+S
GA101+ HI S
0.2
0.1
0.0
0
3.125
6.25
12.5
25
Percent Serum
Figure 10. NK cells adhere to GA101 but not rituximab in the presence of
serum. 96 well EIA plates were coated with either rituximab or GA101 overnight
at 4oC. Serum (S) or heat inactivated serum (HI S) was added to wells and
rinsed, followed by isolated NK cells from human subjects. Plate was spun and
O.D. was measured using spectrophotometer at 405 nm. Data is a
representation of 4 different experiments. An unpaired student’s t-test was
performed with a p-value<0.05.
A
.
.
B
46
A
B
Figure 11. Complement blocks ability of rituximab coated target cells to
activate NK cells, but not GA101 coated Raji cells to activate NK cells. Raji
cells were coated with various concentrations of rituximab, ofatumumab or
GA101 in the presence or absence of 20% normal human serum or heat
inactivated serum from the same donor. NK cell activation, as determined by %
cells CD54 bright was determined. In A, NK activation in the presence of normal
human serum or with heat inactivated serum in B. Cells were stained for CD19CD3-CD56+CD54+CD16+. Data is representative of 5 experiments performed.
47
NK Cell Activation
*
% CD54bright
80
ns
60
40
Rituximab+S
GA101+S
Ofatumumab+S
Rituximab+HI S
GA101+HI S
Ofatumumab+HI S
20
IS
10
1+
tu
H
m
IS
um
ab
+H
IS
O
fa
G
A
ab
+H
im
ab
+S
itu
x
R
tu
m
um
10
1+
S
G
A
O
fa
R
itu
x
im
ab
+
S
0
Figure 12. Complement blocks ability of rituximab and ofatumumab-coated
target cells to activate NK cells, but not GA101-coated cells to activate NK
cells. Daudi cells were coated with 2.5 µg/ml concentrations of rituximab,
ofatumumab or GA101 in the presence or absence of 20% normal human serum
(S) or heat inactivated serum (HI S) from the same donor. NK cell activation, as
determined by % cells CD54bright was determined. NK activation in the presence
of normal human serum or with heat inactivated serum. Cells were stained for
CD19-CD3-CD56+CD54+CD16+. Data is representative of 3 experiments.
48
51
Cr Release Assay
% Specific Lysis
40
Rituximab+ C5 depleted S
GA101+ C5 Depleted S
Rituximab + C5 HI S
GA101 + C5 HI S
Rituximab+ No S
GA101+ No S
30
20
10
0
-10
0:
1
1:
1
12
:1
25
:1
50
:1
-20
Effector:Target
Figure 13. GA101 has enhanced ADCC in the presence of C5-depleted
serum. 51Cr labeled Raji Cells incubated with Allogenic NK Cells For 4 hours with
C5-depleted serum, serum (S) or heat inactivated serum (HI S), or no serum.
Specific Lysis was calculated. Data represent the mean
SD. Data
representative of 5 experiments, each done in triplicate. * Indicates p
** Indicates p<0.001.
0.05.
49
*
*
% B cell depletion
60
Rituximab
GA101
Ofatumumab
40
20
um
ab
10
1
A
fa
tu
m
G
O
R
itu
xi
m
ab
0
mAb Therapy
Figure 14. GA101 has superior B cell depleting capabilities compared to
rituximab and ofatumumab. We compared three different mAbs in their ability
to deplete B cells. Whole blood was treated with either rituximab, GA101 or
ofatumumab at 2.5µg/ml. Cells were then stained for CD45+CD3-CD19+
expression. Percent B cell depletion was calculated and plotted. Controls used
were no Ab (not shown). Data is representative of 3 or more experiments. An
unpaired student’s t-test was performed and the P-value was <0.05.
50
Rituximab
GA101
Ofatumumab
60
40
0
0.008
0.04
0.2
1.0
5
0
0.008
0.2
0.04
1.0
5
0
0.008
0.04
0.2
0
1.0
20
5
% B cell depletion
80
mAb Conc (ug/ml)
Figure 15. GA101 has superior B cell depleting capabilities compared to
rituximab and ofatumumab at various concentrations. We compared three
different mAbs in their ability to deplete B cells. Whole blood was treated with
various concentrations of mAb. Cells were stained for CD45+CD3-CD19+.
Percent B cell depletion was calculated and plotted. Controls used were no Ab.
Data is representative of 3 or more experiments. An unpaired student’s t-test
was performed and the P-value was <0.05.
51
Whole Blood Assay
% B cell depletion
50
Rituximab
RitMAGE
40
30
20
10
0
0
0.04
0.2
1
5
0
0.04
0.2
1
5
-10
mAb conc (ug/ml)
Figure 16. Rituximab-MAGE shows superior ability to deplete B cells
compared to rituximab alone. Percent B cell depletion calculated. Healthy
human blood was incubated overnight with various concentrations of rituximab or
rituximab-MAGE. Stained for cd45+cd3-cd19+. No Ab was used as control.
Isotype and unstained controls not shown.
52
SSC
A
B
C
CD19+
Figure 17. Rituximab depletes more circulating B cells in vivo than
rituximab-MAGE. Three hCD20+ Tg B6 mice were treated IP with rituximabMAGE, rituximab or PBS at 1mg per mouse. Mice were then euthanized and
blood was analyzed by staining for B cells using anti-CD19 APC conjugated
antibody (BD# 522346). This data is representative of circulating B cells in the
blood from each mouse. A. Mice that received no treatment B. Mice that
received rituximab 1mg C. Mice that received rituximab-MAGE 1mg.
53
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