Intralesional Path to Cancer Drug Approval

Fill Factor: the ratio of PV-10 volume to tumor volume to deliver a constant dose of rose bengal sodium per quantity of tumor

Apr 27, 2024

Multiple investigational intralesional (IL) agents have been tested across a range of solid tumor cancer types, in addition to synthetic small molecule PV-10. While the majority of these IL agents have been oncolytic viruses (OVs), several were biologics.

The only IL agent to garner FDA approval to date is Imlygic®, an OV based on a modified herpes simplex virus (HSV) type 1 and designed to selectively replicate in and lyse tumor cells upon IL injection, with secondary potential to promote regional and systemic anti-tumor immunity.^[1] The dosing scheme for Imlygic is predicated on administering sufficient OV into tumor tissue to promote destruction of injected tumors, an approach that appears to be based on a number of predicate IL biologic agents.

Imlygic and its key predicate agents, as well as several agents developed in parallel to it, are reviewed in an overview of strategies utilized during clinical development.

A. Predicate Biologic Agents Cited in Imlygic’s Development

a. BCG

The earliest IL agent to undergo detailed testing in cutaneous melanoma was Bacillus Calmette-Guerin (BCG), led by reports of “objective improvement” in five of 15 patients with Stage III disease receiving single IL injections (3x10⁶ bacilli, volume of injectate not reported) every two weeks for an initial five cycles, with treatment continued if improvement was noted until complete response or progression.^[2] This was followed by a small randomized study in patients with in-transit melanoma (ITM), where BCG was compared to dinitrochlorobenzene (DNCB).^[3] BCG in saline was injected into up to 25 skin lesions (0.1 mL/lesion containing 5x10⁷ bacilli), which was repeated every 4-6 weeks (using 0.1 mL/lesion at 5x10⁶ bacilli) until all lesions were injected, with a few lesions injected twice; DNCB was administered in similar fashion, with injection of 0.1 mL of an acetone formulation into intradermal lesions and small (<1 cm) subcutaneous lesions, with 0.1-0.2 mL administered to larger subcutaneous nodules. Both agents exhibited good local effect (ca. 90% local response) but there was no evidence of systemic benefit. However, BCG was also associated with an unacceptable risk of significant toxicity, with three of 25 patients experiencing disseminated intravascular coagulation (DIC), including one near fatality, while there were minimal adverse experiences in 51 patients receiving DNCB. These observations led to loss of interest in BCG for melanoma although BCG has become a standard immunotherapy for superficial bladder cancer administered via intravesical instillation.^[4]

b. Allovectin-7

Allovectin-7 (velimogene aliplasmid, an immune-stimulating biologic construct composed of a bicistronic plasmid encoding human leukocyte antigen-B7 and beta-2 microglobulin plasmid formulated with cationic lipids intended to recruit macrophages and T cells) was studied in metastatic melanoma in an open-label Phase 2 study (77 patients enrolled at 16 centers).^[5] Patients were given six once-weekly intralesional injections of 10 µg Allovectin-7 (volume not specified) followed by four weeks of observation for up to three treatment cycles. The investigational treatment exhibited minimal adverse events, with modest activity (9% objective response, 4.8 months median duration of response), supporting further testing at higher dose.

A second Phase 2 dose-escalation study evaluated Allovectin-7 in 133 patients with Stage III/IV metastatic melanoma recurrent or unresponsive to prior therapy.^[6] Patients received six weekly intralesional injections followed by three weeks of observation and evaluation; patients with stable or responding disease could receive further cycles of study treatment. The number of injectable lesions was determined by the investigator, and could be cutaneous, subcutaneous, or nodal lesions ≥1 cm² and ≤25 cm². It appears that 1 mL of investigational agent was administered per lesion. A 3 x 3 dose escalation scheme was employed, with cohorts receiving 0.5, 1.0 or 2.0 mg Allovectin-7; based on initial safety observations, remaining patients received 2.0 mg in an efficacy cohort. Patients in the efficacy cohort with multiple injectable lesions could have up to 5 lesions injected in a treatment cycle (i.e., 2 mg Allovectin-7 divided equally among all injected lesions). Thus, it does not appear that a consistent intralesional dose (volume or mg Allovectin-7) was employed. An objective response was observed in 12% of patients, along with acceptable toxicity.

