A Proimmunotoxin Nanodrug Targeting AIDS-associated non-Hodgkin Lymphoma

Abstract

B-cell non-Hodgkin lymphoma (NHL) is the most common hematopoietic malignancy in the United States, with a notably higher incidence and aggressiveness observed in individuals infected with human immunodeficiency virus (HIV), leading to AIDS-associated NHL (AIDS-NHL). The transferrin receptor 1 (TfR1/CD71), a type II transmembrane homodimeric protein, is overexpressed on several cancers, including NHL, providing a meaningful therapeutic target. Our group developed an anti-TfR1 IgG3-avidin fusion protein, ch128.1Av, designed to deliver biotinylated therapeutic agents into cancer cells through receptor-mediated endocytosis. When coupled with biotinylated saporin 6 (b-SO6), a plant-derived protein synthesis inhibitor, the resulting ch128.1Av/b-SO6 immunotoxin is highly effective at killing malignant cells. However, toxicity to normal cells limits its systemic administration. To overcome this problem, we developed a proimmunotoxin nanodrug, named “n(ch128.1Av/b-SO6)-CXCL13”. This strategy involves encapsulating individual immunotoxins within a thin zwitterionic polymer shell, which is stabilized by peptide crosslinkers that only respond to metalloproteinase-2 (MMP-2), a tumor microenvironment-specific enzyme. The nanodrug is further conjugated with a B-cell targeting chemokine CXCL13. This design allows the proimmunotoxin to circulate safely, specifically accumulate and release encapsulated ch128.1Av/b-SO6 intratumorally in response to MMP-2. This approach not only minimizes off-target toxicity but also enhances tissue penetration by enabling choline analogues on proimmunotoxin nanodrugs to bind choline transporters expressed on tumor cells or the blood-brain barrier. Importantly, n(ch128.1Av/b-SO6)-CXCL13 demonstrated antitumor efficacy in an AIDS-associated NHL xenograft mouse model. Taken together, our results suggest that n(ch128.1Av/b-SO6)-CXCL13, or similar proimmunotoxin strategies, represents a promising therapeutic avenue for AIDS-NHL and potentially other malignancies.

1. Introduction

Non-Hodgkin lymphoma (NHL) represents a heterogeneous group of blood cancers originating from lymphocytes, a type of white blood cell crucial for immune system function [1]. As one of the most prevalent hematopoietic malignancies in the United States, NHL encompasses a diverse array of subtypes, each characterized by distinct pathological and clinical features. This heterogeneity poses significant challenges in diagnosis, classification, and treatment, highlighting the need for targeted therapeutic strategies. In 2025, an estimated 80,350 new NHL cases and 19,390 deaths were reported [2]. Among the population, individuals with congenital and acquired immunodeficiencies, such as those with human immunodeficiency virus (HIV) infection, are at increased risk of developing B-cell NHL [3, 4]. Notably, NHL has been classified as an acquired immunodeficiency syndrome (AIDS)-defining cancer since 1985 [5]. Although the incidence of AIDS-associated NHL (AIDS-NHL) has significantly declined in the era of combination antiretroviral therapy (cART), it remains the most common HIV-related malignancy in developed countries [6, 7]. Compared to individuals who are HIV-negative, people living with HIV and AIDS-NHL tend to present with aggressive, advanced-stage disease at a younger median age and frequently exhibit extranodal involvement, including the central nervous system (CNS) [8, 9]. Furthermore, AIDS-NHL is further complicated by its frequent association with oncogenic viruses, such as Epstein-Barr virus (EBV) and human herpesvirus-8 (HHV-8) [10, 11], underscoring the need for more effective and targeted therapeutic strategies.

Transferrin receptor 1 (TfR1, also known as CD71) is a Type II transmembrane, homodimeric glycoprotein responsible for cellular iron uptake via the interaction with iron-loaded transferrin (Tf), the main iron transporter in blood [12–15]. While TfR1 is expressed at low levels in various normal tissues, its expression is significantly elevated in rapidly proliferating cells, such as cancer cells, including NHL [12–18]. Notably, AIDS-NHL exhibits even higher TfR1 mRNA levels compared to NHL cells from non-infected individuals [19, 20]. In general, cancer cells overexpress TfR1, supporting its consideration as a universal cancer marker candidate and both TfR1 and iron have been shown to contribute to cancer cell survival [15] [21]. Given its overexpression on cancer cells, capacity for internalization, and central role in cancer cell pathophysiology, TfR1 presents a compelling target for therapeutic intervention [14, 15].

