A phase I multicenter study of enadenotucirev in combination with nivolumab in tumors of epithelial origin: an analysis of the metastatic colorectal cancer patients in the dose escalation phase

Marwan Fakih1, Ding Wang2, Wael Harb3, Lee Rosen4, Daruka Mahadevan5, Jordan Berlin6, Paul Basciano7, Olajumoke Arogundade8, Chris Cox8, David Krige8, Hilary McElwaine-Johnn8, Richard Brown8.

1 City of Hope Comprehensive Cancer Center, Duarte, California, USA; 2 Henry Ford Hospital, Detroit, USA; 3 Horizon Oncology Research Inc., Lafayette, Indiana, USA; 4 UCLA Medical Center, Los Angeles, California, USA; 5 University of Arizona Cancer Center, Tucson, Arizona, USA; 6 Vanderbilt-Ingram Cancer Center, Nashville, USA; 7 Bristol Myers Squibb, Lawrenceville, New Jersey, USA; 8 PsiOxus Therapeutics Ltd, Abingdon, Oxford, United Kingdom.

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Abstract

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Enadenotucirev (EnAd) is a tumor-selective chimeric Ad11/Ad3 group Boncolytic adenovirus that does not express any transgenes (a so called“unarmed virus”) [Khun et al, 2008]. Following intravenous (IV) dosingof EnAd to patients with primary colon cancer in a phase I study,effective viral delivery has been demonstrated together with intra-tumoral CD8+ T-cell infiltrates [Garcia-Carbonero et al, 2017]. A furtherphase I clinical study identified a well-tolerated dose and regimen forEnAd monotherapy, including in patients with metastatic colorectalcancer (mCRC) [Machiels et al, 2019].Immune checkpoint inhibitors (ICIs) have shown impressive clinicalactivity in several tumor types, including microsatellite instability (MSI)high mCRC [Le et al, 2015]. However, there is still a significantproportion of tumors which appear to have a primary resistance to ICItherapy, including MSI low and microsatellite stable (MSS) mCRC.Combination treatment strategies using ICIs together with immunestimulatory EnAd may help to overcome ICI resistance in MSI low / MSSmCRC and other ICI resistant tumor types.The SPICE study is a phase I study to examine the safety and potentialefficacy of the combination of EnAd and nivolumab in a range ofepithelially derived solid tumor types previously shown to haveprimary or to have developed secondary ICI resistance (squamous cellhead and neck (SCCHN), non-small cell lung (NSCLC), urothelialcarcinoma (UCC) and MSI low / MSS mCRC). The study design is dividedinto a dose escalation phase and a dose expansion phase. Here wepresent an analysis of the 34 mCRC patients dosed to date with EnAdand ICI therapy in the escalation phase of the SPICE study.

1. Background

EnAd was escalated in combination with ICI therapy in patients withmetastatic epithelial tumors in a 3 + 3 design. Subjects receivedincreasing dose levels and/or cycles of EnAd followed by up to 8 cyclesof ICI as shown in Figure 1. In the first cohort the ICI used waspembrolizumab, but from cohort 2 onwards this was switched tonivolumab. Cohort 7 is designed to examine an initial low dose of EnAdfollowed by higher doses. It is hypothesized that the initial low primingdose in this low:high:high (L:H:H) regimen may desensitize forsubsequent doses and thus reduce post-infusional cytokine release andthe associated adverse events.Key Inclusion Criteria include:• Patients 18 years or older• Diagnosis of metastatic or advanced CRC, UCC, SCCHN or NSCLC• Not responding to standard therapy or no standard treatment exists• Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1• No proteinuria and adequate renal, hepatic, bone marrow andcoagulation tests (each as defined per protocol)Key Exclusion Criteria include:• Prior treatment with PD-1 and/or PD-L1 inhibitors• Investigational drug administration in the prior 28 days• Symptomatic brain metastasesThe primary objective of the dose escalation phase was to evaluatesafety of the combination and to identify a recommended dose andregimen for expansion. Secondary endpoints included overall responserate (ORR), median progression free survival (mPFS) and median overallsurvival (mOS).Pharmacodynamic (PD) biomarkers included the longitudinalmeasurement of systemic cytokines and anti-viral antibodies in allpatients. Wherever possible pre- and post-treatment tumor biopsieswere collected and analyzed for signs of enadenotucirev infection andchanges in inflammatory biomarkers. The tumor biomarker analyticalmethodologies included immunohistochemistry (IHC), automated imageanalysis and mRNA analysis by Nanostring®.

