a novel gucy2c-cd3 t cell engaging bispecific construct ... · 1/29/2020 · 1 a novel gucy2c-cd3...

A Novel GUCY2C-CD3 T cell Engaging Bispecific construct (PF-07062119) for the 1

Treatment of Gastrointestinal Cancers 2

Authors and Affiliations: 3

Divya Mathur1*, Adam Root

2, Bozena Bugaj-Gaweda

1, Stephanie Bisulco

1, Xingzhi Tan

1, Wei 4

Fang1, Jessica Kearney

1, Justin Lucas

1†, Magali Guffroy

1‡, Jonathan Golas

1, Cynthia M. Rohde

1, 5

Chad R. Stevens3, Cris Kamperschroer

4, Kerry Kelleher

2, Rosemary Lawrence-Henderson

3, Erik 6

Upeslacis1, Johnny Yao

1, Jatin Narula

2, Edward R. Lavallie

2, Diane R. Fernandez

5, Bernard S. 7

Buetow5, Edward Rosfjord

1, Laird Bloom

2, Lindsay King

3, Lioudmila Tchistiakova

2††, Anhco 8

Nguyen1‡‡, Puja Sapra

1* 9

10

1Pfizer WorldWide Research and Development (R&D), 401 N. Middletown Road, Pearl River 11

NY 10965, USA 12

2Pfizer WorldWide R&D, 610 Main Street, Cambridge MA 02139, USA 13

3Pfizer WorldWide R&D, 1 Burtt Road, Andover, MA 01810, USA 14

4Pfizer WorldWide R&D, Eastern Point Road, Groton, CT 06340 USA 15

5Pfizer WorldWide R&D, 10777 Science Center Drive, La Jolla CA 92121 USA 16

†Current address: Bristol Myers Squibb, Princeton NJ 17

‡Current address: Abbvie, North Chicago, IL 18

††Current address: Third Rock Ventures, Boston, MA 19

‡‡Current address: Fate Therapeutics, San Diego, CA 20

*To whom correspondence should be addressed 21

22

Correspondence: 23

Correspondence to be addressed to Divya Mathur ([email protected], Phone : (845)602-24

5211) and Puja Sapra ([email protected], (845)602-3389). Address: Pfizer Inc., 401 N. 25

Middletown Road, 200-4502, Pearl River NY 10965, USA 26

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Running Title: A GUCY2C-CD3 bispecific targets gastrointestinal cancers. 28

Conflicts of interest: All authors were employees and/or shareholders of Pfizer Inc., a publicly 29

traded company. Pfizer has filed patent applications on aspects of this work 30

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Research. on October 12, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 29, 2020; DOI: 10.1158/1078-0432.CCR-19-3275

mailto:[email protected]


http://clincancerres.aacrjournals.org/

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TRANSLATIONAL RELEVANCE 1

Colorectal cancer (CRC) is an area of high unmet need and a leading cause of cancer related 2

deaths worldwide. The majority of CRC is microsatellite stable (MSS), frequently with mutated 3

KRAS or BRAF oncogenes, resulting in poor prognoses, and shows a lack of response to 4

currently approved immunotherapy. T-cell engaging CD3 bispecific antibodies hold potential as 5

potent therapeutics against solid tumors. Here we demonstrate T-cell mediated anti-tumor 6

activity with PF-07062119, a novel CD3 bispecific against tumors expressing Guanylyl Cyclase 7

C (GUCY2C), a target expressed widely across CRC and other gastrointestinal tumors. PF-8

07062119 shows efficacy in multiple CRC models independent of their KRAS or BRAF 9

mutational status, since bispecific activity is dependent on GUCY2C expression. We also show 10

significant combination benefits with checkpoint blockade, and anti-angiogenesis therapy. PF-11

07062119 has a well-tolerated toxicity profile in cynomolgus monkeys. Accordingly, PF-12

07062119 warrants further clinical investigation for the treatment of CRC and other 13

gastrointestinal cancers. 14

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3

ABSTRACT 1

Purpose: Gastrointestinal cancers remain areas of high unmet need despite advances in targeted- 2

and imumunotherapies. Here we demonstrate potent, tumor selective efficacy with PF-07062119, 3

a T-cell engaging CD3-bispecific targeting tumors expressing GUCY2C, which is expressed 4

widely across CRC and other gastrointestinal malignancies. Additionally, to address immune 5

evasion mechanisms, we explore combinations with immune checkpoint blockade agents, and 6

with anti-angiogenesis therapy. 7

Experimental Design: PF-07062119 activity was evaluated in vitro in multiple tumor cell lines, 8

and in vivo in established subcutaneous and orthotopic human CRC xenograft tumors with 9

adoptive transfer of human T cells. Efficacy was also evaluated in mouse syngeneic tumors using 10

human CD3ε transgenic mice. Immunohistochemistry and mass cytometry were performed to 11

demonstrate drug biodistribution, recruitment of activated T cells, and to identify markers of 12

immune evasion. Combination studies were performed with anti-PD-1/PD-L1 and anti-VEGF 13

antibodies. Toxicity and pharmacokinetic studies were done in cynomolgus macaque. 14

Results: We demonstrate that GUCY2C-positive tumors can be targeted with an anti-15

GUCY2C/anti-CD3ε bispecific, with selective drug biodistribution to tumors. PF-07062119 16

showed potent T-cell mediated in vitro activity and in vivo efficacy in multiple CRC human 17

xenograft tumor models, including KRAS and BRAF mutant tumors, as well as in the 18

immunocompetent mouse syngeneic tumor model. PF-07062119 activity was further enhanced 19

when combined with anti-PD-1/PD-L-1 treatment or in combination with anti-angiogenic 20

therapy. Toxicity studies in cynomolgus indicated a monitorable and manageable toxicity profile. 21

Conclusion: These data highlight the potential for PF-07062119 to demonstrate efficacy and 22

improve patient outcomes in CRC and other gastrointestinal malignancies. 23




4

INTRODUCTION 1

Gastrointestinal malignancies, including colorectal cancer (CRC), gastric cancer and esophageal 2

cancer continue to be areas of high unmet medical need despite advances in targeted therapies 3

(1,2). CRC alone is a worldwide issue affecting both men and women, and responsible for 9.2% 4

of all cancer deaths. The lack of response to targeted therapy, such as anti-EGFR antibodies, has 5

been correlated with mutations in the KRAS and BRAF oncogenes (3-6). Additionally, 6

immunotherapies, such as immune checkpoint inhibitors, have failed to show significant survival 7

benefit in most CRC patients, owing to low tumor mutational burden and reduced density of 8

immune infiltration (7,8). 9

10

The potential of redirected T cell therapies has been demonstrated by the approval of 11

blinatumomab in hematological malignancies, and more recently by reports of early clinical 12

activity with CD3 bispecific antibodies targeting solid tumors, such as colorectal and prostate 13

cancers (8,9). CD3 bispecifics hold potential as potent cancer therapeutics as they recruit and 14

activate a broad repertoire of T cells against tumor cells expressing a tumor associated cell 15

surface antigen (10). They circumvent the need for T cell receptor (TCR) engagement with MHC 16

Class I in complex with antigenic peptide, and instead recruit T cells to target cells expressing 17

cell surface antigen. One arm of the bispecific binds to a tumor associated cell surface antigen, 18

and the other arm binds to the CD3ε protein on T cells, leading to a cytotoxic T lymphocyte 19

(CTL) response against tumor cells. 20

21




5

Given the high potency of CD3 bispecifics, it is important to demonstrate tumor selective 1

activity with an antigen that has minimal or restricted expression in normal tissues. Guanylyl 2

Cyclase C (GUCY2C) is expressed across gastrointestinal malignancies including more than 3

90% of CRC across all stages, and more than 50% of gastric or gastroesophageal junction cancer 4

(11-13). GUCY2C is normally involved in maintaining intestinal homeostasis (14), and is 5

activated by the peptide hormones guanylin and uroguanylin (15,16), as well as by the E. coli 6

heat stable (ST) enterotoxin, which causes familial diarrhea (17,18). GUCY2C expression in 7

normal tissues is largely restricted to the apical side of intestinal epithelial tight junctions (19). 8

Since tumors have disrupted tight junction architecture (20,21), this may allow for preferential 9

uptake of GUCY2C targeted biologics, thereby making it an attractive antigen for targeted 10

therapy approaches. 11

12

Here we show that an anti-GUCY2C/anti-CD3ε bispecific has preferential uptake in tumors 13

versus normal tissues in mouse tumor models of CRC, leading to anti-tumor efficacy in the 14

absence of toxicity to target expressing tissues. The therapeutic candidate, PF-07062119 15

demonstrates potent T cell dependent efficacy in vitro, as well as in vivo in several GUCY2C 16

expressing human cell line- and patient-derived xenograft tumor models, a colon orthotopic 17

xenograft model, as well as in a mouse syngeneic tumor model. Furthermore, the potential 18

benefits of combining PF-07062119 therapy with immune checkpoint inhibitors, as well as anti-19