We previously developed a mouse/human chimeric IgG3-avidin fusion protein specific for human TfR1, ch128.1Av (previously named anti-hTfR IgG3-Av), as a universal vector to deliver a variety of biotinylated agents into cancer cells by receptor-mediated endocytosis (RME) [15, 22, 23]. Chicken avidin was genetically fused to the C_H3 domain of the antibody. We found that the antibody-avidin fusion protein is dimeric in solution [22], due to the natural tetrameric structure of avidin [24], which facilitates the binding of two antibody molecules. Each avidin unit is capable of binding one biotin molecule with high affinity, allowing the dimeric fusion protein to potentially bind up to four biotinylated compounds [25]. Human IgG3 was selected for its unique extended hinge region, which provides spacing and flexibility [26, 27], facilitating antigen and a biotinylated compound binding simultaneously. Notably, both ch128.1Av and its parental antibody, ch128.1, do not inhibit the binding of transferrin or the hemochromatosis protein (HFE: another TfR1 ligand) to TfR1 [28, 29], indicating that the antibody is non-neutralizing [13, 15]. The parental anti-TfR1 antibody (ch128.1/IgG3) exhibits high-affinity antigen binding (K_D 5.7 nM) with a binding site mapped to an epitope located within the apical domain of the human TfR1 [30]. Anti-TfR1 antibodies, like ch128.1/IgG3, can be directly cytotoxic to cells through the inhibition of iron uptake by various mechanisms. Intriguingly, we found that fusing avidin to ch128.1/IgG3 enhances its direct cytotoxicity and induces TfR1 degradation, leading to subsequent lethal iron starvation in certain malignant hematopoietic cells [22, 23, 31]. We also found that administration of ch128.1Av alone results in significant antitumor activity in immunosuppressed mice (SCID-Beige) bearing disseminated human B-cell malignancy multiple myeloma (MM) cells, regardless of the in vitro sensitivity of the cell line [32]. However, the parental antibody without avidin (ch128.1/IgG3) shows superior in vivo antitumor activity, despite its lower direct in vitro cytotoxicity compared to ch128.1Av [32], which is explained by the lower bioavailability of ch128.1Av, as it contains avidin and is cleared faster from blood [33]. This mechanism of antitumor activity in vivo was found to be Fc-mediated (i.e., antibody effector functions), involving macrophages, capable of triggering antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) [34, 35]. The Fc region has also been found to be critical for the in vivo antitumor activity of the IgG1 version of this antibody (ch128.1/IgG1) in xenograft models of MM [34]. More recently and consistent with the studies using human MM cells, we showed that ch128.1/IgG1 and its humanized counterpart, hu128.1, exhibit antitumor activity in SCID-Beige models bearing local or disseminated AIDS-NHL Burkitt lymphoma (BL) cells [2F7-BR44 (EBV+) or JB (EBV-) cells] [36]. However, it is well established that not all tumor types respond equally to anti-TfR1 antibodies, and even those that may eventually develop resistance [15]. In this context, it is of interest to explore the use of TfR1-specific antibodies and their derivatives, conjugated with antitumor agents, for targeted delivery into cancer cells via RME [13, 37].

One promising approach to eliminate malignant cells involves conjugating ch128.1Av with a toxin. Saporin, a plant toxin produced by Saponaria officinalis, is a Type I ribosome-inactivating protein (RIP) [38–40]. This protein synthesis inhibitor is of great interest in cancer therapy [40, 41]. Saporin is a gene family composed of several isoforms. The saporin-6 (SO6) isoform is isolated from the seeds of the plant and is commonly used for in the generation of immunotoxins[42]. Although all isoforms of saporin lack a cell-binding domain and therefore cannot efficiently enter cells, conjugating ch128.1Av with monobiotinylated saporin 6 (b-SO6) to form the immunotoxin ch128.1Av/b-SO6, markedly increases cytotoxicity in malignant B cells sensitive to the direct effects of ch128.1Av and also induces cell death in cells that are not sensitive to the direct effects of the antibody fusion protein [25, 43]. Protein synthesis inhibition but not iron starvation is responsible for the cytotoxicity induced by ch128.1Av/b-SO6 [43]. However, systemic administration of saporin as part of an immunotoxin containing an antibody targeting TfR1 is expected to result in toxicity to normal cells, especially those expressing TfR1. In addition, although saporin does not have a cell-binding chain, it is possible to have non-specific cell uptake without an “escort”, such as through bulk-phase endocytosis [44]. Moreover, the positive charge of avidin increases the likelihood of non-specific interactions with negatively charged structures, such as cell membranes, potentially leading to internalization into normal cells [45, 46].

A potential strategy to mitigate these drawbacks is the use of nanotechnology, which has been broadly explored for this purpose. Attaching polyethylene glycol (PEG) to therapeutic proteins can mask immunogenic epitopes, reduce renal clearance, and prolong circulation time [47]. However, direct PEGylation may compromise the immunotoxin activity [48], and previous attempts showed minimal benefits [49]. Numerous strategies using site-specific PEGylation have been developed, but have not shown success in clinical studies. Alternatively, biodegradable polymeric nanoparticles, designed to improve the therapeutic properties of drugs, provide a practical means to reduce the toxic side effects of immunotoxins. Immunotoxin-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles modified with antibodies have demonstrated improved therapeutic efficacy against breast cancer and increased maximally tolerated doses compared to free immunotoxins [50]. Despite these advancements, achieving tumor-specific release of nanoparticle loaded immunotoxins remains a significant challenge.

In this study, we engineered a proimmunotoxin nanodrug, n(ch128.1Av/b-SO6)-CXCL13, aiming to release the immunotoxin into the tumor microenvironment (TME) of AIDS-NHL and therefore, decreasing off-target toxicities while enhancing tumor targeting. This nanodrug is capable of crossing the blood-brain barrier (BBB) and thus, is expected to be effective again brain metastases. This nanoplatform encapsulates the immunotoxin within a thin polymer shell formed via in situ polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) with a choline analogue, enhancing tumor penetration and BBB translocation [51–54]. The structure is stabilized by peptide crosslinkers responsive to tumor-associated metalloproteinase-2 (MMP-2) [55], enabling controlled release in the TME. Incorporating choline analogues into the shell facilitates enhanced penetration into solid tumors and across the BBB via choline transporters (ChTs). Conjugation with CXCL13, a chemokine targeting CXCR5-expressing B cells, further improves tumor selectivity and mitigates off-target toxicity. Overall, these findings indicate that the present nanocapsule strategy is a promising proimmunotoxin approach against AIDS-NHL and possibly other cancers including those with CNS involvement.

