Immunologic Targeting - How to Channel a Minimal Response for Maximal Outcome

1. Immunologic Targeting - How to Channel a Minimal Response for Maximal Outcome Susan Slovin, MD, PhD Genitourinary Oncology Service December 1, 2005

2. Have we succeeded or failed in our treatments for prostate cancer? SuccessFailure Docetaxel – 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 measures toxicity profile

3. PRO Over-expression and undergycosylation of cell surface molecules Can target receptor-like molecules which can stimulate intracellular signaling pathways. Can prevent target activation. CON Strictly extracellular, no contact with intracellular pathways. Expression varies. Cell can develop “collateral” signaling/survival pathways. Cell can overcome via multiple mechanisms rendering tx inadequate. RATIONALE FOR TARGETED INTERVENTION

5. 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 TARGETS UNDER EXPLORATION

6. 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

10. 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

12. RCC  Bone (10mg)

13. How to Target PSMA? Vaccines Others: • Naked DNA Radionuclide emitter + MoAb (ext domain) • rsPSMA MoAb + maytansinoid • Alphavirus vector Neovasculature?

14. 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

15. 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.

16. Plasmid DNA expression vector used in MSKCC PSMA DNA vaccines

17. MSKCC 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%)

18. Cross-over to break tolerance Cross-over design of clinical trial Human PSMA Human PSMA Mouse PSMA Mouse PSMA

19. 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

21. How to Break Immune Tolerance to PSMA? ActivePassive Vaccines 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)

22. B7x BTLA YYY B7H3 SHP-1 SHP-2 B7h ICOS YY PD-L1 YY PD-1 PD-L2 CD28/B7 superfamily and the paradigm of professional costimulation T cell APC/DC Activation CD28 YYYY B7.1 Induced by Inflammation/ Pathogens Inhibition CTLA-4 YY B7.2 IgC domain IgV domain Y Tyrosine

23. TCR CD28 ~ Antigen B7 MHC TCR CTLA-4 CD28 ~ Antigen B7 MHC TCR CTLA-4 CD28 ~ Antigen B7 MHC Regulation of T cell activation • CTLA-4 : B7 suppression • Termination of response Antigen-specific T cell Activation • TCR : Antigen MHC • CD28 : B7 Co-stimulation Activated T cell • IL-2 secretion • Proliferation • Effector function • Induction of CTLA-4

24. TCR TCR CD28 CTLA-4 CD28 ~ Antigen ~ Antigen B7 MHC B7 MHC CTLA-4 Blockade Augments Antigen Specific T-cell Responses Anti-CTLA-4 mAb

25. 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

26. 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

27. Anti-Murine CTLA-4 mAb Cures Prostate Cancer in Mice Control Ab Anti-CTLA-4 300 Source: Kwon et al. PNAS. 1997(94): 8099 Tumor size (mm 2) (100 ug Ab at days 7, 10, & 13) 0 10 30 50 70 90 Days post tumor injection

28. α-CTLA-4 α-CTLA-4 GVAX

29. Pathology of Autoimmune Breakthrough Events: Dermatitis B A 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

30. Pathology of Autoimmune Breakthrough Events: Colitis D C 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). CD3 E F CD4 CD8 Source: Abstract #3424, ASCO 2003