immunologic targeting - how to channel a minimal response for maximal outcome susan slovin, md, phd...
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Immunologic Targeting - How to Channel a Minimal Response for
Maximal Outcome
Susan Slovin, MD, PhDGenitourinary Oncology Service
December 1, 2005
Have we succeeded or failed in our treatments for prostate cancer?
Success FailureDocetaxel – standard of care Not that many approved Ph III drugs
Multiple targeting pathways Which is the “one” to stop growth
New drugs in the pipeline Too many approved too fast or too few reaching approval status
Responses after 1st line CAB Disease moves too fast
Bone seeking drugs: improved No impact on important measurestoxicity profile
RATIONALE FOR TARGETED INTERVENTION
PRO1. Over-expression and
undergycosylation of cell surface molecules
2. Can target receptor-like molecules which can stimulate intracellular signaling pathways.
3. Can prevent target activation.
CON1. Strictly extracellular, no
contact with intracellular pathways. Expression varies.
2. Cell can develop “collateral” signaling/survival pathways.
3. Cell can overcome via multiple mechanisms rendering tx inadequate.
TARGETS UNDER EXPLORATION
Cell Surface• Mucins, glycolipids,
carbohydrates, glycoproteins [PSA, PSMA, KSA]
• AR• EGFr• Laminin
Intracellular• Vitamin D (calcitriol)• HSP-90• Proteasome• DNA (HDACs)• BCL-2 [α-sense]• Other – Stroma,
neovasculature
What have we learned from MSKCC prostate cancer vaccine trials?
1) chemical mimes of known cell surface molecules were shown to be immunogenic, ie Globo H - first time that a synethetic molecule could break immunologic tolerance in man
2) role of carriers such as KLH and adjuvants such as QS21 in enhancing immunogenicity and facilitating the immune response. QS21 still remains the best adjuvant through all clinical trials.
3) increasing doses of vaccine do not correlate with augmentation of immunogenicity, ie, lower doses appear to be more immunogenic (especially seen in the TF trial)
4) immunologic responses were not immediate but took up to 6 or more months to develop after the last vaccine; no role for boosters unless they were given either every 4-8 weeks.
5) we learned about the use of PSA slopes - no major impact on pts with high risk disease destined to progress within two years.
6) No clear cut immunologic endpoint; controversy as to how to design biologic trials – lead to PSA Working Group Consortium
PSMA
• Type II transmembrane glycoprotein
• Expression on normal and neoplastic prostate epithelial cells, neovasculature
• Functions as a glutamate-preferring carboxypeptidase with two enzymatic activities:
1) Gamma-glutamyl carboxypeptidase (folate hydrolase
2) N-acetylated alpha-linked l-amino dipeptidase (NAALADase), an enzyme involved in regulation of excitation signaling
Prostate Specific Membrane Antigen
PSMA:extracellularform found oncancer cells
A
B
D
E
C
F
cellmembrane
DE CF
PSM’: intracellular formfound in normal cells
COOHCOOHCOOHCOOH
E
Domain A - aa 1-19Domain B – aa 20-39
Prostate Specific Membrane Antigen
PSMA:extracellularform found oncancer cells
A
B
D
E
C
F
cellmembrane
DE CF
PSM’: intracellular formfound in normal cells
COOHCOOHCOOH
COOH
E
Domain A - aa 1-19Domain B – aa 20-39
RCC Bone (10mg)
How to Target PSMA?
Vaccines Others:• Naked DNA Radionuclide emitter
+ MoAb (ext domain)
• rsPSMA MoAb + maytansinoid
• Alphavirus vector Neovasculature?
What is the ideal patient population?What is the appropriate clinical trial endpoint, i.e., does a clinical and/or immunologic endpoint exist?
Metastatic population Rising PSA
High titer Abs High titer Abs
No effect on PSA Change in PSA logslope
Continued POD Disease stabilization
Rationale for targeting signalling cascades and surface receptors…
a) Most prostate cancers have lost PTEN
b) Tumors that have lost PTEN are insensitive to EGFr inhibition;
Restoration of PTEN function (mTOR inhibition) restores sensitivity to EGFR inhibition
c) PTEN negative tumors are sensitive to mTOR inhibition
The combination of mTOR inhibition and EGFR inhibition may be a rationale treatment approach.
Plasmid DNA expression vector used in MSKCC PSMA DNA
vaccines
pING+ HuPSMA
7213 bpCAAT
Intron A
kana cassette
Human PSMA
pCDNA3 polylinker
T7
CMV promoter
ori
TATA
exon 1
PSMA ORF
Why HLA A02.01?