In a pivotal Phase 3 study, Allovectin-7 was tested vs chemotherapy in 390 Stage III/IV melanoma patients (randomized 2:1 against dacarbazine or temozolomide), with administration of 2 mg to a single lesion weekly for six weeks, repeated after each eighth week; the primary endpoint was response rate at 24 weeks aher randomization.^[7] An objective response was achieved in 4.6% of patients receiving Allovectin-7, vs 12.3% receiving comparator, while overall survival was 18.8 months vs 21.3 months. Considering these results, no further studies of Allovectin-7 were conducted in melanoma.^[8] Further details on dosing were not published, but study participants were required to have at least one injectable lesion 1 x 1 cm or greater in size.

c. HSV1716

Phase 1 testing of the IL oncolytic virus HSV1716 is cited as predicate work in reports on initial development of Imlygic. Rampling et al. evaluated toxicity of HSV1716, a Phase 1 study administering the agent intralesionally to relapsed glioma.^[9] Patients received a single treatment with one of three doses of agent (10³, 10⁴, or 10⁵ pfu/mL, N = 3 patients per dose) via stereotactic injection into a single brain tumor (8.6 to 129 cm³ tumor volume) at 6-10 radial locations within the tumor; a total of 1 mL of agent was administered. Hence, no consistent IL dose (volume or pfu of OV) was employed. No encephalitis, adverse clinical symptoms, or reactivation of latent HSV were observed.

d. CV706

CV706 is also cited as predicate work in reports on initial development of Imlygic. Non-clinical testing of CV706 (formerly known as CN706), a replication-competent PSA-selective OV based on an adenovirus planorm, was conducted in nude mice with implanted LNCaP human prostate cancer tumors.^[10] Solitary flank tumors were injected with 0.1 mL of virus suspension (5x10⁸ pfu) when they reached approximately 1 cm diameter (i.e., ca. 0.1 mL / 0.5 cm³ tumor); complete regression was noted in 5 of 10 tumors.

Subsequent Phase 1 testing of CV706 was conducted in patients with locally recurrent prostate cancer following radiation therapy.^[11] Twenty patients were divided into 5 dose cohorts, receiving 1x10¹¹ to 1x10¹³ viral particles delivered via transperineal injection using a modified prostate brachytherapy technique. CV706 was administered as 0.1 mL injections at 20-80 locations (“intraprostatic viral deposits”) using 10-40 needles. Dosing was based on a “viral dosimetric” algorithm derived from non-clinical experience to determine optimal sites for injection to achieve homogeneous coverage of the prostate, where each injection was presumed to “result in ~1 mL of tumor killing.” The primary endpoint was determination of treatment-related toxicity, and the investigational treatment was found to be safe, with no irreversible grade 3 or any grade 4 toxicity. Patients in the highest two dose groups achieved a ≥50% reduction in serum PSA. Further clinical development does not appear to have been done.

e. G207

An HSV-based OV, G207, is also cited as predicate work in reports on initial development of Imlygic. This virus has been modified to selectively target glioblastoma multiforme (GBM) while minimizing infectivity of normal tissue.

An initial Phase 1 dose-escalation study, G207 was administered via stereotactic injection to single treatment-refractory cerebral malignant glial tumors >1 cm diameter in 21 adult patients.^[12] Pre-treatment and post-treatment viable tumor volumes were estimated using an MRI eigenvalue algorithm to determine enhancing tumor volume only, which showed a mean volume of 39 cm³ at baseline. Patients in the initial five cohorts x three patients received 1x10⁶ to 3x10⁸ pfu (0.1 mL administered to a single loci); those in the sixth cohort received 1x10⁹ pfu (0.3 mL administered to a single loci); and those in the final cohort received 3x10⁹ pfu (0.2 mL administered to each of five loci, 1.0 mL total volume). No toxicity or serious adverse events were attributed to G207, while there was some evidence of anti-tumor activity.

In an additional Phase 1 dose-escalation study, G207 was administered to children and adolescents with recurrent or progressive supratentorial brain tumors.^[13] A 3+3 design with four dose cohorts administered 10⁷ or 10⁸ pfu (total volume 2.4 mL) over a period of six hours via 3-4 stereotactically implanted catheters (patients in cohorts 3 and 4 received 5 Gy radiation to the tumor mass 24 hours after G207 administration). At baseline, treated tumors ranged from 2.4 to 6.7 cm in longest diameter (median 3.4 cm). No DLTs aNributed to G207 were observed, with radiographic, neuropathological, or clinical responses observed in 11 of 12 patients.

