Evaluation of antigen agnostic anti-tumor activity and immune memory induced by CBX-15 (alphalex^TM-MMAE) in the rat

ABSTRACT

Aims

To determine whether using a pH-sensitive peptide (CBX-15) for antigen-independent targeting of MMAE to tumors induced anti-tumor efficacy via direct and immunological mechanisms in the rat tumor model.

Methods

CBX-15 was assessed for its ability to kill tumor cells and induce immunogenic cell death (ICD) in female Fischer rats bearing syngeneic 13762 mammary adenocarcinoma tumors via in vivo and ex vivo functional assays.

Results

CBX-15 demonstrated efficacy, safety and anti-tumor immunologic memory, demonstrated by the ability of both CBX-15-cured and vaccinated rats to reject further tumor challenge. Responding rats exhibited the induction of ICD-induced immunomodulatory activity, including recognition of tumor cells ex vivo, recruitment of TILs and increases in memory CD4⁺ T lymphocytes in the bone marrow.

Conclusion

CBX-15 eradicates tumors by a combination of both direct cytotoxic action of MMAE as well as through the induction of ICD, the latter of which results in long term anti-tumor immunological memory. To our knowledge, this is the first work demonstrating both anti-tumor efficacy and induction of long-term anti-tumor immune memory by a tumor-selective drug in the rat, the species used for preclinical toxicological evaluation. This study provides a strong justification for exploring combination therapies with CBX-15 and other immunomodulators.

Plain Language Summary

Some targeted anti-cancer therapies can both directly kill tumor cells as well as indirectly produce anti-tumor activity by causing tumor cells to secrete molecules that result in activation of the immune system against the tumor, a mechanism called immunogenic cell death (ICD). CBX-15 is a novel pH-sensitive peptide-drug agent that targets the microtubule inhibiting drug MMAE universally to all solid tumors while avoiding delivery to healthy tissues. In the rat breast cancer model, CBX-15 demonstrates the ability to eradicate tumors by both direct cytotoxic activity as well as through ICD in a manner that is safe and avoids toxicity to healthy tissues. Furthermore, CBX-15 therapy allows for immune components to recognize and eliminate tumor cells long after CBX-15 treatment is ended. These data support the upcoming clinical trials of CBX-15 in combination with immunotherapy.

1. Introduction

Non-selective oncology treatment modalities induce indiscriminate cell killing, resulting in toxicities in multiple tissues including myelosuppression and gastrointestinal toxicity [1–4]. Recent years have seen the success of antibody-drug conjugates (ADCs), which link potent cytotoxins to a therapeutic antibody. However, this modality has multiple limitations, including population restriction by the target antigen and on target delivery of payload to healthy cells expressing the target antigen, which is exacerbated by the long half-life of the ADC [5–8]. As of January 2024, 13 ADCs have received market approval [9]. These ADCs carry payloads that can elicit immunogenic cell death (ICD), stimulating an active anti-tumor immunological response and memory [10,11]. When combined with immunotherapy, ADCs elicit responses in select populations, however these combinations can result in synergistic toxicities and dose reductions [11,12].

A universal tumor-selective drug delivery system would address the limitations of ADCs while allowing for safe and efficacious combinations with immunotherapy across tumor types. We have developed the alphalex^TM pH-dependent platform to selectively deliver potent payloads in an antigen-independent manner by taking advantage of the Warburg effect, a feature universal to all tumors [13]. The alphalex^TM features a unique variant of a pH-Low Insertion Peptide (pHLIP® [14–19]. In the low pH microenvironment of tumor cells, protonation of the peptide induces alpha helix formation, driving unidirectional transversion of the peptide across the tumor cell membrane. When a small molecule is attached to the C-terminus, the alphalex^TM releases the attached moiety into the tumor cytosol by glutathione cleavage of the plasma-stable linker (Figure 1(a)). In this manner the alphalex ^TM preferentially delivers potent cytotoxic agents to a broad range of neoplasms while avoiding delivery to healthy tissues. We have previously published extensive non-clinical characterization of the alphalex^TM-exatecan conjugate CBX-12, which is currently in a phase 2 clinical trial in platinum-resistant ovarian cancer [17–19]; NCT06315491).

Figure 1.

CBX-15 demonstrates both plasma stability and pH dependent selectivity for tumor tissue in preclinical models. (a) Schematic demonstrating low pH-dependent insertion of CBX-15 within the tumor cell membrane and subsequent release of MMAE within the cytosol. The alphalex comprises three components: peptide, linker, and anti-cancer agent. In the low-pH tumor microenvironment, the peptide forms an alpha helix. The peptide then inserts the C-terminus with anti-cancer agent across the cell membrane. The linker cleaves within the cell and releases the anti-cancer agent directly into the cell cytoplasm. (b) Plasma PK of CBX-15 and released MMAE as assessed by LC-HRMS in Sprague Dawley rats after a 10 mg/kg intravenous bolus of CBX-15. Plasma was isolated from whole blood collected from catheter at 0.033, 0.5, 1, 2, 7 and 24 hours. Data are expressed as mean ± standard error of the mean (SEM) (n = 3). A total of 3 catheterized rats were used in this experiment. (c) Membrane insertion of CBX-15 in the rat 13762 tumor cell line in vitro as assessed by ELISA after a 1-hour incubation period with the indicated concentrations of CBX-15 in either neutral or pH 5.5 tissue culture conditions. Data are expressed as mean ± SEM (n = 3). Significance was determined with a one-way analysis of variance (*p < 0.05 and **p < 0.005). (d) Levels of MMAE detected by LC-HRMS in murine tumor, bone marrow and muscle after an equimolar intraperitoneal dose (either 3 mg/kg CBX-15 or 0.5 mg/kg MMAE) in HCT116 xenograft bearing female nude mice. Data are expressed as mean ± SEM (n = 3 per timepoint). A total of 18 mice were used for this experiment.

To offer an alternate therapeutic modality that addresses the issues faced by ADCs, we have developed and optimized our alphalex^TM peptide platform conjugated to monomethyl auristatin E (MMAE). The resulting alphalex^TM-MMAE conjugate CBX-15 uses the same peptide as CBX-12; however, it delivers a warhead (MMAE) with diverse chemical properties and activity relative to exatecan utilizing a different stable small molecule linker. MMAE is a member of the family of synthetic analogues of auristatins derived from the antineoplastic natural product dolastatin 10 and possesses potent cytotoxic activities by disrupting microtubule assembly [20]. Like CBX-12, CBX-15 demonstrates pH-dependent insertion within the tumor and selective delivery of payload to tumor cells.

