OBI-3424: An Analytical Report on an AKR1C3-Activated Alkylating Prodrug

Executive Summary

OBI-3424 is a first-in-class, small-molecule prodrug designed to be selectively activated by the aldo-keto reductase 1C3 (AKR1C3) enzyme, a target overexpressed in various malignancies but with low expression in most normal tissues. This report provides a comprehensive analysis of OBI-3424, synthesizing its chemical properties, mechanism of action, extensive preclinical data, and its complete clinical development history. The central narrative of OBI-3424's development is one of starkly contrasting outcomes: a highly selective, biomarker-driven agent with a compelling and potent preclinical profile that ultimately demonstrated limited therapeutic potential in advanced solid tumors, leading to the strategic termination of its lead Phase I/II trial (NCT03592264).

The drug's mechanism relies on the AKR1C3-mediated reduction of its nitro-benzene moiety, which releases a potent DNA bis-alkylating agent, OBI-2660, directly within tumor cells. This approach is intended to maximize cytotoxicity at the tumor site while minimizing the systemic toxicities associated with traditional, non-selective alkylating agents. Preclinical studies robustly supported this hypothesis, showing strong AKR1C3-dependent activity in a wide range of solid tumor models, including hepatocellular carcinoma (HCC) and castrate-resistant prostate cancer (CRPC). The most profound preclinical efficacy was observed in models of T-cell acute lymphoblastic leukemia (T-ALL), a disease characterized by particularly high AKR1C3 expression and inherent sensitivity to DNA-damaging agents.

Despite this promising foundation, the clinical program in advanced solid tumors yielded disappointing results. Phase I dose-escalation studies established a manageable safety profile, with dose-dependent, non-cumulative myelosuppression (anemia and thrombocytopenia) identified as the primary dose-limiting toxicities. However, clinical activity was minimal, with the best response being stable disease in the majority of patients and only a single partial response observed across the entire program. This lack of efficacy prompted the termination of the solid tumor trial in March 2024.

In a critical strategic pivot, development continues to focus on hematologic malignancies, specifically relapsed/refractory T-ALL, where the preclinical rationale remains strongest. The ongoing SWOG-led Phase I/II study (NCT04315324) represents the sole remaining path forward for the asset. The development trajectory of OBI-3424 serves as a salient case study on the complexities of translating a highly specific, biomarker-driven therapeutic strategy from preclinical models to clinical success. The divergence in outcomes between the solid and hematologic tumor programs underscores that while a validated biomarker is necessary, it may not be sufficient. The biological context of the target malignancy—such as its intrinsic sensitivity to a specific cytotoxic mechanism—is a critical co-determinant of clinical success. The future of OBI-3424 now rests entirely on its performance in the T-ALL patient population.

OBI-3424: A Profile of a Novel Tumor-Activated Prodrug

Chemical and Physical Properties

OBI-3424 is a small-molecule, synthetic organic compound classified as a nitro-benzene prodrug.[1] It is designed to be inert until it is metabolized within the body. The active component it delivers is a potent nitrogen mustard, specifically N,N'-bisethylenephosphoramidate, a DNA bis-alkylating agent.[1] The molecular formula for OBI-3424 is

, and it has a molecular weight of approximately 460.43 g/mol.[3]

Structural Identifiers and Formulations

OBI-3424 is tracked across scientific literature, regulatory filings, and chemical databases using several key identifiers. It was originally developed by Threshold Pharmaceuticals under the designation TH-3424 and is also referred to as AST-3424 in studies conducted by its partner, Ascentawits Pharmaceuticals.[1] For clinical trials, OBI-3424 is prepared as a liquid formulation for intravenous (IV) infusion.[6]

Table 1: Summary of OBI-3424 Key Identifiers
Identifier	Value
Name	OBI-3424
Synonyms	AST-3424, TH-3424, OBI3424, AKR1C3-activated prodrug AST-3424
Type	Small Molecule
DrugBank ID	DB16057
CAS Number	2097713-68-1
PubChem CID	126961329
Molecular Formula
Molecular Weight	460.43 g/mol
IUPAC Name	3-ethyl]-2-nitrophenoxy]-N,N-dimethylbenzamide
InChI Key	NWGZZGNICQFUHV-OAHLLOKOSA-N
Canonical SMILES	CC@HOP(=O)(N3CC3)N4CC4
Data compiled from sources:

Mechanism of Action: Exploiting a Tumor-Specific Enzymatic Vulnerability

The Aldo-Keto Reductase 1C3 (AKR1C3) Enzyme: A Differentiated Target

The therapeutic strategy of OBI-3424 is entirely dependent on the unique biological characteristics of the aldo-keto reductase 1C3 (AKR1C3) enzyme. AKR1C3 is a monomeric, cytosolic, NAD(P)(H)-dependent oxidoreductase with a molecular weight of 37 kDa. It is also known by several other names, including type 5 17β-hydroxysteroid dehydrogenase (17β-HSD5) and prostaglandin F synthase. Physiologically, AKR1C3 plays a crucial role in the metabolism of steroid hormones, catalyzing the conversion of weak androgens and estrogens into their more potent forms (e.g., androstenedione to testosterone), as well as metabolizing prostaglandins.

The critical feature of AKR1C3 that makes it an attractive cancer target is its differential expression pattern. While it is expressed at low to moderate levels in some normal tissues like the liver, kidney, and small intestine, it is abnormally overexpressed at both the mRNA and protein levels in a wide array of malignant solid and hematologic tumors. High levels of AKR1C3 have been documented in hepatocellular carcinoma (HCC), castration-resistant prostate cancer (CRPC), non-small cell lung cancer (NSCLC), gastric cancer, renal cancer, and T-cell acute lymphoblastic leukemia (T-ALL). In many of these cancers, elevated AKR1C3 expression is associated with tumor progression, metastasis, resistance to standard therapies, and poor patient prognosis. This tumor-specific overexpression provides a therapeutic window, allowing for the design of a prodrug that is selectively activated in cancer cells, thereby sparing healthy tissues.