In a Phase 1b study of G207 in 6 patients with recurrent GBM, a catheter with a solid tip and 16 openings in a circumferential pattern beginning 1 cm from the tip and extending for approximately 2 cm was placed in enhancing tumor; once placed, 1 mL of agent (1.5x10⁸ pfu) was infused over a 10-minute interval, followed by an additional 0.8 mL of saline to clear the catheter.^[14] After administration, the catheter was sealed, and the tumor was subsequently resected 2-5 days later. Immediately after resection, the resected tumor bed was injected at multiple locations with G207 (1x10⁹ pfu, 6 mL total volume, administered in approximately equal portions at a depth of 1 cm space approximately 1 cm apart using a 28G needle). Minimal toxicity was observed with radiologic and neuropathologic evidence suggestive of antitumor activity.

In another Phase 1 trial in nine patients with progressive, recurrent GBM, stereotactic IL administration was used to inoculate multiple sites of the enhancing margin, followed one day later by focal treatment with 5 Gy radiation (6 MV linear accelerator).^[15]The investigational agent (0.2 mL G207 at 1x10⁹ pfu) was injected into 5 different areas “over 2 minutes to avoid possible reflux.” Treatment was well tolerated, with modest evidence of clinical response to the single-dose OV therapy.

f. ONYX-015

A genetically modified attenuated adenovirus, ONYX-015, is also cited as predicate work in reports on initial development of Imlygic. This virus has been modified to selectively replicate and kill cells harboring p53 mutations, including head and neck, gastrointestinal, and pancreatic tumors, and is purported to leave normal cells unaffected.

Initial preclinical testing of ONXY-015 (also known as dl1520) was conducted in tumor xenograph models (C33A human cervical carcinoma and U87 human glioblastoma tumors implanted in flanks of nude mice);^[16] tumors were injected every other day for three doses beginning when they reached palpable size (mean tumor volume 150 µL). In a second series of experiments, implanted flank tumors were injected when they reached 80 µL, receiving 10⁸ pfu suspended in 60 µL of vehicle for five consecutive days, with dose divided equally into each tumor quadrant. C33A tumors exhibited significant regression.

The agent underwent further preclinical testing using a several human xenograph tumor models (HLaC laryngeal carcinoma, in addition to C33A cervical carcinoma and U87 glioblastoma) in nude mice.^[17] Implanted flank tumors were injected daily for five days using 10⁸ pfu of ONYX-015 suspended in 60 µL of vehicle starting when they reached a size of 65-80 µL (5- to 8-mm maximum diameter). Vehicle-only and inactivated virus injection controls were also tested. Improved survival was observed in HLaC and C33A mice but not in U87 mice.^[18]

A Phase 1 trial of ONYX-015 evaluated safety and biological activity of single IL injections in 22 patients with recurrent head and neck cancer.^[19] Tumors were injected based on estimated volume, with dilution of ONXY-015 adjusted so that total injection volume equated to 30% of estimated tumor volume; tumor was mapped into 1 cm² areas, with equal volumes injected into each portion. Patients could receive retreatment at four-week intervals for up to five cycles (64ti received a single cycle, none received 5 cycles). Injected tumors ranged from 2.2 to 20 cm², with a median of 11.8 cm² (i.e., ca. 1.5 to 4.5 cm diameter, median ca. 3.4 cm). Six doseescalation cohorts received 10⁷ to 10¹¹ pfu, with no dose limiting toxicity reached. While no objective tumor responses were observed, MRI scans “were suggestive of tumor necrosis at the site of viral injection in five patients”. Additionally, three patients had satellite tumors injected with diluent alone, all of which progressed. The authors noted that “two patients did experience mild pain on injection…related to the volume of the injected solution” that resolved spontaneously within 1 hour.

