poster #ct141 a phase 1 trial of rtx-240, an allogeneic

A Phase 1 Trial of RTX-240, an Allogeneic Engineered Red Blood Cell With Cell-Surface Expression of 4-1BBL and Trans-Presented IL-15, in Patients With Advanced Solid Tumors

Omid Hamid1, Jason Luke2, Alexander Spira3, Geoffrey Kuesters4, Iga Sienczylo4, Gilad Gordon4, Melissa Johnson5 1The Angeles Clinic and Research Institute, a Cedars Sinai Affiliate, 2University of Pittsburgh, 3Virginia Cancer Specialists Research Institute, US Oncology Research, Johns Hopkins Medicine, 4Rubius Therapeutics, 5Tennessee Oncology, Sarah Cannon Research Institute

The American Association for Cancer Research Annual Meeting/April 10-15, 2021

Figure 3: RTX-240 Phase 1 Clinical Trial in Patients With Solid Tumors Trial Design

Peripheral blood immunophenotyping markers included: CD45, CD56 (NK), CD16 (NK), CD8, CD45RA, CCR7, HLA-DR, CD3, Ki67,

Granzyme B CD45, CD16 (NK), CD56 (NK), NKp30, NKG2D, NKp44, NKG2A, TRAIL, CD3, CD4, CD25, CD127, FoxP3, Ki67.

Immunofluorescence markers included: CD3, CD8, CD4, CD56, CD20, CD68, CD163, FoxP3, PD-1, PD-L1, DAPI, CK/SOX10,

Granzyme B

• Five cohorts are completed in the RTX-240 Phase 1 clinical trial in patients with solid tumors over 2 logs of dosing (1e8 to 1e10 Q4W-Q6W are completed)

• Two cohorts are open to enrollment to further explore dose and schedule (the 3e10 Q4W and a Q3W schedule at the 1e10 dose level are enrolling)

• Two routes of administration were assessed: intravenous (iv) only and 1 exploratory cohort of iv combined with 2 intratumoral (it) doses in Cycle 1

Table 1. Patient Baseline Characteristics

1Lower GI cancers include colorectal cancer (n=2), anal cancer (n=1), pancreatic ductal adenocarcinoma (n=1); melanoma includes

cutaneous melanoma (n=2), ocular melanoma (n=2), mucosal melanoma (n=1); and other cancers (all n=1), gastroesophageal,

mesothelioma, non-small cell lung, ovarian, prostate, soft tissue sarcoma, testicular germ cell tumor

Safety population N=16

Initial data cut-off: Feb 28, 2021

Table 2: Treatment Emergent Adverse Events by Grade Related to RTX-240 Observed in 2 or More Patients

Common Terminology Criteria for Adverse Events (CTCAE v5.0)

Safety population N=16


Observed Immune-Related Adverse Events (irAE)

• Ten irAE of Grade 1/2 were observed among 5 patients, no Grade 3/4 irAEs reported. Observed irAE with a median time to onset of 42 days from the first dose (range, 0-62 days)

• Grade 2 irAE include pneumonitis, adrenal insufficiency and hypothyroidism

• Grade 1 irAE include arthralgia (n=3), myalgia (n=2), AST elevation and ALT elevation

MONOTHERAPYRTX-240 Relapsed/refractory (R/R) or locally advanced solid tumors

Phase 1: Monotherapy in Solid Tumors �16 Patients Evaluable

1e8 �(n=2)

Q4W iv

1e9 �(n=3)

Q6W iv

3e9(n=3)

Q4W iv

1e10

�(n=4)Q4W iv

1e10

�(n=3)Q4W iv/it

1e10

Q3W ivOpen to

�Enrollment

3e10

Q4W iv(n=1)

Open to Enrollment

Further explore dose & schedule

PHARMACOKINETICS/PHARMACODYNAMICS: Fig 8. Peripheral Immunophenotyping

Maximum fold change is defined by the greatest result post treatment divided by baseline value. (A) Activation of NK cells is determined by fold increases in the percentage of NK cells positive for the late activation marker HLA-DR post RTX-240. (B) Expansion of the

NK cells is assessed by fold increases in the absolute number of NK cells post-RTX-240. (C) Activation of memory CD8+ T cells is determined by memory T cell expression of the late activation marker, HLA-DR. (D) Expansion of the memory CD8+ T cell population is

assessed by fold increases (from baseline) in the absolute numbers of circulating memory CD8+ T cells.

