Cancer Therapy: Preclinical
`
`Defining Effective Combinations of Immune
`Checkpoint Blockade and Oncolytic Virotherapy
`Juan J. Rojas1, Padma Sampath1, Weizhou Hou1, and Steve H. Thorne1,2
`
`Clinical
`Cancer
`Research
`
`Downloaded from http://aacrjournals.org/clincancerres/article-pdf/21/24/5543/2029348/5543.pdf by guest on 20 September 2022
`
`Abstract
`
`Purpose: Recent data from randomized clinical trials with
`oncolytic viral therapies and with cancer immunotherapies have
`finally recapitulated the promise these platforms demonstrated in
`preclinical models. Perhaps the greatest advance with oncolytic
`virotherapy has been the appreciation of the importance of
`activation of the immune response in therapeutic activity. Mean-
`while, the understanding that blockade of immune checkpoints
`(with antibodies that block the binding of PD1 to PDL1 or CTLA4
`to B7-2) is critical for an effective antitumor immune response has
`revitalized the field of immunotherapy. The combination of
`immune activation using an oncolytic virus and blockade of
`immune checkpoints is therefore a logical next step.
`Experimental Design: Here, we explore such combinations
`and demonstrate their potential to produce enhanced responses
`in mouse tumor models. Different combinations and regimens
`were explored in immunocompetent mouse models of renal and
`
`colorectal cancer. Bioluminescence imaging and immune assays
`were used to determine the mechanisms mediating synergistic or
`antagonistic combinations.
`Results: Interaction between immune checkpoint inhibitors
`and oncolytic virotherapy was found to be complex, with correct
`selection of viral strain, antibody, and timing of the combination
`being critical for synergistic effects. Indeed, some combinations
`produced antagonistic effects and loss of therapeutic activity. A
`period of oncolytic viral replication and directed targeting of the
`immune response against the tumor were required for the most
`þ
`þ
`beneficial effects, with CD8
`and NK, but not CD4
`cells medi-
`ating the effects.
`Conclusions: These considerations will be critical in the design
`the inevitable clinical
`translation of
`these combination
`of
`approaches. Clin Cancer Res; 21(24); 5543–51. Ó2015 AACR.
`See related commentary by Slaney and Darcy, p. 5417
`
`Introduction
`The last 5 years have seen the emergence of antibody-mediated
`blockade of immune checkpoints as a key new weapon in the
`anticancer arsenal (1, 2). The anti-CTLA4 inhibitor ipilimumab
`has been approved for the treatment of melanoma (3, 4), while a
`panel of monoclonal antibodies targeting the interaction of PD1
`and PDL1 have also demonstrated promising responses in a
`succession of clinical trials (5, 6). Together, these trials have
`demonstrated the clinical need to overcome the tumor's capacity
`to shut down the T-cell response in the creation of an effective
`cancer immunotherapy.
`The field of oncolytic virotherapy has also recently demon-
`strated its potential to produce clinically effective cancer treat-
`ments, with data from several recent randomized trials resulting in
`impressive response rates (7, 8). One factor that has united the
`most successful oncolytic vectors has been the expression of an
`immune-activating transgene (GM-CSF), an indication that a key
`
`1Department of Surgery, University of Pittsburgh Cancer Institute,
`University of Pittsburgh, Pittsburgh, Pennsylvania. 2Department of
`Immunology, University of Pittsburgh Cancer Institute, University of
`Pittsburgh, Pennsylvania.
`
`Note: Supplementary data for this article are available at Clinical Cancer
`Research Online (http://clincancerres.aacrjournals.org/).
`
`Corresponding Author: Steve H. Thorne, University of Pittsburgh Cancer Institute,
`1.46e Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA 15213. Phone:
`412-623-4896; Fax: 412-623-2525; E-mail: ThorneSH@UPMC.edu.
`
`doi: 10.1158/1078-0432.CCR-14-2009
`Ó2015 American Association for Cancer Research.
`
`determinant of the activity of oncolytic viruses is their capacity to
`activate and target the immune response (9, 10). This has since
`been confirmed in a multitude of preclinical studies (11–13),
`such that the oncolytic virus platform might best be considered an
`immunotherapeutic.
`We have previously developed several oncolytic vectors, pri-
`marily focusing on vectors based on vaccinia virus (14–17).