Bioactivation Pathway: From Inert Prodrug to Potent DNA Alkylator

The mechanism of OBI-3424 is a "lock-and-key" bioactivation process. Following intravenous administration, the inert OBI-3424 prodrug circulates systemically and is taken up by cells. In cancer cells that overexpress AKR1C3, the enzyme recognizes OBI-3424 as a substrate. In the presence of the cofactor NADPH, AKR1C3 catalyzes the reduction of the nitro-benzene group on the OBI-3424 molecule. This enzymatic reaction selectively cleaves the prodrug, releasing its active cytotoxic payload, a bis-alkylating aziridine metabolite known as OBI-2660 (or AST-2660).

A crucial aspect of this activation is its efficiency. In vitro kinetic analyses have demonstrated that AKR1C3 exhibits a higher catalytic efficiency () for OBI-3424 compared to its endogenous physiological substrates. This suggests that within the competitive intracellular environment of a cancer cell, the conversion of OBI-3424 may be a highly favored reaction. This preferential processing could create a "substrate trap" effect, leading to the rapid and concentrated accumulation of the cytotoxic OBI-2660 specifically within the target cancer cell, thereby maximizing its therapeutic effect. This rapid conversion may also disrupt the cancer cell's normal steroid and prostaglandin metabolism, which could represent a potential secondary anti-tumor mechanism.

Molecular Cytotoxicity: DNA Cross-linking and Induction of Apoptosis

Once liberated within the cancer cell, OBI-2660 functions as a potent DNA-damaging agent, with a mechanism of action similar to established chemotherapeutics like thiotepa and mitomycin C. As a bis-alkylating agent, OBI-2660 has two reactive sites that enable it to form covalent bonds with DNA. It primarily targets the N7 or O6 positions of guanine bases. This alkylation process results in the formation of both intra-strand (on the same DNA strand) and inter-strand (between the two strands of the DNA helix) crosslinks.

These DNA lesions are highly cytotoxic. Inter-strand crosslinks are particularly damaging, as they physically prevent the separation of the DNA strands, which is a prerequisite for both DNA replication and transcription. This disruption leads to the stalling of replication forks, the introduction of DNA base pair mismatches, and the inhibition of DNA synthesis and repair processes. The overwhelming DNA damage ultimately triggers cell-cycle arrest and activates the intrinsic apoptotic pathway, leading to programmed cell death. Preclinical studies have directly confirmed this molecular mechanism, showing that treatment with OBI-3424 leads to a concentration-dependent increase in DNA cross-linking, as measured by comet assays, specifically in cancer cells with high AKR1C3 expression.

Comparative Analysis: Selectivity Advantages Over Traditional Alkylating Agents

OBI-3424's design offers a significant theoretical advantage over conventional alkylating agents such as cyclophosphamide and ifosfamide. These traditional agents are also prodrugs, but their activation occurs non-selectively, primarily through systemic metabolism by cytochrome P450 enzymes in the liver. This results in the circulation of active alkylating metabolites throughout the body, exposing both cancerous and healthy tissues to their cytotoxic effects. This lack of selectivity is responsible for the significant toxicities associated with these drugs, most notably myelosuppression.

In contrast, OBI-3424's key innovation is its tumor-localized activation. By leveraging an enzyme that is highly overexpressed in cancer cells relative to normal tissue, the generation of the cytotoxic payload (OBI-2660) is theoretically confined to the tumor microenvironment. This targeted activation is designed to achieve a wider therapeutic window by maximizing the concentration of the DNA-damaging agent within cancer cells while minimizing exposure and damage to healthy cells, such as hematopoietic stem cells in the bone marrow. This distinction underpins the potential for OBI-3424 to be a broad-spectrum, yet highly selective, anti-tumor drug with an improved safety profile.

Preclinical Evaluation: Validating the AKR1C3-Targeting Hypothesis

In Vitro Correlates of Activity: AKR1C3 Expression and Cytotoxicity

The foundational principle of OBI-3424 was rigorously tested in vitro across a diverse panel of human cancer cell lines. These studies established a direct and strong correlation between the cytotoxic potency of OBI-3424 and the expression level of AKR1C3, at both the protein and RNA levels. Cell lines with high endogenous AKR1C3 expression were exquisitely sensitive to the drug. For example, the H460 non-small cell lung cancer line exhibited a half-maximal inhibitory concentration (

) of just 4.0 nM. Conversely, cell lines with low or negligible AKR1C3 expression were largely resistant to OBI-3424's effects. This fundamental relationship provided the initial and most critical validation for the drug's proposed mechanism of action and established AKR1C3 as a viable predictive biomarker for patient selection.

In Vivo Efficacy in Solid Tumor Xenograft Models (HCC, CRPC, NSCLC, etc.)

The promising in vitro results were successfully translated into in vivo models. In cell-derived xenograft (CDX) models representing various human cancers with high AKR1C3 expression, OBI-3424 demonstrated significant anti-tumor activity. As a single agent administered at clinically achievable doses, it produced tumor growth inhibition that was either superior or comparable to the respective standard-of-care chemotherapies for those cancer types. This efficacy was observed in models of hepatocellular carcinoma (HepG2), NSCLC (H460), castration-resistant prostate cancer (VCaP), gastric cancer (SNU-16), and kidney cancer (A498).

The biomarker-dependent activity was further confirmed in more clinically relevant patient-derived xenograft (PDX) models. In these studies, OBI-3424 demonstrated excellent anti-tumor efficacy, but only in PDX models derived from patient tumors with high AKR1C3 expression. Models with low AKR1C3 expression did not respond to treatment, reinforcing the enzyme's critical role in mediating the drug's activity and the potential of the biomarker to guide patient selection in a clinical setting.