Safety, PD population: N=16 evaluable


Fig 9. Peripheral Immunophenotyping of NK Population

(A) Maximum fold change in the percent of lymphocytes that are CD16+CD56dim (mature NK cells); (B) Maximum fold change in the percentage of lymphocytes that are CD56bright (immature NK cells). (C) Maximum fold change in the ratio of expression of NKG2D and

NKG2A in NK cell population. (D) Maximum fold change in the percent of NK cells expressing NKp30.

Safety, PD population (high dose only): N=8 evaluable


TARGET CELL TRAFFICKING: Fig 10. Trafficking of NK and T cells to the Tumor Microenvironment

• Trafficking of both T and NK cells was observed in 2/4 patients with on-treatment tumor biopsies (1.8 to 10-fold increases). Shown in patients with metastatic mesothelioma (n=1), metastatic soft tissue sarcoma (n=1). Tumor infiltration was not observed in patients with ovarian cancer (n=1) and heavily pre-treated melanoma (n=1)

• Increased expression of PD-L1 and/or increased ratio of M1/M2 macrophages observed in 3/4 patients with solid tumors – Suggests improved immune-permissive tumor microenvironment, which may enhance innate and adaptive tumor-associated immune cell responses

• One case is shown: A 79-year-old female with mesothelioma. Treated with adjuvant pemetrexed/cisplatin at the initial diagnosis in 2016. Developed metastatic disease in 2017, treated with nivolumab and ipilimumab and palliative radiation. Enrolled in the RTX-240-01 study and was treated at the 1e9 dose level administered

EFFICACY:Table 3. Best Overall Response (BOR)

Patients are considered evaluable for BOR if receiving at least 1 dose of RTX-240 and at least 1 post-baseline imaging

assessment completed. 1 Two PRs observed, 1 confirmed and 1 is unconfirmed with both patients remaining on treatment in the study at the data

cutoff. Time to response is 16 weeks in both patients with PR; duration of response is 1 week and 16 weeks in the unconfirmed

and confirmed PR, respectively.


Efficacy population, N=15 evaluable

Figure 4: Waterfall Plot of Tumor Response by RECIST v1.1


• Scans were performed at baseline and every 8 weeks after the first dose. Best response by RECIST v1.1 is plotted by patient. Patients with prior PD-1/PD-L1 therapy are indicated (*)

• Patients are evaluable for best percent change with at least 1 measurable lesion (per RECIST v1.1) and a baseline and post-treatment imaging (n=14 pts evaluable, 1 patient evaluable for efficacy with non-measurable disease)

Figure 5: Time on Treatment and Response by RECIST v1.1 in All Patients

1 Patient discontinued due to clinical disease progression

Safety population: N=16 evaluable


INTRODUCTION

Immune agonists and recombinant cytokines are promising treatment approaches, but have shown limited success in the clinic.