• HLA-A02.01 allele:– Peptides that match the HLA-A2.01 binding
consensus are found within the huPSMA and muPSMA
– Expressed by 40% of the Caucasian population
• To date: 126 patients typed: 65 + (52%)
MSKCC
Cross-over to break tolerance
Cross-over design of clinical trial
Human PSMA
Mouse PSMA Mouse PSMA
Human PSMA
Why a DNA Vaccine?
• Relatively inexpensive & simple to purify in large quantity
• Avoids complex ex vivo expansion and manipulation of patients’ cells
• Antigen of interest is cloned into a bacterial expression plasmid with a constitutively active promoter.
• Bacterial plasmid DNA itself contains immuno-stimulatory sequences (CpG motifs) that may act as an immunological adjuvant
• Direct entry of the antigen into the intracellular MHC class I processing pathway
How to Break Immune Tolerance to PSMA?
Active PassiveVaccines Others:
• Naked DNA Radionuclide emitter +MoAb (ext domain)
• rsPSMA MoAb + maytansine• Alphavirus vector Neovasculature• Cytokine/GM-CSF (transduced cell line)
• ACP-fusion protein (cellular product)
Y TyrosineIgC domain IgV domain
CD28/B7 superfamily and the paradigm of professional costimulation
T cell APC/DC
B7.1
B7.2
YYYY
YY
CD28
CTLA-4
B7hYYICOS
PD-L1
PD-L2PD-1 YY
Activation
Inhibition
Induced byInflammation/
Pathogens
B7H3
B7xYYYBTLA
SHP-1 SHP-2
Regulation of T cell activation
Antigen
MHC
TCRCD28
B7~
Antigen
CTLA-4
B7MHC
TCRCD28
~
B7MHC
TCRCD28
~Antigen
CTLA-4
Activated T cell IL-2 secretion Proliferation Effector function Induction of CTLA-4
CTLA-4 : B7 suppression Termination of response
Antigen-specific T cell Activation
TCR : Antigen MHC CD28 : B7 Co-stimulation
CTLA-4 Blockade Augments Antigen Specific T-cell Responses
B7MHC
TCR
CD28
~Antigen
B7MHC
TCR
CD28
~Antigen
CTLA-4
Anti-CTLA-4 mAb
CTLA-4
An activated T cell is important in destroying cancer cells or virally infected cells.
CTLA-4 (in red) is sequestered in granules distal to the leading edge of the immunological synapse.
Image courtesy of the Allison lab, University of California, Berkeley
CTLA-4
A T-cell interacts with an antigen presenting cell (APC) and is activated.
Preformed CTLA-4 granules (in red) migrate to the synapse. Signals down-modulate T-cell responses against cancers and other foreign antigens.
Image courtesy of the Allison lab, University of California, Berkeley
Days post tumor injection
Tu
mo
r si
ze (
mm
2 )
0
300
10 30 50 70 90
Source: Kwon et al. PNAS. 1997(94): 8099
(100 ug Ab at days 7, 10, & 13)
Control Ab
Anti-CTLA-4
Anti-Murine CTLA-4 mAb Cures Prostate Cancer in Mice
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
-120 -100 -80 -60 -40 -20 0 20 40 60 80 100 120 140 160 180 200 220 240 260
Days
CA
-125
GVAX
α-CTLA-4
α-CTLA-4
Histopathologic analyses of selected patients experiencing autoimmune events.
(A) Skin rash biopsy from Patient 2 illustrating severe dermatitis with epidermal spongiosis, significant papillary dermal edema and a prominent inflammatory infiltrate in both the superficial and deep dermis (10X magnification).
(B) The cellular infiltrate from Patient 2 reveals the predominance of lymphocytes, monocytes and eosinophils (40X).
Source: Abstract #3424, ASCO 2003
Pathology of Autoimmune Breakthrough Events: Dermatitis
A B
Histopathologic analyses of selected patients experiencing autoimmune events.
(C) Colon biopsy from Patient 9 illustrating severe colitis with infiltration of the lamina propria with neutrophils, lymphocytes, monocytes, plasmacytes and eosinophils. Neutrophils and lymphocytes also infiltrate the crypts; numerous mitotic figures can be seen in the epithelial cells lining the crypts (20X).
Immunohistochemistry evaluating expression of CD3+ (D), CD4+ (E), and CD8+ markers (F) (20X).
Source: Abstract #3424, ASCO 2003
Pathology of Autoimmune Breakthrough Events: Colitis
C D
FE
CD3
CD8CD4