Profound Antileukemic Activity in Preclinical Models of T-ALL

The most compelling preclinical data for OBI-3424 emerged from studies in T-cell acute lymphoblastic leukemia (T-ALL). Analysis of large patient sample cohorts revealed that AKR1C3 mRNA expression was significantly higher in primary T-ALL tumors compared to B-lineage ALL (B-ALL) tumors (). This finding immediately positioned T-ALL as a prime indication for OBI-3424.

In subsequent in vivo studies using a large panel of T-ALL PDX models, many derived from patients with aggressive and fatal disease, OBI-3424 demonstrated profound and superior efficacy. As a monotherapy, it induced deep and prolonged disease regressions, significantly extending event-free survival (EFS) in all nine T-ALL PDX models tested (EFS Treated/Control values ranged from 2.5 to 14.0). The treatment led to dramatic reductions in bone marrow infiltration by leukemic blasts, a key measure of disease burden. Furthermore, OBI-3424 retained its activity upon retreatment in models designed to simulate a relapsed/refractory disease setting. The definitive role of AKR1C3 was elegantly proven through a genetic experiment: a B-ALL PDX model, which is naturally low in AKR1C3 and resistant to OBI-3424, was lentivirally transduced to stably overexpress the enzyme. This single genetic modification rendered the previously resistant leukemia sensitive to OBI-3424 treatment, confirming that AKR1C3 is the key determinant of response in this context.

Emerging Data in Pediatric Cancers: The Case of Hepatoblastoma

Further preclinical investigation by the Pediatric Preclinical In Vivo Testing Consortium (PIVOT) explored OBI-3424's activity in hepatoblastoma, another pediatric cancer with noted AKR1C3 expression. These studies in hepatoblastoma PDX models also showed promising anti-tumor activity. OBI-3424 was well-tolerated and induced objective responses, including partial responses (PRs) and stable disease (SD), while prolonging EFS across the tested models.

However, this research also uncovered a critical layer of complexity. When the degree of clinical response was compared against the level of AKR1C3 protein expression (quantified as an H-score), the correlation was not consistent. For example, a PDX model with a very high AKR1C3 H-score of 255 exhibited progressive disease, whereas another model with a similarly high score of 250 achieved a partial response. This finding suggests that while high AKR1C3 expression is necessary for the initial activation of OBI-3424, it may not be sufficient to guarantee a clinical response in all tumor types. This points to the existence of other, downstream factors—such as the efficiency of DNA repair pathways or resistance to apoptosis—that can modulate a tumor's ultimate sensitivity to the drug's cytotoxic effects. This result serves as an important cautionary note, indicating that the relationship between the biomarker and clinical benefit may be more nuanced than initially hypothesized.

| Table 2: Overview of Key Preclinical Efficacy Studies | | | | | |

| :--- | :--- | :--- | :--- | :--- |

Clinical Development Trajectory and Pharmacokinetics

Overview of the Global Clinical Program

The clinical development of OBI-3424 has been pursued through several parallel and sequential studies across different geographies and patient populations. The cornerstone of the initial strategy was the first-in-human, Phase I/II dose-escalation and expansion study in patients with advanced solid tumors (NCT03592264), sponsored by OBI Pharma. A similar Phase I study was initiated in China by Ascentawits Pharmaceuticals, evaluating the drug as AST-3424 (NCT06239155). Following the compelling preclinical data in leukemia, a separate Phase I/II study was launched in collaboration with the Southwest Oncology Group (SWOG) to investigate OBI-3424 in patients with relapsed or refractory T-ALL and T-cell lymphoblastic lymphoma (T-LBL) (S1905, NCT04315324). As of 2024, the solid tumor trial (NCT03592264) has been terminated, while the T-ALL trial (NCT04315324) remains the primary focus of the drug's development.

Table 3: Summary of Major Clinical Trials for OBI-3424/AST-3424
Trial ID	Sponsor	Phase	Status	Indication(s)	Dosing Schedule(s)	Key Objective(s)	Reported Outcome(s)
NCT03592264	OBI Pharma	I/II	Terminated	Advanced Solid Tumors (incl. HCC, CRPC)	Sch. A: D1, D8 q21d; Sch. B: D1 q21d	Safety, MTD/RP2D, PK, Preliminary Efficacy	RP2D: 12 mg/m² (Sch. B); Best response: SD (54%); Terminated due to lack of efficacy.
NCT04315324 (S1905)	SWOG / NCI	I/II	Recruiting	Relapsed/Refractory T-ALL / T-LBL	D1, D8 q21d	Safety, MTD, Response Rate (CR/CRi)	Phase I complete; Phase II ongoing. No efficacy results published.
NCT06239155	Ascentawits	I/II	Recruiting	Advanced Solid Tumors (incl. HCC)	D1, D8 q21d	Safety, MTD/RP2D, PK, Preliminary Efficacy	RP2D: 6.0 mg/m²; Best response: SD (42.9%); Phase II expansion ongoing in HCC.
Data compiled from sources:

Human Pharmacokinetics: Metabolism and Excretion of OBI-3424 and OBI-2660

Pharmacokinetic (PK) data collected from the Phase I clinical trials have provided a clear picture of how OBI-3424 and its active metabolite behave in humans. The parent prodrug, OBI-3424 (or AST-3424), exhibits linear pharmacokinetics across the tested dose range of 1.0 to 14.0 mg/m², with minimal drug accumulation observed after repeated dosing. A key characteristic of the prodrug is its very short plasma half-life (

), which ranges from 0.16 to 0.40 hours (approximately 10–24 minutes). This indicates that the administered prodrug is either rapidly cleared from circulation or, more likely, quickly converted into its active form.

