BCL6 expression is deregulated in B cell lymphomas'

1
BCL6 expression is deregulated in B cell
lymphomas.
Translocation puts BCL6 under the control of
heterologous promoters.
More than 20 promoters are linked to BCL6 through
translocation.
Chromosome 3
3
1
2
BCL6
breakpoints
Chromosome 14
Cm
Sm
Im
IgH
Em
JH
Class switch recombination
3
t(314)
2
Im
Em
JH
Hybrid Im-BCL6 mRNA
2
Disruption of BCL6 regulation by somatic
hypermutation
BCL6
1
3
4
2
Negative auto-regulatory element
Aberrant somatic hypermutation
BCL6
1
3
4
2

Somatic hypermutation
3
Mouse model of DLBCL
Em
Im
Sm
Cm
IgH
Sm
Cm
TK
BCL6
neo
Knock-in of BCL6 into IgH
Im
Em
Sm
Cm
BCL6
IgH / IgH-BCL6
BCL6 knock-in mice
4
Increased GC formation in BCL6 knock-in mice
BCL6 and PNA staining of spleen sections
BCL6 staining
5
Development of DLBCL in BCL6 knock-in mice
DLBCL
Kaplan-Meyer plot
Ig V region contains somatic hypermutation
The lymphoma is derived from GC.
6
Deregulation of BCL6 expression prevents the
differentiation of plasma cells.
BCL6
BCL6
BCL6 expression turned off
BCL6 continuously on
Blimp-1 off
p53 repressed (less apoptosis)
Blimp-1 on
Lymphoma
normal
7
Malignant transformation may require multiple
mutations.
Mutations of BCL6 and other oncogenes can be
found in normal B cells.
IgH-Bcl2 translocation can be detected in healthy
people.
8
Monoclonal antibody
epitopes
Cell lines producing individual antibody
serum
Polyclonal antibody
Monoclonal antibody
9
Monoclonal antibody
Polyclonal antibody
Non-specific interaction
Control pre-immune serum
Specific
Limited supply
Control peptide containing the epitope
Unlimited supply
10
Immunogen
Phylogenetic difference foreign
Large size and complex composition more
interaction with T, B cells
Particulate or aggregated phagocytosis, slow
release and clearing
Protein or protein conjugates T cell help
Dose
Too low or high dose can induce tolerance.
IgG High affinity
Boost injection
IgG Medium affinity
IgM Low affinity
Ab
time
11
Adjuvant enhancer immune response
Freunds complete adjuvant oil-water emulsion
with mycobacteria component
Freunds incomplete adjuvant oil-water emulsion
Alum aluminum hydroxide gel
Alum plus Bordetella pertussie aluminum
hydroxide gel with killed B. Pertussis
Slow release of antigen
Aggregate antigen to facilitate phagocytosis
Induce inflammation to activate and attract APCs
12
Animal
Mouse and rat for monoclonal antibody
Rabbit and goat for polyclonal antibody
Route of injection
Intraperitoneal (ip) into the peritoneal cavity
Subcutenous (sc) beneath the skin
Intravenous (iv) into the vein
Intradermal (id) into the skin
Intramuscular (im) into the muscle
13
Immunization for monoclonal antibody
Several mice
Primary injection (D0, ip with adjuvant)
Boost injection (D14, ip with adjuvant)
Test serum (D24, ELISA)
Additional boost injection and test
Final boost (iv or ip) for the best responder (3
weeks from the previous injection)
Isolate spleen 3 days (iv) or 5 days (ip) later
14
Fusion of B cells with myeloma cells
Mice
Mineral oil
Immunized mouse
Plasmacytoma (plasma cell tumor)
No Ig production
Myeloma
Spleen B cells
Ig secretion apparatus
Ig production
Immortal
Nonviable in culture
Fusion by PEG (polyethylene glycol)
Hybridoma
Ig production
Ig secretion apparatus
Immortal
15
Drug selection of hybridoma
de novo pathway (inhibited by HAT)
Nucleotide synthesis
Salvage pathway (HPRT)
HAT selection
Hybridoma
Myeloma
B cell
de novo
de novo
de novo
Salvage (HPRT-)
Salvage (HPRT)
Salvage (HPRT)
nonviable
survival
Nonviable (primary cell)
16
Hybridoma Screening
hybridoma
B cells
Myeloma
fusion
ELISA screening with Ag
Hybridoma clone producing desired antibody
17
Monoclonal antibody (mAb) in cancer therapy
NK cells, Mf
ADCC
Tumor cell
mAb against Tumor antigen
complement
Tumor antigen
MAC
18
The role of Fc?R in ADCC against tumor
Fc?RIIIA CD16
IgG1, IgG3 (human), IgG2a (mouse)
FcgRIIIB CD16
Fc?RIIB
Fc?RIIA
Fc?RI
Ig domain
membrane
?-GPI
?
?
ITIM
?
ITAM
?
?2
?
?2
?