In another Phase 1 dose-escalation trial, ONYX-015 was administered every four weeks to primary pancreatic tumors intraoperatively (N = 1 patient) or under CT guidance (N = 22 patients) using a 22-ga needle.^[20] Diluted virus (10⁸ to 10¹¹ pfu) was delivered at 10% to 20% of estimated tumor volume, with number of injections dependent on size of tumor: ≤3 cm received one central injection; >3 to≤6 cm received two injections to the respective centers of the tumor halves; >6 cm received 3 injections. The volume of injected agent was distributed equally among injection sites. All dose levels were tolerated but no objective responses were observed nor was there evidence of viral replication.

In a Phase 1/2 trial, ONYX-015 was administered to 21 patients with locally advanced or metastatic pancreatic adenocarcinoma (mPDAC), but with no or minimal liver metastases; patients received 2x10¹⁰ or 2x10¹¹ pfu in a 3+3 dose escalation scheme, with an additional 15 patients receiving the higher dose; ONYX-15 was administered to the primary tumor weekly under ultrasound guidance over 8 weeks, with gemcitabine given concurrently with the final four treatments.^[21] The volume of injected virus was adjusted to comprise one-tenth of tumor volume. The authors concluded that the treatments were generally well tolerated with partial or minor regression of the injected tumor observed in four subjects (19% of subjects).

In a Phase 2 trial, ONYX-015 was tested in patients with recurrent/relapsed SCC of the head and neck (HNSCC).^[22] The agent was administered intralesionally to a single identified tumor on five consecutive days repeated every three weeks (30 patients in a standard treatment cohort) or twice daily on 14 consecutive days repeated every three weeks (10 patients in a hyperfractionated treatment cohort) with a total of 533 injections administered. The agent was administered at 10¹⁰ pfu into each of five equally spaced, equally sized tumor quadrants, with concentration adjusted so that total injection volume equated to 30% of estimated tumor volume. Partial or complete response was observed in 14% of patients in the standard treatment cohort, while 41ti exhibited stable disease; in the hyper-fractionated cohort, 10% of patients achieved complete response with another 62% achieving stable disease. In addition, acceptable safety was demonstrated.

B. Imlygic Pre-Clinical Development and Testing

Imlygic (also known as talimogene laherparepvec, JS1/ICP34.5-/ICP47-/GM-CSF, OncoVEX^GM-CSF, or T-Vec) was tested in vitro then in vivo in mouse xenograph models using human tumor cell lines.^[23] Flank tumors were injected three times (2-3 days apart) with an intermediate test article (an OV that did not contain all the coding features of T-Vec): implanted HT-29 colon, Fadu carcinoma, and U87MG glioma tumors were injected with 50 µL of agent when they reached a size of 5 mm diameter (i.e., 50 µL injected into approximately 65 µL of tumor volume) and tumor growth followed for several weeks after final injection. A bilateral flank model of A20 lymphoma was employed to evaluate T-Vec injected into one flank tumor (5 mm diameter, 50 µL injected three times every other day) at 10⁶, 10⁷, and 10⁸ plaque forming units (pfu) / mL with growth followed thereafter. Because T-Vec is built on an HSV backbone, the role of prior HSV infection was studied and found to be irrelevant.

C. Imlygic’s Phase 1 Study

Imlygic was studied in a dose escalation study in patients with cutaneous or subcutaneous solid tumors who had failed prior therapy.^[24] Thirteen patients were enrolled in a single-dose group and received Imlygic at 10⁶, 10⁷, or 10⁸ pfu/mL (N = 4, N = 5, and N = 4, respectively), while another 17 patients were in a multidose group testing a number of dose regimens (these received an initial dose of 10⁶ pfu/mL followed by 10⁷ or 10⁸ pfu/mL, N = 3 and N = 14, respectively). High dose treatments (10⁸ pfu/mL) were limited to patients that were seropositive for HSV at baseline. In all groups, Imlygic was administered based on diameter of the injected tumor:

tumors ≤ 1.5 cm received up to 1 mL of Imlygic,
tumors > 1.5 to ≤ 2.5 cm received up to 2 mL, and
tumors > 2.5 cm received up to 4 mL.

It is notable that the study initially was designed to include all patients (i.e., HSV-seronegative and HSV-seropositive) in 10⁸ pfu/mL exposure groups, but results in the early cohorts identified unacceptable local inflammatory reactions at 10⁷ pfu/mL in HSV-seronegative patients; thus, high-dose groups were limited to HSV-seropositive patients.