VEGF therapy are described. An exploratory toxicity study in cynomolgus monkeys showed T 20

cell activation and elevations in cytokines, and in-life findings that were manageable with oral 21

and/or subcutaneous fluids. No erosions, ulcerations, evidence of overt necrosis or loss of 22

overlying epithelium were observed in the intestinal tract, where GUCY2C is expressed. These 23




6

studies collectively provide rationale for clinical evaluation of PF-07062119 for the treatment of 1

gastrointestinal cancers. 2

3

MATERIALS AND METHODS 4

Generation of anti-GUCY2C antibodies 5

Anti-GUCY2C antibodies were generated using hybridoma technology. Mouse lymphoma 6

300.19 cells over-expressing human GUCY2C (300.19/huGUCY2C) were injected as 7

immunogens into eight-week old female Balb/c mice for the generation of hybridomas. Balb/c 8

mice were immunized with 5 x 106 300.19/huGUCY2C cells twice per week for one month 9

without adjuvant through intraperitoneal (IP) injection. Antibodies derived from mouse 10

immunizations were humanized using conventional grafting methods (22). 11

Characterization of KRAS and BRAF mutational status of Colorectal Cancer CLX and 12

PDX models 13

Mutational status of KRAS and BRAF oncogenes was determined for cell line xenograft (CLX) 14

and patient-derived xenograft (PDX) models used for evaluating anti-tumor activity of PF-15

07062119. The OASIS 3.0 database was queried for all cell lines and PDX models, and KRAS or 16

BRAF mutations in these models were identified (23). KRAS and BRAF mutational status in 17

LS174T has been described earlier (24,25) 18

PBMC Collection and Isolation of Human T cells for in vitro and in vivo studies 19

Whole blood was collected from healthy donors and immediately treated with the anti-coagulant 20

10 mM Ethylenediaminetetraacetic acid (EDTA). All peripheral blood mononuclear cell 21

(PBMC) and T cell isolations were carried out at room temperature. Each 25 mL of blood was 22

mixed with 10 mL of DPBS containing 2 mM EDTA and layered above the frit of a 50 mL 23




7

Accuspin tube pre-loaded with 15 mLs of Histopaque-1077 density gradient media. Tubes were 1

spun at 800 x g for 20 minutes, and the material above the frit was decanted into a fresh 50 ml 2

conical tube and spun again at 800 x g for 10 minutes. Following centrifugation, the supernatant 3

was discarded, and the pellet was resuspended in DPBS containing 2 mM EDTA. PBMCs were 4

spun at 200 x g for 10 minutes, the supernatant was discarded, and the cells were resuspended to 5

5 x107 cells/mL in Robosep buffer. T cells were isolated using the EasySep human T cell 6

enrichment kit (Stem Cell Technologies) according to the manufacturer’s protocol. T cells were 7

used at this stage for in vitro experiments or expanded and activated further for in vivo studies. T 8

cells were activated using a Human T cell activation/ Expansion kit (Miltenyi) following the 9

manufacturer’s protocol using T cell media containing X-Vivo 15 with 5% human serum AB, 10

1% Penn/Strep, 0.01 mM 2-mercaptoethanol. After 48 hours of T cell activation, T cells were 11

transferred to a G-Rex cell culture device (Wilson Wolf) for expansion, and human IL-2 12

(Shenandoah Biotechnology) was added to the media at a final concentration of 5 ng/mL and 13

replenished after 2 days. T cells were harvested 5 days after expansion. At the time of harvest, 14

beads were removed with a magnet, and cells were resuspended in DPBS at 1x107 cells/mL for 15

in vivo adoptive transfer studies. 16

Immunohistochemistry 17

Tumor samples were fixed in 10% neutral buffered formalin for 48 to 96 h, trimmed, and 18

processed or dehydrated overnight through a series of xylene, graded alcohols (100%, 95%, 70% 19

reagent alcohol), and paraffin. Samples were then embedded in paraffin and cooled prior to being 20

sectioned on a microtome at 5 µm. Sections were floated in deionized water and placed on glass 21

slides and allowed to dry. Slides were placed in a baking oven at 60°C for approximately 30 22




8

minutes, then deparaffinized in xylene and rehydrated through a graded series of alcohols to 1

deionized water. 2

3

For Hematoxylin and Eosin (H & E) staining, sections were placed in CAT hematoxylin 4

(Biocare Medical) for 1 minute followed by rinsing in tap water to blue. Sections were 5

differentiated with 80% reagent alcohol, placed in Edgar Degas Eosin (Biocare Medical) for 90 6

seconds, dehydrated in 95% then 100% reagent alcohol, and cleared in xylene. Slides were 7

coverslipped using Permount medium. 8

9

For bispecific biodistribution staining, following euthanasia and prior to tissue collection, 10

animals underwent cardiac perfusion with saline to remove systemic therapeutic, which might 11

interfere with IHC detection. To immunohistochemically detect the bispecific molecule, sections 12

underwent heat-induced epitope retrieval in Borg Decloaker (Biocare Medical) for 30 minutes, 13

followed by peroxidase block in Peroxidazed 1 (Biocare Medical) for 10 minutes, and protein 14

block with Background Punisher (Biocare Medical) for 10 minutes. Primary antibody rabbit 15

mAb anti-Human IgG (EPR4421, Abcam ab109489) was applied at 1:200 (0.41 µg/ml) for 1 16

hour followed by MACH2 Rabbit HRP-Polymer (Biocare Medical) for 30 minutes. Color was 17

developed using Betazoid DAB (Biocare Medical) chromogen for 5 minutes. After a rinse in 18

water, the sections were counterstained for 30 seconds in Tacha’s Hematoxylin (Biocare 19

Medical), dehydrated in 100% reagent alcohol, and cleared in xylene. Slides were coverslipped 20

using Permount medium. 21

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9

For GUCY2C staining, sections underwent heat-induced epitope retrieval in Borg Decloaker for 1

30 minutes, followed by peroxidase block in Peroxidazed 1 for 10 minutes, and protein block 2

with Background Punisher for 10 minutes. The primary antibody anti-GUCY2c (9H3-rabbit 3

IgG) was applied at 2 µg/mL for 60 minutes followed by the labeled polymer MACH2 Rabbit 4

HRP-Polymer for 30 minutes. Color was developed using Betazoid DAB chromogen for 5 5

minutes. After a rinse in water the sections were counterstained for 10 seconds in Tacha’s 6

hematoxylin, dehydrated in 100% reagent alcohol, and cleared in xylene. Slides were 7

coverslipped using Permount medium and examined under a microscope for Histo score (H-8

score) evaluation. The H-score was calculated by multiplying the estimated percent of tumor 9

cells with membrane (apical, or complete circumferential, or incomplete circumferential) 10

staining of each staining intensity (0 to 3; 0 = negative, 1 = low, 2 = medium, 3 = high) by the 11

intensity value (0 to 3) and then adding together all 4 scores for a single score. The highest H-12

score obtainable by this method is 300, or 100% of cells staining at an intensity of 3. Colorectal 13

tumors were also described by their grade of differentiation status as "well differentiated," 14

"moderately differentiated" or “poorly differentiated” by examining the H & E stained sections 15

of all tumors. All slides were evaluated by a trained pathologist. 16

17

For CD3, Granzyme B and PD-L1 expression in the LS0134 adoptive transfer efficacy study, 18

tumors were harvested from mice treated with PF-07062119 at 1 mg/kg and 0.03 mg/kg, and 19

from the vehicle-treated group. Tumors (n = 5/group) were formalin fixed and paraffin 20

embedded as described above. Following epitope retrieval in Borg Decloaker, peroxidase block 21

and protein block as mentioned above, sections were stained with rabbit mAb anti-CD3 (SP162, 22

Abcam ab135372) at 1:200 (0.075 µg/ml) or rabbit mAb anti-PD-L1 (SP142, Abcam ab228462) 23




10

at 1:200 (0.385 µg/ml) for 1 hour followed by MACH2 Rabbit HRP-polymer for 30 minutes. 1

Color was developed using Betazoid DAB chromogen for 5 minutes. For Granzyme b and CD3 2

sequential co-staining, the tumor sections were incubated in mouse mAb anti-Granzyme B 3

(11F1,Leica Biosystems) at 1:50 (0.94 µg/ml) for 1 hour, followed by MACH2 Mouse HRP-4

Polymer (Biocare Medical) for 30 minutes and Betazoid DAB chromogen for 5 minutes. 5

Sections were rinsed in deionized water, and endogenous peroxidase activity was inactivated 6

again with Peroxidazed 1 for 10 minutes. Non-specific protein interactions were blocked again 7

for 10 minutes with Background Punisher. Sections were incubated with anti-CD3 as described 8

above, followed by MACH2 Rabbit HRP-polymer for 30 minutes and Vina Green (Biocare 9

Medical) chromogen for 12 minutes. Immunostained sections were rinsed with deionized water, 10

counterstained for 30 seconds in Tacha’s hematoxylin, washed in tap water, dehydrated in 11

alcohol, and cleared in xylene. All slides were coverslipped using Permount medium. 12