In contrast, the active cytotoxic metabolite, OBI-2660 (or AST-2660), has a significantly longer plasma half-life, ranging from 1.59 to 2.08 hours. This PK profile is highly favorable for a prodrug of this nature. The short half-life of OBI-3424 suggests a "hit-and-run" delivery mechanism, where the prodrug acts as a transient vehicle to transport the payload to the target site. Once inside AKR1C3-expressing cells, it is rapidly converted. The resulting active metabolite, OBI-2660, then persists for a much longer duration, providing a "sustained-effect" profile. This longer half-life allows the active agent sufficient time to engage with its target DNA, form cytotoxic crosslinks, and execute its anti-cancer function within the tumor cell, while systemic exposure to the inert parent compound is minimized. This kinetic relationship also provides a rationale for the intermittent dosing schedules used in clinical trials (e.g., once every 3 weeks or on Days 1 and 8 of a 21-day cycle), which allow for the full cytotoxic effect of the metabolite to be realized, followed by a recovery period for normal tissues like the bone marrow.

Clinical Findings in Advanced Solid Tumors: A Trajectory of Promise and Disappointment

Phase I Dose Escalation (NCT03592264 & NCT06239155): MTD and RP2D Determination

The first-in-human study of OBI-3424 (NCT03592264) in 39 patients with advanced solid tumors meticulously evaluated two different dosing schedules to optimize safety and tolerability. The initial regimen, Schedule A, involved intravenous administration of OBI-3424 on Days 1 and 8 of a 21-day cycle. Under this schedule, dose-limiting toxicities (DLTs), primarily Grade 3-4 thrombocytopenia and anemia, were observed at the 12 mg/m² dose level, establishing the maximum tolerated dose (MTD) for this schedule at 8 mg/m².

Due to these hematologic toxicities, the protocol was amended to explore a less frequent dosing regimen, Schedule B, which involved administration only on Day 1 of each 21-day cycle. This schedule was better tolerated. The MTD was not reached even at the highest dose tested (14 mg/m²), and the recommended Phase 2 dose (RP2D) was determined to be 12 mg/m² once every three weeks. The parallel Phase I study in China (NCT06239155), which used the Day 1 and 8 schedule, enrolled 21 patients and established a more conservative MTD and RP2D of 6.0 mg/m² based on similar hematologic DLTs.

Clinical Activity and Efficacy Outcomes

Despite the strong preclinical rationale and the establishment of a tolerable dose, the clinical activity of OBI-3424 in patients with advanced, heavily pre-treated solid tumors was profoundly disappointing. In the 39-patient NCT03592264 trial, there was only one confirmed partial response. The best outcome for the majority of evaluable patients (21 of 33, or 64%) was stable disease. The results from the NCT06239155 trial in China were similar; among 15 evaluable patients, there were no objective responses (PR or CR), and the best response observed was stable disease in 9 patients (42.9%). This lack of meaningful anti-tumor activity stood in stark contrast to the robust tumor growth inhibition seen in preclinical xenograft models.

Analysis of Program Termination: Strategic Rationale and Implications

The culmination of these disappointing efficacy results was the decision by OBI Pharma to halt the development of OBI-3424 in solid tumors. On March 11, 2024, the company officially announced the termination of the OBI-3424-001 trial (NCT03592264). The company's press release cited a "strategic portfolio review" and stated that the drug had "shown only limited therapeutic potential" and had "not demonstrated therapeutic potentials in these cancer patients". The official reason listed on the ClinicalTrials.gov registry is concise and unambiguous: "Little evidence of clinical activity in tumor types enrolled".

This outcome represents a significant setback for the program and highlights a critical disconnect between the preclinical models and the clinical reality of treating advanced solid malignancies. The decision involved reallocating an estimated $10 million USD in resources to other priority projects within the company's pipeline. While the solid tumor program was terminated, OBI Pharma affirmed its commitment to continue collaborations for the drug's development in hematologic malignancies, signaling a clear strategic pivot.

Clinical Investigation in Hematologic Malignancies: A Renewed Focus on T-ALL

Rationale for the Pivot to Relapsed/Refractory T-ALL/T-LBL

Despite the failure in solid tumors, the clinical development of OBI-3424 continues with a focused effort in T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL). This strategic pivot is grounded in a distinct and compelling scientific rationale that differentiates the T-ALL indication from the solid tumor settings.

First, the expression of the target enzyme, AKR1C3, is exceptionally high and more uniformly expressed in T-ALL compared to many solid tumors, providing a stronger biomarker signal. Second, the preclinical efficacy observed in T-ALL PDX models was not merely positive but profound, with OBI-3424 inducing deep and durable remissions in highly aggressive, patient-derived models. These PDX models are generally considered to have higher predictive value for clinical success than the CDX models used for many of the solid tumor studies. Third, and perhaps most importantly, is the fundamental biology of the disease. Hematologic malignancies, and ALL in particular, are known to be preferentially sensitive to DNA-damaging agents. This intrinsic vulnerability to the drug's specific mechanism of action—DNA alkylation—provides a more favorable biological context for OBI-3424 to succeed, a condition that may not have been present in the more heterogeneous and potentially multi-resistant advanced solid tumors.

Design and Objectives of the SWOG-led S1905 Study (NCT04315324)

The investigation in this new strategic direction is being conducted through the S1905 clinical trial (NCT04315324), a Phase I/II study led by the National Cancer Institute-sponsored Southwest Oncology Group (SWOG). The trial is enrolling patients aged 12 years and older with T-ALL or T-LBL that has either relapsed after a prior remission or was refractory to initial treatment—a population with a dire prognosis and high unmet medical need.

