An approach to discovering new immunotherapy targets for oncology is introduced and examples presented. New programs from biotech and pharma are discussed.

1. Paul D Rennert, Founder & Principal
SugarCone Biotech LLC
Exploring evolution of diverse immune
checkpoint pathways
ImVacs, August 11-12, 2014

2. www.sugarconebiotech.com
How can we discover novel immune
checkpoints?
• Focus on specific gene familes that we know
contribute to immune checkpoint modulation
• Query these target collections using an evolutionary
perspective, to derive specific new therapeutic
hypotheses
• Why?
Ad hoc sampling of such families, especially the
large ones, is time-consuming and expensive
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3. www.sugarconebiotech.com
This is what we usually
see ... and it’s useful
Drew M Pardoll, 2012. Nat Rev Cancer
• e.g. we know that CTLA4, an Ig-
superfamily member, is a critical
immune checkpoint target
• We’ve found out through the very hard
work of many labs that PD-1 is another
critical immune checkpoint target
• But, can we use this level of information
to guide our investigation of other Ig-
superfamily members?

4. Using clustering to inform hypotheses
• The prior figure is an example of functional clustering and can only inform us
about members whose function has been elucidated
• Highly refined distance methods are of interest, and by design use distance
cutoff algorithms to unlink related families
Yap et al. 2013 JMB 4
Butyrophilins Siglecs
B7s PD-Ls
TIMs

5. www.sugarconebiotech.com
What is this showing us?
• Relatedness is a term of convenience/relativity
• We “look” for relatedness to CD28 and B7s when we identify
novel proteins
! PD-1 is cited as CD28/CTLA4 relative: PD-1 binds PD-L1 and 2 and
B7-1; the receptor has inhibitory activity
! In strict distance analyses, PD-1 is a singleton, with distant homology
to CD8 and the antigen receptor Ig domains
! ICOS can be aligned with CD28 and CTLA4 using pairwise
comparison methods
• ICOS-L, B7-H3 and B7-H4 align more closely with
butyrophilins than B7s
• VISTA is a weak PD-L1 homologue, structurally unique
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6. www.sugarconebiotech.com
Where do B7-1 & 2 and CD28 lead us?
ICOS! CTLA4!CD28!
PD-1!
VISTA!
????!
?"
?"
B7-H3 !
B7-H4 ! ICOS-L !
?"
????!
?"
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7. www.sugarconebiotech.com
We have a big butyrophilin/B7 family
B7-H3 !
B7-H4 ! ICOS-L !
We have a small CD28 receptor family
ICOS! CTLA4!CD28!
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8. www.sugarconebiotech.com
Interesting New Directions
• Focus on new targets
! VISTA, B7-H3, B7-H4
• Novel families
! Butyrophilins are likely underexploited targets
• Different cell types
! NK cells, NKT cells, γ/δ T cells
• An insight:
! There is no a priori reason to anticipate high homology
when looking for the receptors/ligands of orphan
proteins (e.g. IgSF BTLA binds TNFRSF HVEM)
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9. www.sugarconebiotech.com
B7-H3 and B7-H4
B7-H3
• Biology poorly understood; low expression in normal tissues
• Macrogenics (MGNX) and Servier are co-developing MGA271, a
humanized, Fc-optimized mAb that is in a Phase 1 trial, recruiting
refractory prostate cancer and melanoma patients based on abundant
expression of the target on these tumors
B7-H4
• Biology poorly understood, marks diverse normal and cancer cell types
• Well-established biomarker of tumor progression
• B7-H4 is expressed on infiltrating myeloid cells, vasculature, and tumor
cells in melanoma, NSCLC, prostate, ovarian, pancreatic, breast, renal
and other cancers
• Amplimmune (now owned by the Medimmune division of Astra Zeneca;
AZN) had listed B7-H4 in its’ portfolio
• Five Prime Therapeutics (FPRX) also has promoted this target
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10. www.sugarconebiotech.com
VISTA
• Normally expressed on hematopoietic cells including myeloid
cells and T cells
• In multiple cancer models VISTA is found at particularly high
levels on tumor-infiltrating myeloid cells
• Appears to be a negative regulator of T cell responses, may
suppress myeloid cell responses in the tumor microenvironment
• Anti-VISTA antibody treatment blunts tumor development even in
the presence of high PD-1 expression
• Antibodies to VISTA are being developed by a private company,
ImmuNext, in collaboration with Johnson&Johnson (JNJ)
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11. www.sugarconebiotech.com
New “B7s”
• you can always force a
phylogeny by limiting
the input sequences,
as shown here
• utility: can highlight
relationships of
suspected novel family
members
• B7H6, tumor-specific
in human?, binds
NKp30, function poorly
understood, an outlier
• B7H7/HHLA2 – likely
related to butyrophilins
PD-L2
PD-L1
B7-H7
B7-H4
B7-H3
ICOS-L
B7-1
B7-2
VISTA
B7-H6
Flajnick et al. 2012. immunogenetics