13

IHC analysis of CD3 positive T cells in the anti-VEGF-A and PF-07062119 combination study 14

in PDX-CRX-11201 was done on tumors harvested on day 7 after the first bispecific dose, using 15

the CD3 staining method described above. 16

Human T Cell Adoptive Transfer Established Tumor Model 17

Female NOD-scid IL-2Rγ null (NSG) animals (Jackson Laboratory) were used for experiments 18

under approval by the Institutional Animal Care and Use Committee (IACUC), and all applicable 19

animal care and use regulations, guidelines and policies were followed. For xenograft studies, 20

NSG mice were inoculated with LS1034, HT55, LS174T or HCT116 cells in the flank in a total 21

injection volume of 0.2 mL. For the LS1034, HT55 and HCT116 subcutaneous CLX models, 5 x 22

106 cells per mouse were administered in 50% Matrigel Basement Membrane Matrix. For the 23




11

LS174T CLX model, 2 x 106 cells per mouse were administered in 50% Matrigel Basement 1

Membrane Matrix. PDX-CRX-11201 (Asterand) tumor fragments (4 mm x 4 mm) generated 2

from 500-800 mm3 tumors, expanded in NSG mice, were implanted subcutaneously in the flank 3

of NSG mice. 4

5

Tumor measurements were collected using Vernier caliper, and volumes were calculated by use 6

of the modified ellipsoid formula ½ x length x width2. Mice were randomized and staged at 7

tumor size of 150-200 mm3. An initial dose of PF-07062119, a non-targeted CD3 bispecific 8

control, or PBS (vehicle) was administered to animals on Day 0, and 2 x 106 cultured activated 9

pan human T cells (containing CD8 and CD4 T cells) were inoculated the following day. Mice 10

were dosed in 0.2 mL bolus injections weekly for 3 doses in all studies except in the single agent 11

efficacy study in PDX-CRX-11201, which was dosed weekly for 4 doses. In combination 12

studies, combination agents were administered starting on Day 0. Anti-human PD-L1 was 13

administered at 10 mg/kg every 3 days for 6 doses, anti-human PD-1 was dosed at 5 mg/kg 14

weekly for 3 doses, and anti-VEGF-A mAb (G6-31) that blocks both human and mouse VEGF 15

(26) was dosed at 5 mg/kg every 3 days for 4 doses. All compound and T cell administrations 16

were intravenous via the lateral tail vein of each animal. Tumor measurements were collected 17

twice weekly along with continuous monitoring for signs of a graft versus host response. 18

19

Tumor volume data was log-transformed after an adjustment was made for zero tumor size. A 20

separate ANOVA analysis using all groups was performed for each day. From each analysis, a 21

one-sided p-value from a Student’s T pairwise comparison of each group with the control was 22

reported. For observed differences between the combination agent treatments and their respective 23




12

single agent treatments, statistical analysis was performed to determine statistical significance (p-1

values) for each difference. In the studies using anti-PD-L1 and anti-PD-1, the tumor size was 2

log-transformed and a one-sided t-test of the log-transformed tumor sizes was used to assess 3

significance. Since the difference observed varied with day, this assessment was performed 4

separately for each day. For the study using anti-VEGF-A, many tumors had zero size, so a non-5

parametric Wilcoxon ranked sum test (one-sided with t-approximation) of tumor sizes was used 6

in place of a parametric t-test. The p-values reported in sections 7.4 and 7.5 are those for the last 7

day of measurements since that p-value is similar (in most case identical) across the last five 8

days of measurements. A separate p-value is computed for each single agent (when compared 9

with the combination agent), and the more conservative (higher) of the two p-values is reported 10

for each combination being evaluated. 11

LS1034 Colorectal Orthotopic Tumor Model 12

5x106 LS1034 tumor cells expressing firefly luciferase were subcutaneously inoculated in female 13

NSG donor mice. Three weeks after cell inoculation, the subcutaneous tumors (250 to 300 mm3) 14

were harvested, and tumor fragments (4 x 4 mm) were prepared for orthotopic implants. 15

Laparotomy was performed through a midline incision, and the cecum was exposed. Then 16

LS1034-luc tumor fragment was sutured to the cecum adjacent to the ascending colon. The mice 17

with orthotopic tumor implants were monitored weekly with the tumor growth assessed by In 18

Vivo Imaging System (IVIS) (Perkin Elmer) scans of luciferin bioluminescence using Living 19

Image version 4.3.1 software. Animals were staged to an average bioluminescence level of 24 to 20

26 x 106 photons/s and dosed weekly intravenously for 5 doses via tail vein injection with PF-21

07062119 at 0.3, 0.1 and 0.03 mg/kg, or non-targeted CD3 bispecific at 0.3 mg/kg. 22




13

Activated/expanded T cells were administered as described earlier for adoptive T cell transfer, 24 1

hours after dosing with bispecifics. 2

CT26-mGUCY2C efficacy study in human CD3ε transgenic mice 3

To generate CT26-mGUCY2C cells, mouse GUCY2c nucleotide sequence (NM_001127318) 4

encoding the mature extracellular domain, transmembrane domain, and the first three amino 5

acids of the cytoplasmic domain (nucleotide sequence 194-1507 from GenBank accession # 6

NM_001127318, corresponding to amino acid sequence V20-Y457 of GenBank accession # 7

NP_001120790) was inserted into a mammalian expression vector downstream of the synthetic 8

CAGGS promoter. The vector also contained the neomycin resistance gene for growth selection. 9

CT26 cells (ATCC) were transfected with this expression construct, and a G418-resistant, single 10

cell clonal population was generated using limiting dilution. 11

12

CT26-mGUCY2C tumor cells were cultured in vitro with RPMI medium supplemented with 13

10% fetal bovine serum in a humidified chamber at 37°C under 5% CO2 atmosphere. Cells were 14

harvested during the exponential growth phase, and 1 x106 cells were inoculated in each human 15

CD3ε transgenic mouse (CrownBio) subcutaneously in the right rear flank region in 0.1 ml of 16

PBS mixed with matrigel (1:1) for tumor development. Mice were randomized (n = 5 17

mice/group) when mean tumor size reached approximately 68 mm3. On the day of 18

randomization, mice were dosed with PF-07062119 at 3, 1, or 0.3 mg/kg, or with the non-19

targeted-CD3 bispecific control at 3 mg/kg every 3 days for 6 doses. Tumor volumes were 20

measured twice a week using calipers, and animals were monitored for changes in bodyweight, 21

behavior or signs of toxicity. 22




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Pharmacokinetic Measurements of GUCY2C(M)-CD3 in LS1034 Adoptive Transfer Model 1

A qualified quantitative ligand-binding assay was used to measure GUCY2C(M)-CD3 in NSG 2

mouse diluted whole blood, using the Meso-Scale Discovery (MSD) assay platform. The assay 3

used biotinylated goat anti-human IgG (Southern Biotech, 2049-08) coated on the surface of the 4

MSD plate to capture GUCY2C(M)-CD3. After washing the bound drug was detected with a 5

mouse anti-human IgG Fc (Clone JDC10 Southern Biotech, 9040-01), which was labeled with 6

ruthenium. After a final set of washes, tripropylamine was added and plates read on the MSD 7

SECTOR Imager 6000. The electrochemiluminescent signal generated was proportional to the 8

amount of bound drug. Sample concentrations were determined by interpolation from a standard 9

curve that was fit using a 4-parameter logistic equation with 1/y^2 weighting. The range of 10

quantitation in 100% diluted whole blood was 80.0 ng/mL to 5120 ng/mL. The pharmacokinetic 11

parameters were determined from individual animal data using non-compartmental analysis in 12

Watson LIMS (Version 7.5, Thermo, Inc. Philadelphia, PA) Concentrations below the limit of 13

quantitation (BLQ) were not used in the calculations. In addition, PK data was also analyzed 14

using a 2-compartment PK model with linear elimination from the central compartment using 15

Phoenix 64®

Win Non Lin®

(Certara L.P.). 16

Cell Culture 17

T84, LS1034, LS174T, and HCT116 cells were obtained from the American Type Culture 18

Collection (ATCC). HT55 cells were obtained from the European Collection of Authenticated 19

Cell Cultures (ECCAC, Source: Sigma-Aldrich). T84 cells were cultured in a 1:1 mixture of 20

Ham's F12 medium and Dulbecco's modified Eagle's medium (DMEM) with Glutamax and 10% 21

fetal bovine serum. LS1034 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 22

with 10% fetal bovine serum. HT55 cells were cultured in modified Eagles’s medium with 20% 23




15

fetal bovine serum. LS174T cells were cultured in modified Eagles’s medium with Glutamax and 1

10% fetal bovine serum. HCT116 cells were grown in McCoy’s 5a medium with 10% FBS. 2