2. Material and methods

2.1. Materials

All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA), unless otherwise specified. Cell culture materials were obtained from Thermo Fisher Scientific (Waltham, MA, USA), unless otherwise specified. NHS-PEG₄-Azide, Zeocin^®, Hoechst 33342, HRP-conjugated goat anti-mouse IgG Fc cross-adsorbed secondary antibody and mouse IgG total ELISA Ready-SET-Go! Kit were purchased from Thermo Fisher Scientific. ELISA reagents, including anti-human IgG antibody, HRP-conjugated donkey anti-human IgG, and 3, 3’, 5, 5’ tetramethyl benzidine (TMB), were purchased from Biolegend (San Diego, CA, USA). The CellTiter-Blue^® Cell Viability Assay and the CytoTox-Glo^™ Cytotoxicity Assay were purchased from Promega Corporation (Madison, WI, USA). Recombinant human CXCL13 chemokine was purchased from Peprotech (Cranbury, NJ, USA), and Matrigel^® matrix basement membrane was purchased from Corning (Corning, NY, USA). [³H]-thymidine was obtained from MP Biomedicals (Santa Ana, CA, USA) for the thymidine incorporation assay.

2.2. Synthesis of nanodrugs

Production of the anti-TfR1 antibody-avidin fusion protein (ch128.1Av) in murine myeloma cells has been described previously [25]. An isotype control IgG3-avidin fusion protein (IgGAv) specific for the hapten dansyl ([5-dimethylamino naphthalene-1-sulfonyl chloride) was also produced in murine myeloma cells [25]. b-SO6 was manufactured by Advanced Targeting Systems (Carlsbad, CA, USA). The ch128.1Av/b-SO6 and the isotype control IgGAv/b-SO6 immunotoxins were obtained through the avidin-biotin interaction between the fusion protein and b-SO6 at the molar ratio of 1:1 on ice for 30 mins as described [25]. Nanodrugs of model protein bovine serum albumin (BSA) or immunotoxin ch128.1Av/b-SO6 were synthesized. Monomer and crosslinker solutions were freshly prepared, degassed, and added to 1 mg/mL of ch128.1Av/b-SO6 or BSA in 50 mM HEPES buffer at a molar ratio of MPC:APM:crosslinker:ch128.1Av/b-SO6 or BSA (12000:100:1000:1, n/n). Degradable nanodrugs were synthesized using vinyl-functionalized peptide crosslinkers (PQGIAGQ), whereas non-degradable nanodrugs were prepared using N,N’-methylene-bisacrylamide (BIS). Free radical polymerization was initiated using ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED), with an APS:ch128.1Av/b-SO6 or BSA ratio of 500:1 (n/n) and a TEMED:APS ratio of 2:1 (w/w), carried out on ice for two hrs. The synthesized n(ch128.1Av/b-SO6) or n(BSA) was purified and concentrated using Spectrum^™ Dialysis Membrane Tubing (Thermo Fisher Scientific, MWCO:12 kDa) at 4 °C overnight. The proimmunotoxin n(ch128.1Av/b-SO6) or n(BSA) was conjugated with CXCL13 via copper-free click chemistry to synthesize n(ch128.1Av/b-SO6)-CXCL13 or n(BSA)-CXCL13. n(ch128.1Av/b-SO6) or n(BSA) and CXCL13 were functionalized with DBCO-SS-NHS and NHS-PEG4-Azide, respectively, at a 10:1 molar ratio on ice for one hr. Purified DBCO-modified n(ch128.1Av/b-SO6) or n(BSA) and azide-modified CXCL13 were reacted at 4° C for 17 hrs at a 1:1 molar ratio. The conjugation was confirmed using UV-VIS absorption with a OneC Microvolume Nanodrop (Thermo Scientific). The resulting n(ch128.1Av/b-SO6)-CXCL13 or n(BSA)-CXCL13 was purified using a Vivaspin^® 500 centrifugal concentrator. The synthesis was confirmed via Transmission electron microscopy (TEM) images acquired using a 120 kV T12 cryo-electron microscope (FEI), SDS-PAGE, and Dynamic light scattering (DLS) using a Zetasizer Nano ZS instrument (Malvern Panalytical, Malvern England).

2.3. Cell culture

The parental 2F7 AIDS-NHL cell line that harbors the Epstein-Barr virus (EBV) was a kind gift from Dr. Otoniel Martinez-Maza (University of California Los Angeles, Los Angeles, CA) [56]. 2F7 cells were genetically modified to express both mCherry fluorescent protein and a luciferase reporter (firefly). After in vivo selection for brain metastases, the 2F7-BR44 single cell clone was selected [51, 52]. 2F7-BR44 cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM; Gibco) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (P/S), and 50 μg/mL Zeocin^®. To generate 3D tumor spheroids, 2F7-BR44 were cultured in growth media containing 5% (w/v) Matrigel^® for 5 days. Jurkat cells, a human immortalized T lymphocyte cell line derived from a patient with T-cell leukemia, were purchased from ATCC (TIB-152^™, Manassas, VA, USA) and cultured in IMDM supplemented with 10% FBS and 1% P/S.

2.4. In vitro stability assays

The proteolytic stability of ch128.1Av/b-SO6 and n(ch128.1Av/b-SO6) was evaluated by incubating each formulation with trypsin (25 mg/mL) at 37 °C for up to 6 days. Samples were collected at 6 hrs, 12 hrs, and on Days 1, 2, 3, 4, 5, and 6. Following incubation, samples were digested overnight in IMDM supplemented with 10% FBS and 1% P/S, and the released native ch128.1Av/b-SO6 was quantified using ELISA. To assess the functional cytotoxic activity of ch128.1Av/b-SO6 and n(ch128.1Av/b-SO6) following prolonged trypsin exposure, both formulations were pre-incubated with trypsin (25 mg/mL) at 37 °C for 6 days. The released immunotoxin was then incubated with 2F7-BR44 cells (10⁴ cells/well) in culture medium at 37 °C for 24 hrs. Cell viability was determined by flow cytometry based on cell counts.

The storage stability of n(ch128.1Av/b-SO6) was evaluated at 4 °C. Nanocapsule samples were stored at 4 °C for 7 days, and hydrodynamic diameter was measured daily using DLS to monitor potential aggregation or structural changes.