`These provide several advantages as immunotherapies beyond
`their long historical use as vaccines: (i) They can induce an
`adaptive immune response raised against tumor antigens as a
`result of their selective replication within the tumor microen-
`vironment (18, 19). This in situ vaccination effect results in
`production of CTL targeting relevant tumor antigens without
`the need for any prior interrogation of the tumor. (ii) Viral
`replication within the tumor can at least transiently overcome
`localized immunosuppression, something that most traditional
`vaccine approaches fail to achieve. However, in many cases,
`once the oncolytic virus is cleared by the host immune response,
`the immunosuppressive environment is apparently restored and
`the tumor relapses. The combination of oncolytic virus and the
`blockade of
`immune checkpoint
`inhibitor therefore is an
`appealing strategy.
`Although there has been much interest in this combination,
`including the proposed clinical combinations of the oncolytic
`HSV T-Vec (Amgen) and ipilimumab (Yervoy, Bristol Myers
`Squibb) in the treatment of melanoma (Clinical Trials.gov
`NCT01740297), there have been very little supportive data
`reported to date. Here, we examine the combination of oncolytic
`vaccinia with several different immunotargeting monoclonal
`antibodies.
`
`www.aacrjournals.org
`
`5543
`
`Replimune Limited Ex. 2017 - Page 1
`Transgene and Bioinvent International AB v. Replimune Limited
`PGR2022-00014 - U.S. Patent No. 10,947,513
`
`

`

`Downloaded from http://aacrjournals.org/clincancerres/article-pdf/21/24/5543/2029348/5543.pdf by guest on 20 September 2022
`
`Rojas et al.
`
`Translational Relevance
`Two of the most promising novel therapeutic platforms for
`the treatment of cancer are blockade of immune checkpoints
`and oncolytic viral therapies. Here, we look to combine these
`in preclinical mouse tumor models. The realization that inhi-
`bition of immune checkpoints is a critical need for successful
`immunotherapy and that the immune response activated by
`oncolytic viral therapies provide their most potent antitumor
`effects, means that the combination of these approaches is
`likely to result in significant clinical benefit. The enthusiasm in
`this combination is seen with the ongoing clinical combina-
`tion of the oncolytic T-Vec with ipilimumab. However, there
`has been almost no preclinical data reported to support this
`combination to date. In this article, we not only demonstrate
`that clinically relevant combinations can produce significantly
`enhanced responses in mouse tumor models, but also provide
`mechanistic insight into why some combinations are syner-
`gistic and others resulted in complete loss of therapeutic
`advantage.
`
`Materials and Methods
`Cell culture and viruses
`Renca (murine renal adenocarcinoma) cell line was obtained
`from ATCC. MC38 cell line (murine colon adenocarcinoma) was
`a kind gift from Dr. David Bartlett (University of Pittsburgh
`Cancer Institute, Pittsburrgh, PA). Cell lines were maintained in
`
`recommended culture media containing 5%–10% FBS at 37
`C,
`5% CO2. Cell lines have not been authenticated by the authors
`beyond their ability to form tumors in syngeneic mouse models.
`All recombinant vaccinia strains used in this work are derived
`from the Western Reserve (WR) strain (BEI Resources). The
`double-deleted strains vvDD and WR.B18R-.TK- (B18R- in short)
`have been described previously (15, 20). These contain deletions
`in the tk gene and in the vgf or B18R viral genes, respectively. In
`addition, both strains express the firefly luciferase gene from the
`synthetic vaccinia promoter pE/L (21), which allows monitoring
`of luciferase expression as a surrogate indicator of viral replication
`(22). Viruses were titered, manufactured, and purified as previ-
`ously described (23).
`
`Animal models
`All animal studies were approved by the University of Pitts-
`burgh Institutional Animal Care and Use Committee. C57/BL6
`and BALB/c female mice (6–8 weeks old) were purchased from
`The Jackson Laboratory. Renca or MC38 tumor cell lines were
`implanted subcutaneously at 5  105 cells per mouse into BALB/c
`or C57/BL6 mice, respectively. Oncolytic vaccinia viruses were
`injected intravenously (tail vein) at 2  108 pfu/mouse when
`tumors reached approximately 50 to 100 mm3.
