Genetic engineering has substantially improved antibodies used in the treatment of cancer and related diseases, initially by providing more compatible reagents (chimeric, humanized, human) for patients but more recently by improving their clinical efficacy.1  In this issue of Blood, Gupta et al have used a novel genetic engineering method, termed “dock and lock,”2-4  to construct a HexAb bispecific antibody directed against 2 B-cell epitopes (CD20, CD74). By so doing, they have tapped into a distinctive B-cell homeostatic mechanism that controls B-cell growth and proliferation.5 

Earlier it was observed that the agglutination of malignant B cells by certain antibodies induced the movement of bound antigens into cell surface lipid rafts located sporadically on B cells to give a speckled membrane pattern of antibody binding by immunofluorescence microscopy.6  For example, it is now known that anti-CD20 antibodies fall into 2 groups: type I and type II.7,8  Type I antibodies represented by Rituxan and Veltuzumab used in this study produce a ring pattern on lymphoma cells after binding and by themselves do not produce significant apoptosis. By comparison, type II anti-CD20 antibodies represented by tositumomab produce a speckled membrane pattern and are very effective in inducing cell death by a caspase-independent mechanism. Type I antibodies can be induced to produce this type of apoptosis after cross-linking by antibodies that bind Rituxan or using multivalent antibody polymers to simulate this cross-linking phenomenon.9 

In the paper by Gupta et al, the authors have found another method of inducing B lymphoma cell death using a bispecific antibody that cross-links 2 different B-cell antigens, CD20 and CD74.2  By testing different compositions of HexAbs, they determined that 2 combinations are clinically active, necessitating additional studies to determine which is most efficacious in patients. As shown in the figure, the cross- linking of CD74, which is directed against the invariant chain of the HLA-Dr molecule,10  and CD20, which is highly expressed on B-cell neoplasms but whose function is still not fully understood, produced cell clustering that is associated with actin reorganization, antigen migration into lipid rafts, swelling of mitochondria and lysosomes to cause influx of calcium and release of capthepsin B, respectively, down-regulation of Bcl-xL and pAkt, and the rapid and sustained phosphorylation of ERK and JNK second signals culminating in cell death.

Biologic effect of cross-linking multivalent anti-CD20/CD74 bispecific antibodies in B-cell lymphomas. Aggregation of lymphoma cells by bispecific antibodies causes reorganization of actin and migration of antigen (red) into lipid rafts that in turn induces the release of lysosomal cathepsin B and reactive oxygen species (ROS) and influx of calcium ions into mitochondria. Swollen lysosomes and mitochrondria are accompanied by the deactivation of the PI3K/Akt signaling pathway and the rapid and sustained activation of ERK, JNK, and MAPKs associated with cell death. Nonaggregated cells remain viable in the absence of antibody binding.

Biologic effect of cross-linking multivalent anti-CD20/CD74 bispecific antibodies in B-cell lymphomas. Aggregation of lymphoma cells by bispecific antibodies causes reorganization of actin and migration of antigen (red) into lipid rafts that in turn induces the release of lysosomal cathepsin B and reactive oxygen species (ROS) and influx of calcium ions into mitochondria. Swollen lysosomes and mitochrondria are accompanied by the deactivation of the PI3K/Akt signaling pathway and the rapid and sustained activation of ERK, JNK, and MAPKs associated with cell death. Nonaggregated cells remain viable in the absence of antibody binding.

Close modal

Because Veltuzumab is a type I anti-CD20 and by itself is not apoptotic, it would be of interest to determine whether substituting a type II anti-CD20 in the HexAb might be more effective. CD74 is a good choice for the HexAb because it binds a critical molecule for B-cell survival and proliferation.10  Nonetheless, the emergence of this novel genetically engineered bispecific antibody should be a welcome new product for the treatment of mantle zone lymphoma, chronic lymphocytic leukemia, and other difficult-to-treat B-cell tumors expressing both CD20 and CD74 antigens. The ingenuity and creativity displayed in this study demonstrates further the ever-increasing role of genetic engineering in the development of promising new antibody reagents for cancer immunotherapy.

Conflict-of-interest disclosure: The author declares no competing financial interests. ■

1
Beck
 
A
Wurch
 
T
Bailly
 
C
Coraia
 
N
Strategies and challenges for the next generation of therapeutic antibodies.
Nat Rev Immunol
2010
, vol. 
10
 
5
(pg. 
345
-
352
)
2
Gupta
 
P
Goldenberg
 
DM
Rossi
 
EA
, et al. 
Dual-targeting immunotherapy of lymphoma: potent cytotoxicity of anti-CD20/CD74 bispecific antibodies in mantle cell and other lymphomas.
Blood
2012
, vol. 
119
 
16
(pg. 
3767
-
3778
)
3
Rossi
 
EA
Goldenberg
 
DM
Cardillo
 
TM
, et al. 
Stably tethered multifunctional structures of defined composition made by the dock and lock method for use in cancer targeting.
Proc Natl Acad Sci U S A
2006
, vol. 
103
 
18
(pg. 
6841
-
6846
)
4
Chang
 
CH
Rossi
 
EA
Goldenberg
 
DM
The dock and lock method: a novel platform technology for building multivalent, multifunctional structures of defined composition with retained bioactivity.
Clin Cancer Res
2007
, vol. 
13
 
18 Pt 2
(pg. 
5586s
-
5591s
)
5
Hofmeister
 
JK
Cooney
 
D
Coggeshall
 
KM
Clustered CD20 induced apoptosis: Src-family kinase, the proximal regulator of tyrosine phosphorylation, calcium influx, and caspase 3-dependent apoptosis.
Blood Cells Mol Dis
2000
, vol. 
26
 
2
(pg. 
133
-
143
)
6
Tobin
 
C
DeNardo
 
S
Zhang
 
N
Epstein
 
AL
Liu
 
C
DeNardo
 
G
Combination immunotherapy with anti-CD20 and anti-HLA-Dr monoclonal antibodies induces synergistic anti-lymphoma effects in human lymphoma cell lines.
Leuk Lymphoma
2007
, vol. 
48
 
5
(pg. 
944
-
956
)
7
Cragg
 
MS
Glennie
 
MJ
Antibody specificity controls in vivo effector mechanisms of anti-CD20 reagents.
Blood
2004
, vol. 
103
 
7
(pg. 
2738
-
2743
)
8
Beers
 
SA
Chan
 
CH
James
 
S
, et al. 
Type II (tositumomab) anti-CD20 monoclonal antibody out performs type I (rituximab-like) reagents in B-cell depletion regardless of complement activation.
Blood
2008
, vol. 
112
 
10
(pg. 
4170
-
4177
)
9
Zhang
 
N
Khawli
 
LA
Hu
 
P
Epstein
 
AL
Generation of rituximab polymer capable of inducing hyper-crosslinking-induced apoptosis in non-Hodgkin's lymphomas.
Clin Cancer Res
2005
, vol. 
11
 
16
(pg. 
5971
-
5980
)
10
Shachar
 
I
Haran
 
M
The secret life of an innocent chaperone: The story of CD74 and B cell/chronic lymphocytic leukemia cell survival.
Leuk Lymphoma
2011
, vol. 
52
 
8
(pg. 
1446
-
1454
)

Sign in via your Institution