D. Imlygic’s Phase 2 Study

Phase 2 testing of Imlygic was conducted in 50 patients with Stage III/IV melanoma.^[25] Patients received an initial dose of up to 4 mL of Imlygic (10⁶ pfu/mL) that was followed 3 weeks later with up to 4 mL at 10⁸ pfu/mL administered q2w thereaher for up to 24 treatments. The initial low dose was intended to seroconvert HSV-negative patients to HSV-positive status, thereby avoiding unacceptable reactions in HSV-negative patients. Up to 10 lesions were injected per treatment cycle, using a dosing schema modified from Phase 1:

tumors 0.5 to 1.5 cm in diameter received 0.5 mL of Imlygic,
tumors 1.5 to 2.5 cm received up to 2 mL, and
tumors > 2.5 cm received up to 2 mL.

A maximum of 4 mL of Imlygic could be administered per cycle. Response was assessed by RECIST, starting after six treatment cycles, then every 12 weeks. Overall, 26% of patients achieved an objective response (8 CR, 5 PR, the majority of these having Stage III or M1a disease), and adverse experiences were limited primarily to transient flu-like symptoms.

E. Imlygic’s Phase 3 Trial

Pivotal testing compared IL Imlygic to subcutaneous GM-CSF in 436 patients (randomized 2:1) with unresectable Stage IIIB to IV melanoma.^[26] Patients were required to have at least one cutaneous, subcutaneous, or nodal lesion or aggregation of up to 5 lesions with a sum of diameters ≥10 mm that were suitable for injection. As in phase 2, patients received initial injection at 10⁶ pfu/mL followed by subsequent doses at 10⁸ pfu/mL q2w starting 3 weeks aher the initial dose. The dose schema was again modified from prior work, using the following algorithm:

tumors up to 0.5 cm in diameter received up to 0.1 mL of Imlygic,
tumors 0.5 to 1.5 cm received up to 0.5 mL,
tumors 1.5 to 2.5 cm received up to 1.0 mL,
tumors 2.5 to 5 cm received up to 2.0 mL, and
tumors > 5 cm received up to 4 mL.

No minimum size was set for injection, while tumors >10 cm, or patients with tumor burden in excess of 20 cm (sum of longest diameters), required prior approval of the study medical monitor.^[27] A maximum of 4 mL per lesion and per treatment cycle was retained from prior work. The primary endpoint was durable response rate (DRR, defined as an objective response lasting continuously ≥6 months), with OS as a key secondary endpoint. DRR in the Imlygic arm was 16.3% vs 2.1% for SQ-GM-CSF (P < 0.001), while median OS was 23.3 months vs 18.9 months (HR = 0.79, P = 0.051). Imlygic was approved based on the results of this study.

F. Imlygic PI

The prescribing information for Imlygic specifies a dose schema identical to the Phase 3 schema.

G. Additional OVs

These studies have led to study of more recently of OVs that leverage predicate work and contemporary development of Imlygic. As with these other examples, dose of OV in pfu is adjusted to maximize oncolytic effect, generally with volume of injectate adapted to facilitate delivery of the desired viral titer.

a. Myb34.5

Gayral et al. tested an investigational oncolytic virus (a targeted HSV-1, Myb34.5) in mouse models of PDAC (human PDAC-derived Mia PACA-2 or Mia PACA-2 Lucia cells implanted in the tail of the pancreas of athymic mice).^[28] Tumors were injected once with the OV (10⁶ to 10⁸ pfu, volume not reported) when they reached 150 mm³ (control animals received PBS). Tumor necrosis, hemorrhage, viral replication, and cancer cell apoptosis were observed within two weeks of injection. When the OV was combined with chemotherapy (gemcitabine at 125 mg/kg q3d x 2wk), marked reduction in tumor growth was noted. Neither regimen elicited evidence of significant toxicity.