2.5. In vitro BBB transwell penetration assay

The in vitro BBB transwell assay with bEnd.3 cells (ATCC) was established according to the protocol previously developed by our group [51–53]. Briefly, bEnd.3 cells (10⁵ cell per well) were seeded on the transwells with permeable polyester membrane and the integrity of bEnd.3 monolayer was confirmed by transepithelial electrical resistance (TEER). When the TEER of the bEnd.3 transwells reached above 600 Ω·cm², transcytosis of the nanocapsules across the bEnd.3 monolayer was performed. Native BSA and fluorescein isothiocyanate (FITC)-labelled n(BSA) were added to the apical compartment and incubated at 37 °C. The fluorescence intensity of basolateral compartment was analyzed at various time points by an Infinite 200 PRO plate reader (Tecan, Männedorf, Switzerland).

2.6. In vitro cytotoxicity activity

Cell proliferation under immunotoxin treatment was monitored using the [³H]-thymidine incorporation assay as described [25]. 2F7-BR44 cells were incubated with 0.1 or 1 nM ch128.1Av, b-SO6, ch128.1Av/b-SO6, IgGAv/b-SO6, or ch128.1 for 48 hrs in tissue culture conditions. The cells were incubated with0.5mCi/well [³H]-thymidine for the final 16 hrs of the treatment period. Radioactivity was determined as described previously [25]. Data are presented as a percentage of [³H]-thymidine incorporated into control cells treated with buffer only.

In vitro cytotoxicity activity was also tested using tumor spheroids. ch128.1Av/b-SO6 or n(ch128.1Av/b-SO6) was added to the 2F7-BR44 spheroid culture media. Fluorescent images of 2F7-BR44 spheroids treated with phosphate-buffered saline (PBS), ch128.1Av/b-SO6, or n(ch128.1Av/b-SO6) were taken at 24 and 48 hrs to evaluate cell viability by monitoring mCherry expression. Fluorescent imaging of cells and spheroids was imaged using a Leica SP8 fluorescent microscope or a Leica TCS-SP8-SMD confocal microscope (Leica, Boston, MA). Spheroid size under different treatments was measured using ImageJ software [57]. The penetration capability of ch128.1Av/b-SO6 and n(ch128.1Av/b-SO6) into 3D tumor spheroids was examined using a Hoechst dye staining assay. 0.1 μM Hoechst 33342 was added for 15 mins, enabling differentiation of the core and outer layer based on the staining intensity at 10⁴ relative fluorescent units (RFU) by flow cytometry analysis performed on the Fortessa (BD Biosciences, San Jose, CA, USA) or Attune NxT flow cytometer (Thermo Fisher Scientific).

Dual-chamber co-culture using the ibidi μ-Slide VI system (ibidi, Gradelfing, Germany) was employed to evaluate the targeting efficacy of proimmunotoxin nanodrugs. Targeted B-cell AIDS-NHL 2F7-BR44 cells were seeded in one chamber, while non-target Jurkat cells were seeded in an adjacent chamber, with both chambers connected by a central microchannel that permits passive diffusion. After culture medium was removed post 1-day culture, ch128.1Av/b-SO6, n(ch128.1Av/b-SO6), and n(ch128.1Av/b-SO6)-CXCL13 were introduced with 500 μL from non-target chamber and allowed to diffuse across the channel for 4 hrs. The samples were washed out by adding 1 mL fresh medium from non-target chamber and collecting medium from targeted chamber. The cell viability of each chamber was monitored by CytoTox-Glo^™ cytotoxicity assays at Days 3, 5, 7 post-treatment and the specificity was calculated as: Cytotoxicity_2F7-BR44 / Cytotoxicity_Jurkat.

The microfluidic platform was established to mimic the environment of blood vessels in order to evaluate the targeting efficacy of the proimmunotoxin nanodrugs. The microfluidic device was fabricated using soft lithography with an SU-8 2000 series photoresist on a silicon wafer substrate. The wafer was developed in SU-8 developer (PGMEA) for five mins, rinsed with isopropanol, and dried with nitrogen gas. Polydimethylsiloxane (PDMS) was prepared by mixing the base and curing agent at a 10:1 ratio, degassed, and poured over the SU-8 mold, followed by overnight curing at room temperature. The cured PDMS layer was then peeled off and bonded to a glass slide or another PDMS layer using plasma treatment. The final device was inspected for channel integrity and tested for fluid flow using a syringe pump programmed to inject at 15 μL/min. Once the channel integrity was verified, 2F7-BR44 spheroids were introduced into the device at a flow rate of 15 μL/min using a syringe pump and incubated overnight in designated wells to establish individual tumor microenvironments.

2.7. Animal studies

All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, under a protocol approved by the UCLA Chancellor’s Animal Research Committee (ARC), in compliance with the guidelines of the National Institutes of Health (NIH) and the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

To evaluate safety, six-week-old male BALB/c mice purchased from The Jackson Laboratory (Bar Harbor, ME, USA) (n = 6) received a single intravenous (i.v.) dose of PBS, ch128.1Av/b-SO6 (4 mg/kg), or n(ch128.1Av/b-SO6) (4 mg/kg) through the tail vein, with dosing normalized to the ch128.1Av content. Blood samples were collected on Days 0, 1, 3, 7, 10, 14. Plasma ALT (alanine transaminase) levels were measured using an enzyme activity assay kit (Abcam, Cat. No. ab105134) according to the manufacturer’s instructions to assess hepatic toxicity.

