1. The University of Texas Health Science Center
PRIME / BOOST
A trans-atlantic collaboration studying T cell priming and boosting with BCG vaccination
Rob S. Svatek, MD, MSCI
The University of Texas Health Science Center
San Antonio

2. Prime/Boost Participants
Europe
Cyril Rentsch (University Hospital of Basel, Switzerland)
Matthew Albert (Institut Pasteur, Paris)
George Thallman (Inselspital, Bern, Switzerland)
EORTC
Richard Sylvester (Senior Statistician)
Julie Hermans (Operations)
Sanofi Pasteur
Andrew Farrow (Director, Franchise Liaison)
SAKK
Celine Genton (SAKK)
SWOG
Rob Svatek (San Antonio)
Seth Lerner (Houston)
Cathy Tangen (Senior Statistician)
This is a trans-atlantic collaboration that was inspired by the work of Matthew Albert’s laboratory which will be highlighted here. Matthew is the Director of Research INSERM and Head of the Laboratory of Dendritic Cell Immunobiology at the Institut Pasteur.

3. BCG adjuvant therapy remains the standard-of-care for high-risk superficial bladder cancer
Inner muscle layer
Lamina propria
Mucosa
Prostate gland
Urethra
Carcinoma in situ
Papillary tumor
Outer muscle layer
1 wk
BCG
surgery

4. BCG – something extraordinary….
Carcinoma-in-situ (CIS)
Complete Resolution
BCG Treatment
Induction
(6 weeks)
Maintenance (3weeks)
remarkable
surgery
BCG induction
BCG maintenance
week 4
week 6
week 1
week 12

5. BCG therapy: one of the few examples of successful immunotherapy in the clinic
1.0
0.9
0.8
0.7
0.2
0.0
Progression-free survival 5 10 15 20
years
0.6
0.5
0.4
0.3
0.1
surgery + BCG
surgery alone
Superficial cancer (T1G3 stage)
BCG is the mainstay for treatment of intermediate and high-risk superficial bladder cancer. There have been multiple RCT comparing BCG to intravesical chemotherpay and BCG consistently wins.
Adapted from Herr, 1997

6. Current proposed model of BCG therapy
in bladder cancer
BCG
tumor cells
Bladder
urothelium
1 attachment and invasion
of the urothelium
4 cytolysis of bladder tumor
cells by CD8+ T cells
T cells
Neutrophils
Monocytes
What is known: In response to mycobacterial stimulation, urothelial cells secrete a limited array of proinflammatory cytokines, which initiate a first wave of innate immune cells, mainly neutrophils followed by monocytes/macrophages which will add more chemokines and cytokines to the tumor microenvirnoment. This is in turn responsible for an influx of various lymphocyte subsets, among which include CD4+ T cells, and a Th1 polarization of the response. The subsequent effective eradication of tumor cells is thought to depend mostly on cytolytic T cells and natural killer cells. However the precise mechanisms of killing are not known.
2 attraction of innate immune cells
and release of cytokines / chemokines
3 attraction and activation of T cells
Adapted from Albert, 2014

7. BCG adjuvant therapy remains the standard-of-care for high-risk superficial bladder cancer
Inner muscle layer
Lamina propria
Mucosa
Prostate gland
Urethra
Carcinoma in situ
Papillary tumor
Outer muscle layer
It is unclear why Morales chose to repeat instillations weekly when he established BCG therapy for bladder cancer (and Zbar used only a single injection of BCG in his preclinical model).
1 wk
BCG
surgery

8. Boosted cytokines after repeated instillations of BCG
surgery
BCG
week 4
week 6
week 1
Measurement of urine cytokines in patients treated with BCG reveal “boost” in immune responses evident from moelcuarl analyte profiling of urine samples.
Others have confirmed this phenomenon.

9. PRIME/BOOST Concept
Intradermal vaccine prior to intravesical instillation could improve resonse to intravesical BCG
There is strong rationale that pre-existing BCG-specific immunity will improve the anti-tumor response and an initial complete response is strongly associated with survival. Putting things into perspective. Currently a trial is being developed in Europe to evalaute this concept. However, A significant proportion of European patients are PPD negative, despite childhood vaccination So in Europe, they propose a boosting trial using Parenteral immunization of these patients prior to intravesical therapy to improve response to therapy.
In the U.S. We have a unique opportunity because the vast majority of our patietns are not immunized and therefore we can study the effects of priming.