HCT116-hGUCY2C cells were generated by transducing HCT116 cells with human GUCY2C 3

lentiviral particles (Origene, RC213901L3V), and selecting puromycin resistant clonal 4

populations. 5

T84, LS1034, LS174T, HT55 and HCT116 tumor cells were transduced with pantropic 6

retrovirus produced from pMSCVpuro_LucSh or pMSCVneo_LucSh retroviral transfer vectors 7

to introduce firefly luciferase followed by selection of drug-resistant pools puromycin or 8

geneticin. HCT116 and T84 cells expressing luciferase were maintained under 1 µg/ml of 9

puromycin drug selection. LS174T and LS1034 luciferase cells were maintained 200 µg/ml and 10

HT55 luciferase cells were maintained with 500 µg/ml of Geneticin. All cells were grown in a 11

humidified chamber at 37°C under 5% CO2 atmosphere. 12

GUCY2C Receptor Density Measurements 13

Tumor cell lines were dissociated using enzyme free cell dissociation buffer (Gibco) for 14

saturation flow cytometry. GUCY2C receptor density in cell lines was measured using an anti-15

GUCY2C (9H3-human IgG, Pfizer) conjugated at a 1:1 ratio to phycoerythrin (Thermo 16

Scientific). Cells (1x105) were stained with 10 µg/ml of the PE labeled anti-GUCY2C mAb on 17

ice for 1 hour. Cells were washed and resuspended in FACS buffer (PBS, 3% BSA) with 18

4′,6-diamidino-2-phenylindole (DAPI) and acquired using BD LSRII Fortessa with FACS Diva 19

software version 8.0.1. QuantiBRITE PE labeled beads were used in the same acquisition PE 20

voltage settings to calculate the number of PE labeled antibodies per cell (ABC) based on the 21

background corrected PE geometric median fluorescence intensity (MFI). 22




16

Characterization of PF-07062119 Binding to human T cells and GUCY2C expressing 1

tumor cells 2

Freshly isolated human T cells, tumor cells (HCT116, HCT116-human GUCY2C, CT26, CT-26-3

mouse GUCY2C) dissociated with CDB (1 x 106 cells per sample) were were blocked and 4

washed in cold FACS buffer containing PBS and 3% BSA. Cells were stained in flow buffer 5

containing PF-07062119 (0.25µM) on ice for 45 minutes. Cells were washed with cold PBS and 6

resuspended in 100 µl of cold FACS buffer containing 1:100 dilution of goat anti-human PE 7

(Jackson Immunoresearch, 109-115-098) and incubated in the dark on ice for 30 minutes. Cells 8

were washed with cold PBS and resuspended in 200 µl of cold PBS with 3 µM DAPI for data 9

acquisition using a BD Fortessa X-20 or BD LSR II Fortessa with FACS Diva software (BD 10

Biosciences). Data analysis was performed using Flowjo software v10.4.1 (Treestar Inc.). 11

Cytotoxic T Lymphocyte Assay 12

To test PF-07062119 sensitivity on a panel of colorectal tumor cell lines endogenously 13

expressing GUCY2C, monolayer cultures of firefly luciferase expressing tumor cell lines were 14

isolated from cell culture flasks using Tryp LE dissociation reagent. Tumor cells were 15

resuspended in R10 medium (RPMI, 10% FBS, 3 ml of 45% glucose). Five thousand tumor cells 16

per well were plated into clear bottom white wall 96-well plates (Costar). Human T cells were 17

also resuspended in R10 media and added to tumor cells at an Effector to Target ratio (E: T ratio) 18

either 5:1. The cells were treated with serial dilutions of PF-07062119 or non-targeted-CD3 19

control bispecific and incubated at 37°C under 5% CO2 for 48 hours. The luciferase signal in 20

viable cells was measured in relative lights units (RLU) using the neolite reagent read on a 21

Victor plate reader (Perkin Elmer) at 0.1 seconds/well. EC50 values were calculated in Graphpad 22

PRISM v7.04 using variable slope four parameter non-linear regression analysis of percent 23




17

cytotoxicity versus Log10 concentration of bispecifics. For each tumor cell line, n = 3 separate 1

donor CD3+ lymphocytes were used in cytotoxicity assays, and the average EC50 for each cell 2

line was calculated. PF-07062119 activity was also measured GUCY2C expressing tumor cells 3

in the absence of T cells. 4

IFNγ Induced In Vitro by PF-07062119 5

To test PF-07062119 induced cytokine release, supernatants were collected at the end of the CTL 6

assays described above and analyzed using a Milliplex MAP Human Cytokine/Chemokine 7

Magnetic Bead Panel according to the manufacturer’s guidelines, read on a Luminex 200 8

(Luminex Xmap Technology) with Luminex xPONENT software 3.1, and analyzed using 9

MILLIPLEX ANALYST v5.1.0.0 to measure human interferon (IFN)- levels. 10

Mass Cytometry Deep Phenotyping 11

The mass cytometry panel included the following metal conjugated antibodies from Fluidigm- 12

anti-CD45-89Y (3089003B), anti-CD127-176Yb (3176004B), anti-FoxP3-162Dy (3162011A), 13

anti-PD-1-175Lu (3175008B), anti-CCR4-158Gd (3158006A), anti-Tim-3-153Eu (3153008B), 14

anti- Lag3-150Nd (3150030B). Additionally, the following antibodies were conjugated using 15

Maxpar X8 antibody labeling kits (Fluidigm) for their corresponding metal conjugate – anti-16

CD3-154Sm (Tonbo Biosciences, 70-0038-U100), anti-CD4-145Nd (Tonbo Biosciences, 70-17

0049-U100), anti-CD8-168Er (Tonbo Biosciences, 70-0088-U100), anti-CD25-149Sm (BD 18

Biosciences, 340739), anti-PD-L1-159Tb (Pfizer, Avelumab), anti-41-BB-161Dy (Pfizer 19

proprietary, PF-05082566), anti-OX40-163Dy (Pfizer proprietary, PF-8600), anti-CD27-167Er 20

(Tonbo Biosciences, 70-0279-U100), anti-CD45RA-171Yb (Tonbo Biosciences, 70-0458-U100) 21

and anti-GITR-170Er (Pfizer proprietary, 10H2). These metal conjugated antibodies were used 22

in an antibody mastermix to label input T cells, as well as tumors harvested from the LS1034 23




18

adoptive transfer efficacy study in groups treated with PF-07062119 at 1 mg/kg and 0.03 mg/kg, 1

7 days after the first bispecific dose. Tumors were dissociated into single cell suspensions using 2

a tumor dissociation kit (Miltenyi 130-096-730) according to the manufacturer’s guidelines. 3

Viability staining was performed with 5 µM final concentration of Cisplatin (Fluidigm) for five 4

minutes in 1 mL of PBS (Ca/Mg free). Cell suspensions were washed and then stained with the 5

antibody mastermix with 1-3 million cells in 100 µL of staining buffer. Cells were incubated at 6

room temperature for 30 minutes before being washed and resuspended in nuclear antigen 7

staining buffer (Maxpar Nuclear Antigen Stain Buffer Set, Fluidigm). Cells were washed in 8

Maxpar Nuclear Antigen Staining Perm buffer before staining for intracellular antigens. After 9

washing in PBS, cells were fixed in 2% paraformaldehyde (Electron Microscopy Sciences) 10

overnight. Cells were pelleted and resuspended in freezing media containing 90% FBS and 10% 11

DMSO and stored at -80ºC until acquisition. Prior to acquisition, samples were thawed at 37 °C 12

and washed in Maxpar Staining Buffer. Cell pellets were resuspended in Maxpar Fix/Perm 13

buffer with 0.05 µM Cell-ID Intercalator-Ir to stain DNA for cell-ID. After incubation at room 14

temperature for 30 minutes, Cell-ID™ 20-Plex Pd Barcoding Kit (Fluidigm) was added to 15

corresponding samples and mixed by pipetting up and down and incubated for an additional 30 16

minutes. Cells were washed in PBS twice, then washed in water prior to resuspending in water 17

with 1:10 EQ™ Four Element Calibration Beads (Fluidigm). The concentration of the cell 18

suspension was adjusted so samples are acquired at a rate of 250 to 500 events per second. A 19

CyTOF 2 instrument with Helios upgrade was tuned using Maxpar tuning solution and quality 20

controlled using EQ™ Four Element Calibration Beads. Samples were acquired, normalized, 21

concatenated, and debarcoded using Fluidigm’s CyTOF software version 6.7.1014. Debarcoded 22

samples were uploaded to the Cytobank cloud-based analysis program for manual gating. 23




19

CD45+ populations were exported from Cytobank and uploaded to a Pfizer cytometry analysis 1

portal. This portal utilizes Cytofkit as the base for Rphenograph analysis and resulting tSNE 2

maps. Data generated through the portal was then used for statistical proportion differential to 3

determine clusters that differentially shift with treatment when compared to a control group. 4