2. Methods

Of the 40 patients enrolled in the study, 34 patients had a diagnosis of mCRCwith the characteristics as summarized in Table 1. The EnAd dose rangedbetween 1x1012 and 3x1012 virus particles infused IV for one or two cycles,followed by 360mg of nivolumab every 3 weeks, as shown in Figure 2.

3. Patient characteristics and disposition

Figure 1: Study design

The SPICE study is conducted at 6 clinical sites in the United States of America. The dose

escalation phase of the study uses a 3+3 design to escalate the dose and regimen of

enadenotucirev in combination with nivolumab. The progression through the 7 cohorts

investigated to date are shown below:

Treatment in each cohort consists of up to 8 x 21-day cycles.

Enadenotucriev is administered on

• Days 1, 3 & 5 of cycle 1 (cohorts 2, 3 & 7), or

• Days 1 ,3 & 5 of cycle 1 & 2 (in cohorts 1, 4, & 5)

• Days 1, 3, 5, 8, 10 & 12 of cycle 1 (cohort 6)

Nivolumab is administered on

• Day 8 of each cycle (cohorts 4 & 5), or

• Day 15 of each cycle (all other cohorts)

Table 1: Patient characteristicsA summary of the key characteristics of the 34 mCRC patients recruited to date in the

dose escalation phase of the SPICE study is summarized in the table below.

Figure 2: Patient disposition and follow-up

The disposition and follow-up of the 34 mCRC patients across the 7 dose escalation cohorts

are shown in the swim lane plot below. The cut-off date for this data is 6th September 2019

and follow-up is ongoing. RECIST v1.1 assessments shown are from the central reader where

available.

Table 2: Adverse event summary

The common treatment-emergent adverse event incidence for all 40 SPICE patients

dosed to date (including the 34 mCRC patients) is summarized in the table below:

Note: The prolonged aPTT is believed to be due to Lupus Anticoagulant. Five patients with

prolonged aPTT experienced a total of 4 bleeding events (one post study due to progressive

appendix cancer) and 1 clotting event (pulmonary embolism during study). Two patients with

normal or near-normal aPTT experienced 2 bleeding events and 1 clotting event (1 DVT post

study). The incidence is not considered to be excessive considering the population treated,

but patients will continue to be monitored.

*

* Occurring in ≥20% patients overall

Table 3: Serious Adverse Event summary

The serious adverse event incidence for all 40 SPICE patients dosed to date (including

the 34 mCRC patients) is summarized in the table below

Figure 3: Patient case studyThe patient with a confirmed PR shown in Figure 2, is a 46yo female with stage IVb, MSI-Low

mCRC (MSI status confirmed by both sequencing and immunohistochemistry) with a BRAF

V600E mutation, KRAS wild type and low mutational burden (5 mutations per megabase)

phenotype.

This patient had received three prior lines of treatment: (1) FOLFOX (Best response: stable

disease); (2) FOLFIRI + bevacizumab (Best response: unknown); (3) Cetuximab + encorafenib

+ binimetinib (Best response: unknown). The start of nivolumab treatment was delayed in this

patient compared to the rest of the mCRC patients.

Panel 3a Revised nivolumab dosing schedule for this patient

Panel 3b Investigator assessment of tumor response by RECIST v1.1

Panel 3c Example target lesion at screening

Panel 3d Example target lesion at week 18

3a 3b

3c 3d

The adverse event profile is summarized in Tables 2 and 3. Flu-likesymptoms were reported in the majority of patients following EnAdadministration. Adverse events of infusion reaction and hypoxia meetingDLT criteria were reported on C2D1 administration of EnAd requiringincreased prophylaxis. Cases of severe acute renal injury (includingmembrano-glomerulonephritis indicative of immune complex deposition)and severe acute lung injury have been reported. Measures to prevent andmitigate against these adverse events have been added to the ongoingprotocols. Additional urinalysis monitoring revealed changes suggestive ofpost-infectious GN in ~25% of patients after 3-4 weeks of EnAd dosing.These were largely asymptomatic and resolved without treatment.Prolonged aPTT has been observed in 6 patients but the laboratoryevidence and incidence of thrombotic and hemorrhagic events suggestthat this is an in vitro artefact associated with Lupus Anticoagulant, aspreviously described with other adenoviral vectors [Malaeb 2005].