In the present study, we characterize the activity of CBX-15 in a rat syngeneic model of mammary adenocarcinoma and show that CBX-15 therapy enhances tumor immunogenicity as demonstrated by the development of long-term anti-tumor immunological memory, both in vivo and ex vivo. We also dissect the mechanism of tumor immunogenicity caused by CBX-15 and demonstrate the induction of immunogenic cell death in the rat.

2. Materials and methods

2.1. Cells and cell culture

The rat 13762 MAT B III breast adenocarcinoma cell line (ATCC, Manassas, VA) was maintained in McCoy’s 5a (Hyclone, Logan, UT) tissue culture media (TCM) supplemented with 10% fetal bovine serum, 50 IU/mL Penicillin and 50 µg/ml Streptomycin, GlutaMax and 1 mm HEPES (Gibco, Grand Island, NY) at 37°C in a humidified chamber with 5% CO_2. The cells were subcultured twice weekly and harvested with TrypLE (Gibco, Grand Island, NY) during the exponential growth phase for tumor inoculation. The human HCT116 colorectal carcinoma (ATCC, Manassas, VA) was maintained in DMEM (Hyclone, Logan, UT) TCM supplemented with 10% fetal bovine serum, 50 IU/mL Penicillin and 50 µg/ml Streptomycin, GlutaMax and 1 mm HEPES (DMEM complete TCM), grown and sub-cultured twice a week and prepared for inoculation as described below. Cells were verified to be mycoplasma negative prior to all experiments.

2.2. Animals

All animal studies were approved by the Institutional Animal Care and Use Committee. Animal study plans including dosing details and samples to be collected were prepared and approved by Cybrexa prior to the initiation of experiments in accordance with guidelines from the Institutional Animal Care and Use Committee. Animal details are in Tables 1–3.

Table 1.

Catheterized and uncatheterized Fischer rats.

Species/strain	Rat/Fischer inbred 344/NHsd
Gender	Female
Origin	Envigo; Cat# F344/NHsd
Surgery	femoral vein cannulation
Weight on arrival	225–250g
Weight at study start	280–300g
Food	Soy protein free extruded rodent diet (Envigo #2020X)
Water	Autoclaved water ad libitum
Light Cycle	12:12 (lights off at 7:00 pm and lights on at 7:00 am)
Housing	3 per cage on corn cob bedding
Caging	High temperature polycarbonate (HTPC)
Randomization Protocol	Syngeneic tumor volumes were measured 2–3 times weekly until a minimal volume of 200mm3 was obtained. Rats were assigned to different treatment groups to ensure a similar tumor volume between groups
Body Weight and Tumor Volume	Animals were weighed and their tumor volumes were measured 2-3x weekly to evaluate the effect of compound administration on body weight and tumor growth. Rats were removed from study if body weight loss exceeded 20% of baseline body weight or tumor volume reached 6000mm3
IACUC	In vivo studies were approved by the Cybrexa Animal Use and Care Committee following regulations in the Guide for the Care and Use of Laboratory Animals (National Academy Press, 2011)

Table 2.

Catheterized Sprague Dawley Rats.

Species/strain	Rat/Hsd:Sprague Dawley®SD®
Gender	Female
Origin	Envigo; Cat# 68000-F
Surgery	Jugular vein cannulation. Catheterized rats were used in experiments involving multiple sequential blood draws.
Weight on arrival	225–250g
Weight at study start	280–300g
Food	Soy protein free extruded rodent diet (Envigo #2020X)
Water	Autoclaved water ad libitum
Light Cycle	12:12 (lights off at 7:00 pm and lights on at 7:00 am)
Housing	3 per cage on Alpha-Dri bedding when off-study Single housed during dosing and blood collection
Caging	High temperature polycarbonate (HTPC)
IACUC	In vivo studies were approved by the Cybrexa Animal Use and Care Committee following regulations in the Guide for the Care and Use of Laboratory Animals (National Academy Press, 2011)

Table 3.

Nude mice.

Species/strain	Mouse/Athymic Nude Foxnnu
Gender	Female
Origin	Taconic Labs; Cat# NCRNU-F
Weight on arrival	20–23g
Weight at study start	23–25g
Food	Soy protein free extruded rodent diet (Envigo #2020X)
Water	Autoclaved water ad libitum
Light Cycle	12:12 (lights off at 7:00 pm and lights on at 7:00 am)
Housing	5 per cage on Alpha-Dri bedding
Caging	100% PET, recyclable plastic IVC cage; BPA-Free
Randomization Protocol	Xenograft tumor volumes were measured 1–3 times weekly until a minimal volume of 300mm3 was obtained. Mice were assigned to different treatment groups to ensure a similar tumor volume between groups.
IACUC	In vivo studies were approved by the Cybrexa Animal Use and Care Committee following regulations in the Guide for the Care and Use of Laboratory Animals (National Academy Press, 2011)

Female 6-week-old Fischer 344 rats (Envigo, Indianapolis, IN) and Sprague Dawley rats (Envigo, Indianapolis, IN) were acclimated for a minimum of 10 days prior to use in experiments. Female athymic nude mice were used for drug tissue distribution studies (Taconic Labs, Germantown, NY). For terminal experiments, animals were euthanized by exsanguination under anesthesia according to regulations in the National Research Council Guide for the Care and Use of Laboratory Animals (USA).

Based on NCI guidelines as well as internal data on tumor growth, 5–10 animals per group were deemed necessary for statistical significance of efficacy experiments. For efficacy studies, animals were randomized into treatment groups based on initial tumor size to ensure balanced distribution at the start of the experiment. Treatment or tumor measurements were tracked by animal ID but were not formally blinded during data collection. While cage location or the order of treatments/measurements were not randomized, potential biases were accounted for by standardizing procedures- e.g., consistent personnel/timing for dosing and measurements. No animals were excluded from final in vivo analyses.

2.3. The effects of pH on CBX-15 peptide cellular incorporation

13762 cells in log phase growth were washed once with complete DMEM and 2 × 10⁶ cells in 8 mL of TCM were seeded into 100 mm tissue culture dishes (Corning, NY). Following incubation for 48 h in a 5% CO₂ humidified chamber at 37°C, non-adherent cells were harvested, pelleted, and replenished with 8 mL of fresh media without FBS and added back to the tissue culture dishes. Next, 20 mL of the CBX-15 peptide dilutions were added to each plate resulting in final concentrations of 12.5, 3.75, 1.25 and 0.375 uM. The cultures were then incubated for 5 min in a 5% CO₂ humidified chamber at 37°C and 2 mL of citrate buffered TCM was added to immediately lower the pH to 5.5. The neutral pH groups received 2 mL of neutral media resulting in a final CBX-15 alphalex^TM peptide concentration of 10, 3, 1 and 0.3 uM. The drug-treated tumor cells were incubated for an additional hour in a 5% CO₂ humidified chamber at 37°C. All cells were collected by scraping and washed four times with the appropriate media (pH 5.5 or 7.4). Cells were counted, homogenized in RIPA Buffer (Thermo Fisher Scientific, Waltham, MA) supplemented with HALT protease inhibitor (Thermo Fisher Scientific, Waltham, MA) at 10 × 10⁶/mL, and placed into tissue grinding tubes (Benchmark Scientific, Sayreville, NJ) for complete cell disruption using a Precellys Evolution at 7200 rpm (Bertin Technologies, France). The cell preparation was disrupted over 2 cycles for 30 s per cycle. Homogenized samples were centrifuged at 14,000 × g for 5 min and the supernatant was diluted either 1:5 or 1:10 with ELISA buffer for cell associated alphalex^TM peptide analysis.

