Targeting of CCR8 induces antitumor activity as a monotherapy that is further enhanced in com- bination with a Listeria-based immunotherapy

Daniel O Villarreal, Susan R Armington, Andrew L’Huillier, Cristina Mottershead, Kimberly Ramos, Elena V Filippova, Dipti M Kelkar, Brandon D Coder, Robert G Petit, Michael F Princiotta Advaxis, Inc., Princeton, NJ USA

ABSTRACT RESULTS (cont.) RESULTS (cont.) CCR8 is a receptor that is expressed principally on regulatory T cells (Tregs) Blocking CCR8 Reduces Treg Conversion and Treg Function and known to be critical for Treg function, as CCR8+ Tregs can drive immunosuppression. CCR8 mAb Treatment Impairs Tumor Growth in Solid Tumor Models Recent studies have demonstrated that CCR8 is uniquely upregulated in human tumor- A A resident Tregs of breast, colon, and lung cancer patients compared to normal tissue- resident Tregs. Therefore, CCR8 tumor-resident Tregs are rational targets for cancer im- munotherapy. Here, we demonstrate that monoclonal antibody therapy targeting CCR8 significantly suppressed tumor growth and improved long-term survival in two different tumor-bearing mouse models. The antitumor activity could be correlated with an in- B crease in tumor-specific T cells, enhanced infiltration of CD4+ and CD8+ T cells, and a significant decrease in the frequency of intratumoral Tregs. Initial studies explored a 1 0 0 combinatorial regimen using anti-CCR8 mAb therapy and a Listeria-based cancer immu- B C

8 0

n

o i t * *

notherapy. Anti-CCR8 mAb therapy synergized with the Listeria-based immunotherapy a CT26

r 6 0

e

f

i l

o 4 0

to significantly delay tumor growth and induce complete regression in 20% of the mice. r

P

These results suggest that CCR8 represents a promising target for cancer immunothera- % 2 0

0 py, either as a single agent or in combination with other forms of immunotherapy. C D 8 C D 8 T r e g T r e g +  C C R 8 + C D 8 + C D 8 +  C C R 8 INTRODUCTION C D E + + C D 4 T C e ll D e a th C D 8 T C e ll D e a th • Tumor-infiltrating Foxp3 CD4 regulatory T cells (Tregs) are a major immune cell 6 0 n s

6 s

s

l

l l

l *

e

e

C

C

population that contribute to the establishment of an immunosuppressive TME. g

g

n

n i

i 4 4 0

y

y

D

D

r r

CCR8 is predominantly expressed on Tregs. o

o

d

MC38

d a

a 2 2 0

e e

+ D

D

• f

Tregs expressing CCR8, (CCR8 Tregs) are major drivers of immunosuppression, criti- f

o

o

% cal for Treg function and suppression. % 0 0  C C R 8 - +  C C R 8 - + C D 3 + + C D 3 + + • + high CCR8 has been shown to be a specific marker selectively upregulated by tumor- + high % A n n e x in -V a n d /o r V ia b ility D ye % A n n e x in -V a n d /o r V ia b ility D ye resident Tregs from several tumor types. Figure 2. CCR8 mAb therapy enhances antitumor efficacy. Tumor-bearing mice treated with aCCR8 (4ug) Figure 5. Blocking CCR8 reduces generation of Tregs, Treg function and enhances . (A) CCR8 + • Advaxis Lm platform can induce antigen-specific CD8 effector T cells in mice. decreased rates of tumor growth and improved long-term survival in two different solid tumor models. (A). blocking mAb (aCCR8) reduces generation of Tregs from Foxp3-CD4+ T cells; whereas, the isotype control (IgG) does not. Naïve CD4+ T cells (1x106) were cultured under specific conditions (including aCD28 and IL- • Lm–based immunotherapy has been shown, in preclinical cancer models and in the Schematic representation of the treatment regimen. (B) Tumor growth curve and survival of CT26 implanted + + mice. (C) Tumor growth curve and survival of MC38 implanted mice. 2) and generation of Tregs (frequency of Foxp3 CD4 T cells) was measured by flow cytometry. (B) αCCR8 clinic, to change the local TME. reduces the capacity of Tregs to suppress CD8+ proliferation. Culture induced Tregs were cocultured • These studies were designed to evaluate whether (1) CCR8 represents a promising CCR8 mAb Treatment Increases the Frequency and Functionality with purified CD8+ T cells (1:1 ratio) with or without aCCR8 (10 ug). CD8+ T cells were cultured for 3 d on new target to selectively reduce tumor-resident Tregs and if (2) aCCR8 mAb would of CD8+ T Cell Responses plates coated with αCD3 in the presence or absence of Tregs or αCCR8. Addition of αCCR8 strongly re- + synergize with an Lm-based immunotherapy to enhance antitumor activity and pro- versed Treg suppressive function. (C) Blocking CCR8 using an αCCR8 mAb does not affect CD4 T cell prolif- A B eration as determined by CFSE staining and flow cytometry. (D) The effects of 72 h treatment with αCCR8 on long survival in tumor-bearing mice. Treg apoptosis and (E) on CD8+ T cell apoptosis were determined by staining with Annexin V and viability MATERIALS AND METHODS dye using flow cytometry. *P<0.01; ***P<0.001; ****P<0.0001.