• Limitations of agonist antibodies and recombinant cytokines include: – Severe toxicity leading to a narrow therapeutic index – Diminished activity of an agonistic antibody compared with the natural ligand, – Lack of multiple signals needed to effectively activate most cell types in the anti-tumor immune response

• RTX-240 is an allogeneic cellular therapy using genetically engineered red blood cells to express 4-1BB ligand (4-1BBL) and IL-15/IL-15Rα fusion (IL-15TP) in their natural conformation on the cell surface

• RTX-240 mimics human biology by broadly stimulating adaptive and innate immunity to generate an anti-tumor response

– Improved safety due to the restricted biodistribution of red blood cells to the vasculature and spleen

• Proposed increased efficacy given synergistic and complementary effects of 4-1BBL and IL-15TP when administered together

The Phase 1/2 clinical trial of RTX-240 is designed to determine the safety and tolerability, pharmacokinetics, maximum tolerated dose, and a recommended Phase 2 dose and dosing regimen of RTX-240 in adult patients with relapsed/refractory or locally advanced solid tumors or with relapsed/refractory acute myeloid leukemia (Phase 1 only). The trial is also assessing the pharmacodynamics of RTX-240 measured by changes in T and NK cell number and function relative to baseline and anti-tumor activity.

• Initial clinical results from the solid tumor portion of the Phase 1 study are reported here

Figure 1: RTX-240 Structure

• 4-1BBL and the cytokine interleukin-15 (IL-15TP, trans-presented) activate and expand natural killer (NK) and CD8 T cells

• 4-1BBL can drive T and NK cell proliferation and activation and interferon γ (IFNγ) production

• IL-15 and 4-1BB both bridge innate and adaptive immunity by promoting T and NK cell proliferation, and NK cell cytotoxicity

Figure 2. The RED PLATFORM® is Designed to Generate Allogeneic, Off-the-Shelf Cellular Therapies

• Red Cell Therapeutics™ (RCTs™) are a new class of allogeneic, off-the-shelf cellular therapeutic candidates for the treatment of cancer and autoimmune diseases

• RCTs are enucleated red blood cells that are genetically engineered to express hundreds of thousands of copies of multiple biotherapeutic proteins on the cell surface

• Scalable and consistent manufacturing process

RTX-240 Preclinical Observations

• RTX-240 increased CD8 T cell and NK cell expansion and activation in vitro when compared to the combination of a 4-1BB agonist antibody and rIL-15

• Expansion was directly correlated with the percentage of 4-1BBL and IL-15TP expressed on the cell surface

• RTX-240 expanded key NK subsets including CD56dim NK cells, induced the expression of key NK cytotoxicity receptors in vitro, and promoted NK cell killing of the K562 myeloid leukemia cell line

• A murine surrogate for RTX-240, mRBC-240, was effective in multiple murine tumor models, promoting significant expansion of CD8 T cells and NK cells in vivo as early as Day 4 post-treatment and the expansion of differentiated NK cells in the tumors

• RTX-240 demonstrated potent immune stimulation, antitumor activity, and no observed animal toxicity, leading to the potential for a wide therapeutic window in clinical studies

4-1BBL

IL-15TP

4-1BBL

IL-15TPBROAD IMMUNE

SYSTEM STIMULATION

T CELL

RTX-240 | (4-1BBL + IL-15TP)

NATURAL KILLER (NK) CELL

Figure 6: Confirmed PR in Metastatic Squamous Cell Cancer of the Anus (ASCC)

60-year-old female with metastatic ASCC, multiple nodal metastases. Diagnosed in May 2016, treated with surgery and adjuvant mitomycin C and 5FU. Developed nodal metastases (August 2017). First-line therapy with LY3300054 (anti-PD-L1). Second- line therapy with atezolizumab and KY1044 (anti-ICOS). PD-L1 status unknown. Disease burden includes 2 nodal target lesions (images) and 2 non-target nodal lesions (not included in images).

Treated with RTX-240 in the third-line setting. No adverse events reported. Time to response 16 weeks, duration of response 16 weeks. 54% reduction in target lesions. Patient remains on study at time of data cut-off.