Phenotyping of these clusters was determined using heatmaps of each cluster for all markers 5

used in the Rphenograph clustering. Resulting data from the clustered samples were used to 6

generate heatmaps and tSNE maps in Tibco Spotfire software. The heatmap depicts expression 7

of Arcsinh transformed median intensities of each marker to represent the phenotypes of each 8

cluster. 9

10

RESULTS 11

GUCY2C Expression in Gastrointestinal Cancer and Normal Tissues 12

Although GUCY2C expression in colorectal, gastric, esophageal and pancreatic cancers has been 13

evaluated previously (11,12), both membrane and cytoplasmic expression of the target has been 14

observed across these indications. Since our bispecific targeting modality relies on binding to 15

cell surface GUCY2C, we sought to specifically characterize membrane expression of GUCY2C 16

in primary gastrointestinal tumors and in normal tissues (sources: Indivumed, Proteogenix, 17

Avaden, University of Michigan and Cornell University) using immunohistochemistry with an 18

anti-GUCY2C mAb (Figure 1, Table S1). GUCY2C was expressed in the majority of colorectal 19

adenocarcinomas across all stages, including liver metastases (Figure1A-F). It was also 20

expressed in gastric (Figure 1A, G, Table S1) and esophageal adenocarcinomas (Figure 1A, H, 21

Table S1). Moderately to well differentiated tumors tended to be associated with higher 22

membrane expression of GUCY2C compared with those that were poorly differentiated (Table 23




20

S1). Relatively heterogenous and low incidence of membrane expression was observed in 1

pancreatic adenocarcinomas (Figure 1A, Table S1). In normal tissues, GUCY2C expression was 2

primarily observed on the apical side of the colon (Figure 1I) and small intestinal epithelium 3

(Figure 1J). A lower level of expression was present in prostatic apical epithelium, and all other 4

tissues evaluated were negative (Figure S1) 5

6

Tumor selective targeting with an anti-GUCY2C/anti-CD3ε bispecific 7

8

To test the ability to target GUCY2C expressing tumors with an anti-GUCY2C/anti-CD3ε 9

bispecific, we developed a heterodimeric diabody Fc-fusion protein comprised of two single 10

chain Fv (scFv) domains, one targeting GUCY2C and the other targeting CD3ε. The VH of one 11

binding domain pair is joined with the VL of the other binding domain pair such that when the 12

two constructs are co-expressed, the result is the formation of a diabody. These scFv domains 13

are fused to the Fc domain of human IgG1 (Figure 2A). Mutations (L234A, L235A and G237A 14

EU numbering) were introduced in the CH2 region of the Fc to reduce binding to Fc gamma 15

receptor (FcγR) (27). Additionally, knob-in-hole (KIH) mutations in the CH3 region of the Fc 16

domain were used to facilitate correct heterodimeric pairing and bispecific purification (28,29). 17

The Fc domain fusion was also designed to extend the bispecific half-life and allow for less 18

frequent dosing than antibody fragment based bispecifics such as BiTEs (e.g., blinatumomab), 19

which require constant intravenous infusion into patients via a pump. BiTEs contain only two 20

tandem antibody variable domains (scFv-scFv) and no Fc domain. The diabody-Fc format is 21

similar to the BiTE format in that it contains two scFv domains. However the variable domains 22

in the diabody are more tightly packed together than those of the BiTE, allowing for more potent 23




21

killing activity (30) and introduction of the Fc further extends half-life from 1 hr to 1

approximately 1 week (31). 2

3

To illustrate both tumor selective targeting and efficacy, we generated an anti-GUCY2C/anti-4

CD3ε bispecific (GUCY2C(M)-CD3), which had equivalent binding affinity to human and 5

mouse GUCY2C (Table S2). NOD-scid IL-2Rnull (NSG) mice bearing LS1034 CRC cell line 6

xenograft (CLX) tumors were treated GUCY2C(M)-CD3 at two different dose levels of 1 and 7

0.3 mg/kg, along with adoptive human T cell transfer. Pharmacokinetic analyses demonstrated 8

that the Fc domain fusion design of GUCY2C(M)-CD3 led to a systemic estimated half-life of 9

eleven days in these tumor-bearing mice, which enabled weekly dosing with the bispecific 10

(Figure 2B). Even though GUCY2C expression was observed in both the LS1034 tumors as well 11

as in the normal intestinal tract epithelium of the same mice (Figure 2C, left panel), only tumors 12

showed uptake of GUCY2C(M)-CD3 (Figure 2C, middle panel) and infiltration of human CD3-13

positive T cells (Figure 2C, right panel), while no drug or human CD3 T cell infiltration could be 14

detected in the intestinal tract. Importantly, anti-tumor efficacy was observed with 15

GUCY2C(M)-CD3 at 1 and 0.3 mg/kg but not with a non-targeted CD3 bispecific (Figure 2D). 16

No changes in body weight were noted in response to bispecific treatment (Figure S2). These 17

observations support the hypothesis that GUCY2C can be selectively targeted on tumors with an 18

anti-GUCY2C targeting agent. 19

20

PF-07062119 elicits target dependent CTL activity in vitro 21




22

After establishing the ability to preferentially target GUCY2C expressing tumors, we developed 1

a fully humanized and lead-optimized anti-GUCY2C/anti-CD3ε therapeutic bispecific (described 2

herein as PF-07062119). PF-07062119 was generated in the diabody Fc fusion format shown in 3

Figure 2A and characterized in vitro for target binding and T cell-mediated cytotoxicity. PF-4

07062119 showed cell surface binding to human T cells, as well as HCT116 tumor cells 5

overexpressing GUCY2C, but not to HCT116 cells that are GUCY2C negative (Figure 3A, 6

Figure S3). To understand the range of GUCY2C expression capable of eliciting T cell-mediated 7

cytotoxicity, cell surface receptor density was quantitatively measured across a panel of 8

colorectal tumor cell lines. These cells showed receptor densities ranging from 875 to 8067 9

receptors per cell (Figure 3B). Treatment of these cells with PF-07062119 and human T cells 10

showed dose-dependent and GUCY2C expression dependent cytotoxicity, as measured by tumor 11

cell survival (Figure 3C). Consistent with cytotoxicity measurement, supernatants harvested from 12

these cytotoxicity assays showed release of IFNγ in a PF-07062119 dose-dependent manner 13

(Figure 3D). No change in tumor viability or IFNγ release was observed in GUCY2C-negative 14

HCT116 cells, confirming the need for GUCY2C expression for induction of PF-07062119-15

mediated T cell effector function. 16

17

Since the GUCY2C pathway regulates intestinal fluidity, PF-07062119 was also characterized 18

for its potential to modulate GUCY2C signaling by measuring production of the downstream 19

effector, cyclic guanosine monophosphate (cGMP). While the GUCY2C pathway agonist 20

bacterial enterotoxin STp increased cGMP in a dose-dependent manner in GUCY2C-expressing 21

T84 tumor cells, cGMP production was not enhanced by the addition of increasing 22

concentrations of PF-07062119 (Figure S4). A non-targeted CD3 bispecific control also did not 23




23

show cGMP production. Additionally, PF-07062119 did not affect cGMP production induced by 1

STp. These findings indicate the PF-07062119 is not a GUCY2C pathway agonist and does not 2

neutralize GUCY2C pathway function in the presence of ligand. 3

4

PF-07062119 demonstrates anti-tumor efficacy in GUCY2C positive human xenograft and 5

mouse syngeneic tumors 6

After characterizing the in vitro range of activity of PF-07062119, we next evaluated in vivo 7

efficacy with this bispecific in cell line xenograft (CLX) and patient derived xenograft (PDX) 8

models of CRC. GUCY2C expression was characterized in four CLX models and one PDX 9

model by immunohistochemistry to generate an H-score reflective of cell surface expression of 10

GUCY2C (Table 1 and Figure 4A). The tumors were also characterized for their mutational 11

status of KRAS and BRAF oncogenes, as well as their differentiation status (Table 1). Amongst 12

the models tested, LS1034 and HT55 CLX models showed relatively high GUCY2C expression, 13

reflective of human colorectal tumors with high GUCY2C expression (Figure 1A, Table S1). 14

LS1034 and HT55 were mutant for KRAS and BRAF, respectively (Table 1). PDX-CRX-11201 15

also had relatively high GUCY2C expression and was mutant for KRAS. The LS174T CLX 16

model was mutant for KRAS and showed lower GUCY2C expression, representative of human 17

tumors expressing GUCY2C below the median H-score level in CRC (Table 1, Figure 1A). The 18

HCT116 model was negative for GUCY2C. Both LS174T and HCT116 were poorly 19

differentiated tumors. Apically enhanced expression of GUCY2C was noted in all tumors with 20

high GUCY2C expression, and higher differentiation state. 21

22




24

To assess PF-07062119 activity in vivo, NSG mice were implanted subcutaneously with the 1

characterized colorectal cancer cell lines and PDX-CRX-11201 fragments. An initial dose of PF-2

07062119, non-targeted-CD3 negative control bispecific, or vehicle was administered to animals 3

with established tumors, along with adoptive transfer of human T cells, followed by weekly 4

dosing with PF-07062119 or control agents. In the LS1034 model, which had the highest 5

expression of GUCY2C, PF-07062119 treatment at 0.1 mg/kg or higher doses led to complete 6

tumor regressions (p-value<0.0001) (Figure 4B). The anti-tumor activity was dose-dependent, 7

since partial reduction in tumor volumes was observed at 0.03 mg/kg of PF-07062119. The 8