`Anti-mouse CTLA4 (9D9) and anti-mouse CD25 (PC-61.5.3)
`antibodies (BioXCell) were injected intraperitoneally at 100 or
`200 mg/mouse/dose, respectively, with treatments consisting of
`3 doses each 3 days apart. Mouse IgG2b k Isotype Control
`(BioXCell) was used as a control. For depletion experiments,
`anti-mouse CD8 (2.43), anti-mouse CD4 (GK1.5), and anti-
`IFNg
`mouse
`(XMG1.2) were purchased from BioXCell,
`and anti-mouse Asialo-GM1 was purchased from Wako Pure
`
`Chemicals (Richmond, VA). Mice were injected intraperitoneally
`with 500 mg at days 1 and 2 after tumor implantation, followed by
`250 mg injection every 5 days till the end of the experiment.
`Tumor volume was monitored by caliper measurement and
`defined by V(mm3) ¼ p/6  W2  L, where W and L are the width
`and the length of the tumor, respectively. Data are expressed as
`tumor size relative to the beginning of the therapy (100%). For
`Kaplan–Meier survival curves, end point was established at 750
`mm3. Animals whose tumor size never achieved the threshold
`were included as right-censored information.
`
`Bioluminescence imaging
`Viral gene expression was determined through biolumines-
`cence imaging of luciferase expression in vivo. A dose of 4.5 mg
`of D-luciferin (GoldBio) was injected intraperitoneally per mouse
`before imaging on an IVIS2000 (PerkinElmer; 2% isoflurane).
`Images were analyzed using LivingImage software (PerkinElmer).
`
`IFNg ELISPOTs
`For ELISPOT assays, splenocytes were mixed with tumor cells or
`splenocytes from na€ve mice infected with UV-inactivated vaccinia
`virus at 5:1 ratio. Na€ve splenocytes were used as control. 96-well
`membrane filter plates (EMD Millipore) coated with 15 mg/mL of
`monoclonal anti-mouse IFNg antibody AN18 (Mabtech, Inc.)
`were used. Cells were maintained for 48 hours at 37 oC and spots
`were detected using 1 mg/mL of biotinylated anti-mouse INFg
`antibody R4-6A2-biotin (Mabtech). Plates were developed using
`an ABC kit and an AEC substrate kit for peroxidase (Vector
`Laboratories, Inc.). Specific spots were counted and analyzed using
`an ImmunoSpot Analyzer and ImmunoSpot software from CTL.
`
`Flow cytometry
`Tumors were harvested from mice and mechanically disaggre-
`gated and digested with triple enzyme mixture (Collagenase type
`IV, DNase type IV, and Hyaluronidase type V, Sigma-Aldrich)].
`Cell surface and intracellular immunostaining analyses were
`performed using a Gallios Flow Cytometer (Beckman Coulter,
`Inc.). For intracellular staining, cells were fixed and permeabilized
`using a Foxp3 Fix/Perm Buffer Set (eBioscience). Tumor-disag-
`gregated cells were stained using PE-Cy7 anti-mouse CD3 (BD
`Biosciences), eFluor450 anti-mouse NKp46, APC anti-mouse
`NKg2D, FITC anti-mouse CD4, PerCP-Cy5.5 anti-mouse CD8,
`PE anti-mouse CD25, and APC anti-mouse Foxp3 antibodies
`(eBioscience).
`
`Statistical analysis
`Standard Student t tests (two-tailed) were used throughout
`this work, except for the comparison of survival curves, where a
`log-rank test was used. In all cases, significance was achieved if
`P< 0.05.