Cell associated alphalex^TM peptide was analyzed by a peptide competition ELISA. NUNC^TM MaxiSorp ELISA plates (Thermo Fisher Scientific, Waltham, MA) were coated with 0.1 mM of alphalex^TM peptide in 100 mL of Buph Carbonate-Bicarbonate buffer, pH 9.4 (Thermo Fisher Scientific, Waltham, MA), incubated overnight at 4°C and washed 4 times with a wash buffer consisting of PBS (Gibco, Grand Island, NY) and 0.05% Tween 20. The plates were blocked with a wash buffer containing 5% milk (VWR, Radnor, PA) for 2 h at room temperature and then washed 4 times in preparation for sample analysis. Samples were mixed 1:1 with an alphalex ^TM peptide monoclonal mouse antibody in antibody diluent (wash buffer containing 2% milk) and incubated for 30-min prior to addition to the ELISA plate. To measure the amount of peptide in the samples, a standard curve was set up in 13762 control cell matrix with an anti-alphalex^TM peptide monoclonal mouse antibody mixed with known concentrations of the alphalex^TM peptide. Subsequently, the peptide/antibody mixture (100 uL) was added to the ELISA plate and incubated an additional 60 min. Bound antibody was detected using a peroxidase-conjugated goat, a mouse IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA), incubated for 1 h at room temperature, washed 4 times and developed with SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA). The luminescent signal was read on a Cytation plate reader (BioTek, Shoreline, WA).

2.4. CBX-15 in vivo plasma stability

The goal of this experiment was to quantify by mass spectrometry the release of MMAE from CBX-15 in rat circulation. Female Sprague Dawley rats were administered a single intravenous bolus of CBX-15 at 10 mg/kg via a jugular vein catheter at a volume of 2 mL/kg (600 mL/300 g rat). Blood samples (100 µL) were collected into EDTA coated tubes at 0 and 0.033, 0.5, 1, 2, 4, 7 and 24-h post exposure to the drug. Samples were centrifuged at 2200 rpm for 20 min at 4°C, and plasma was collected and transferred into a 96 well Lo-Bind polypropylene plate (Thermo Fisher Scientific, Waltham, MA). Isolated samples were stored at −80°C until analyzed by LC-HRMS on a Waters I-class Acquity mass spectrometer (Manassas, Va) equipped with a Waters Acquity UPLC protein BEH C4 column to determine plasma CBX-15 and MMAE concentrations.

For CBX-15, a 20 µL volume of each matrix sample was added to the wells of a clean 1 mL 96-well LoBind polypropylene plate followed by 20 µL of 4% phosphoric acid in water. The plates were covered with a silicone cap mat and fortified samples were vortexed at 700 rpm for 2 min. Samples were subsequently centrifuged at 1500 rpm for 1 min at 4°C, and a 20 µL volume of working internal standard was added to wells containing matrix samples, while 20 µL of water: acetonitrile: formic acid (500:500:1, v:v:v) was added to double blank wells. A 180 µL volume of acetonitrile: methanol: formic acid (500:500:1, v:v:v) was added to each well, and the plate was covered with a silicone cap mat. Fortified samples were vortexed at 700 rpm for 2 min and subsequently centrifuged at 3000 rpm for 10 min at 4°C. Supernatants (50 µL) were transferred to a clean 0.700 mL 96 well LoBind polypropylene plate followed by the addition of 100 µL water: acetonitrile: formic acid (900:100:1, v:v:v). The sample plate was covered with a silicone cap mat, and the final samples were vortexed at 700 rpm for 2 min. Analysis was accomplished by injecting 5–10 µL onto the LCHRMS system.

For MMAE, a 180 µL volume of acetonitrile: formic acid (1000:1, v:v) containing MMAE-D8 internal standard was added to the wells of a 96 well protein precipitation plate resting on top of a 0.700 mL 96 well LoBind polypropylene plate. Double blank and carryover sample wells received 200 µL of acetonitrile: formic acid (1000:1, v:v) without internal standard. A 20 µL volume of each matrix sample was added to the protein precipitation plate wells containing internal standard. Fortified samples were covered and vortexed at 700 rpm for 1 min followed by centrifugation at 3000 rpm for 2 min at 4°C. The protein precipitation plate was discarded. The samples were covered with a silicone cap mat, and final samples were vortexed again 700 rpm for 2 min. Analysis was accomplished by injecting 1 µL onto the LCHRMS system.

2.5. MMAE in vivo tissue distribution

The goal of this experiment was to quantify by mass spectrometry the distribution of MMAE in mouse tissues after dosage of equimolar amounts of either CBX-15 or unconjugated MMAE. Female athymic nude mice bearing HCT116 xenografts were administered an intraperitoneal injection of 3 mg/kg CBX-15 or 0.5 mg/kg MMAE diluted in vehicle of 5% mannitol in citrate (equimolar 0.7 µmoles/kg doses of MMAE). At 4-, 24- and 48 h post-injection, mice were euthanized by exsanguination under anesthesia, according to regulations in the National Research Council Guide for the Care and Use of Laboratory Animals (USA) and tumor, muscle and bone marrow cells were harvested for the determination of tissue MMAE levels. Xenograft tumors and quadricep muscle tissues were weighed and cut into smaller pieces with a scalpel blade. To obtain bone marrow cells, the femurs were isolated, the ends were clipped off, and bone marrow was collected by passing cold PBS through the marrow cavity by means of a 23-gauge needle. Bone marrow aggregates were disbanded by repeated gentle pipetting followed by passing the suspension through a 70 µm cell strainer. Cell preparations were washed once with cold PBS, resuspended in TCM, and were counted using Trypan blue exclusion. Matrix preparations were stored at −80°C until analyzed by LC-HRMS on a Waters I-class Acquity mass spectrometer equipped with a Waters Acquity UPLC protein BEH C4 column to determine tissue MMAE concentrations.