HT55 CLX model and PDX-CRX-11201 (Figure 4B), which both had similar H-scores, showed 9

complete tumor regressions at doses as low as 0.15 mg/kg of PF-07062119 (p-value<0.0001). 10

The LS174T CLX model, which had low GUCY2C expression showed a small but significant 11

reduction in tumor burden only at the high dose of PF-07062119 treatment (1 mg/kg, p-12

value<0.0001) (Figure 4B). The GUCY2C-negative HCT116 colorectal cancer model was not 13

responsive to PF-07062119 at any dose tested (Figure 4B). In summary, tumors with the highest 14

H-scores showed complete tumor regressions with 0.1-0.15 mg/kg or higher doses of PF-15

07062119, whereas tumors with lower GUCY2C tumors had a moderate anti-tumor response 16

with a higher dose of 1 mg/kg. In all models, treatment with the PBS vehicle control, or with a 17

non-targeted CD3 bispecific at the same or higher doses that were efficacious with PF-07062119, 18

did not result in any anti-tumor activity. These data demonstrate GUCY2C expression dependent 19

anti-tumor efficacy with PF-07062119, independent of the KRAS or BRAF mutational status of 20

the tumor. 21

22




25

We also examined PF-07062119 activity in a colon orthotopic model to evaluate the ability of 1

the drug to target tumors located within the gastrointestinal tract. LS1034 cells expressing 2

luciferase were generated to monitor tumor growth in vivo using bioluminescence. GUCY2C 3

expression in this model was confirmed by immunohistochemistry analyses and found to be 4

relatively comparable between LS1034 tumors and the adjacent mouse colon epithelium (Figure 5

4C, top right). Similar to subcutaneous models, treatment of these established orthotopic tumors 6

with PF-07062119 following adoptive human T cell transfer led to complete tumor regressions at 7

0.3 mg/kg, as measured by bioluminescence imaging scans (p-value<0.0001) (Figure 4C, top 8

left). Additionally, necropsy findings at the end of the study indicated that no tumors were 9

observed at secondary sites in mice treated with PF-07062119 at doses above 0.1 mg/kg. 10

However, in animals treated with PF-07062119 at 0.03 mg/kg, 2 out 9 mice had tumors spread to 11

the liver and 2 other mice had secondary spread at the abdominal wall. In the non-targeted CD3 12

treatment group, 2 out of 9 animals had tumor spread to the liver. No body weight loss was 13

observed in the bispecific treated animals (Figure S5). These data indicate that PF-07062119 has 14

the ability to target primary tumors located in the intestinal tract and could potentially prevent 15

tumor spreading to secondary sites. 16

17

While the above experiments demonstrate the ability of PF-07062119 to target tumors in 18

immunodeficient mice using adoptive human T cell transfer, we also sought to examine PF-19

07062119 driven anti-tumor activity in an immunocompetent mouse model. While PF-07062119 20

binds to mouse GUCY2C albeit with lower affinity than to human GUCY2C (Table S2), it does 21

not bind to mouse CD3. Therefore, CT26 mouse syngeneic tumor cells were engineered to 22

express mouse GUCY2C (CT26-mGUCY2C), and a transgenic mouse model expressing human 23




26

CD3ε on mouse T cells was used for efficacy studies. PF-07062119 showed specific binding to 1

CT26-mGUCY2C cells but not to GUCY2C-negative CT26 cells (Figure 4D). GUCY2C 2

expression on the tumor cell surface was confirmed by immunohistochemistry in CT26-3

mGUCY2C subcutaneous tumors established in the human CD3ε transgenic model (Figure 4E). 4

Similar to NSG models with CLX and PDX tumors, PF-07062119 treatment showed reduction of 5

tumor burden at all doses tested and resulted in complete regressions at doses above 1 mg/kg 6

(Figure 4F). No body weight loss was observed with PF-07062119 treatment (Figure S6) 7

suggesting that PF-07062119 has tumor selective activity in an immunocompetent model, 8

thereby showing the ability to harness T cell effector function in the setting of cancer immune 9

escape. 10

11

PF-07062119 treatment increases TIL infiltration and activation, and enhanced efficacy with 12

checkpoint blockade 13

To better understand the mechanism of action of PF-07062119-mediated human T cell activity, 14

the LS1034 subcutaneous adoptive transfer model was used to evaluate changes in tumors and 15

tumor infiltrating lymphocytes (TILs) in vivo. Tumors treated at 1 mg/kg of PF-07062119 16

showed increased T cell infiltration compared to tumors treated at the minimally efficacious dose 17

of 0.03 mg/kg, whereas tumors in the vehicle treatment group did not show any T cell infiltration 18

(Figure 5A, top row). Tumor infiltrating lymphocytes (TILs) in PF-07062119 treated tumors 19

showed expression of granzyme B that was polarized towards tumor cells, suggesting that these 20

TILs were poised to form immune synapses and kill tumor cells (Figure 5A, second row). 21

Tumors treated with PF-07062119 also showed upregulation of PD-L1 at both 1 mg/kg and 0.03 22

mg/kg doses (Figure 5A, third row). The upregulation of PD-L1 was specific to PF-07062119-23




27

treated tumors, and was not observed in vehicle treated, indicating that checkpoint mechanisms 1

that could dampen T cell activity were being induced with bispecific treatment. These PF-2

07062119 induced changes in LS1034 tumors were concomitant with tumor necrosis (Figure 5A, 3

bottom row). Additionally, tSNE analyses on CYTOF data from TILs from these tumors 4

identified two T cell clusters that were unique to the PF-07062119 treatment groups. These 5

clusters were CD4 and CD8 positive T cells that were PD-1+ 41-BB

+ Tim-3

+ Lag-3

+, indicating 6

that these TILs were activated, but also expressed activation-induced checkpoint markers, which 7

if chronically expressed could negatively regulate T cell effector function (Figure 5B, C). While 8

these data imply that PF-07062119 treatment induced T cell infiltration into tumors in a dose-9

dependent manner, the upregulation of PD-L1 on tumors, and PD-1 on TILs suggests that a PD-10

1/PD-L1 axis that could dampen T cell activity is induced with PF-07062119 treatment. 11

12

Based on the rationale that PF-07062119 treatment induced expression of PD-1/PD-L1 on TILs 13

and tumors, we tested if checkpoint blockade with anti-PD-1 or anti-PD-L1 antibodies could 14

enhance anti-tumor efficacy of PF-07062119. LS1034 subcutaneous tumors treated with a sub-15

optimally efficacious PF-07062119 of 0.03 mg/kg showed significant improvement in efficacy in 16

combination with either an anti-PD-L1 or an anti-PD-1 antibody (p-value 0.0001 and 0.0004 17

respectively), while these agents were not efficacious as single agents in this model (Figure 5D). 18

These findings indicate that while checkpoint mechanisms may limit efficacy of PF-07062119, 19

the bispecific activity can be further enhanced in combination with checkpoint blockade agents. 20

21

Anti-VEGF blockade enhances PF-07062119 anti-tumor activity 22




28

Anti-angiogenesis treatment is currently used for the treatment of CRC and is also reported to 1

improve responses to T cell mediated therapy by enhancing T cell infiltration into the tumor. 2

Since suboptimal doses of PF-07062119 showed reduced T cell infiltration, we tested the effects 3

of combining anti-VEGF-A blockade with PF-07062119 treatment in vivo. PDX-CRX-11201 4

was used for this evaluation since this tumor model is only partially responsive to G6-31, an anti-5

VEGF-A mAb that blocks murine VEGF-A. A combination of anti-VEGF-A treatment with 6

PF-07062119 administered at a suboptimal dose of 0.05 mg/kg led to a significant combination 7

benefit leading to complete tumor regressions (p-value < 0.0001) (Figure 6A). Additionally, 8

immunohistochemistry analyses showed that tumors that received the combination treatment had 9

notably increased T cell infiltration compared to tumors treated with either PF-07062119 or anti-10

VEGF-A alone (Figure 6B). Therefore, these data suggest that apart from the combination 11

benefit observed with checkpoint blockade agents, anti-angiogenesis treatment could further 12

enhance the efficacy observed with PF-07062119. 13

14

Toxicology and Pharmacokinetics of PF-07062119 in cynomolgus monkey 15

Cynomolgus monkeys were selected as relevant species for toxicity studies after confirming 16

binding of PF-07062119 to cynomolgus T cells and GUCY2C protein (Table S2, Figure S7). 17

Nonclinical safety and pharmacokinetics of PF-07062119 were evaluated in cynomolgus 18

monkeys in an exploratory intra-animal dose escalation (2 doses, once weekly) toxicity study at 19

30 µg/kg followed a week later by 100 µg/kg, and 60 µg/kg followed a week later by 180 µg/kg 20