Figure 7: Unconfirmed PR in Metastatic Uveal Melanoma

54-year-old male with metastatic uveal melanoma, multiple hepatic metastases. Diagnosed in August 2018, treated with enucleation and radiation. Developed liver metastases approximately 1 year later (July 2019). First-line therapy with nivolumab/ipilimumab (8 months prior to enrollment in RTX-240-01) with slow progression over the 8 months. Ipilimumab discontinued due to Grade 3 hepatitis. Treated with RTX-240 in the second-line setting. Overall burden of disease with 1 target lesion of 10 mm and >14 other evaluable lesions. Developed adrenal insufficiency and hypothyroidism early in Cycle 3 (Day 60 following the first dose of RTX-240, managed with steroids).

Complete regression of 14/15 hepatic metastases at 16 weeks with a single non-target hepatic lesion remaining. Time to response 16 weeks, duration of response, 1 week. Patient remains on study at time of data cut-off.

RED PLATFORM®

ONE �HEALTHY�O- DONOR

EXPANSION & �DIFFERENTIATION

PROGENITOR �CELL COLLECTION

LENTIVIRAL VECTORENCODING OF A

CO-STIMULATORYMOLECULE & CYTOKINE

ENUCLEATION & MATURATION

100-1000’s �OF DOSES

RED CELL THERAPEUTIC

METHODS

PARAMETERN=16 EVALUABLE

Age (median, range) 55 (23-80)

8/8

11

5

4

5

7

11

3 (1-10)

5

12

7

Gender (Male/Female)

Race/Ethnicity

Disease setting1

Colorectal/lower GI cancers, n

Prior anti–PD-1/PD-L1

Prior ipilimumab + nivolumab

Prior chemotherapy

Prior radiation

Non-Hispanic/Non-Latino

Hispanic/Latino

Melanoma, n

Other cancers, n

Lines of prior therapy in the metastatic setting, median (range)

RESULTS

ADVERSE EVENT PREFERRED TERMGRADE 1

n (%)GRADE 2

n (%)GRADE 3

n (%)GRADE 4

n (%)ANY GRADE

n (%)

Fatigue

Chills

Decreased Appetite

Arthralgia

Nausea

Pyrexia

Myalgia

Dysgeusia

Hyperhidrosis

2 (13)

3 (19)

3 (19)

3 (19)

3 (19)

2 (13)

2 (13)

2 (13)

2 (13)

2 (13)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4 (25)

3 (19)

3 (19)

3 (19)

3 (19)

2 (13)

2 (13)

2 (13)

2 (13)

RESPONSE EVALUATIONN=15 EVALUABLE

Complete response

Partial response

Stable disease

Progressive Disease

Disease control (SD, >12 weeks or PR)

0

21

6

7

6

100

50

0

-50

-100

* * * * * * * * * *

cPR(ongoing)

uPR(ongoing)

*

Be

st P

erc

en

t C

han

ge

in T

um

or

Siz

e fr

om

Bas

elin

e

0 50 100 150 200 250

Time of Treatment (days)

Partial response

Metastatic Anal Cancer

Metastatic Uveal Melanoma

Stable disease

Progressive disease

Treatment ongoing with RTX-240

Treatment arm: 1e8 Q4W 1e9 Q6W 3e9 Q4W 1e10 iv Q4W 1e10 iv/it Q4W 3e10 iv Q4W

BaselineJune 2020

C7D1Dec 2020

-51% targets (PR)

C9D1Feb 2021

-54% targets (PR)

C3D1Aug 2020

-17% targets (SD)

C5D1Oct 2020

-34% targets (PR)

BaselineOct 2020

Target Lesion (10mm)

Cycle 3 Day 1Dec 2020

Target Lesion (SD, 10 mm)

Cycle 5 Day 1Feb 2021

Target Lesion (-100%, PR)