To assess the immunogenicity of n(ch128.1Av/b-SO6) and its native counterpart ch128.1Av/b-SO6, plasma samples at Day 14 post-treatment from biosafety studies were analyzed by ELISA. Total IgG levels were measured using the mouse IgG total ELISA Ready-SET-Go! Kit according to the manufacturer’s instructions. Anti-drug antibodies (ADA) against the b-SO6 component of ch128.1Av/b-SO6 were quantified using a sandwich ELISA. High-binding 96-well plates were coated overnight with soluble b-SO6, blocked with 1% BSA, and then incubated with plasma samples (diluted to 1:50 with PBS). Bound ADA was detected using an HRP-conjugated goat anti-mouse IgG Fc cross-adsorbed secondary antibody, followed by TMB substrate development and absorbance measurement at 450 nm.

To evaluate the in vivo antitumor activity, six-week-old male NOD/SCID/IL-2Rγnull (NSG) mice were purchased from the The Jackson Laboratory and maintained in a pathogen-free facility at UCLA. NSG mice were xenografted intravenously (i.v.) via the tail vein with 10⁶ 2F7-BR44 cells. In animal study set I, designed to assess the therapeutic efficacy of n(ch128.1Av/b-SO6), mice were randomized into three groups and treated i.v. with either PBS, 0.1 mg/kg of ch128.1Av/b-SO6, or n(ch128.1Av/b-SO6) (n = 6) on Days 7 and 21 post-tumor xenograft. The study was repeated in mice treated with PBS (n = 3), 0.1 mg/kg of ch128.1Av/b-SO6 (n = 4), or n(ch128.1Av/b-SO6) (n = 4). In animal study set II, aimed at evaluating the efficacy of n(ch128.1Av/b-SO6)-CXCL13, mice were randomized into four groups and treated i.v. via the tail vein with either PBS, 1 mg/kg ch128.1Av/b-SO6, n(ch128.1Av/b-SO6), or n(ch128.1Av/b-SO6)-CXCL13 (n = 6), on Days 7 and 21 post-tumor xenograft. The study was repeated in mice treated with PBS (n = 3), 1 mg/kg of ch128.1Av/b-SO6 (n = 6), n(ch128.1Av/b-SO6) (n = 7), or n(ch128.1Av/b-SO6)-CXCL13 (n = 7).

Longitudinal tumor progression in AIDS-NHL mice was monitored weekly for four weeks using an IVIS Lumina II Imaging System (Revvity, Waltham, Massachusetts, USA) at the UCLA Crump Preclinical Imaging Technology Center and analyzed with Living Image Software (Revvity, Waltham, Massachusetts, USA). In vivo bioluminescence imaging was performed following intraperitoneal (i.p.) injection of 4.5 mg D-luciferin (Gold Biotechnology, Cat. No. LUCNA) and tumor burden was quantified as total bioluminescent intensity (BLI) per second within a region of interest (whole body or head area); identically sized regions of interest were used for all measurements. Mice were euthanized upon meeting any of the following criteria: I) total BLI exceeding 10¹¹ (photons/sec/cm²/sr), II) body weight loss exceeding 20%, or III) at the end of the study (Day 35). Survival was assessed based on the time from tumor challenge to euthanasia, and differences in survival were determined using a log-rank test. Tumor-bearing organs, including the kidneys, bone marrow (BM), brain, spleen, were harvested and processed for flow cytometry analysis of mCherry-expressing 2F7-BR44 cells. Set II plasma samples collected at end of the study were used to monitor liver toxicity using enzyme activity assay kits for ALT (Abcam, Cat. No. ab105134), AST (aspartate aminotransferase) (Abcam, Cat. No. ab105135), and ALP (lkaline phosphatase) (Abcam, Cat. No. ab83369) according to the manufacturer’s instructions.

To assess the tumor targeting of n(ch128.1Av/b-SO6)-CXCL13 in vivo, mice were i.v. administered Cy5.5-labeled BSA or n(BSA)-CXCL13 at a dose of 1 mg/kg via at Week 2 post-tumor xenograft (n = 3). Both tumor burden monitored by bioluminescence imaging and label samples tracked by fluorescence imaging were performed 24 hrs post-injection using an IVIS Lumina II system (Revvity), and data were analyzed using Living Image software (Revvity).

2.8. Statistical analysis

Measurements in this study were obtained from distinct samples. Prism 10 (GraphPad Software, Boston, MA) was used to perform all statistical analysis. All data are presented as the mean ± standard deviation (SD). Statistical significance was calculated via an unpaired two-tailed Student’s t-test (*p <0.05, **p <0.01, ***p <0.005, and ****p <0.001), Mann-Whitney U test (*p <0.05, **p <0.01, ***p <0.005, and ****p <0.001) or two-way ANOVA (*p <0.05, **p <0.01, ***p <0.005, and ****p <0.001). The survival curve was generated using the Kaplan-Meier method, and statistical significance was assessed using the log-rank test.

Results

Synthesis and characterization of proimmunotoxin nanodrugs

The native immunotoxin, ch128.1Av/b-SO6, was prepared via an avidin-biotin interaction between ch128.1Av and b-SO6 with a molar ratio of 1:1. The fusion protein consists of an IgG3 molecule fused to two avidin molecules through the C_H3 domains (Supplemental Fig. S1) [25]. Notably, the aggressive AIDS-NHL 2F7-BR44 cell line, which is resistant to the direct cytotoxic effects of ch128.1Av, was confirmed to be highly susceptible to ch128.1Av/b-SO6 (Supplemental Fig. S2).

To mitigate the concerns regarding toxicity of ch128.1Av/b-SO6 to normal cells expressing TfR1, we synthesized a proimmunotoxin nanodrug using a nanotechnology-based approach, whereby individual macromolecules are encapsulated within a thin polymer shell, termed a nanocapsule [51–54]. Inspired by the active transport of choline from blood into the brain and tumors [58, 59], we developed a nanocapsule formulation incorporating choline analogues and zwitterionic polymers, enabling increased stability and interaction with ChTs that are highly expressed on the BBB and in tumors. This ChT-mediated uptake mechanism facilitates effective penetration of tumors and crossing of the BBB [51–54].