The study is designed in two parts. The initial Phase I dose-escalation portion has been completed and established the MTD of OBI-3424 for this patient population. The trial is now in its Phase II portion, which is focused on efficacy. The primary objective is to assess the overall response rate, defined as the percentage of patients who achieve either a complete remission (CR) or a complete remission with incomplete hematologic recovery (CRi). Secondary objectives include evaluating the safety profile, event-free survival (EFS), relapse-free survival (RFS), and overall survival (OS). A critical component of the study is its translational medicine objectives, which aim to prospectively assess AKR1C3 expression levels in patients and evaluate the association between biomarker expression and clinical outcomes, such as response rates and achievement of minimal residual disease (MRD) negativity.

Potential Role in the T-ALL Treatment Landscape

The standard of care for patients with relapsed/refractory (R/R) T-ALL is challenging, and long-term survival rates are dismal, often below 10% for adults. Current treatment paradigms typically involve intensive multi-agent reinduction chemotherapy regimens (such as DHAP or ICE) or targeted agents like nelarabine, with the goal of achieving a second remission to allow for a potentially curative allogeneic hematopoietic stem cell transplant. However, many patients do not respond to salvage therapy or relapse after transplant.

If the S1905 trial demonstrates that OBI-3424 is both safe and effective, it could fill a significant gap in the T-ALL treatment landscape. As a novel, biomarker-driven agent with a distinct mechanism of action, it could provide a new therapeutic option for this high-risk population. Preclinical data also suggested potential synergy when OBI-3424 was combined with nelarabine, hinting at future possibilities for combination therapies. A successful outcome in this trial would validate the strategic pivot to hematologic malignancies and could establish OBI-3424 as a valuable new tool for treating R/R T-ALL.

Comprehensive Safety and Tolerability Profile

Analysis of the Phase I Safety Population

The safety and tolerability of OBI-3424 have been characterized through the Phase I dose-escalation portions of the NCT03592264 study (39 patients) and the NCT06239155 study (21 patients) in advanced solid tumors. Across these studies, the drug was generally described as well-tolerated. In the larger OBI Pharma study, treatment-related adverse events (TRAEs) of any grade were reported in 82% (32/39) of patients. Serious TRAEs were reported in a minority of patients, and importantly, no fatal TRAEs were attributed to the study drug.

Dose-Limiting Toxicities: Characterization of Hematologic Events

The consistent and primary dose-limiting toxicity (DLT) observed across all clinical studies of OBI-3424 was myelosuppression. Specifically, Grade 3-4 thrombocytopenia (low platelet count) and Grade 3-4 anemia (low red blood cell count) were the events that defined the maximum tolerated dose. These hematologic toxicities were observed to be dose-dependent, meaning their frequency and severity increased with higher doses of OBI-3424. Encouragingly, these effects were also characterized as non-cumulative, suggesting that the bone marrow was able to recover between treatment cycles. The nadir, or lowest point, for platelet counts typically occurred on Day 15 or Day 22 of the 21-day cycle, consistent with the expected timeline for alkylating agent-induced myelosuppression.

This safety profile is notably different from what was predicted by preclinical toxicology studies. A non-GLP study in cynomolgus monkeys identified severe gastrointestinal toxicity (diarrhea and intestinal pathology) as the dose-limiting factor, leading to euthanasia at a dose of 1 mg/kg. In contrast, the human clinical trials clearly established hematologic toxicity as the DLT, with gastrointestinal events like nausea being common but less severe. This species-specific difference in the primary toxicity is a significant finding that underscores the limitations of non-human primate models in perfectly predicting human safety profiles for certain classes of drugs. The observed human DLTs are more typical for a DNA alkylating agent and are generally considered manageable within the standard practice of oncology.

Spectrum of Treatment-Related Adverse Events

Beyond the dose-limiting hematologic events, the overall safety profile of OBI-3424 was characterized by a range of common, mostly low-grade adverse events. The most frequently reported TRAEs of any grade in the NCT03592264 study were anemia (64%), thrombocytopenia (51%), nausea (26%), and fatigue (21%). In the NCT06239155 study, the most common Grade ≥3 TRAEs were anemia (33.3%), decreased platelet count (19.0%), decreased white blood cell count (9.5%), and decreased neutrophil count (9.5%). Across both studies, the rate of Grade ≥3 TRAEs was in the range of 38-49%.

| Table 4: Consolidated Safety Profile from Phase I Solid Tumor Trials | | | | |

| :--- | :--- | :--- | :--- |

| Anemia | 64 | 33–41 | Yes | |

| Thrombocytopenia | 51 | 19–26 | Yes | |

| Nausea | 26 | <5 | No | |

| Fatigue | 21 | <5 | No | |

| Leukopenia / Decreased WBC Count | Not Reported | 9.5 | No | |

Percentages are drawn from the most comprehensive reports (NCT03592264 and NCT06239155) and represent the range or most frequently cited value.

Strategic Analysis and Future Outlook

The AKR1C3 Biomarker Strategy: Successes and Challenges

The development of OBI-3424 was predicated on a sound and elegant biomarker strategy: target an enzyme with highly differential expression between tumor and normal tissue to enable selective activation of a potent cytotoxin. This strategy was successful in its initial stages. It identified a viable target in AKR1C3, allowed for the rational design of a selective prodrug, and was strongly validated in a broad range of preclinical models. However, the clinical failure in advanced solid tumors, coupled with the inconsistent biomarker-response correlation observed in preclinical hepatoblastoma models, has revealed the limitations of this approach. It is now clear that high AKR1C3 expression is a necessary prerequisite for drug activity but is not, by itself, a sufficient guarantee of clinical benefit. The future of this biomarker-driven strategy now rests entirely on the T-ALL program, where the confluence of high biomarker expression and the inherent biological sensitivity of the malignancy to DNA alkylation may finally provide the right context for success.