Tumor models, tumor vaccine, and treatments: CT26 (300,000) and MC38 (300,000) cells were im- Lm-Ah1 and CCR8 Combination Therapy Enhanced Antitumor Efficacy planted subcutaneously (s.c.) in the right flank of mice. For therapeutic treatments, mice were treated with intraperitoneal (i.p.) injections of anti-CCR8 (4ug; clone: SA214G2) or control antibody (rat IgG2b,k,

Clone LTF-2) as indicated. For combination studies, CT26 tumor-bearing mice were immunized i.v. twice 2 5 0 0 1 0 0 Ig G P B S /N a iv e ) * * *

8 C D 3 l on day +1 and +12 post-tumor implantation, with either Lm-AH1 (1 x 10 CFU), an Lm vector express- m

2 0 0 0 L m d d A -2 7 4 * a L m d d A -2 7 4 m

v 7 5 (

* * * * i

v e ing the AH1 antigen (gp70423-431, SPSYVYHQF) expressed on the CT26 murine tumor cell line that is rec-  C C R 8 r  C C R 8 1 5 0 0 u

m * * *

s

u t l L m -A H 1 5 0 L m -A H 1

+ 8 n o * *

ognized by CD8 T cells in the BALB/c mouse or LmddA-274 (1 x 10 CFU), an Lm vector expressing no e

V 1 0 0 0

L m -A H 1 +  C C R 8 c L m -A H 1 +  C C R 8

r

r

e o

tumor-specific antigen. P 2 5

m 5 0 0

u T

Flow analysis: Tumors were enzymatically dissociated into single cell suspensions using a Stomacher 0 0 0 10 13 17 20 24 27 0 2 0 4 0 6 0 8 0 machine (Steward) with Collagenase IV (Stem Cell Technologies). The resulting single-cell suspensions D a y D a y s a fte r C T 2 6 im p la n ta tio n were immunophenotyped with the following antibodies using standard staining procedures: anti-CD45, anti-CD4, anti-CD8, anti-CCR8 (clone SA214G2; Biolegend), anti-CCR8 (GeneTex), anti-CD44, anti- Figure 6. CCR8 mAb synergizes with a Listeria-based therapy to enhance therapeutic outcome. Naïve CD25, anti-CD107a, anti-CTLA4, anti-PD-1, anti-TCR, MHC class I peptide AH1 Tetramer, anti-IFNγ, E  mice (n=10 per group) were implanted with CT26 tumor cells (300,000) on the right flank. Combination ther- anti-TNFα, annexin V, and Invitrogen LIVE/Dead fixable Violet Fluorescent Reactive Dye. For IFN stain- apy was given sequentially. On day 4 post-inoculation, mice were treated with CCR8 (administered for ing, cells were stimulated with either SIINFEKL peptide, AH1 peptide (gp70 , SPSYVYHQF), and/or 423-431 eight consecutive days) and on day 11 post-inoculation mice were treated with Lm-AH1, alone or in combina- cell stimulation cocktail plus transport inhibitors (Invitrogen.). Events were acquired using the At- tion with CCR8, as indicated. tune flow cytometer (Fisher Scientific) and analyzed using FlowJo software (Tree Star). Tumor infiltrating lymphocytes (TILs) were defined as CD45+ cells. Combination Lm-AH1/αCCR8 Therapy Increases CD8+ T cell Responses in the TME In vitro assays: For Treg induction, 2 x 106 CD4+ T cells were isolated from spleens of BALB/c mice through negative selection (StemCell). The cells were seeded on plates pretreated with 2 ug/ml anti- A B CD3. Cells were incubated for 3 days with aCD28 (1ug/mL), IL-2 (100U/mL), and TGF-1 at 5ng/ml. 6 0 5 0 4 0 * * * * * *

+ * n s o Figure 3. CCR8 mAb treatment increases the frequency of CT26 antigen-specific CD8 T cells. Spleens )

4 0 i *

t s

The percent of converted Tregs was evaluated by flow cytometry on the third day. For inhibition of con- s 3 0 a