Measurement of tissue MMAE concentrations was accomplished in xenograft tumor and quadricep muscle homogenates thawed on wet ice. Briefly, matrices were adjusted to 100 mg/mL with ice-cold RIPA buffer based on tissue weight. Samples were homogenized on a Precellys Evolution machine at 7200 rpm for 2 × 30-s cycles with a 10-s pause in between each cycle. Tissue homogenates were centrifuged at 14,000 rpm for 5 min at 4° C and the supernatants were transferred to clean 2 mL LoBind Eppendorf tubes. Matrix sample (100 µL) was added to a clean 2 mL 96 well polypropylene plate followed by 100 µL of ammonium formate buffer, pH 6.9, and 25 µL of working internal standard. Double blank control wells received 25 µL of water: acetonitrile (1:1, v:v) + 0.1% formic acid without internal standard. Fortified samples were covered with a silicone cap mat and vortexed at 700 rpm for 2 min. Working on a negative pressure manifold, 200 µL of fortified matrix samples were added to a supported liquid extraction (SLE) plate, and samples were allowed to percolate through the plate frit under a negative pressure of ~650–700 torr for up to 1 min. Samples were allowed to completely absorb into the SLE plate for 5 min. Prior to sample elution, a 2-mL 96 well TrueTaper collection plate was placed within the vacuum manifold as the collection vessel. Elution was accomplished by applying 1000 µL of MTBE (methyl tert-butyl ether) to the system and allowing the solvent to flow under gravity for 5 min. Negative pressure of ~650 torr was applied for 1030 s or until the wells were completely evacuated. Elutions were evaporated under a heated stream of nitrogen at 40°C and reconstituted in 100 µL of acetonitrile:water:200 mm ammonium formate, 92:3:5, v:v:v. The collection plate was covered with a silicone cap mat and vortexed at 900 rpm for 2 min. Final samples were centrifuged at 3000 rpm for 5 min at 4°C and analysis was accomplished by injecting 1 µL onto the LCHRMS system.

To determine MMAE concentrations in the bone marrow, thawed bone marrow sample pellets kept on wet ice were adjusted to a final concentration of 1.0 × 10⁷ cells/mL with ice-cold RIPA buffer. Samples were homogenized on a Precellys Evolution machine at 7200 rpm for 2 × 30-s cycles with a 10-s pause in between each cycle. Bone marrow cell homogenates were centrifuged at 14,000 rpm for 5 min at 4°C, and the supernatants were transferred to clean 2 mL LoBind Eppendorf tubes. A 200 µL volume of each bone marrow cell homogenate was added to the wells of a clean 2 mL 96 well polypropylene plate followed by 175 µL of ammonium formate buffer, pH 6.9, and 25 µL of working internal standard. Double blank controls received 25 µL of water: acetonitrile (1:1, v:v:v) only without internal standard. Fortified samples were covered with a silicone cap mat and vortexed at 700 rpm for 2 min. Working on a negative pressure manifold, 400 µL of fortified matrix samples were added to a supported liquid extraction (SLE) plate, and samples were allowed to percolate through the plate frit with a negative pressure of ~650–700 torr for up to 1 min. Samples were allowed to completely absorb into the SLE plate for 5 min. Prior to sample elution, a 2 mL 96 well TrueTaper collection plate was placed within the vacuum manifold as the collection vessel. Elution was accomplished by applying 900 µL of MTBE (methyl tert-butyl ether):ethyl acetate (1:1, v:v) to the system and allowing the solvent to flow under gravity for 5 min. Negative pressure of ~650 torr was applied for 1030 s or until the wells were completely evacuated. The elution process was repeated as described. Samples were evaporated under a heated stream of nitrogen at 40°C and reconstituted in 25 µL of water: acetonitrile: formic acid (900:100:1, v:v:v). The collection plate was covered with a silicone cap mat and vortexed at 900 rpm for 2 min. Final samples were centrifuged at 3000 rpm for 5 min at 4°C, and analysis was accomplished by injecting 2 µL onto the LCHRMS system.

2.6. CBX-15 efficacy in the Fischer 344 rat

The primary goal of this experiment was to assess the impact of CBX-15 on tumor growth inhibition in the rat. The secondary goal of this experiment was to assess the results of tumor rechallenge on the opposite flanks of cured rats. Female 6-week-old Fischer 344 rats were inoculated subcutaneously with 13762 tumor cells (0.1 × 10⁶) in 0.1 mL of a 1:1 dilution PBS:Matrigel vehicle (Corning, Corning, NY). Compound or vehicle control was administered intraperitoneally or intravenously when tumor sizes reached approximately 200 mm³. Mouse xenograft size achieved 5-days after inoculation, a timepoint which aided in the most consistent tumor grouping, was used to experimentally extrapolate and determine the number of rat 13762 cells to use for animal inoculation. Tumors were measured by calipers, and volume was calculated using the equation for ellipsoid volume: Volume = π/6 × (length) × (width)².

For intraperitoneal (IP) and intravenous (IV) dosing, the CBX-15 stock was initially prepared at 0.025 mg/μL in 100% DMSO (Sigma, Lavonia, MI) and was subsequently diluted using 5% mannitol (JT Baker, Phillipsburg, NJ) citrate buffer vehicle. A single IP dose of the vehicle or 2.5, 5, and 10 mg/kg CBX-15 was given once every 5 days for a two-cycle treatment for the first experiment. For the second experiment, a single IV bolus of a 10 mg/kg dose was administered via the jugular vein catheter. Following dosing, tumor growth was monitored by taking measurements with calipers, and data was recorded as described above to demonstrate tumor suppression by CBX-15 therapy. Cured animals from each study were rechallenged with live tumor cells to validate the generation of CBX-15 ICD activity in vivo. Cured rats treated with an IP injection of 5 or 10 mg/kg doses were rechallenged with live 13762 tumor cells (0.1 × 10⁶) in their opposite flank 50 days after the initial tumor implantation and monitored for tumor growth over the next 40 days. Those animals that were cured after receiving one IV injection of a 10 mg/kg dose were also rechallenged with live tumor cells (0.1 × 10⁶) in the opposite flank 57 days post the initial 13762 tumor implantation and were monitored for tumor growth over the next 10 days. At the termination of each study, the animals were sacrificed for ex vivo studies in accordance with the guidelines in the National Research Council Guide for the Care and Use of Laboratory Animals (USA).