Bridging the Gap: Deconstructing the Preclinical-to-Clinical Disconnect in Solid Tumors

The stark disconnect between the robust preclinical efficacy in solid tumor models and the lack of meaningful clinical activity warrants careful analysis. Several factors likely contributed to this translational failure. First, preclinical xenograft models, particularly subcutaneous CDX models, often fail to fully recapitulate the complex tumor microenvironment, drug penetration barriers, and extensive heterogeneity of advanced, metastatic human cancer. Second, the patient population enrolled in the Phase I trials was heavily pre-treated, having failed multiple prior lines of therapy. These tumors may have acquired broad, pleiotropic drug resistance mechanisms (e.g., enhanced DNA repair capacity, upregulated drug efflux pumps) that were not present in the treatment-naïve preclinical models. Finally, it is possible that the AKR1C3 expression threshold used for patient selection (e.g., an immunohistochemistry H-score ≥135) was not optimal, or that significant intratumor heterogeneity in AKR1C3 expression allowed for the survival and outgrowth of AKR1C3-low resistant clones.

Competitive Landscape and Standard of Care in Key Indications

OBI-3424 was initially developed to address unmet needs in several major cancer types, but the therapeutic landscapes in these areas have evolved significantly, raising the bar for new entrants.

Advanced Hepatocellular Carcinoma (HCC): The standard of care for first-line treatment has rapidly shifted from single-agent tyrosine kinase inhibitors (TKIs) like sorafenib and lenvatinib to more effective immunotherapy-based combinations, primarily atezolizumab plus bevacizumab and durvalumab plus tremelimumab. With its modest single-agent activity, OBI-3424 was unlikely to be competitive in this dynamic and increasingly effective treatment landscape.
Metastatic Castration-Resistant Prostate Cancer (mCRPC): The mCRPC treatment paradigm is crowded and multifaceted, including potent next-generation androgen receptor pathway inhibitors (abiraterone, enzalutamide), taxane chemotherapy (docetaxel, cabazitaxel), PARP inhibitors for patients with homologous recombination repair gene mutations, and targeted radioligand therapy (Lutetium-177-PSMA-617). OBI-3424 would have faced immense difficulty finding a viable position in this market.
Relapsed/Refractory (R/R) T-ALL: In contrast, the landscape for R/R T-ALL remains an area of profound unmet medical need. Outcomes are exceptionally poor, and therapeutic options are limited, consisting mainly of intensive salvage chemotherapy and nelarabine, primarily as a bridge to allogeneic stem cell transplant. This setting represents the clearest and most compelling opportunity for a novel agent like OBI-3424 to demonstrate clinical value.

Regulatory and Corporate History: Key Milestones and Partnerships

The asset now known as OBI-3424 was originally designated TH-3424 and was acquired by OBI Pharma from Threshold Pharmaceuticals in a deal announced on June 1, 2017. Under the agreement, OBI Pharma obtained worldwide development and commercial rights, with the exception of several key territories in Asia and the Middle East (including China, Japan, and South Korea), where rights are held by Ascentawits Pharmaceuticals.

OBI-3424 achieved key regulatory milestones early in its development. The U.S. Food and Drug Administration (FDA) granted it Orphan Drug Designation for the treatment of HCC in July 2018, followed by a second Orphan Drug Designation for the treatment of Acute Lymphoblastic Leukemia (ALL) in September 2018. These designations provided potential incentives for development in these rare or difficult-to-treat diseases. A critical component of the current strategy is the collaboration with SWOG, sponsored by the National Cancer Institute (NCI), to conduct the pivotal S1905 trial in T-ALL.

Concluding Assessment: The Future Viability and Niche Potential of OBI-3424

The initial ambition for OBI-3424 as a broad-spectrum, biomarker-driven therapy for solid tumors has not been realized. The termination of the NCT03592264 trial marks the end of that strategy. The future of OBI-3424 as a therapeutic agent is now narrowly but acutely focused on the high-risk, high-reward indication of relapsed/refractory T-ALL. The success or failure of the ongoing NCT04315324 trial will be the pivotal, make-or-break event for the entire program. If the trial yields positive results, demonstrating a meaningful response rate in this population with a manageable safety profile, OBI-3424 could be successfully repositioned as a valuable niche oncology product for a patient population with critically few effective options. If the trial is negative, the asset's path forward would become highly uncertain. The ultimate question that remains is whether the profound and unambiguous preclinical efficacy signal in T-ALL will finally translate into clinical benefit where the solid tumor data did not.