`
`Results
`Anti-CTLA4 antibody hinders vaccinia virus replication in mice
`Mice harboring syngeneic subcutaneous mouse renal adeno-
`carcinomas (Renca cells) were injected with a single intravenous
`dose of oncolytic vaccinia virus and with three intraperitoneal
`doses of 100 mg of mouse anti-CTLA4 antibody at days 0, 3, and 6
`after virus injection. The schedule and doses of anti-CTLA4
`antibody used were determined on the basis of previously
`
`5544
`
`Clin Cancer Res; 21(24) December 15, 2015
`
`Clinical Cancer Research
`
`Replimune Limited Ex. 2017 - Page 2
`Transgene and Bioinvent International AB v. Replimune Limited
`PGR2022-00014 - U.S. Patent No. 10,947,513
`
`

`

`Downloaded from http://aacrjournals.org/clincancerres/article-pdf/21/24/5543/2029348/5543.pdf by guest on 20 September 2022
`
`Oncolytic Virus Combination with Checkpoint Inhibitors
`
`published preclinical studies (24–26). Initially, we looked to
`determine the safety of the combination and the effects of injec-
`tion of anti-CTLA4 antibody on the replication of vaccinia in the
`tumors. We monitored viral luciferase transgene expression with
`bioluminescence imaging as ourselves and others have shown this
`to directly correlate with viral replication (22, 27). Anti-CTLA4
`antibody significantly reduced viral luciferase expression from
`within the tumors (at days 3 and 5 after virus injection, >5- and 40-
`fold reduction was detected; Fig. 1A). A similar depletion in viral
`replication was also observed in a second tumor model (MC38
`tumors implanted subcutaneously in C57/B6 mice, Supplemen-
`tary Fig. S1B), demonstrating that this was not a cell line- or mouse
`strain–associated effect. Reduced viral replication did, however,
`correlate with enhanced immune activation, as seen with the
`increased numbers of CTLs recognizing vaccinia epitopes detected
`in the spleens of the mice (Fig. 1B). Although increased antiviral
`CTL appeared as early as day 3 after treatment, it is likely that
`innate immune responses may also be enhanced with the com-
`bination as viral replication is reduced as early as 24 hours after
`treatment. This indicated a more robust immune response was
`raised in mice when anti-CTLA4 antibody was injected together
`with the viral therapy.
`
`Delayed administration of anti-CTLA4 antibody improves
`antitumor efficacy
`A novel schedule for oncolytic vaccinia and anti-CTLA4 anti-
`body combination was therefore designed to permit an initial
`phase of viral oncolytic activity before anti-CTLA4 antibody
`administration (Fig. 1C). Anti-CTLA4 doses were therefore
`injected at days 4, 7, and 10 after virus injection, allowing an
`initial phase of unhindered viral replication and spread within the
`tumor (Supplementary Fig. S1C). Whereas simultaneous injec-
`tion of vaccinia virus and anti-CTLA4 antibody resulted in no
`significant antitumor benefit (compared with mice treated with
`single vaccinia therapy), when we delayed administration of the
`blocking antibody until after the peak of viral replication, a greater
`than 3-fold reduction (P < 0.04) in tumor volume was observed
`(isotype control antibody had no effect on tumor growth; Fig. 1D
`and Supplementary Fig. S1A). In addition, this novel treatment
`schedule was able to significantly increase survival of mice relative
`to groups treated either with single vaccinia therapy or vaccinia
`injected concurrently with anti-CTLA4 (Fig. 1E).
`
`Combination of anti-CD25 antibody with vaccinia provided no
`therapeutic benefit
`As an alternative to immune checkpoint blockade therapy
`(anti-CTLA4), we looked to test whether other approaches that
`also target tumor immunosuppression [such as depletion of
`regulatory T cells (Treg) with anti-CD25 therapy] also synergized
`with oncolytic vaccinia therapy. We again initially monitored
`virus replication in tumors via luciferase bioluminescence imag-
`ing after anti-CD25 administration. As with anti-CTLA4 combi-
`nation, we observed a reduction in viral kinetics when antibody
`therapy began on the same day as viral treatment; however,
`differences were not significant (Fig. 2A). Furthermore, when the
`antitumor effects of vaccinia/anti-CD25 combination therapy
`were tested, neither regimen (injecting anti-CD25 antibody con-
`currently with virus or after viral replication peak) resulted in
`improved efficacy relative to single oncolytic vaccinia therapy
`(Fig. 2B and Supplementary Fig. S2B). As a further test, anti-CD25
`antibody was also added before viral therapy (Supplementary
`
`Fig. S2C), however, again no therapeutic advantage was seen (Treg
`depletion with the anti-CD25 regimen used was also confirmed;
`Supplementary Fig. S2A). Finally, a direct comparison of the
`anticancer activity of vaccinia/anti-CD25 versus vaccinia/anti-
`CTLA4 combination therapies confirmed the enhanced efficacy
`of combining oncolytic virus with blockade of CTLA4 (Fig. 2B).