2.7. CBX-15 13762 whole cell vaccine

The goal of this experiment was to assess whether rats vaccinated by CBX-15-killed cells would mount an immune response resulting in tumor rejection upon subsequent challenge, which is diagnostic of immunogenic cell death. Rat 13762 cells were seeded at 5 × 10⁶ cells per T150 flask (Costar, Washington, DC) and cultured overnight in complete McCoy 5a media (Hyclone, Logan, UT). Cells were then treated with 40 uM CBX-15 for 72 h. Cells growing in suspension were collected and combined with adherent cells that were detached using warm TrypLE (Gibco, Grand Island, NY). Harvested cells were then washed with cold complete McCoy 5a media and pelleted by centrifugation at 1200 rpm for 5 min at 4°C. Cells were washed a second time with 50 mL of cold PBS and resuspended in cold PBS at a final concentration of 30 × 10⁶ cells/mL. Next, the cell vaccine was kept on ice until administration to the left flanks of rats by subcutaneous injection at a volume of 0.1 mL per rat (3 × 10⁶ cells/rat). The same procedure was performed for the preparation of a booster 13762 cell vaccination and was administered 2 weeks after the primary cell vaccination. For the live tumor cell challenge 13762 cells (0.1 × 10⁶) were inoculated subcutaneously in 0.1 mL of a 1:1 dilution PBS:Matrigel into the opposite flank 7 days after the booster vaccination.

2.8. Freeze-thaw 13762 whole cell vaccine

Rat 13762 cell cultures were grown to 50% confluence in T150 culture flasks (Costar, Washington DC). Cells growing in suspension were harvested and combined with adherent cells that were detached with warm TrypLE. The cells were washed with cold complete McCoy 5a media and pelleted by centrifugation at 1200 rpm for 5 min at 4°C. Pelleted cells were counted and resuspended in PBS at a final concentration of 30 × 10⁶ cells/mL. The cells were then subjected to six freeze/thaw cycles in an ice bucket containing liquid nitrogen plus methanol and checked by trypan blue exclusion to ensure they possessed 0% viability. The freeze-thaw 13762 whole cell vaccine was kept on ice until administration to the left flanks of rats by subcutaneous injection at a concentration of 0.1 ml per rat (3 × 10⁶ cells/rat). Booster vaccinations were administered 2 weeks after the initial vaccination using fresh freeze/thaw cell vaccine prepared as described above. For the live tumor cell challenge 13762 cells (0.1 × 10⁶) were inoculated subcutaneously in 0.1 mL of a 1:1 dilution PBS:Matrigel into the opposite flank 7 days after the booster vaccination.

2.9. Ex vivo tumor draining lymph node and splenocyte tumor cell stimulation for cytokine measurement

Tumor draining lymph nodes (DLNs) and spleens were harvested under sterile conditions and placed into individual wells of a six well plate (Falcon, Corning, NY) each containing 5 mL of cold PBS. Single cell suspensions (SCS) were prepared by pressing each organ in between autoclaved frosted glass slides (VWR, Radnor, PA) and passing the processed organs through 40 µm and 70 µm cell strainers (Cell Treat, Pepperell, MA) into a 50 mL tube. The volume was adjusted to 50 mL using cold sterile PBS, and cells were pelleted by centrifugation at 1200 rpm for 5 min at 4°C. The supernatant was discarded, and the cell pellet was resuspended in cold complete tissue culture media consisting of RPMI 1640 (Cytiva, Marlborough, MA) supplemented with heat inactivated 10% Defined FBS (Cytiva, Marlborough, MA), penicillin at 10 units/mL, streptomycin at 10 µg/mL, GlutaMAX, HEPES at 1.0 mm and 50 µM 2-β-mercaptoethanol (Gibco, Grand Island, NY). DLN and splenocyte suspensions were adjusted to 1 × 10⁶ cells/mL in complete RPMI 1640 tissue culture medium.

DLN cells or splenocyte preparations were plated into 96 well round bottom polystyrene tissue culture plates (Falcon, Corning, NY) in volume of 100 µL/well (1×10⁵ cells/well). Stimulator 13762 cells were pre-treated with 30 µM Mitomycin C (Sigma, Livonia, MI) for 2.5 h, washed four times with 50 mL cold PBS, and resuspended in complete RPMI 1640 TCM. The cells were diluted at an Effector-to-Stimulator ratio of 5:1 and cultured at 37°C in a 5% CO₂ humidified chamber. Tissue culture supernatants were harvested at various time points to determine rat IL2 and IFNγ production by ELISA (R&D Systems, Minneapolis, MN).

2.10. CBX-15-induced tumor infiltrating lymphocyte phenotyping by flow cytometry

The goal of this experiment was to assess the impact of CBX-15 on tumor immune cell composition. Female 6-week-old Fischer 344 rats were inoculated subcutaneously with 13762 tumor cells (0.1 × 10⁶) in 0.2 ml of PBS. The tumors were monitored for 6–10 days until they reached approximately 850–900 mm³ (larger than the 200 mm³ starting size of an efficacy experiment in order to obtain enough intact cells for flow cytometry after tumor dissociation). Subsequently, the rats were treated with an IP injection of either 20 mg/kg CBX-15 or vehicle control prepared as previously described.

Tumors were harvested five-day post treatment and tumor single cell suspensions (SCSs) were prepared for flow cytometry by mechanical dissociation with scissors and forceps followed by enzymatic treatment using a Tumor Dissociation Kit as described by the manufacturer (Miltenyi Biotec, Auburn, CA). An equal volume of cold complete RPMI 1640 tissue culture medium was added to each sample which was passed through 70 µm and 40 µm cell strainers. The SCSs were washed with cold FACS Wash Buffer (FWB) which consisted of PBS containing 2% fetal bovine serum and 0.02% w/v sodium azide (VWR, Radnor, PA) and resuspended to a concentration of 3 × 10⁶ cells/mL in cold FWB. Next, 100 μL of each sample was added to the wells of a 96 well-roundbottom polypropylene plate (Greiner, Monroe, NC). The samples were pelleted, and the liquid was aspirated prior to cell staining.

The cells were stained in 200 µL of cold FWB for the identification of T lymphocytes and natural killer cells. T cells were stained with a rat CD3-FITC antibody (BD Biosciences, Franklin Lakes, NJ) or an isotype mouse IgG3-FITC control (BD Biosciences, Franklin Lakes, NJ). Natural killer cells were stained using a rat CD335-FITC antibody (Biolegend, San Diego, CA) or a mouse IgG1-FITC isotype control (Biolegend, San Diego, CA). Incubation proceeded for 30 min in the dark at 4°C. The labeled cells were washed 3X with 200 mL of cold FWB, centrifuged at 1200 rpm for 5 min, and resuspended in a 200 μL volume of FWB. Cell suspensions were analyzed by Flow Cytometry using a Becton Dickinson Accuri C6 Sampler Flow Cytometer (BD Biosciences, Franklin Lakes, NJ) and collecting 10,000 events. FSC vs SSC gating was used to identify the cell population based on size and granularity, respectively, to gate out the cell debris.