As illustrated in Fig. 1A, we produced, for the first time, nanocapsules containing the immunotoxin ch128.1Av/b-SO6 to form the proimmunotoxin n(ch128.1Av/b-SO6) in two steps. First (Step I), MPC monomers, containing a choline analogue and zwitterionic structure, along with tumor-specific peptide crosslinkers (PQGIAGQ), which are selectively cleaved by MMP-2 [55], were concentrated around individual ch128.1Av/b-SO6 molecules. Next (Step II), in situ polymerization was performed to encapsulate ch128.1Av/b-SO6, forming the proimmunotoxin nanodrug n(ch128.1Av/b-SO6). The resulting nanodrug is expected to be inert and highly stable, resistant to nonspecific protein adsorption, and show reduced immunogenicity, thereby effectively shielding the cargo from environmental degradation and off-target toxicity. Upon systemic administration, n(ch128.1Av/b-SO6) is expected to efficiently penetrate tumors and crosse the BBB, both of which are mediated by ChTs. ch128.1Av/b-SO6 is selectively released in the TME upon MMP-2-mediated cleavage of the peptide crosslinkers, ensuring localized activation of the immunotoxin while minimizing off-target effects.

Fig. 1. Design and characterization of proimmunotoxin nanodrugs.

(A) Illustration of the design and mechanism of action of the proimmunotoxin nanodrug. The process begins with MPC monomers and MMP-2-sensitive peptide crosslinkers enriched around individual immunotoxin ch128.1Av/b-SO6 molecules (Step I). ch128.1Av/b-SO6 is encapsulated in a thin polymer shell via in situ polymerization to form n(ch128.1Av/b-SO6) (Step II). Upon systemic administration, the choline analogue of n(ch128.1Av/b-SO6) facilitates BBB penetration via ChTs. In the TME, MMP-2-mediated cleavage of crosslinkers triggers the targeted release of the immunotoxin. (B) DLS analysis confirms the size differences between ch128.1Av/b-SO6 and n(ch128.1Av/b-SO6). (C) TEM imaging shows the structural integrity and spherical morphology of n(ch128.1Av/b-SO6).

The successful synthesis of n(ch128.1Av/b-SO6) was confirmed through physicochemical characterization. The hydrodynamic radius of n(ch128.1Av/b-SO6) was measured using dynamic light scattering (DLS). Compared to ch128.1Av/b-SO6, which has an average diameter of approximately 9 nm, n(ch128.1Av/b-SO6) exhibited an increased diameter of 25 nm, indicating the formation of polymer shells around the immunotoxins (Fig. 1B). This observation was further validated by transmission electron microscopy (TEM), which confirmed the structural integrity and spherical morphology of the nanocapsules (Fig. 1C).

Nanocapsules showed markedly enhanced stability against proteolysis and storage-induced aggregation. Upon incubation with trypsin, a model protease, ch128.1Av/b-SO6 rapidly lost structural integrity and functional activity within 12 hrs, whereas n(ch128.1Av/b-SO6) retained protein structure and robust activity even after 6 days of continuous exposure (Supplemental Fig. S3A,B). The nanocapsules also demonstrated excellent storage stability, with no detectable aggregation at 4 °C for up to 7 days (Supplemental Fig. S3C).

Subsequently, to assess whether the synthesis of n(ch128.1Av/b-SO6) preserved the cytotoxic efficacy of ch128.1Av/b-SO6 while enhancing tumor penetration, the antitumor efficacy of n(ch128.1Av/b-SO6) was evaluated in vitro using 2F7-BR44 spheroids. Spheroids were treated with PBS (control), ch128.1Av/b-SO6, or n(ch128.1Av/b-SO6), and cell viability was assessed by tracking the loss of mCherry expression at 24- and 48-hrs post-treatment. Representative fluorescent images revealed a reduced radius of spheroids in both ch128.1Av/b-SO6- and n(ch128.1Av/b-SO6)-treated groups compared to the PBS control, indicating that the encapsulation did not compromise the cytotoxic potential of ch128.1Av/b-SO6 (Fig. 2A). Notably, only n(ch128.1Av/b-SO6) led to observable cell loss in the spheroid core, as indicated by penetrating holes (circled in white), suggesting enhanced deep-tissue penetration. To quantitatively compare tumor penetration, spheroid radii were measured across groups, confirming that both ch128.1Av/b-SO6 and n(ch128.1Av/b-SO6) exhibited comparable cytotoxicity in the outer layers where immunotoxin access was unrestricted (Fig. 2B). However, to directly assess penetration into deeper tumor regions, a Hoechst dye staining assay was employed [60]. Hoechst dye diffusion was used to determine cell positioning within spheroids, as the outer layers exhibited higher dye concentration, while the inner regions showed lower Hoechst intensity (Fig. 2C). Measurement of mCherry expression at the spheroid core further confirmed that n(ch128.1Av/b-SO6) effectively induced cell death in the inner layers, demonstrating improved tumor penetration (Fig. 2D).

Fig. 2. In vitro evaluation of the cytotoxic activity of n(ch128.1Av/b-SO6) in 2F7-BR44 spheroids and its BBB penetration in a transwell assay.