4. SafetyFigure 4: Overall survival

Figure 5: Progression free survival

Figure 6: Immune cell infiltrates

8 patients with a diagnosis of mCRC had matching pre- and post-treatment tumor biopsy samples with

sufficient tumor and stromal tissue for IHC staining and automated image analysis. Cell counting by automated

image analysis demonstrates evidence of increased inflammation in 6 out of 8 of these patients. Post-

treatment biopsies were taken ~5 weeks after first viral dosing.

Panel 6a IHC staining for intratumoral CD8+ cells: example pre- versus post treatment

Panel 6b IHC staining for PD-L1 + immune cells: example pre- versus post treatment

Panel 6c Intratumoral CD8+ cell infiltrates: Median 4.7 fold increase from baseline (mean 34 fold increase)

Panel 6d Stromal CD8+ cell infiltrates. Median 2 fold increase from baseline (mean 1.3 fold increase)

Panel 6e Percentage infiltrating PD-L1+ immune cells. Median 2 fold increase from baseline (mean 2.2 fold

increase)

6c

6d

6e

6a

6b

Pre-treatment Post-treatment

Pre-treatment Post-treatment

There was one confirmed partial response (ORR = 2.9%) and stable diseaseof 4 or more months was seen in 7/34 (21%) patients. The patient with aconfirmed PR shown in Figure 3, is a 46yo female with stage IVb, MSI-LowmCRC (MSI status confirmed by both sequencing andimmunohistochemistry) with a BRAF V600 mutant, KRAS wild type and lowmutational burden (5 mutations per megabase) phenotype. As of 6thSeptember 2019, with a median follow-up of 11 months, mOS is 14.0months (95% CI = 11.0m, 16.5m) and mPFS is 1.6 months (95% CI = 1.5m,2.8m). The Kaplan Meier estimates are shown in Figures 4 and 5respectively. For the subset of 26 patients with confirmed MSS/MSI lowstatus, the mOS was 15.4 months (95% CI 11.8m, 16.5m).

5. Efficacy

PD biomarkers show evidence of inflammation in 6 out of the 8 patientswith matched pre- and post-treatment tumor biopsy samples [Figure 6].Intratumoral CD8+ T cell infiltration showed a median 4.7 fold increase frompre-treatment levels whilst the stromal levels increased by a median of 2fold. There was also evidence in some patients of increases in CD8+ T cellproliferation (Ki67) and cytolytic activity (Granzyme B) as well as increasesin the number of PD-L1+ immune cells. Kinetics, cytokines and anti-drugantibody responses were consistent with known profiles for both agents.Initial data from cohort 7 suggests that a L:H:H dosing regimen may reducethe levels of acute cytokines released following administration and may beassociated with an improved tolerability profile.

6. Biomarkers

The combination of enadenotucirev and nivolumab in patients with heavilypre-treated MSI low or MSS mCRC has a manageable tolerability profile andis associated with preliminary but encouraging signals of efficacy, includinga median overall survival estimate of 14 months. These data support furtherinvestigation of this combination in epithelial tumors with primary orsecondary resistance to checkpoint inhibitors. These data with thegenetically unmodified oncolytic enadenotucirev virus are also supportiveof a range of follow-on genetically modified or “armed” viruses usingenadenotucirev as a cancer gene therapy vector. These gene therapyproducts are expected to have the oncolytic and pro-inflammatory activityof enadenotucirev but with the enhancement of the added co-delivery oftherapeutic transgenes. Several such Tumor Specific Immuno-gene Therapy(T-SIGn) products are currently in phase I clinical trials.

7. Discussion

Khun I et al, 2008: Directed Evolution Generates a Novel Oncolytic Virus for the Treatment of Colon Cancer. PLoSONE 3(6): e2409.Garcia-Carbonero R et al, 2017: Phase 1 study of intravenous administration of the chimeric adenovirusenadenotucirev in patients undergoing primary tumor resection. Journal for Immuno Therapy of Cancer Vol 5, Article71Machiels JP et al, 2019: A phase 1 dose escalation study of the oncolytic adenovirus enadenotucirev, administeredintravenously to patients with epithelial solid tumors (EVOLVE). Journal for Immuno Therapy of Cancer Vol 7, Article20Le DT et al, 2015. PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. N Engl J Med. 372(26):2509-20Malaeb BS et al. 2005: Elevated activated partial thromboplastin time during administration of first-generationadenoviral vectors for gene therapy for prostate cancer: identification of lupus anticoagulants. Urology. 66(4):830-4

References

Clinical trial information: EUDRACT Number 2017-001231-39