`
`Immunogenicity-enhanced oncolytic vaccinia vectors improve
`synergistic effects with anti-CTLA4 antibody
`As a next step, we looked to examine the importance of the viral
`vector used in these combination approaches. Two different
`double-deleted oncolytic vaccinia viruses were compared in com-
`bination with anti-CTLA4 antibody therapy. vvDD (vgf and tk
`double-deleted vaccinia virus) has demonstrated highly tumor-
`restricted replication (28) that is equivalent in level and selectivity
`to the B18R- strain. B18R- (B18R and tk double-deleted vaccinia
`virus) also demonstrated highly tumor-restricted replication but
`this was coupled with enhanced immunogenicity relative to vvDD
`(including increased production of cytokines and chemokines
`within the tumor; ref. 29). This is due to the loss of B18R, that
`encodes a secreted type I IFN-binding protein (14). When
`both viral strains were compared for anticancer effects in combi-
`nation with anti-CTLA4 antibody (Fig. 3), B18R-/anti-CTLA4
`treatment induced a more than 3.6-fold (P < 0.009) reduction
`in tumor size at sacrifice compared with PBS treatment, while
`in this model vvDD/anti-CTLA4 combination only induced a
`1.4-fold inhibition.
`
`B18R- oncolytic vaccinia virus exhibits potent antitumor
`efficacy in optimized combination with anti-CTLA4 antibody
`therapy
`We next looked to test in more detail the most effective
`combination of viral vector (B18R-), antibody (anti-CTLA4) and
`regimen (antibody treatment beginning 4 days after viral therapy)
`determined from the previous studies.
`Mice carrying either Renca (renal adenocarcinoma) or MC38
`(colon adenocarcinoma) tumors were injected with a single
`intravenous dose of B18R- at 2  108 pfu per mouse. At days
`4, 7, and 10 after virus injection, an intraperitoneal dose of 100 mg
`of mouse anti-CTLA4 antibody was administrated. PBS or single
`therapy treatments were used as controls. At the time of sacrifice,
`combination therapy resulted in a reduction of more than 2.7-
`(P < 0.035) and 1.3-fold (P < 0.02) in Renca and MC38 tumor
`models, respectively, relative to single B18R- therapy (Fig. 4A).
`The combination induced a reduction of more than 2.8-fold
`(P < 0.04) in tumor volume compared with singe anti-CTLA4
`therapy at day 42 after treatment in Renca models. Importantly,
`B18R-/anti-CTLA4 combination therapy induced 3 of 12 com-
`plete responses in this model. For MC38 tumors, B18R-/anti-
`CTLA4 combination therapy did not produce as dramatic an
`effect, but still reduced tumor volume 1.5-fold (P < 0.045)
`compared with single anti-CTLA4 therapy, a significant improve-
`ment by day 24 after treatment.
`
`Vaccinia/anti-CTLA4 combination therapy resulted in
`enhanced systemic and tumor-specific cellular immune
`response.
`To evaluate the mechanisms driving the most effective combi-
`nation of oncolytic vaccinia and anti-CTLA4 antibody, we exam-
`ined the immune response raised against and within the tumor.
`Mice bearing Renca tumors were treated as before. Controls
`
`www.aacrjournals.org
`
`Clin Cancer Res; 21(24) December 15, 2015
`
`5545
`
`Replimune Limited Ex. 2017 - Page 3
`Transgene and Bioinvent International AB v. Replimune Limited
`PGR2022-00014 - U.S. Patent No. 10,947,513
`
`

`

`Rojas et al.
`
`Downloaded from http://aacrjournals.org/clincancerres/article-pdf/21/24/5543/2029348/5543.pdf by guest on 20 September 2022
`
`Figure 1.
`Combining oncolytic vaccinia virus and anti-CTLA4 antibody therapies. A, anti-CTLA4 antibody injection reduces vaccinia virus replication in the tumor in vivo. Balb/c
`mice with subcutaneous Renca tumors (renal adenocarcinoma) were randomized and injected with a single intravenous dose of 2  108 plaque-forming units (pfu)
`per mouse of oncolytic B18R- vaccinia virus (VV). In the combination group, 100 mg of mouse anti-CTLA4 antibody was injected intraperitoneally on days
`0, 3, and 6 after virus administration. Bioluminescence imaging was used to follow viral luciferase transgene expression from within the tumor. Mean values of
`9 to 10 animals þ SD are plotted. Representative luciferase signals at day 3 after injection are also depicted (tumors are circled). B, viral/anti-CTLA4 combination
`results in increased levels of vaccinia-specific cytotoxic T cells (CTL). Mice were treated as in A, adding PBS and single therapy with anti-CTLA4 antibody as
`additional controls. At days 3 and 8 after virus injection, spleens were harvested and quantified by the IFNg ELISpot assay for vaccinia-reacting T cells. Values of
`individual mice and means  SEM of the different treatments are plotted. C, alternative schedule for vaccinia virus and anti-CTLA4 antibody combination.