12. The “true” Butyrophilins
• The failure to
identify clear
ligand/receptor
interactions has
limited drug
development
• A reasonable
prediction is that
further study of
the butyrophilin
family will yield
novel targets
Arnett & Viney. 2014.
Nat Rev Immunol.

13. Underexploited Ig-families
• Precedence here may come from the distinct but instructive LIR/KIR and NKG2
families of NK cell surface proteins
• Despite a huge complexity of family members and confusing biology, useful
targets are emerging, notably from Innate Pharma (IPH)
• Bristol-Myers Squibb (BMY) is sponsoring trials of lirilumab in combination with
ipilimumab (anti-CTLA4) and nivolumab (anti-PD-1) in patients with solid tumors.
These trials will start to read out over the next 2 years.
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14. www.sugarconebiotech.com
Lets try this again: TIM family proteins
• TIM-1 identified as a critical T cell control protein (2001)
• Member of a small subfamily of receptors featuring IgV
domains atop mucin domains and short stalks
! Human proteins are TIM-1, TIM-3, TIM-4
• We knew as early as 2002 that these were Siglec
homologues
! TIM-1 cell binding was cation-sensitive suggesting a carbohydrate
component but a screen by he Consortium for Functional
Glycomics failed
! Based on publications we chased semaphorins, CD300b, others
! Galectin-1,8 identified as binding proteins, unclear specificity?
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15. www.sugarconebiotech.com
TIM family proteins: nearest neighbors
• Extensive homology modeling places TIM-1 between
Siglecs and Butyrophilins
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Rubenstein et al. 2013 Structure.

16. TIM-1 relationship to Siglecs
• TIM-1 IgV domain resembles a Siglec with a modified sialic acid
binding motif
• Minimal energy modeling suggest carbohydrate docks as in a typical
Siglec
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*: Miyanishi et al. Nature 450: 435; Kobayashi et al. Immunity 27: 927; Santiago
et al. Immunity 27: 941; Ichimura et al. 2008. J. Clin. Immunol. 118: 1657.
• TIM-1, TIM-4 and TIM-3 bind PS
• PS binding site: a cation buried in a charged
cavity
• The sialic-acid binding residues lie atop the
PS-binding site.
The real key to unlocking TIM-family function
was phosphotydylserine (PS)