(Figure S8A). The main in-life effects observed with PF-07062119 treatment included emesis, a 21

slight increase in body temperature, decreased activity, hunched posture, reduced appetite, 22

dehydration, body weight loss (≤ 7%), and soft or liquid feces. However, animals in both cohorts 23




29

tolerated the treatment with administration of oral and/or subcutaneous fluids. Importantly, 1

histopathological evaluations of the intestinal tract, which is the main site of GUCY2C 2

expression in normal tissues, showed only minimal to mild infiltrates and crypt hyperplasia in 3

multiple segments of the large and small intestine, along with typically minimal to mild villous 4

atrophy in the small intestine (Figure S8B. However, there were no erosions, ulcerations, or other 5

evidence of overt necrosis or loss of overlying epithelium. 6

7

PF-07062119 treatment induced pharmacodynamic effects in the monkeys, as measured by 8

increases in the percentage of activated (CD69+) CD8+ T cells (Figure S7C), and in peripheral 9

cytokines, including IFN-γ, IL-2, IL-10 and IL-6 (Figure S7D). Cytokine induction observed 10

after the first dose in each cohort was dampened after the second dose administration of PF-11

07062119, indicative of a priming effect. Toxicokinetic analysis of PF-07062119 in both 12

treatment cohorts, assessed by maximum observed concentration (Cmax) and area under the 13

concentration-time curve (AUC), showed dose-proportional systemic exposure, as well as a 14

linear PK profile (Figure S7E). No sex-related differences in systemic exposure were noted in 15

this study. 16

17

DISCUSSION 18

Despite the benefit and approval of checkpoint blockade agents for the treatment of Micosatellite 19

Instability (MSI)-High colorectal tumors, immunotherapy has had limited success in the majority 20

of gastrointestinal cancers. Attempts to enhance T cell activity against these cancers with CD3 21

bispecifics have shown early signs of activity in the clinic but have been limited by target 22




30

expression on normal tissues (32,33). Here we have described the expression of GUCY2C across 1

gastrointestinal cancers, particularly in moderately- to well-differentiated colorectal, gastric and 2

esophageal adenocarcinomas, and provided support for the hypothesis that GUCY2C-expressing 3

tumors can be selectively targeted with an anti-GUCY2C/anti-CD3ε bispecific, owing to the 4

restricted expression of the target in the apical intestinal epithelium. For this evaluation, a 5

bispecific with equivalent affinity to mouse and human GUCY2C was used to show selective 6

biodistribution to xenograft tumors expressing human GUCY2C versus the intestinal tract of the 7

host mice expressing mouse GUCY2C. In further support of this hypothesis, anti-tumor efficacy 8

was observed in this study in the absence of any tissue damage to the intestinal tract (as 9

evaluated by histopathology), suggesting that GUCY2C positive tumors can be preferentially 10

targeted with an anti-GUCY2C/anti-CD3ε bispecific T cell redirection approach. 11

12

Based on these data, PF-07062119, a fully humanized and lead-optimized anti-GUCY2C/anti-13

CD3ε bispecific was developed and characterized in vitro and in several in vivo tumor models for 14

pharmacological activity. Both immunodeficient mice with human T cell adoptive transfer and 15

immunocompetent mice were used in a complementary manner to characterize in vivo efficacy 16

with PF-07062119. Notably, PF-07062119-mediated anti-tumor activity was observed in all 17

models in a dose dependent manner and was associated with the level of GUCY2C expressed in 18

human xenograft models, suggesting a potential to obtain activity through dose modulation in the 19

clinic. The human xenograft tumors evaluated for in vivo efficacy included those with mutations 20

in the KRAS or BRAF oncogenes, which are clinically associated with a lack of response to 21

currently approved EGFR targeted therapies, suggesting that PF-07062119 can target tumors 22

regardless of the mutational status of these genes. 23




31

1

Furthermore, a colon orthotopic xenograft model showed that PF-07062119 was effective at 2

selectively eliminating primary tumors in the intestinal tract without damaging the normal 3

adjacent tissue (as assessed by histopathology) and had the potential to prevent tumor spreading 4

to secondary sites, such as the liver. While these T cell adoptive transfer studies highlighted PF-5

07062119-mediated redirected T induced cell killing, we also demonstrated that dose-dependent 6

efficacy could be achieved in a mouse syngeneic tumor model, in the presence of a functional 7

immune system. Even with the caveat that both mouse GUCY2C and human CD3ε proteins are 8

over-expressed in the syngeneic system, this model allows for long term evaluation of anti-tumor 9

responses, whereas the duration of adoptive transfer tumor models is limited by the eventual 10

onset of graft vs host disease. 11

12

We further explored the mechanisms of PF-07062119 activity using the T cell adoptive transfer 13

model and showed that activity was mediated through recruitment of TILs into GUCY2C 14

expressing tumors. These T cells had granzyme B expression, which was polarized towards 15

target expressing tumor cells, suggesting that they are poised to form immune synapses with the 16

tumor cells and initiate cell killing. Such analyses of T cell infiltration and activity could be 17

informative in the clinic as pharmacodynamic biomarkers of PF-07062119 mediated activity. 18

Further evaluations showed that tumors treated with PF-07062119 also upregulated PD-L1, and 19

TILs from these tumors had expression of activation markers including PD-1, indicating 20

initiation of checkpoint mechanisms that could dampen T cell mediated cytotoxicity. Based on 21

these observations, PF-07062119 was combined with checkpoint blockade via anti-PD-1 and 22

anti-PD-L1 antibodies. Combinations of anti-PD-1 or anti-PD-L1 blocking antibodies with a 23




32

suboptimal dose of PF-07062119 showed improved efficacy compared to single agents, 1

indicating that checkpoint blockade combinations will be important to consider during clinical 2

evaluation of PF-07062119. A limitation of murine models is that due to their lack of sensitivity 3

to systemic cytokine release, the sequencing of combinations and their effects on cytokine 4

release syndrome, will need to be evaluated in the clinic. 5

6

Recent preclinical and clinical studies with anti-angiogenesis agents have been reported to 7

improve responses to immunotherapy through various mechanisms, including enhancing T cell 8

infiltration into tumors (34-36). For example, the FDA recently approved combinations with 9

axitinib (VEGFR inhibitor) and avelumab (anti-PD-L1 mAb) in renal cell carcinoma (37), and 10

with bevacizumab (anti-VEGF mAb) and atezolizumab (anti-PD-L1 mAb) in non-small cell lung 11

cancer (38), due to significant improvements in progression free survival and overall response 12

rate. Based on these observations, we tested a combination of PF-07062119 with an anti-VEGF 13

mAb, which induced complete tumor regressions, while single agents were only minimally 14

active in the PDX model tested. Although increased T cell infiltrate was observed in the 15

combination treatment group, further mechanistic studies will elucidate whether this increase in 16

TILs was due to changes in the tumor vasculature or was a consequence of increased PF-17

07062119 uptake following anti-VEGF blockade. A limitation of the adoptive transfer model is 18

that T cell engraftment in the tumor is dependent on bispecific-mediated recruitment of T cells, 19

thereby resulting in minimal T cell engraftment with anti-angiogenesis treatment alone. 20

Therefore, additional combination studies in immunocompetent mice will be pursued to further 21

elucidate the mechanisms by which VEGF blockade improves T cell infiltration in PF-07062119 22

treated tumors. 23




33

1

Finally, the exploratory toxicity study with PF-07062119 in cynomolgus monkeys suggests a 2

clinically monitorable and manageable toxicity profile. PF-07062119 treatment did not induce 3

any erosions, ulcerations, or evidence of overt necrosis or loss of overlying epithelium, and the 4

overall in-life findings were tolerated with administration of oral and/or subcutaneous fluids. 5

Furthermore, the gastrointestinal findings of minimal to mild immune infiltrates, crypt 6

hyperplasia and villous atrophy would all be expected to resolve following cessation of 7

treatment. Pharmacologic activity in the monkeys was shown by CD69 upregulation on CD8+ T 8

cells and increases of systemic cytokines (Figure S7); although there are polymorphisms in 9

macaque CD3 that abolish activity of a specific anti-CD3 antibody (FN18) in a subset of 10

animals (39-41), PF-07062119 has shown binding and pharmacologic activity in all monkeys 11

tested (Figures S7 and S8 and Table S2). Additionally, pharmacokinetic analyses in cynomolgus 12

monkeys estimated the terminal half-life of PF-07062119 to be similar to a regular mAb. A 13

longer time-course PK study would be needed to calculate the precise terminal half-life. 14

15

Collectively, these studies demonstrate that GUCY2C-positive tumors can be preferentially 16

targeted with an anti-GUCY2C/anti-CD3ε bispecific. Our lead clinical candidate, PF-07062119 17

has shown potent single agent anti-tumor efficacy with PF-07062119 in multiple in vitro and in 18

vivo models. This activity can be further enhanced with immune checkpoint blockade agents, 19

which prevent T cell exhaustion, as well as with anti-angiogenesis agents, which could increase 20