`Anti-CTLA4 antibody doses were administrated at days 4, 7, and 10 after virus injection, in an approach designed to permit an initial period of viral replication.
`D, injection of anti-CTLA4 antibody after vaccinia virus replication improves therapeutic activity of combination therapy. Mice (Balb/c bearing Renca tumors)
`were treated as before or in combination with anti-CTLA4 antibody as depicted in C. Relative tumor growth and Kaplan–Meier survival curves (E) are plotted. For
`survival curves, the end point was established at a tumor volume 750 mm3. Mean values of 7 to 8 mice/group þ SE are plotted. , P < 0.05, compared
`with the VV group; f, P < 0.05, compared with the PBS group; c, P < 0.05, compared with the anti-CTLA4 group; #, P < 0.05, compared with the VVþanti-CTLA4
`day 0 group.
`
`5546
`
`Clin Cancer Res; 21(24) December 15, 2015
`
`Clinical Cancer Research
`
`Replimune Limited Ex. 2017 - Page 4
`Transgene and Bioinvent International AB v. Replimune Limited
`PGR2022-00014 - U.S. Patent No. 10,947,513
`
`

`

`Oncolytic Virus Combination with Checkpoint Inhibitors
`
`Downloaded from http://aacrjournals.org/clincancerres/article-pdf/21/24/5543/2029348/5543.pdf by guest on 20 September 2022
`
`Figure 2.
`Combination of vaccinia virus with anti-CD25 antibody did not provide any therapeutic advantage. A, anti-CD25 antibody therapy effect on vaccinia virus replication.
`Balb/c mice bearing Renca tumors were injected intravenously with 2  108 pfu/mouse of oncolytic vaccinia virus (VV, strain B18R-). For the combination
`group, a dose of 200 mg of mouse anti-CD25 antibody was also injected intraperitoneally at days 0, 3, and 6 after virus administration. Viral luciferase expression
`from within the tumor was quantified at indicated time points by bioluminescence imaging. Mean values of 7 to 8 animals þ SD are plotted. Bioluminescence
`signals from one representative animal of each group at day 3 after administration are also shown (tumors are circled). B, mice (Balb/c with subcutaneous
`Renca tumors) were treated intravenously with 2  108 pfu of VV (n ¼ 10–12 per group). For combination groups, anti-CTLA4 or anti-CD25 antibodies were
`injected with 100 or 200 mg/mouse, respectively, at days 4, 7, and 10 after virus injection. PBS was injected intraperitoneally as a control. Tumor growth was
`followed by caliper measurements. Meansþ SE are plotted. , P < 0.05, compared with the PBS group; #, P < 0.05, compared with the VV group; f, P < 0.05, compared
`with the VVþ anti-CD25 day 4 group.
`
`included PBS, single B18R- therapy, or single anti-CTLA4 therapy
`(injected at days 0, 3, and 6). Mice were sacrificed at day 11 after
`virus administration and evaluated for specific CTLs in the spleen
`by the ELISpot assay and for immune cell populations in tumors
`by flow cytometry. Combination therapy was able to significantly
`increase the numbers of CTLs recognizing tumor cell antigens
`þ
`þ
`compared with any of the controls (Fig 4B). When CD3
`CD4
`populations in tumors were quantified, a significant percentage
`increase was observed after treatment with B18R-/anti-CTLA4
`combination therapy relative to any other treatment (Fig. 5A and
`þ
`þ
`C). An increase in the percentage of CD3
`CD8
`cells infiltrating
`the tumor was also observed, but appeared to be more closely
`associated with replication of the virus in the tumor (Fig. 5B and
`C), with both virus-treated groups displaying high levels of these
`þ
`þ
`cells. Finally, to ensure that the increased CD3
`CD4
`population
`infiltrating the tumors did not represent Tregs, additional staining
`for CD25 and FoxP3 was used (Fig. 5D). We observed that in the
`þ
`þ
`control group, about 40% of the CD3
`CD4
`cells present a Treg
`þ
`þ
`phenotype
`(CD25
`Foxp3
`). Anti-CTLA4 treatment barely
`reduced this percentage, but treatment with B18R- virus dropped
`amounts to 17%, and this improved further to only 13% when
`
`anti-CTLA4 was combined with oncolytic virus. Although very few
`NK or NK-T cells were detected in the tumor (<0.01% of cells), this
`number was also significantly increased only when the combi-
`nation of B18R- virus and anti-CTLA4 antibody was used in
`combination (Fig 5E and Supplementary Fig. S4).