2.11. Assessment of resident CD4⁺ bone marrow T lymphocytes by flow cytometry

Bone marrow cells collected from the femurs of female Fischer rats as previously described were washed with cold FWB and resuspended in FWB at a concentration of 3 × 10⁶ /mL. Subsequently, 100 uL of the cell preparation was added to wells of a 96 well-rounded bottom polypropylene plate (Greiner, Monroe, NC) and washed once as previously described in preparation for cell viability and cell surface marker staining.

The cells were pelleted, supernatant removed, and cells stained with Live/Dead stain as described by the manufacturer (Thermo Fisher, Waltham, MA). Cells were washed and incubated with 1 ug/well of a fluorochrome labeled rat CD4-PE antibody (BD Biosciences, Franklin Lakes, NJ) or an isotype control mouse IgG2a-PE (BD Biosciences, Franklin Lakes, NJ) in 100 uL of cold FWB for 30 min in the dark at 4°C. The plate was washed twice, and cells were resuspended in 200 uL of cold FWB and analyzed by Flow Cytometry using the Becton Dickinson Accuri C6 Sampler Flow Cytometer and collecting 10,000 events. FSC vs SSC gating of the viable cells was used to identify the cell population based on size and granularity, respectively, to gate out the cell debris.

2.12. Detection of extracellular ATP in the rat 13762 tumor cell line treated with CBX-15

Extracellular release of ATP was detected in tissue culture supernatants using the RealTime-Glo Extracellular ATP Luminescence kit according to the manufacturer’s instructions (Promega, Madison, WI). Briefly, 200 µL of tumor cells in complete McCoy’s TCM were seeded into 96-well flat bottom solid white tissue culture plates at 1563 cells/well (Corning, New York, NY) and incubated overnight in a 5% CO₂ humidified chamber at 37°C. Media supernatant (50 uL) was removed, and 50 µL of a 4X concentration of CBX-15 was added to each well at the indicated final concentrations. Following an additional 24-h incubation, 10 µL/well of a 20X concentration of Glo Luminescent Reagent reconstituted in McCoy 5a complete medium was added to the wells. The cultures were monitored for extracellular ATP release over 18 h in a 5% CO_2, 37^o C humidified chamber using a Cytation3 luminescence reader (BioTek, Shoreline, WA). GraphPad Prism (San Diego, CA) was used for all statistical analyses.

3. Results

3.1. pH-dependent tumor targeting of CBX-15 results in tumor selective delivery of MMAE

CBX-15 was developed to safely deliver efficacious levels of the microtubule inhibitor MMAE to solid tumors in an antigen-independent manner using a unique variant of the pHLIP® peptide [14,15,17–19]. CBX-15 consists of the pH-dependent peptide, glutathione cleavable linker, and MMAE, in a 1:1 molar ratio (Figure 1(a)). In vivo plasma stability was assessed by a single intravenous bolus injection in the rat. Analysis of intact conjugate and unconjugated MMAE by LC-HRMS demonstrated that there was less than 0.5% release of the warhead over a 24-h period in systemic circulation (Figure 1(b)). The half-life of intact CBX-15 was 2.8 h, which is significantly shorter than the weeks long half-life of an ADC [7].

We next demonstrated pH-dependent insertion of CBX-15 in vitro using the rat 13762 cell line. A significant dose-dependent increase in cell associated CBX-15 was demonstrated at pH 5.5, while minimal insertion was detected at pH 7.4 (Figure 1(c)). The pH-dependent insertion of CBX-15 was further evaluated in vivo through profiling of CBX-15 tissue pharmacokinetics in the mouse. In these studies, after intraperitoneal administration of CBX-15, levels of free warhead were profiled in tumor, muscle, and bone marrow of nude mice bearing HCT116 xenograft tumors. Healthy tissues profiled were chosen to reflect the frequent dose-limiting toxicities of MMAE-bearing ADCs, which include severe neutropenia and motor neuropathy. These data demonstrated the tissue selectivity of CBX-15, with released MMAE detected specifically in the tumors but not in bone marrow or muscle (Figure 1(d)). In contrast, an equimolar dose of unconjugated MMAE warhead (a dose which is also the MTD of MMAE in the mouse) exhibited nonspecific distribution as evident in the high levels found in tumor, bone marrow and muscle (Figure 1(d)).

3.2. Antigen independent tumor targeting by CBX-15 mediates complete tumor regression in the rat syngeneic breast cancer model

We further demonstrate that CBX-15 safely delivers efficacious levels of MMAE in the rat with multiple dosing paradigms. We utilized the 13762 flank tumor model derived from mammary adenocarcinoma metastasis in the Fischer rat. CBX-15 therapy mediated complete regressions in a dose-dependent manner in tumor bearing rats (Figures 2(a,b)). Doses of CBX-15 in these efficacy experiments were chosen to cover the range profiled in both the plasma PK experiments as well as the equimolar dose relative to the MTD of MMAE covered in the tissue PK experiments. Interestingly, CBX-15 also exhibited delayed efficacy in two of the rats at the 10 mg/kg dose, with partial regressions of large tumors 2–3 weeks post dosing. Due to the partial nature of the regression and aggressive nature of the tumor, the larger of the tumors underwent regrowth at a similar rate to controls in the absence of further dosing (Figure 2(a)).

Figure 2.

CBX-15 efficacy in Fischer 344 rats bearing syngeneic 13762 mammary adenocarcinoma flank tumors receiving two different dosing regimens. (a) Tumor volumes of 13762 tumor-bearing Fischer rats after receiving a total of two IP doses of CBX-15 five days apart at 2.5, 5.0 or 10 mg/kg relative to vehicle treated rats (n = 5/group). A total of 20 rats were used in this experiment. (b) Body weights of animals described in figure a. Data are expressed percent change in body weight as mean ± SEM. (c) Tumor volumes of 13762 tumor-bearing Fischer rats after receiving a single IV 10 mg/kg dose of CBX-15 or vehicle (N = 6/group). A total of 12 rats were used in this experiment. (d) Body weights of animals described in figure c. Data are expressed percent change in body weight as mean ± SEM. CBX-15 treatment began when the tumor size reached approximately 200 mm³ in all experiments and the arrows indicate the time of dosing. (e, f) The 13762-tumor infiltrating lymphocyte population was analyzed by FACS analysis in Fischer 344 rats that were treated with a single CBX-15 IP injection 20 mg/kg (n = 8) or the vehicle control (n = 7) five days post therapy. CBX-15 therapy induced a significant increase in the percentages of CD3⁺ T cells (e) and NK cells (f) over the vehicle controls (**p < 0.005). (g) Bone marrow was assessed by FACS at 10 days post-tumor rechallenge for the levels of CD4⁺ T cells and a significant increase in the percentage of CD4⁺ bone marrow resident T cells was observed in the cured rats as compared to control groups (**p < 0.005). Significance was determined with one-way analysis of variance. Data are expressed as mean ± SEM (N = 5/group).