2 x 106
2 x 105
2 x 106
5 x 105
Ka (M-1)
108
NK cells, M?, neutrophils, eosinophils, DC, mast
cells
IgA1, IgA2
IgE
Fc?RI
Fc?RI
?
?
?
?
5 x 107
Ka (M-1)
1010
Neutrophils, M?, eosinophils
Mast cells, basophils
19
Anti-tumor function of mAb requires Fc?R
Proc. Natl. Acad. Sci. 95, 652-656 (1998)
FcR? chain-/-
Wild-type or
14-17 days
iv
melanoma
Count lung metastasis
iv
melanoma
Count lung metastasis
mAb against melanoma Ag
Control mAb
or
Passive protection
melanoma
iv
ip
Count lung metastasis
Tumor Ag (gp75)
Complete Freunds adjuvant
Ab against gp75
3 x boost 2 week intervals
Active protection
20
Fc?R is required for passive protection.
UPC control antibody
TA99 anti-melanoma antibody
Fc?R is required for active protection.
Sf9 control
Sf9-gp75 tumor antigen
21
Macrophage ADCC of tumor cells
Bacillus Calmette-Guerin
lymphoma
Complete Freunds adjuvant
TNP
51Cr
activated macrophage
51Cr
mAb against TNP
ADCC
51Cr
Opsonized tumor cell
lysis
51Cr released into culture supernatant Quantified
by counting ?-irradiation
22
Humanizing mAb
Mouse or rat mAb can induce immune response in
human.
(human anti-mouse antibodies, HAMAs)
Chimeric antibodies
Tumor antigen
Hybridoma producing mAb against tumor antigen
23
Chimeric antibody
VH(m)
CH(m)
Clone VH and VL (mouse) of the hybridoma
VL(m)
CL(m)
VH(m)
C?1, C?3
CH(h)
V mouse
Make hybrid genes
VL(m)
C human
CL(h)
transfection
Complement activation
myeloma
IgG1, IgG3
Fc?RI, III (NK, M?)
24
Humanized antibody
CDR mouse
VH(m)
CH(m)
Clone VH and VL (mouse) of the hybridoma Determin
e the sequences of CDRs
VL(m)
CL(m)
CDRs
VH(h/m)
CH(h)
Make synthetic genes Replace human CDRs with
mouse CDRs
VL(h/m)
CL(h)
25
Human mAb
Make transgenic mice with human Ig genes on yeast
artificial chromosome (YAC)
Human IgH on YAC
Human Ig? on YAC
Human Ig? on YAC
IgH(h)
Ig?(h)
Ig?(h)
breeding
IgH(m)
IgL(m)
immunize
hybridoma
IgH(h), Ig?(h), Ig?(h)
Human mAb
IgH(m), IgL(m)
26
Chimeric anti-CD20 antibody (Rituxan, rituximab)
CD20 is a marker expressed on B cells and B cell
lymphomas.
Anti-CD20 mAb is used to treat non-Hodgkins
lymphoma (NHL)
6 complete
166 patients
42 partial response
Median response duration is 12 months.
Mild toxicity
Approved by FDA in 1997
27
Humanized mAb against ERBB2 (HER2/NEU)
Herceptin
HER2/NEU is a tumor-associated antigen that is
over-expressed by tumor cells in metastatic
breast cancer.
8 complete response
222 patients
26 partial response
Median response duration is 9 months.
Approved by FDA in 1998
28
Deletion of inhibitory Fc?R enhances
anti-tumor activity of mAb.
Nature Medicine 6, 443-446 (2000)
Xenograft human tumor models
Human B cell lymphoma
Human breast carcinoma
tumor
tumor
Nude mice (nu/nu)
Nude mice (nu/nu)
No thymus No graft rejection
No thymus No graft rejection
Human breast carcinoma
Human B cell lymphoma
Tumor inhibtion
Tumor inhibtion
Herceptin
Rituxan
Weekly iv injection
Weekly iv injection
29
The role of activating and inhibitory Fc?R
PBS control
Subtheraputic dose
Herceptin
Anti-tumor activity is enhanced by knockout of
Fc?RIIB
Fc?RI and III
30
Interaction between mAb Fc with Fc?R is required
for anti-tumor activity
mutation
Asp265 to Ala265 (D265A)
4D5
Does not bind Fc?R
ADCC assay
NK cells
Breast carcinoma cells (51Cr)
Effector (NK) / target ratio
31
Direct arming mAb
mAb-toxin conjugate
Toxicity in clinical trials
Mylotarg
Humanized anti-CD33-calicheamicin
Treat CD33 acute myeloid leukemia
30 response rate
Radioimmunoconjugates
32
Problem with superagonist antibody drug
anti-CD28 mAb
Treat autoimmune disease?
CD4CD25Treg
CD28
CD4TH
Anti-CD28 Ab
Activation without TCR interaction
No activation in animal tests
Cytokine storm
Organ failure
CD4TH
Anti-CD28 Ab
Activation of CD4TH in humans
33
Relevant part in book Monoclonal antibody
p99-100 Antibody engineering p128-133