(A) Representative fluorescent images of 2F7-BR44 spheroids treated with PBS, ch128.1Av/b-SO6, or n(ch128.1Av/b-SO6) (4.6 μg/mL) for 24 and 48 hrs. White circles indicate penetration holes in the spheroids. (B) Spheroid size was quantified by measuring the spheroid radius, and comparisons were made across treatment groups (n = 5). Data are presented as means ± S.D., with significance calculated using the Student’s t-test. *** p< 0.001. (C) Hoechst 33342 staining assay was used to assess tumor penetration. 2F7-BR44 spheroids were stained with 0.1 μM 33342 dye, dissociated into single-cell suspensions, and analyzed via flow cytometry for Hoechst intensity and mCherry expression. Hoechst intensity below 10⁴ was used to identify cell positioning within the spheroid core, while mCherry expression was used to monitor cell viability. (D) Tumor penetration efficacy of ch128.1Av/b-SO6 and n(ch128.1Av/b-SO6) was compared based on cell viability within the spheroid core. Data are presented as means ± S.D., with significance calculated using the Student’s t-test. ns: not significant. (E) Accumulative penetration efficiency of BSA and n(BSA) labeled with FITC through a monolayer of bEnd.3 cells in an in vitro BBB transwell assay (n = 3). The fluorescence intensity in the basolateral compartment was monitored. Data are analyzed with two-way ANOVA. ns: not significant, * p < 0.05, *** p< 0.001.

The ability of the nanocapsules to penetrate the BBB was evaluated using a widely adopted in vitro transwell model, in which a dense monolayer of mouse brain endothelial cells (bEnd.3) was cultured on the apical surface of the transwell insert. Monolayer integrity was verified by fluorescence microscopy and TEER measurements (Supplemental Fig. S4). To avoid endothelial damage from ch128.1Av/b-SO6, FITC-labeled bovine serum albumin (BSA) was used as a model protein to synthesize n(BSA). BSA or n(BSA) was added to the apical chamber, and penetration was quantified based on fluorescence intensity in the basolateral compartment. As shown in Fig. 2E, n(BSA) exhibited time-dependent translocation across the endothelial monolayer, reaching 20.2% penetration at 48 hrs, whereas native BSA showed minimal transport (1.5%).

Enhanced safety and decreased immunogenicity of proimmunotoxin nanodrugs

Non-target toxicity from immunotoxins and their delivery vehicles remains a major safety concern in immunotoxin therapies. To evaluate the biosafety of the polymer shells used in the proimmunotoxin nanodrugs and their ability to shield encapsulated immunotoxins before release, a non-degradable variant, n(ch128.1Av/b-SO6)_BIS, was synthesized using BIS as a crosslinker. Compared to ch128.1Av/b-SO6, n(ch128.1Av/b-SO6)_BIS induced negligible cell death, confirming both the biocompatibility of the polymer shells and their protective function (Supplemental Fig. S5). Moreover, n(ch128.1Av/b-SO6), when exposed to MMP-2 present in culture medium of 2F7-BR44 cells, exhibited delayed but ultimately comparable cytotoxicity to ch128.1Av/b-SO6. These findings demonstrate that polymer encapsulation effectively prevents toxicity before immunotoxin release, thereby enhancing the biosafety of the proimmunotoxin nanodrug in vivo.

To further evaluate the biosafety of n(ch128.1Av/b-SO6), BALB/c mice were administered a single i.v. injection via the tail vein of 4 mg/kg, a dose that previously resulted in non-specific toxicity. In the absence of tumor inoculation, n(ch128.1Av/b-SO6), designed to release the immunotoxin only within the tumor microenvironment, was expected to show negligible release in healthy mice. As shown in Fig. 3A, mice treated with the native ch128.1Av/b-SO6 immunotoxin displayed a reduction in body weight. In contrast, mice treated with n(ch128.1Av/b-SO6) maintained stable body weight throughout the duration of the experiment (14 days), similar to the PBS group, consistent with the protective shielding conferred by encapsulation of the immunotoxin. Hepatic toxicity, a major concern for immunotoxin-based treatments [47, 61], was assessed by plasma ALT measurements. Mice treated with ch128.1Av/b-SO6 showed markedly elevated ALT levels relative to those receiving n(ch128.1Av/b-SO6), indicating that encapsulation effectively mitigated hepatic toxicity (Fig. 3B).

Fig. 3. Assessment of off-target toxicity and immunogenicity of n(ch128.1Av/b-SO6) in vivo.

BALB/c mice received a single i.v. dose of PBS, ch128.1Av/b-SO6 (4 mg/kg) or n(ch128.1Av/b-SO6) (4 mg/kg) via the tail vein (n = 6). (A) Body weight plots show the weight change in groups administered with PBS, ch128.1Av/b-SO6 or n(ch128.1Av/b-SO6). Data are shown as means ± S.D., with significance determined using the two-way ANOVA. ns: not significant, * p < 0.05. (B) Plasma ALT levels were compared between treatment groups. Data are presented as means ± S.D., with significance calculated using the two-way ANOVA. ns, not significant, * p < 0.05, ** p < 0.01. (C) The ADA levels in plasma on Day 14. (D) Total IgG level in plasma on Day 14. Data are presented as means ± S.D., with significance calculated using the unpaired one-tailed Student’s t-test. ns, not significant, *** p < 0.001.

Finally, both anti-drug antibody (ADA) levels and total plasma IgG levels were assessed on Day 14 to evaluate the potential immunogenicity of ch128.1Av/b-SO6 (native or part of the nanodrug). Because the immunogenicity of immunotoxins is primarily driven by the toxin component, ADA responses against b-SO6 were quantified using a b-SO6-specific ELISA. As shown in Fig. 3C, mice treated with ch128.1Av/b-SO6 demonstrated markedly increased ADA levels, whereas n(ch128.1Av/b-SO6) effectively prevented ADA response. Moreover, consistent with the expected immunogenicity of saporin as a plant protein, ch128.1Av/b-SO6 induced a strong immune response with significantly increased IgG levels, whereas n(ch128.1Av/b-SO6) produced IgG levels comparable to PBS controls (Fig. 3D), suggesting that the polymer shell of n(ch128.1Av/b-SO6) was biocompatible and prevented cargo-induced immunogenicity prior to release. This experiment was independently repeated using retro-orbital administration. Consistent safety outcomes confirmed that n(ch128.1Av/b-SO6) mitigated the elevated ALT levels and acute ocular mucosal inflammation induced by ch128.1Av/b-SO6 (Supplemental Fig. S6).