`
`Vaccinia/anti-CTLA4 combination therapy synergistic effects
`þ
`þ
`require CD8
`T cells, NK cells, and IFNg, but not CD4
`T cells
`To define the host factors critical for the therapeutic advantage
`seen with the B18R-/anti-CTLA4 combination, viral replication
`and antitumor effect experiments were repeated in Renca tumor-
`þ
`þ
`bearing mice depleted for CD4
`T cells, CD8
`T cells, or NK cells
`þ
`(Fig. 6). It was seen that both CD8
`T cells and NK cells were
`required for the therapeutic advantage (while antitumor effects
`þ
`were maintained after depletion of CD4
`T cells; Fig. 6B). Deple-
`þ
`þ
`T cells but not NK cells or CD4
`T cells also
`tion of CD8
`significantly enhanced viral replication, indicating this cell lineage
`was responsible for both reduced viral replication and enhanced
`antitumor effects during B18R-/anti-CTLA4 combination (Fig.
`þ
`6A). Interestingly CD8
`T cells appeared responsible for reduced
`viral replication in the tumor, even at times as soon as 1 day after
`
`www.aacrjournals.org
`
`Clin Cancer Res; 21(24) December 15, 2015
`
`5547
`
`Replimune Limited Ex. 2017 - Page 5
`Transgene and Bioinvent International AB v. Replimune Limited
`PGR2022-00014 - U.S. Patent No. 10,947,513
`
`

`

`Downloaded from http://aacrjournals.org/clincancerres/article-pdf/21/24/5543/2029348/5543.pdf by guest on 20 September 2022
`
`Rojas et al.
`
`Discussion
`It is evident that the blockade of immune checkpoint alone is
`rarely curative, but has the capacity to synergize with other
`therapies that selectively activate the immune response. As such,
`the realization that the immune response raised by oncolytic viral
`therapies is a critical mechanism mediating their therapeutic
`activity means that the combination of these two platforms would
`be logical and appealing (30). However, despite the fact that
`clinical trials have been proposed combining the oncolytic HSV T-
`Vec with ipilimumab, little preclinical data have been reported on
`such combinations (31).
`Here, we examine approaches to combine oncolytic vaccinia
`viruses with different monoclonal antibodies that target cancer-
`mediated immunosuppression. Significantly improved antitu-
`mor responses were demonstrated in several mouse tumor mod-
`els, providing strong support for the clinical translation of this
`approach. However, it was initially seen that careful consideration
`was needed to identify the correct combination of antibody, viral
`strain and especially in the timing of application of the different
`treatments. Incorrect combination resulted in a loss of benefit and
`potentially antagonistic effects.
`In initial studies combining anti-CTLA4 blocking antibody
`with vaccinia virus, no therapeutic benefit was seen (Fig. 1D). In
`these studies the antibody treatment was begun at the same time
`as viral inoculation, and imaging of viral luciferase transgene
`expression demonstrated that viral gene expression was reduced
`by more than 40-fold relative to virus used alone (Fig. 1A). This
`indicated that a robust antiviral immune response was being
`raised leading to premature clearance of the virus. Indeed, this
`
`Figure 3.
`Therapeutic activity of oncolytic vaccinia in combination with anti-CTLA4
`antibody is viral strain dependent. A total of 2  108 pfu of oncolytic vaccinia
`virus (B18R- or vvDD) were administrated intravenously to Balb/c mice
`bearing subcutaneous Renca tumors. At days 4, 7, and 10 after virus injection,
`a dose of 100 mg of anti-CTLA4 antibody was injected intraperitoneally.
`B18R- displayed greater inhibition of tumor growth relative to vvDD. Relative
`tumor volume after virus administration is plotted (n ¼ 12–15 mice/group þ
`SE). , P < 0.05, compared with the PBS group; f P < 0.05, compared with
`the vvDDþanti-CTLA4 day 4 group.
`
`þ
`T cells was supported
`viral treatment. The importance of CD8
`through depletion of IFNg, which also resulted in loss of thera-
`peutic advantage and enhanced viral tumor-specific replication
`(Supplementary Fig. S5). NK cells appear to be required for the
`antitumor effect, but do not limit viral replication.
