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