b. ParvOryx

Hajda et al. conducted a Phase 2 study of an oncolytic H-1 parvovirus (H-1PV, ParvOryx) at 3 escalating doses (10⁹, 5x10⁹, and 10¹⁰ pfu total administered dose) in 7 adult patients with confirmed PDAC and at least one hepatic metastasis who had progressed on FOLFIRINOX and were candidates for gemcitabine.^[29] Patients received an initial infusion of H-1PV (40ti of total dose given in equal fractions qd x 4) then the remaining dose was administered intralesionally under ultrasound guidance 6-13 days later (administered “as slowly as possible”, injectate volume not specified).^[30] Gemcitabine was commenced on day 28 at 1000 mg/m² BSA, with Nab-paclitaxel (125 mg/m² BSA) added in the event of disease progression. All dose levels were tolerated, with a median PFS of 2.4 months and a median OS of 5.8 months. While the authors don’t disclose details on injection volume, in predicate non-clinical models, HA-RPC tumors implanted in the pancreatic tail of immunocompetent rats were injected intraoperatively with 100 µL of H-1PV when they reached a size of 150 mm³.^[31]

c. IL-12 OV

Barton et al. report on a phase 1 dose escalation study where a replication-competent oncolytic adenovirus modified to express IL-12 (Ad5-yCD/mutTKSR39rep-hIL-12 adenovirus) was injected into metastatic PDAC tumors under ultrasound guidance; 12 subjects received 10¹¹ to 10¹² pfu administered to their primary tumor in a volume equivalent to 10ti of tumor mass, with 5fluorcytosine followed by FOLFIRINOX or GEM/NAB starting 21 days after OV injection.^[32] The study MTD was not reached, with a median OS of 18.1 months (range 3.5-20.0) observed in the high-dose cohort.

d. HF10 (TaKaRa Bio Inc.)

HF10 is a replication-competent OV derived from HSV-1. Andtbacka et al. reported results from a Phase 2 study of HF10 combined with ipilimumab in patients with Stage IIIB-IV cutaneous melanoma.^[33] HF10 was administered at 10⁷ pfu/mL, at up to 5.0 mL, with injection volume adjusted based on size of tumor mass (no further details provided). The authors concluded that the regimen “demonstrated a favorable benefit/risk profile and encouraging antitumor activity.”

H. Additional Non-OV IL Agents

a. SD-101 (Dynavax)

SD-101 is a Toll-like receptor 9 agonist that has been administered via IL injection to several solid tumor types.

Cohen et al. reported results from a Phase 2 study of SD-101 in combination with pembrolizumab in treatment-naïve HNSCC.^[34] SD-101 was administered into two dose cohorts as 2 mg injected into 1-4 lesions or 8 mg injected into a single lesion weekly x 4 doses then q3w x 7 doses (28 patients received 2 mg and 23 received 8 mg per injection). One quarter of patients experienced grade ≥3 treatment emergent adverse events (TEAEs), with an ORR of 24% (2 CR and 10 PR).

Similar results were reported by Ribas et al. for the phase 1b portion of a phase 1b/2 study (co-sponsored by Dynavax and Merck as Keynote-184/SYNERGY-001) of SD-101 in combination with pembrolizumab in 22 patients with advanced melanoma; in phase 1b, SD-101 was administered at 1, 2, 4, or 8 mg weekly x 4 doses then q3w x 7 doses.^[35] Adverse events appear to have been mostly mild to moderate, with modest clinical outcome (an estimated 12-month PFS of 88% and an ORR of 15%).

Further specifics on treatment parameters were not provided.

b. Tilsotolimod (Idera)

Tilsotolimod (IMO-2125) is an investigational synthetic oligonucleotide which binds to TLR9, improving tumor antigenicity.

Haymaker et al. reported results of a phase 1/2 trial in combination with ipilimumab in 62 patients with anti-PD-1-resistant advanced melanoma.^[36] In Phase 1, patients received tilsotolimod at 4 mg (N = 3 patients), 8 mg (9), 16 mg (3), and 32 mg (3), while in phase 2 tilsotolimod was administered to 44 patients at 8 mg; IL injections were administered weekly x 3 weeks then q3w x 3 cycles, then q6w, for a total of nine doses. The total injected dose remained constant, with injection volume adjusted depending on size of injected tumor (no additional details provided). Safety was manageable (48% of patients experienced grade 3 or 4 TEAEs) with a 22% overall response rate in Phase 2.