Works cited

Definition of AKR1C3-activated prodrug AST-3424 - NCI Drug ..., accessed October 6, 2025, https://www.cancer.gov/publications/dictionaries/cancer-drug/def/akr1c3-activated-prodrug-ast-3424
OBI-3424 | Ligand page | IUPHAR/BPS Guide to PHARMACOLOGY, accessed October 6, 2025, https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=12770
AST-3424 - Drug Targets, Indications, Patents - Patsnap Synapse, accessed October 6, 2025, https://synapse.patsnap.com/drug/e5b3274bcc2e42f5be47524235edc563
OBI-3424 | CAS# 2097713-68-1 | MedKoo, accessed October 6, 2025, https://www.medkoo.com/products/38999
OBI-3424: Uses, Interactions, Mechanism of Action | DrugBank Online, accessed October 6, 2025, https://go.drugbank.com/drugs/DB16057
NCT03592264 | A Phase I/II Study of OBI-3424 in Subjects with Advanced Solid Tumors, accessed October 6, 2025, https://clinicaltrials.gov/study/NCT03592264
A Phase I/II Study of OBI-3424 in Subjects with Advanced Solid Tumors | ClinicalTrials.gov, accessed October 6, 2025, https://www.clinicaltrials.gov/study/NCT03592264?lead=OBI%20Pharma,%20Inc&rank=3
DrugMapper, accessed October 6, 2025, https://idaapm.helsinki.fi/CompoundInfo?compound_id=2127578
Obi-3424 | C21H25N4O6P | CID 126961329 - PubChem, accessed October 6, 2025, https://pubchem.ncbi.nlm.nih.gov/compound/126961329
A novel selective AKR1C3-activated prodrug AST-3424/OBI-3424 exhibits broad anti-tumor activity - PMC, accessed October 6, 2025, https://pmc.ncbi.nlm.nih.gov/articles/PMC8332853/
Original Article A novel selective AKR1C3-activated prodrug AST-3424/OBI-3424 exhibits broad anti-tumor activity - e-Century Publishing Corporation, accessed October 6, 2025, https://e-century.us/files/ajcr/11/7/ajcr0131972.pdf
AKR1C3 in carcinomas: from multifaceted roles to therapeutic strategies - Frontiers, accessed October 6, 2025, https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2024.1378292/full
OBI-3424, a novel AKR1C3-activated prodrug, exhibits potent efficacy against preclinical models of T-ALL - PubMed Central, accessed October 6, 2025, https://pmc.ncbi.nlm.nih.gov/articles/PMC6635081/
Safety, pharmacokinetics, and clinical activity of OBI-3424, an AKR1C3-activated prodrug, in patients with advanced or metastatic solid tumors: A phase 1 dose-escalation study. - ASCO Publications, accessed October 6, 2025, https://ascopubs.org/doi/10.1200/JCO.2022.40.16_suppl.3030
A novel selective AKR1C3-activated prodrug AST-3424/OBI-3424 exhibits broad anti-tumor activity - PubMed, accessed October 6, 2025, https://pubmed.ncbi.nlm.nih.gov/34354865/
OBI Pharma Granted FDA Orphan Drug Designation for OBI-3424 for the Treatment of Hepatocellular Carcinoma (HCC), accessed October 6, 2025, https://www.obipharma.com/news/obi-pharma-granted-fda-orphan-drug-designation-for-obi-3424-for-the-treatment-of-hepatocellular-carcinoma-hcc/
Selective and Broad Anti-Tumor Activity of AKR1C3-Activated Prodrug AST-3424/OBI-3424, accessed October 6, 2025, https://www.ascentawits.com/uploadfiles/2021/12/20211221161346703.pdf
A first-in-man phase I/II study of OBI-3424, an AKR1C3-selective bis-alkylating agent prodrug, in subjects with advanced cancer, including hepatocellular carcinoma (HCC) and castrate-resistant prostate cancer (CRPC). - ASCO Publications, accessed October 6, 2025, https://ascopubs.org/doi/10.1200/JCO.2020.38.15_suppl.TPS3658
Safety, pharmacokinetics, and clinical activity of OBI-3424, an AKR1C3-activated prodrug, in patients with advanced or metastatic solid tumors: A phase 1 dose-escalation study. - ResearchGate, accessed October 6, 2025, https://www.researchgate.net/publication/361120176_Safety_pharmacokinetics_and_clinical_activity_of_OBI-3424_an_AKR1C3-activated_prodrug_in_patients_with_advanced_or_metastatic_solid_tumors_A_phase_1_dose-escalation_study
Safety, tolerability, pharmacokinetics and clinical activity of AST ..., accessed October 6, 2025, https://ascopubs.org/doi/10.1200/JCO.2024.42.16_suppl.3121
OBI-3424, a Novel AKR1C3-Activated Prodrug, Exhibits Potent ..., accessed October 6, 2025, https://aacrjournals.org/clincancerres/article/25/14/4493/81788/OBI-3424-a-Novel-AKR1C3-Activated-Prodrug-Exhibits
OBI Pharma Announces Acquisition of TH-3424 from Threshold ..., accessed October 6, 2025, https://www.obipharma.com/news/obi-pharma-announces-acquisition-of-th-3424-from-threshold-pharmaceuticals/
OBI Pharma Granted FDA Orphan Drug Designation For OBI-3424 For The Treatment Of Acute Lymphoblastic Leukemia (ALL) - Drug Discovery Online, accessed October 6, 2025, https://www.drugdiscoveryonline.com/doc/obi-pharma-granted-fda-orphan-drug-designation-for-obi-for-the-treatment-of-acute-lymphoblastic-leukemia-all-0001
(R)-Odafosfamide (OBI-3424) | DNA-alkylating Agent - MedchemExpress.com, accessed October 6, 2025, https://www.medchemexpress.com/obi-3424.html
Abstract 7050: Preclinical evaluation of the AKR1C3-activated alkylator OBI-3424 in hepatoblastoma - AACR Journals, accessed October 6, 2025, https://aacrjournals.org/cancerres/article/85/8_Supplement_1/7050/760569/Abstract-7050-Preclinical-evaluation-of-the-AKR1C3
Preclinical evaluation of the AKR1C3-activated alkylator, OBI-3424, in hepatoblastoma – A report from the Pediatric Preclinica, accessed October 6, 2025, https://preclinicalpivot.org/wp-content/uploads/pivot_2025_2010_obi3424.pdf