`
`Figure 4.
`Optimized combination therapy
`results in synergistic anticancer
`activity. A, Renca (left) or MC38 (right)
`tumors were implanted into Balb/c or
`C57/Bl6 mice, respectively. Mice were
`injected with PBS or 2  108 pfu of
`B18R- oncolytic vaccinia virus (VV)
`through the tail vein. For the
`anti-CTLA4 group, 100 mg of
`anti-CTLA4 antibody was injected
`intraperitoneally at days 0, 3, and 6.
`For the combination group, anti-
`CTLA4 antibody doses were
`administrated at days 4, 7, and 10 after
`virus injection. Tumor volumes were
`measured, and relative tumor volume
`þ SE of the 12–15 mice/group is
`plotted. B, combination therapy
`increases cytotoxic T cells recognizing
`tumor antigens. Cellular immune
`responses to tumor cells was
`evaluated by the IFNg ELISpot assay.
`At day 11 after virus administration,
`spleens were harvested from Balb/c
`mice bearing Renca tumors and
`treated as in A. Splenocytes were
`evaluated for CTLs recognizing Renca
`cells. Values of individual mice and
`means  SEM are depicted. , P < 0.05,
`compared with the PBS group; f,
`P < 0.05, compared with the VV group;
`#, P < 0.05, compared with the
`anti-CTLA4 group.
`
`5548
`
`Clin Cancer Res; 21(24) December 15, 2015
`
`Clinical Cancer Research
`
`Replimune Limited Ex. 2017 - Page 6
`Transgene and Bioinvent International AB v. Replimune Limited
`PGR2022-00014 - U.S. Patent No. 10,947,513
`
`

`

`Oncolytic Virus Combination with Checkpoint Inhibitors
`
`Downloaded from http://aacrjournals.org/clincancerres/article-pdf/21/24/5543/2029348/5543.pdf by guest on 20 September 2022
`
`Figure 5.
`Altered T-cell repertoire in the tumor
`after vaccinia/anti-CTLA4 combination
`therapy. Balb/c mice with subcutaneous
`Renca tumors were treated as before
`(Fig. 4), and tumors were harvested at
`day 11 after virus injection and evaluated
`for lymphocyte populations by flow
`þ
`þ
`CD4
`(A)
`cytometry. Numbers of CD3
`þ
`þ
`and CD3
`CD8
`(B) cells per 200,000
`total cells are plotted. C, representative
`þ
`þ
`distributions of CD4
`and CD8
`þ
`populations within CD3
`population
`within the tumor. D, percentage of Tregs
`þ
`þ
`þ
`þ
`(CD25
`Foxp3
`) within the CD3
`CD4
`population of the tumor. Values for
`individual tumors and means þ SEM are
`plotted. E, numbers of NK cells
`þ
`þ
`
`(NKp46
`NKg2D
`CD3
`) and NK-T cells
`þ
`þ
`þ
`(NKp46
`NKg2D
`CD3
`) per 200,000
`events within the tumor. , P < 0.05,
`compared with the PBS group;
`#, P < 0.05, compared with the anti-
`CTLA4 group; f, P < 0.05, compared
`with the B18R- group.
`
`combination was also shown to result in a significant increase in
`the level of antiviral CTL (Fig. 1B) and viral replication was
`þ
`T cells or IFNg (Fig. 6A
`restored after depletion of either CD8
`and Supplementary Fig. S4A). This is potentially important as
`several groups are looking to express antibodies blocking immune
`checkpoints directly from oncolytic vectors (32). We have previ-
`ously used exogenous regulation of cytokine transgene function
`to down regulate cytokine function for a period of around 4 days
`after initial treatment (22, 33). This allowed an initial phase of
`viral oncolytic activity and unhindered replication within the
`tumor, prior to a secondary phase of immunotherapeutic activity
`that could be enhanced through subsequent stabilization of the
`cytokine function. Using a similar tactical approach, it was felt that
`addition of anti-CTLA4 antibody at later times after viral therapy
`could result in improved therapeutic activity.
`This was indeed confirmed (Fig. 1D), withinitiationof anti-CTLA4
`therapy 4 days after viral delivery found to result in significantly
`improved antitumor effects in mouse syngeneic tumor models.
`
`Several different monocl