10. MB49 animal model for BCG studies
High-resolution ultrasound:
Baseline
Week 1
BCG Instillation
Dwell time: 2hrs
BCG Dose:
~ 3 x 106 CFUs per mouse (2% of human dose)
Week 1
BCG
Week 2
Week 3

11. BCG s.c. 21 days prior to instillation
s.c. immunization prior to intravesical challenge results in rapid bladder T cell infiltration at 1st intravesical instillation
BCG s.c.
Repeated
Weeks 1 to 4 *
-21 7 14 21
33-35
Single
Week 1 *
-21
33-35
Single
Week 4 * 21
33-35
Instillation:
PBS
BCG
Week(s) of treatment:
W1-4 W1 W4 Ø
BCG s.c. 21 days prior to instillation
Biot et al. Science Transl Med. 2012

12. Intravesical PBS or BCG
Pre-existing BCG-specific immunity improves anti-tumor response in a mouse model for bladder cancer
80,000 MB49 (+ poly-L-lysin)
BCG s.c.
Intravesical PBS or BCG *
-19 2 9 16 23 30
days
BCG s.c.
Intravesical ttt + -
BCG (n=9)
BCG (n=8)
PBS (n=9)
PBS (n=8) **
Here they immunized the mice sc with BCG and 3 weeks later, tumor cells were implanted in the bladder. BCG therapy was initiated 2 days later. Strikingly 100% mice that received BCG sc prior to intravesical BCG therapy survived, while in comparison, mice treated with intravesical BCG alone succumbed within 50d (a few days later in average than PBS control group).
Biot et al. Science Transl Med. 2012

13. Pre-existing BCG-specific immunity improves anti-tumor response in patients
Patients with high-risk bladder tumor
PPD test
Clinical outcome?
Surgery
BCG therapy
PPD + (n=23)
PPD - (n=32) * +
Censored
Time until recurrence (months) 10 20 30 40 50 60 70 80 90
100
Recurrence-free survival (%)
Here a cohort of patient with high-risk bladder tumors that had undergone a PPD test before surgery and BCG therapy. A positive PPD test is the signature of previous exposure and active cellular immune response to mycobacteria (through a DTH reaction in which proteins purified from a mycobacterial culture are injected in the arm, after which the size of induraction at injection site is measured). When patients were stratified according to their PPD status prior to therapy (cutoff 10mm), it was observed that patients with a positive PPD test had a significantly better recurrence-free survival than patients negative for PPD test.
These patients were tested for their PPD reaction as a mean to exclude from therapy those patients who over-react to the test, therefore potentially allergic.
Biot et al. Science Transl Med. 2012

14. PRIME
SWOG Trial Schema

15. PRIME/BOOST PRIME U.S. PPD negative BOOST Europe PPD negative/positive
A significant proportion of European patients are PPD negative, despite childhood vaccination
Stratify – PPD expression
Putting things into perspective. significant proportion of European patients are PPD negative, despite childhood vaccination So in Europe, bParenteral immunization of these patients prior to intravesical therapy could boost response to therapy.

16. PRIME/BOOST
Trial Schema

17. Correlative Studies Impact of prime/boost on immune responses
Monitor blood and urine protein biomarkers using multiplexed immunoassays
Validate prior models and identify novel molecular determinates of BCG responsiveness
Systemic/local immune responses following therapy
Urine based Cell-lineage specific gene expression profiles
Tissue-based IHC
we are very excited about the opportunity for correlative studies within this trial. In particular the idea of predicting the response to BCG based on cell lineage gene expression patterns. In addition, Dr. Albert’s group proposes some very exciting assays for development of means to measure the response to BCG including BCG specific T-cells using point-of care tests.
Although BCG therapy has been applied for >30 years and represents one of the most effective immunotherapies, the exact mechanisms leading to tumor clearance are poorly understood. The trial will help to test our model for immune mediated tumor immunity – thus providing important fundamental insight into the mechanism of an effective form of tumor immunity; and our secondary objectives will help to establish novel methods for monitoring immune responses during immunotherapy protocols and identify correlates of clinical occurring immune escape:
Develop markers/tools predicting treatment outcome:
Ia: Monitor plasma and urine protein biomarkers using multiplexed immunoassays
Ib: Quantitate BCG-specific, common- and neo- tumor antigen-specific CD8+ T cell responses using multimer assays
II) Develop tools for the clinical correlation of cancer immune escape
a. genetic changes associated with recurrent tumors as compared to the primary tumor respecting intratumoral heterogeneity followed by next generation genomic analysis (array-CGH, NGS).

18. Conclusions PRIME / BOOST
BCG immunotherapy is effective but currently we have no reliable determinants of therapy response
Intradermal BCG vaccination – a cost-effective means to improve response?
Opportunity for correlative studies
PRIME / BOOST
A trans-atlantic collaboration studying T cell priming and boosting with BCG vaccination