Phase I/II Study of AKR1C3-Activated Prodrug OBI-3424 (OBI-3424) in Patients with Relapsed/Refractory T-Cell Acute Lymphoblastic Leukemia (T-ALL)/ T-Cell Lymphoblastic Lymphoma (T-LBL), accessed October 6, 2025, https://www.masseycancercenter.org/clinical-trials/details/phase-iii-study-of-akr1c3-activated-prodrug-obi-3424-obi-3424-in-patients-with-relapsedrefractory-t-
Study Details | NCT04315324 | Study to Test OBI-3424 in Patients ..., accessed October 6, 2025, https://clinicaltrials.gov/study/NCT04315324
CLINICAL TRIAL / NCT04315324 - UChicago Medicine, accessed October 6, 2025, https://www.uchicagomedicine.org/find-a-clinical-trial/clinical-trial/cirb210385
A Phase I/II Study of OBI-3424 in Subjects with Advanced Solid Tumors, accessed October 6, 2025, https://clin.larvol.com/trial-detail/NCT03592264
OBI announces the termination of OBI-3424-001 trial while continuing the collaboration of OBI-3424 with partners - OBI Pharma, accessed October 6, 2025, https://www.obipharma.com/announcement/obi-announces-the-termination-of-obi-3424-001-trial-while-continuing-the-collaboration-of-obi-3424-with-partners/
Safety, pharmacokinetics, and clinical activity of OBI-3424, an AKR1C3‑activated prodrug, in patients with advanced or metastatic solid tumors: A phase 1 dose-escalation study. - ASCO, accessed October 6, 2025, https://www.asco.org/abstracts-presentations/ABSTRACT376692
Phase 1 dose-escalation study evaluating the safety ... - PubMed, accessed October 6, 2025, https://pubmed.ncbi.nlm.nih.gov/37173365/
Study to Test OBI-3424 in Patients with T-Cell Acute Lymphoblastic Leukemia (T-ALL) or T-Cell Lymphoblastic Lymphoma (T-LBL) - National Cancer Institute, accessed October 6, 2025, https://www.cancer.gov/research/participate/clinical-trials-search/v?id=NCI-2020-00768
Testing the Drug OBI-3424 in People with T-Cell Acute ... - SWOG, accessed October 6, 2025, https://www.swog.org/patients/trials-open-patients/testing-drug-obi-3424-people-t-cell-acute-lymphoblastic-leukemia-or-t
NCT04315324 | Study to Test OBI-3424 in Patients With T-Cell Acute Lymphoblastic Leukemia (T-ALL) or T-Cell Lymphoblastic Lymphoma (T-LBL) | ClinicalTrials.gov, accessed October 6, 2025, https://www.clinicaltrials.gov/study/NCT04315324
Management of adults with T-cell lymphoblastic leukemia - Fred Hutchinson Cancer Center, accessed October 6, 2025, https://www.fredhutch.org/content/dam/stripe/lymphoma-tumor-board/publications/Management%20of%20adults%20with%20T-cell%20lymphoblastic%20leukemia.pdf
Nelarabine: when and how to use in the treatment of T-cell acute ..., accessed October 6, 2025, https://ashpublications.org/bloodadvances/article/8/1/23/496638/Nelarabine-when-and-how-to-use-in-the-treatment-of
Treatments for relapsed or refractory acute lymphoblastic leukemia, accessed October 6, 2025, https://cancer.ca/en/cancer-information/cancer-types/acute-lymphoblastic-leukemia-all/treatment/relapsed-or-refractory
T-cell acute lymphoblastic leukemia - PMC - PubMed Central, accessed October 6, 2025, https://pmc.ncbi.nlm.nih.gov/articles/PMC6142501/
Adult T-Cell Leukemia/Lymphoma: Relapsed/Refractory - LRF, accessed October 6, 2025, https://lymphoma.org/understanding-lymphoma/aboutlymphoma/nhl/atll/relapsedatll/
OBI-3424 completes its phase 1 safety evaluation and demonstrates ..., accessed October 6, 2025, https://www.obipharma.com/announcement/obi-3424-completes-its-phase-1-safety-evaluation-and-demonstrates-its-safety-with-no-major-concerns-proceeding-to-phase-2-as-planned/
Advanced hepatocellular carcinoma treatment strategies: Are transarterial approaches leading the way? - PMC, accessed October 6, 2025, https://pmc.ncbi.nlm.nih.gov/articles/PMC11664626/
Systemic Therapy for Advanced Hepatocellular Carcinoma: ASCO Guideline, accessed October 6, 2025, https://ascopubs.org/doi/10.1200/JCO.20.02672
Systemic Therapy for Advanced Hepatocellular Carcinoma: ASCO ..., accessed October 6, 2025, https://ascopubs.org/doi/10.1200/JCO.23.02745
Treatments for castration-resistant prostate cancer | Canadian ..., accessed October 6, 2025, https://cancer.ca/en/cancer-information/cancer-types/prostate/treatment/castration-resistant-prostate-cancer
State of the Art: Personalizing Treatment for Patients With Metastatic Castration-Resistant Prostate Cancer - ASCO Publications, accessed October 6, 2025, https://ascopubs.org/doi/10.1200/EDBK-25-473636
OBI Pharma Announces Acquisition of the Novel First-in-class Anti-Cancer Drug OBI-3424 from Threshold Pharmaceuticals to be Developed as Potential Anti-Cancer Therapy for Cancers with High AKR1C3, accessed October 6, 2025, https://www.obipharma.com/announcement/obi-pharma-announces-acquisition-of-the-novel-first-in-class-anti-cancer-drug-obi-3424-from-threshold-pharmaceuticals-to-be-developed-as-potential-anti-cancer-therapy-for-cancers-with-high-akr1c3/
OBI Pharma Granted FDA Orphan Drug Designation for OBI-3424 for the Treatment of Acute Lymphoblastic Leukemia (ALL), accessed October 6, 2025, https://www.obipharma.com/announcement/obi-pharma-granted-fda-orphan-drug-designation-for-obi-3424-for-the-treatment-of-acute-lymphoblastic-leukemia-all-2/
New Drug OBI-3424 An 'Orphan Drug' To Treat Acute Leukaemia | CCI, accessed October 6, 2025, https://www.ccia.org.au/blog/new-drug-obi-3424-an-orphan-drug-to-treat-acute-leukaemia

AI-Powered Research

Premium Access

Deudextromethorphan Advanced Drug Monograph

Generic Name