In an ongoing Phase 1 study in the Netherlands (PANFIRE III), a single IL injection of tilsotolimod (8 mg) is being studied in combination with irreversible electroporation (IRE) and nivolumab in patients with mPDAC; IRE is conducted 1 week after tilsotolimod administration, with nivolumab commenced 2 weeks later.^[37] A total of 18 patients are planned for three total arms of the study, with tilsotolimod administered using a perforated needle (ProFusion).

c. Darleukin (L19-IL2, Philogen)

Darleukin is a synthetic immunocytokine comprised of the antibody L19 and the cytokine IL2 designed to accumulate in tissue where angiogenesis occurs and the EDB-FN antigen is expressed.

Weide et al. reported results from a Phase 2 study of darleukin administered to 25 patients with Stage IIIB-IIIC cutaneous melanoma.^[38] Darleukin was administered once weekly via IL injection at a dose of 10⁶ IU of IL2 equivalents dissolved in 6 mL of vehicle, with total daily dose divided between all injectable lesions (all lesions present at screening were injected). A CR by modified immune-related response criteria (irRC) was achieved in 6 patients, and 44ti of injected lesions achieved a CR. Toxicity was similar to systemic IL2 (Aldesleukin).

Danielli et al. reported results from a phase 2 study of darleukin administered to 22 patients with Stage IIIC-IVM1a cutaneous melanoma.^[39] Darleukin (10⁶ IU IL2 equivalents) and Fibromun (78-312 µg of L19-TNF at investigator’s discretion) were mixed to a final volume of 4.2 mL, and administered once weekly for up to 4 weeks via IL injection, with total daily dose divided between all injectable lesions (all lesions present at screening were injected, along with new lesions that occurred within the regional field during the treatment phase). CR was observed in 1 patient, with PR in an additional 10 patients. Adverse events were mostly mild to moderate.

PV-10

Similar to the algorithms used for ONYX-015 (A.f. above) and Imlygic (C-E above) , PV-10 dosing is based on a space filling model where the ratio of PV-10 volume is matched to the tumor volume (“fill factor”) to deliver a constant dose of rose bengal sodium (RBS) per quantity of tumor (mg RBS / mg tumor).

Non-clinical Fill Factor

In non-clinical testing, Provectus showed that PV-10 yielded maximal benefit in a dose-dependent manner in two sponsored studies of A375 radiation-resistant human melanoma xenographs that documented clinical outcome and overall survival for investigational formulations ranging from 0.1% to 10% RBS (Study A375-PH-10.03, Study A375-e64, and Study 040216.01C), with volume equivalent to approximately 50% of tumor volume. This work established 10% RBS as the standard concentration for PV-10 in all ensuing studies.

Research on PV-10 by the University of Calgary in xenograph models of relapsed and refractory human neuroblastoma substantiated — i.e., reproduced/repeated — that IL injection of PV-10 at approximately 0.4 to 0.8 µL/mm³ estimated tumor volume (i.e., administration of a volume of PV-10 equivalent to 40% to 80% of estimated tumor volume) increased survival in a dose-dependent manner.^[40]

Clinical Fill Factor

In initial clinical testing, Thompson et al. reported results from a 20-patient Phase 1 study of PV-10 administered as single IL injections to up to 20 cutaneous melanoma lesions at a dose of 0.5 mL PV-10/cm³ lesion volume (10% RBS; volume equivalent to 50% of tumor volume).^[41]

In a follow-up 80-patient study, Thompson et al. reported results from Phase 2 testing of PV-10 administered as repeat IL injections (1-4 injections per lesion administered 4 or more weeks apart); up to 20 cutaneous melanoma lesions were injected at a dose of 0.5 mL PV-10/cm³ lesion volume (10% RBS; volume equivalent to 50% of tumor volume), with a limit of 15 mL (1,500 mg of RBS) at each treatment cycle.^[42]

A Phase 1 Study to Assess the Safety and Tolerability of PV-10 in Combination with Gemcitabine/Nab-paclitaxel in Metastatic Pancreatic Adenocarcinoma

a. Dose escalation/dose expansion flow diagram

PV-10 dosing (i.e., fill factor) will escalate to a “high dose” volume equivalent of 50% of tumor volume.

b. Timeline of study treatment and assessments

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[2] Mastrangelo MJ, Sulit HL, Prehn LM, et al. Intralesional BCG in the treatment of metastatic malignant melanoma. Cancer. 1976 Feb;37(2):684-92. https://doi.org/10.1002/1097-0142(197602)37:2<684::aidcncr2820370212>3.0.co;2-y.

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