We also tested the efficacy of a single intravenous bolus in the rat. Femoral vein catheterized rats bearing 13762 flank tumors received a single intravenous 10 mg/kg dose and were monitored for tumor growth. Most notably 5 of 6 of the rats treated with CBX-15 exhibited complete responses, while 100% of the vehicle controls were euthanized by day 8 due to rapid tumor growth (Figures 2(c,d)). CBX-15 again exhibited the ability to regress larger tumors, with one 3000 mm³ tumor exhibiting sudden and complete regression three-weeks post dose, suggesting involvement of the immune system. Together, these data indicate that CBX-15 is highly active and safe in the rat model.

3.3. CBX-15 enhancement of tumor immunogenicity in the rat

The delayed ability of CBX-15 to completely regress large tumors in the rat long after the termination of drug therapy was suggestive of an active immunomodulatory component of its mechanism (Figures 2(a,c)). One of the hallmarks of the generation of tumor immunogenicity is an increase in the number of tumor infiltrating leukocytes (TILs) into the tumor. Two key players that cooperate in rejecting cancer cells include T lymphocytes and natural killer (NK) cells [21]. Concordantly, tumors of CBX-15-treated rats exhibited a significant increase in the infiltration of CD3⁺ T cells and NK cells into 13762 tumors 5 days after therapy (Figures 2(e,f)).

In addition to the immune activity at the site of the tumor, healthy bone marrow serves as a reservoir for immunological activity [22–24]. A recent study has reported that the long-term resting resident bone marrow memory CD4⁺ T lymphocytes are reactivated and expand upon a rechallenge to circulating antigen [22]. Given that CBX-15 avoids bone marrow toxicity, we monitored for any changes in the numbers of bone marrow CD4⁺ lymphocytes in tumor bearing rats cured by CBX-15 treatment and then subsequently rechallenged 40 days later with live 13762 cells. There was a significant increase in the population of bone marrow CD4⁺ T lymphocytes that occurred 10 days post-tumor rechallenge (Figure 2(g)). While additional work needs to be done to confirm the function of these cells, these data support the observations of Siracusa et al. in the mouse [22]. In addition, these data further demonstrate the tumor selective delivery of MMAE by CBX-15, as exposure to unconjugated warhead would have eliminated this population due to toxicity.

3.4. CBX-15 therapy induces long-term anti-tumor immunologic memory in the rat

In addition to mediating direct cellular cytotoxicity by inhibiting cellular metabolism and DNA synthesis, some cytotoxic drugs induce ICD, stimulating active tumor immunity and ensuring long-lasting anti-tumor immune memory [25–27]. We first confirmed that CBX-15 could induce ICD in vitro by monitoring for ATP release by dying 13762 cells, which acts as a Damage-Associated Molecular Pattern Molecule (DAMP) by directing immune cells to the site of active inflammation [25]. Indeed, CBX-15 induced significant ATP release into the tissue culture medium (Figure 3(a)). Further in vitro profiling of rat DAMPs was hampered by the lack of verified anti-rat antibodies, and as a result, additional experiments focused on conclusively demonstrating ICD in vivo.

Figure 3.

Induction of anti-tumor immunologic memory after CBX-15 therapy. (a) Extracellular ATP DAMP release was measured in vitro from dying 13762 tumor cells 18 hours post CBX-15 exposure. Significance was compared to the controls using one-way analysis of variance (*p < 0.05 and ****p < 0.0001). Data are expressed as mean ± SEM. (b) CBX-15 cured 13762 tumor bearing Fischer rats shown in Figure 2(a) (n = 5) were rechallenged with an injection of live 13762 tumor cells on the opposite flank 50 days post drug treatment and followed for tumor growth. Growth was compared to a rechallenge control group with newly injected tumors (n = 5). A total of 10 rats were used in the rechallenge experiment. (c–f) The production of TH1 cytokines measured ex vivo by ELISA from cured animals (10 mg/kg group n = 3; 5 mg/kg group n = 2) and controls (n = 5/group) 10 days post tumor rechallenge. Splenocyte (c and d) or DLN (e and f) single cell suspensions from cured and control rats were stimulated for 10 days with Mitomycin C treated 13762 cells. DLN and splenocytes from non-treated tumor bearing animals and naïve, non-tumor bearing animals were used as controls. Significance was compared to the tumor controls for cytokine production using unpaired T test (*p < 0.05, **p < 0.005, ***p < 0.001, and ****p < 0.0001). Data are expressed as mean ± SEM.

Given that CBX-15 showed evidence of ICD induction in vitro, we confirmed that CBX-15 can generate in vivo anti-tumor immune memory utilizing two different methodologies. The first method involved direct in situ killing of established tumors by CBX-15 therapy and the second approach utilized a prophylactic CBX-15-killed tumor cell vaccine to protect animals from tumor challenge, the gold standard for demonstrating ICD [27].

The first approach involved rechallenging rats that were cured with two doses of CBX-15 therapy (Figure 2(a)). The cured animals were rechallenged on the opposite flank with live 13762 tumor cells 50 days after receiving the last CBX-15 dose. The live tumor challenge was rejected by all five of the rats cured by CBX-15 therapy, while untreated tumor controls developed rapidly growing 13762 tumors and were euthanized by day 10 (Figure 3(b)). In addition to the in vivo data, an active immune response was demonstrated ex vivo. Secondary lymphoid organs were harvested, and single cell suspensions were stimulated with mitomycin C treated syngeneic 13762 tumor cells as a source of tumor antigen. Immune splenocytes and draining lymph node (DLN) cultures were monitored for the production of T Helper 1 (TH1) cytokines. Splenocytes and DLNs produced significant levels of IFNγ and IL2 in both CBX-15 therapy dose groups when stimulated with 13762 cells, while naïve and tumor bearing controls showed no production for either cytokine (Figure 3(c–f).

The second approach used to validate the ability of the CBX-15 to generate ICD in vivo was carried out by syngeneic tumor cell vaccination. In this approach, 13762 tumor cells were killed in vitro by exposure to CBX-15 to generate an ICD inducing prophylactic vaccine. The vaccination approach consisted of two immunizations with 13762 tumor cells killed by either a rapid freeze/thaw as a control or by treatment with CBX-15. Seven days after the last vaccination, rats were challenged with 13762 tumors. Animals were monitored for tumor growth for 42 days, and individual rat data is presented as a spider plot (Figure 4(a)). Unlike control rats, CBX-15-treated 13762 whole cell vaccinations resulted in complete tumor rejection in 5 of 8 rats, confirming that CBX-15 is an ICD inducer. ICD-induced immune memory was measured ex vivo 42 days post live tumor challenge by measuring the ability of immune splenocytes to produce TH1 cytokines when exposed to tumor antigen. Significant IFNγ and IL2 production was observed in the supernatants from CBX-15 13762 whole cell vaccine protected rats over both control groups (Figure 4(b,c)). Together, the in vivo and ex vivo results demonstrate the capability of CBX-15 to induce ICD, resulting in the generation of long-term anti-tumor immune memory.