18. www.sugarconebiotech.com
• A reasonable hypothesis is that TIM-3 antagonism is
efficacious in preclinical tumor models because it prevents
T-cells from receiving inhibitory signals delivered by PS
expressed by tumor cells and tumor associated
macrophages (MDSC)
! Whether the putative TIM-3 ligand Galectin-9 also plays a role is
unclear
• Novartis (NVS)/CoStim, Bristol-Myers Squibb (BMS),
Tesaro (TSRO)/Anaptsys all have active programs
targeting TIM-3, with many others coming along ... why?
PS, tumors and TIM-3
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19. www.sugarconebiotech.com
TIM-3 preclinical data are compelling
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20. www.sugarconebiotech.com
Not so TIM-1: our evolving view of TIM-1
expression
• Focal, bright expression on mucosal endothelium
• Expressed on kidney epithelium (lumenal), and highly
expressed in the injured kidney
• Highly (drastically) upregulated not only on kidney and
ovarian carcinoma but also non-cancerous kidney and liver
cells after chemo/radiation
• It’s reasonable to predict that the anti-TIM-1-ADC being
developed by Celldex’ (CLDX) will have a difficult time
finding a safe therapeutic window in the context of standard
of care
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21. www.sugarconebiotech.com
Other programs targeting cell death pathways
• Cell surface expression of PS is triggered by apoptosis; the
appearance of sufficient PS on the cell surface triggers
phagocytosis
• However, PS expression is quite widespread even on
normal cells
• SIRPα/CD47 is an explicitly anti-phagocytic signaling
mechanism
• However, CD47 is widely expressed, and is abundantly
expressed on platelets
• Both targets seem problematic, however...
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22. www.sugarconebiotech.com
Other programs targeting apoptotic signaling
These independent lines of evidence support the
hypothesis that TIM-3 is acting in the context of PS-binding
• Peregrine Pharmaceutical’s (PPHM) anti-PS antibody bavituximab has
advanced to Phase 3 with fast-track desination in r/r solid tumors
including NSCLC (+ platinum-based chemo)
• In Phase 2, the ORR was only 17%, those responding had a median
increase in OS of just over 4 months. It is predictable that this program
will seek rational combination therapy trials.
• Celgene (CELG) handed privately held Inhibrx LLC a 50MM USD
upfront with potential royalties running to 500MM for an anti-CD47
antibody specifically designed not to activate platelets
• Additional CD47 and SIRPα programs are under development
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23. www.sugarconebiotech.com
• uh-oh...
One more example...
• shown are two proteins that
been investigated in the
context of immune oncology:
• TIGIT
• CD200
• The fact that these cluster in
the complex Nectin family is a
little worrisome: these family
members homo and hetero-
dimerize fairly promiscuously,
and this may complicate
analysis and use of biologics
targeting these proteins
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24. www.sugarconebiotech.com
• CD200 was being targeted by an antibody ALXN6000/
samalizumab (Alexion, ALXN) although there are no recent
clinical updates - the company abandoned oncology for rare
diseases
• TIGIT is a relatively new IgSF protein, with clear cell inhibitory
function. There is defined binding to PVR and Nectin-2, potentially
leading the way to influencing both T cells and NK cells through
inhibition of this pathway
• However, proteins in the nectin family may have diverse binding
partners, complicating development, e.g. the TIGIT ligands may
also bind DNAM
Targeting “Nectins”?
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25. Finally ... such analyses go only so far
LAG3
BTLA
FLT3
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26. www.sugarconebiotech.com
other applications ...
• this type of analysis can be productively applied to
the TNF and TNFR superfamilies ... which are much
smaller
• allowing us to look for interesting and distinct
pathways ...
• recent work has highlighted TNFRSF25 as a critical
node in the immune response to tumors as you will
hear more about from Taylor Schreiber
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27. www.sugarconebiotech.com
Acknowledgements
Slides will be available on SLIDESHARE
Link through the sugarconebiotech.com blog
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• TIM-1 program: BiogenIdec
• TIM-1 oncology: CoStim
• TIM-1 homology and binding specificity: Alexey Lugovskoy (Merrimack), Yen-
Ming Hsu (AbBio), Veronique Bailly (BIIB), Patricia McCoon (AZN), Gabriela
Constantin (U Verona, IT)
• TIM-1 virology:
! Ebola, Marburg: Wendy Maury (U Iowa), Rob Davies (Tx Biomed)
! HIV: Shan-Lu Liu (U Missouri)