L *

r

I

L

4 0 * I

T

g T ( 3 0 * 6

and TILs from tumor-bearing CT26 mice were harvested 17 days after tumor implantation. (A) The frequency * e

+

5 r version, aCCR8 was added at 10 ug/ml. To measure suppression, 1 x 10 5 day culturally induced 8

2 0

4

T

D

/

D

C 2 0 * *

6 C + 6 * 8

of AH1-specific IFNγ (spot forming cells/10 splenocytes) responses determined by IFNγ ELISpot assay in re- 2 0 * % Tregs were incubated with freshly isolated CD8 T cells (2 x 10 ) in a 1:1 ratio, on plates pretreated D

% 1 0

1 0 C with 2 ug/ml aCD3. The Fresh isolated CD8+ T cells were stained with 1uM CFSE for 10 minutes, sponse to stimulation with CT26 AH1 peptide (SPSYVYHQF). (B-C) Bar scatter plot graphs show the percent- * 0 0 0

1 4 8 1 8 4 8 8 4 8 1 8 G 7 G 7 H G 7 R H R R R R H R + g 2 Ig -2 A g 2 quenched with R10 medium and washed prior to being plated. Co-cultured cells were incubated for 3 ages of AH1 peptide tetramer-positive CD8 T cells in the spleens and in the tumors of tumor-bearing mice. (D) I - C -A C C - C I - C -A C A A A C C C m C C C d  m  d   d  m  d L + d L + d L + m 1 m 1 m 1 L H L H L H days with aCD28 (1ug/mL), IL-2 (100U/mL) and indicated samples were treated with 10ug of aCCR8. + A A A Column graphs showing intracellular cytokine staining for IFNγ in CD8 TILs following AH1 peptide stimulation - - - m m m L L L or (E) PMA/ION stimulation. 5 0 C 8 0 * * * *

RESULTS s

* * *

L s

* * I 4 0

L T

I 6 0

T

+

8 +

8 3 0

CCR8 mAb Treatment Alters the TME in the CT26 Tumor Model D

D

C +

C 4 0 +

 2 0 F

 * * *

N

N

F T

I * *

CCR8 is Highly Expressed by Tumor-resident Tregs 2 0 1 0 % A B % 0 0 4 1 4 1 G 7 8 8 G 7 8 8 R H R R H R Ig -2 A Ig -2 A C - C C - C A C C A C C d m d m  L +   L +  d d 1 1 m m L H L H -A -A m m L L

1 0 0 Figure 7. Lm-AH1 and αCCR8 combination therapy increases effector CD8+ TIL numbers. (A) TILs (n = 4-5 + 8 * * * * + R 8 0 per group) were harvested at day +22, followed by analysis of frequency of CD45 leukocyte infiltrate and C C D + + + + + +

C CD8 as percentage of total CD45 cells. (B) The ratio of CD8 effector T cells to Tregs (CD4 Foxp3 CD25 ) +

3 6 0

p in the tumors. (C) IFNγ and TNFα expression by CD8+ TILs following peptide incubation with PMA/ION stimu-

x o

F 4 0 lation. Error bars indicate SEM. *P<0.01; ***P<0.001; ****P<0.0001.

+

4 D

C 2 0

SUMMARY % 0 S p le e n T u m o r • CCR8 mAb therapy enhanced antitumor efficacy and improved long-term survival.

• CCR8 mAb increased tumor-specific T cells and significantly decreased tumor-resident CCR8+ Tregs.

• CCR8 blocking mAb prevented generation and suppressive function of Tregs. Figure 4. CCR8 mAb treatment alters the TME. TILs (n = 5-6 per group) from tumors mice were harvested 17 days after CT26 tumor implantation. (A) CD45+ leukocyte infiltrate and CD8+ T cells as a percentage of • Lm-AH1 and αCCR8 combination therapy enhanced antitumor efficacy and prolonged survival relative to Figure 1. CT26 tumor-infiltrating Tregs exhibit high expression of CCR8. Cells from spleens and tu- total CD45+ cells are shown in treated versus untreated groups. (B) Frequency of CTLA-4 and PD-1 expres- Lm-AH1 or αCCR8 alone. mors of CT26 mice were harvested 17 days after tumor implantation. Representative scatter plots and + + + + sion on tumor infiltrating CD8 T cells (C) Tumor-resident CD4 Tregs (Foxp3 CD25 ) and the ratio of CD8/ • Lm-AH1 and αCCR8 combination therapy increased CD8+ T cell responses and induced changes in the im- graphs show frequency of CCR8 expression by Foxp3+CD4+ Tregs. Tregs in the tumor. (D) CCR8 expression by tumor-resident Tregs (Foxp3+CD4+). *P<0.05; **P<0.01. mune effectors in the TME.