T cell infiltration (Figure S9). Further evaluation of PF-07062119 along with these combination 21

agents will be pursued in clinical studies to examine their ability to reduce tumor burden in 22

patients with gastrointestinal cancers. 23




34

Acknowledgments: 1

2

We would like to thank Patrick Gaffney for support with statistical analyses, Andrew Gifford for 3

pharmacokinetics analyses support, Susan Benard for BIAcore analysis, Dawn DeThomas for 4

graphical illustrations, John Kreeger for pathology support, and John Hill, David Schaer, Steven 5

Pirie-Shepherd for discussions on the study design and manuscript. All funding for this study 6

was provided by Pfizer Worldwide R&D. 7

8

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29

Author Contributions: D. Mathur, A. Root, L. King, E. Rosfjord, E. Lavallie, L. Tchistiakova, 30

L. Bloom, A. Nguyen, P. Sapra designed the experiments. B. Bugaj-Gaweda, X. Tan, W. Fang, 31

S. Bisulco, J. Golas, J. Lucas, C. Stevens, R. Lawrence-Henderson, K. Kelleher, J. Kearney, E. 32

Upeslacis, J. Yao performed the experiments. D. Mathur, A. Root, L. King, J. Narula, C. Rohde, 33

C. Kamperschroer, B. Buetow, M. Guffroy, D. Fernandez analyzed the data. D. Mathur wrote the 34

manuscript. All authors have reviewed the manuscript. 35

36

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37

Table 1. Characterization of Colorectal Cancer Xenograft Models Tested for In Vivo 1

Efficacy 2

3

Tumor H-Score

(GUCY2C)

KRAS mutation BRAF mutation Differentiation

Status

LS1034 210 A146T WT Moderately

differentiated

HT55 180 WT N581Y Moderately

differentiated

LS174T 105 G12D WT Poorly differentiated

HCT116 0 G13D WT Poorly differentiated

PDX-CRX-

11201

185 G12V WT Moderately

differentiated

4

FIGURE LEGENDS 5

6

Figure 1. GUCY2C expression in gastrointestinal tumors and normal tissues. (A) H-scores 7

depicting GUCY2C cell surface expression across colorectal, pancreatic, gastric and esophageal 8

tumors (Red line = median H-score). Immunohistochemistry showing GUCY2C expression 9

(brown) in (B) Colorectal adenocarcinoma Stage I; (C) Colorectal adenocarcinoma Stage II; (D) 10

Colorectal adenocarcinoma Stage III; (E) Colorectal adenocarcinoma Stage IV; (F) Liver 11

Metastasis of colorectal adenocarcinoma (H = Hepatocytes; T = Tumor; dashed line = 12

tumor/normal boundary); (G) Gastric adenocarcinoma; (H) Esophageal adenocarcinoma; (I) 13

Normal colon and (J) Normal small intestine. Scale bar = 200 µm. 14

15




38

Figure 2. An anti-GUCY2C/anti-CD3ε bispecific selectively targets tumors versus normal 1

tissue. 2

(A) Schematic of an anti-GUCY2C/anti-CD3ε bispecific human IgG1 FcγR- diabody. (B) 3

Pharmacokinetics of a murine GUCY2C cross-reactive bispecific GUCY2C(M)-CD3 at 1 and 4

0.3 mg/kg showing an estimated half-life of 11 days in LS1034 tumor bearing female NSG mice 5

with human adoptive T cells transfer. (C) Immunohistochemistry showing GUCY2C expression 6

(left panel, brown staining) in LS1034 cell line xenograft tumor (top) and in the colon from an 7

LS1034 tumor bearing mouse (bottom). Middle panel shows biodistribution of the bispecific 8

GUCY2C(M)-CD3 (brown) in the same LS1034 tumor (top) and lack of bispecific uptake in the 9

colon (bottom), 7 days after dosing in an adoptive transfer study. Right panel shows infiltration 10

of human CD3 positive T cells (brown) in the bispecific treated tumors (top) but not in colons 11

from the same mice (bottom); Scale bar = 200 µm. (D) GUCY2C(M)-CD3 showed anti-tumor 12

activity in the LS1034 human T cell adoptive transfer model at 1 and 0.3 mg/kg (n = 10 13

mice/group, Q3DX3 dosing, p-value < 0.0001). 14

15

Figure 3. PF-07062119 demonstrates in vitro cytotoxic T lymphocyte mediated killing in 16

GUCY2C-positive tumor cells. 17

(A) Flow cytometry assay shows binding of PF-07062119 to naïve human T cells and to 18

HCT116-hGUCY2C cells, but not to GUCY2C negative HCT116 parental cells. (B) Receptor 19

density of GUCY2C in tumor cells measured using a flow cytometry based QuantiBrite assay 20

usign an anti-GUCY2C mAb labeled 1:1 with phycoerythrin. (C) PF-07062119 shows CTL 21

mediated killing in GUCY2C positive tumor cell lines (E:T ratio = 5:1, 48h assay). (D) PF-22




39

07062119 elicits dose dependent IFNγ release in GUCY2C positive tumor cells in the presence 1

of T cells (E:T ratio = 5:1, 48h post treatment). NB = no binding; NR = not reached. 2

3

4

Figure 4. PF-07062119 mediates anti-tumor efficacy in multiple in vivo models (A) 5

GUCY2C expression in LS1034 (CLX), HT55 (CLX), PDX-CRX-11201 (PDX), LS174T 6

(CLX), HCT116 (CLX) tumor models (Scale bar = 200 µm). (B) PF-07062119 induced anti-7

tumor efficacy in LS1034, HT55, PDX-CRX-11201, LS174T and lack of efficacy in GUCY2C-8

negative HCT116 subcutaneous established models using adoptive human T cell transfer. Mice 9

with established tumors of 150-200 mm3 were dosed with PF-07062119, a non-targeted CD3 10

bispecific control or PBS (vehicle) weekly upto three times for all models, and upto four times 11

for PDX-CRX-11201. Human T cells were administered intravenously 24h after the first 12

bispecific or vehicle dose (C) LS1034 colon orthotopic established tumor model using adoptive 13

human T cell transfer described in (B) with bispecific dosed weekly up to five doses. GUCY2C 14

expression (brown) in LS1034 orthotopic tumor and normal adjacent tissue. H&E staining 15

(purple) shows tumor and normal tissue boundary. Bioluminescence imaging showing complete 16

tumor regression with PF-07062119 at 0.3 mg/kg. (D) Flow cytometry assay showing PF-17

07062119 binding to CT26-mGUCY2C and lack of binding to CT26 cells (110 nM Ab 9H3-h 18

used for binding). (E) CT26-mGUCY2C tumors show membrane expression of GUCY2C 19

(brown) by immunohistochemistry (Scale bar = 200 µm). (F) PF-07062119 mediated efficacy in 20

CT26-mGUCY2C tumors in hCD3ε mice. Tumors staged to 50-80 mm3 were treated with PF-21

07062119 or non-targeted CD3 every 3 days up to 6 doses. 22

23




40

Figure 5. PF-07062119 recruits TILs to mediate cytotoxicity and shows combination benefit 1

with checkpoint blockade. 2

(A) PF-07062119 treatment shows dose dependent infiltration of CD3+ Tcells (top row; brown; 3

Scale bar = 300 µm). Granzyme B (second row, brown, Scale bar = 200 µm) was observed in 4

these CD3+ T cells (second row, brown, Scale bar = 300 µm) and was polarized towards 5

adjacent tumor cells (inset). PF-07062119 dose dependent upregulation of PD-L1 was observed 6

on tumors (third row, brown), concomitantly with tumor cell necrosis (bottom row, H&E; Scale 7

bar = 200 µm). (B) tSNE of CYTOF analyses from tumor infiltrating lymphocytes at Day 7 post 8

dosing, identified unique clusters 11 and 12 in PF-07062119 tumors. (C) Heatmap depicts 9

expression of Arcsinh transformed median intensities of each marker to represent the phenotypes 10

of each TIL cluster shown in (B). Expression values range from 0-8 with max expression 11

represented in red, median expression in white, and min expression in black (D) Combination of 12

a minimally active dose of PF-07062119 with Anti-PD-1 or Anti-PD-L1 shows combination 13

benefit compared to single agent alone. 14

15

Fig. 6. Combination study of PF-07062119 with Anti-VEGF-A mAb. 16

(A) Combination of a minimally efficacious dose of PF-07062119 in PDX-CRX-11201 tumor, 17

which are partially responsive to Anti-VEGF-A mAb treatment, results in a significant 18

combination benefit compared to single agents. (B) Increased CD3+ T cells (brown) were 19

observed in the PF-07062119 + Anti-VEGF-A combination treatment group compared to single 20

agents or non-targeted-CD3 treatment. 21




Published OnlineFirst January 29, 2020.Clin Cancer Res Divya Mathur, Adam R Root, Bozena Bugaj-Gaweda, et al. (PF-07062119) for the Treatment of Gastrointestinal CancersA Novel GUCY2C-CD3 T cell Engaging Bispecific construct

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