BaselineOct 2020

Non-Target Lesion


Non- Target Lesion


Non-Target Lesion

BaselineOct 2020

Non-Target Lesion


Non- Target Lesion


Non-Target Lesion

Hepatic target lesion

A. Maximum Fold Change in the Percent of NK Cells Expressing HLA-DR

Max

imu

m F

old

Ch

ang

e f

rom

Bas

elin

e 4

3

2

1

Low Dose High Dose

0

Baseline

Baselin

e

Post-tr

eatment

Baselin

e

Post-tr

eatment

B Maximum Fold Change in the Absolute Number of Circulating NK Cells

Max

imu

m F

old

Ch

ang

e f

rom

Bas

elin

e 3

2

1

0

Baseline

Baselin

e

Post-tr

eatment

Baselin

e

Post-tr

eatment

Low Dose High Dose

C Maximum Fold Change in the Percent of Memory CD8+ T Cells

Expressing HLA-DR

Max

imu

m F

old

Ch

ang

e f

rom

Bas

elin

e 6

4

2

0

Baseline

Baselin

e

Post-tr

eatment

Baselin

e

Post-tr

eatment

Low Dose High Dose

D Maximum Fold Change in the Absolute Number of Circulating

Memory CD8+ T Cells

Max

imu

m F

old

Ch

ang

e f

rom

Bas

elin

e 4

3

2

1

0

Baseline

Baselin

e

Post-tr

eatment

Baselin

e

Post-tr

eatment

Low Dose High Dose

Low dose = 1e8-3e9 cells/dose (n=8);

High dose = 1e10-3e10 cells/dose (n=8)

A BMaximum Fold Change in the Percent of Lymphocytes

that are CD16+CD56dim

Maximum Fold Change in the Percent of Lymphocytes that

are CD56bright

Max

imu

m F

old

Ch

ang

e f

rom

Bas

elin

e

Max

imu

m F

old

Ch

ang

e f

rom

Bas

elin

e

4

5

3

2

1

High Dose

0

20

1510

5

0

Baseline

Baseline

Baselin

e

Post-tr

eatment

Baselin

e

Post-tr

eatment

High Dose

C DMaximum Fold Change in the Ratio of Expression of

NKG2D/NKG2A of All NK Cells

Maximum Fold Change in the Percent of NKp30+NK Cells

High Dose

Max

imu

m F

old

Ch

ang

e f

rom

Bas

elin

e 5

4

3

2

0

1 Baseline

Baselin

e

Post-tr

eatment

High Dose

Max

imu

m F

old

Ch

ang

e f

rom

Bas

elin

e 3

2

1

0

Baseline

Baselin

e

Post-tr

eatment

High dose = 1e10-3e10 cells/dose (n=8)

Archival diagnostic biopsy On-treatment biopsy Day 34 (2 iv doses) On-treatment biopsy Day 34 (2 iv doses)

GmzB CD3 (T cells) CD8 (T cell) CD56 (NK cell)

• 10-fold increase in % of NK cells

• More than 4-fold increase in CD8+ T cells

• Improved immune-permissive environment via increase in PD-L1 expression

On-treatment, % (fold change)Baseline, %

% NK (CD56) of all cells

% All T (CD3) of all cells

% Cytotoxic T (CD8) of all cells

PD-L1+ cells

0.4

4

2

29

4.5 (10-fold)

16 (4-fold)

8.8 (>4-fold)

51 (1.8-fold)

CONCLUSIONS

• RTX-240 has a favorable safety profile at the dose levels tested with no related Grade 3/4 adverse events, minimal irAEs and no dose limiting toxicities observed to date

• RTX-240 activated and expanded both T and NK cells, which led to 2 partial responses in anal cancer and metastatic uveal melanoma

• RTX-240 induced tumor trafficking of both NK and T cells into the tumor microenvironment in solid tumors

• RTX-240 induced activation and expansion along with tumor trafficking of the two key target cell subsets in the innate and adaptive immune system: the NK cells and memory CD8+T cells

• Enrollment continues in the Phase 1 portion of the study. RTX-240 is expected to enter a Phase 1 combination trial with an anti-PD-1 therapy in 2H'21 and a Phase 2 trial in specific tumor types in 2022

Poster #CT141

poster #ct141 a phase 1 trial of rtx-240, an allogeneic

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