Figure 4.

Induction of immunogenic cell death by vaccination with CBX-15 killed 13762 cells. (a) The growth of live tumor cell challenge of Fischer 344 rats 7 days after vaccination with 13762 cells killed in vitro either by CBX-15 treatment (N = 8) or by freeze thawing (N = 5). Rats were immunized with two immunizations two weeks apart and a naïve control group (N = 4) was included in the study. A total of 17 rats were used in this experiment. (figures b and c) Splenocyte single cell suspensions were stimulated with Mitomycin C treated 13762 cells from rats described in (a). Tissue culture supernatants were tested for IFNγ(b) and IL2 (c) production by ELISA. Significance was compared to the tumor controls for cytokine production using unpaired T test (*p < 0.05, **p < 0.005). Data are expressed as mean ± SEM.

4. Discussion

This study describes the ability of CBX-15 to deliver MMAE selectively to established tumors in a pH based, antigen agnostic manner as well as to eradicate actively growing tumors in a preclinical rat mammary adenocarcinoma cancer model. While MMAE has been previously described to be an ICD inducer in the mouse model [28], to our knowledge, this is the first report describing anti-tumor and ICD activity of a tumor selective therapeutic modality in a relevant toxicology species, the rat. The ability to evaluate efficacy, immunomodulatory activity, and toxicity in the same species to be used in IND-enabling toxicology allows for the assessment of relative effects at the same exposure levels, thereby allowing for more accurate modeling and better predictive translation of nonclinical data to the clinical setting.

The gold standard for the ability of a cytotoxin to trigger the development of ICD in vivo is the induction of long-term tumor-specific immune memory as demonstrated by the rejection of live tumor after vaccination with drug treated tumor [25]. In our study, we demonstrated that a CBX-15 treated cell-based vaccine protected rats from live tumor challenge leading to long-term immune memory, a result that has been reported for murine preclinical tumor models for various ICD inducing cytotoxins [27]. In addition to this observation, we found that CBX-15 therapy directly induced ICD in situ as tumor bearing rats that were cured generated immune activity that led to protective immune memory and ex vivo tumor-specific lymphocyte cytokine production.

We further show that CBX-15 induced multiple hallmarks that characterize an ICD cytotoxic drug. One such observation includes the release of ATP from treated tumor cells, a key ICD DAMP involved in immune cell trafficking to the tumor site [25,29]. In addition, CBX-15 therapy increased tumor immunogenicity which was demonstrated by the infiltration of TILs into the tumor and expansion of CD4⁺ lymphocytes numbers in the bone marrow after CBX-15 therapy in tumor bearing rats. Further studies are needed to confirm whether the CD4⁺ expanded T cells are from a pool of tumor-specific resident memory cells. The bone marrow has been shown to harbor greater than 80% of the long-term memory CD4⁺ and CD8⁺ memory T cell populations [22,23]. Future studies will further explore anti-tumor activities of these TILs.

5. Conclusion

In summary, we demonstrate that CBX-15 represents a new therapeutic modality that is well differentiated from an ADC, allowing for safe delivery of MMAE and mediating antigen-independent tumor-specific cytotoxicity in a rat tumor model. CBX-15 treatment results in the eradication of tumors in the rat by a combination of both direct cytotoxic action of MMAE as well as through the induction of ICD, the latter of which results in long term anti-tumor immunological memory. The demonstration of a broad range of immunomodulating activities, both in vivo and ex vivo, along with the tumor selectivity and safety of CBX-15, provides the preclinical rationale to examine combinations with both checkpoint inhibitors and other immunomodulators in clinical trials initiating in 2025.

Funding Statement

This paper was funded by Cybrexa Therapeutics.

Article highlights

Introduction

• While antibody drug conjugates (ADCs) have revolutionized cancer therapy, activity is limited to specific subsets of the population that express the target antigen, requiring the redevelopment of new ADCs to address different populations and indications.

• The CBX-15 alphalex^TM-MMAE conjugate was developed to selectively deliver MMAE to tumor in an antigen agnostic manner by relying on tumor acidity, a universal feature of all tumors due to the Warburg effect.

Methods

• This study characterizes the in vivo stability and tissue pharmacokinetics of CBX-15 as well as the immunomodulatory activities of CBX-15 in the 13762 syngeneic rat model of mammary adenocarcinoma.

Results

• CBX-15 demonstrates pH selective insertion in vitro as well as plasma stability and tumor selectivity in vivo.

• CBX-15 treatment results in rapid and complete tumor regressions in the 13762 rat tumor model when dosed either intraperitoneally or intravenously, resulting in increased tumor infiltrating lymphocytes post treatment and long-term resistance to additional tumor challenges in cured animals.

• CBX-15 results in immunogenic cell death in the rat tumor model as evidenced by both in vitro induction of extracellular ATP as well as through the ability of rats vaccinated with CBX-15 killed 13762 cells to resist tumor challenge.

Conclusion

• This study demonstrates that CBX-15 treatment results in pH-dependent, tumor selective activity in vivo that results in complete tumor suppression in the 13762 rat syngeneic model of mammary adenocarcinoma.

• In addition to direct killing of tumor cells by selective delivery of the MMAE payload, this study demonstrates that CBX-15 treatment results in enhancement of tumor immunogenicity and the development of long term anti-tumor immunological memory through immunogenic cell death.

Author contributions

T. Paradis: Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing. Q. Zhang: Investigation, Methodology. L. Tylaska: Investigation, Formal analysis. C. Hagen: Investigation. T. Pasqualini: Investigation. V. Paralkar: Supervision, Conceptualization, Writing – review and editing. S. Gayle: Supervision, Project administration, Conceptualization, Methodology, Writing – review and editing.

Disclosure statement

T. Paradis, Q. Zhang, L. Tylaska, C. Hagen, T. Pasqualini, V. Paralkar and S. Gayle are employees of Cybrexa Therapeutics.The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical disclosure

The authors state that they have obtained appropriate institutional review board approval for all animal experimental investigations. All animal studies were approved by the Cybrexa Institutional Animal Care and Use Committee under Animal Use Protocols CBXAUP005, CBXAUP006, and CBXAUP007.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article.