Improved antitumor efficacy of proimmunotoxin nanodrugs in a lymphoma xenograft model

The tumor-killing efficiency of n(ch128.1Av/b-SO6) was compared to the native immunotoxin in a xenograft AIDS-NHL model established by our group. Tumors were established in the CNS and BM within the first week, forming aggressive malignancies in the brain, spinal cord, femur, and kidneys at Week 2 [51, 52]. Based on the kinetics of lymphoma progression, treatment was administered at Weeks 1 and 3 with PBS, ch128.1Av/b-SO6, or n(ch128.1Av/b-SO6) (Fig. 4A). To minimize toxicity, a safe dose of 0.1 mg/kg of ch128.1Av/b-SO6 or an equivalent dose of n(ch128.1Av/b-SO6) was administered i.v. via the tail vein. Tumor progression was monitored weekly using in vivo bioluminescent imaging to measure the increase in bioluminescent intensity (BLI) from 2F7-BR44 cells, with all animals euthanized on Day 35 for endpoint analysis.

Fig. 4. Evaluation of antitumor efficacy of n(ch128.1Av/b-SO6) in a xenograft AIDS-NHL mouse model.

(A) Treatment scheme in the xenograft AIDS-NHL mouse model. NSG mice were inoculated i.v. via the tail vein with 10⁶ 2F7-BR44 cells and then treated i.v. via the tail vein with PBS, ch128.1Av/b-SO6 (0.1 mg/kg), or n(ch128.1Av/b-SO6) (0.1 mg/kg, antibody-equivalent dose) via tail vein on Days 7 and 21 post-tumor xenograft and euthanized on Day 35 (n = 6). (B) Kaplan-Meier plot shows survival of all groups after cell inoculation. Statistical significance was calculated using log-rank test compared to the PBS control group. ** p < 0.01. (C) Longitudinal tumor progression was monitored weekly using bioluminescent imaging. Red X indicate deceased mice. (D) Tumor burden was quantified by flow cytometry as the percentage of 2F7-BR44 cells (mCherry+) among total cells isolated from bone marrow (BM), kidney, spleen, and brain. Data are presented as means ± S.D., with significance determined using the Student’s t-test with Welch’s correction. ns: not significant, *** p < 0.001, **** p < 0.0001.

AIDS-NHL xenografted mice treated with n(ch128.1Av/b-SO6) exhibited extended survival (Fig. 4B) and a significantly lower tumor burden (Fig. 4C) by more than one week compared to those treated with PBS or ch128.1Av/b-SO6. Although ch128.1Av/b-SO6 slightly prolonged survival in a subset of mice, its tumor burden reflected by bioluminescence in both regions remained comparable to PBS controls during Weeks 1–3, suggesting limited tumor suppression at the tested dose. In contrast, n(ch128.1Av/b-SO6) exhibited consistent therapeutic efficacy in both systemic and CNS tumors throughout the experiment. To further confirm tumor suppression across different metastatic sites, major organs were harvested, and mCherry-expressing tumor cells were quantified using flow cytometry (Fig. 4D). Consistent with BLI results, n(ch128.1Av/b-SO6) significantly suppressed tumor growth in major metastatic sites, with particularly strong effects in the kidneys and BM compared to PBS- and ch128.1Av/b-SO6-treated groups. The tumor growth rates per week were calculated based on the BLI data to evaluate the tumor suppression efficacy by treatment. Compared to ch128.1Av/b-SO6, n(ch128.1Av/b-SO6) demonstrated improved tumor suppression across all weeks. However, its suppression rate declined after the second dose, highlighting the need for further optimization of drug delivery to tumor sites. This experiment was independently repeated to confirm reproducibility (Supplemental Fig. S7).

Enhanced tumor targeting and antitumor efficacy of CXCL13-conjugated proimmunotoxin nanodrugs

Proimmunotoxin nanodrugs can be functionalized with ligands to enhance target specificity, directing them to lymphoma cells and thereby increasing the concentration of ch128.1Av/b-SO6 at tumor sites. This targeted approach is expected to enhance both potency and specificity while minimizing off-target effects. To achieve this, we successfully synthesized n(ch128.1Av/b-SO6)-CXCL13 by conjugating n(ch128.1Av/b-SO6) with human CXCL13, a chemokine that interacts with CXCR5, a receptor expressed on mature B cells as well as B-cell NHL [51, 56]. The conjugation was performed using copper-free click chemistry at a molar ratio of 1:1. The specific elimination of 2F7-BR44 cells was compared among ch128.1Av/b-SO6, n(ch128.1Av/b-SO6), and n(ch128.1Av/b-SO6)-CXCL13 using a dual-chamber co-culture system comprising 2F7-BR44 (targeted cells) and Jurkat cells (non-targeted cells) cultured separately but interconnected by a diffusion chamber (Fig. 5A). Jurkat is a human T-cell leukemia cell line characterized by expression of TfR1 and absence of CXCR5 expression. The specificity of targeted cell death in 2F7-BR44 was determined by calculating the ratio of 2F7-BR44 cell death to Jurkat cell death. Given that both cell lines express high levels of human TfR1 and secrete MMP-2, ch128.1Av/b-SO6 and n(ch128.1Av/b-SO6) showed similar cytotoxic effects against both malignant cell lines, resulting in a specificity ratio of approximately 1. However, with CXCL13 conjugation, n(ch128.1Av/b-SO6)-CXCL13 showed specific killing of 2F7-BR44 by Day 3, confirming enhanced specificity for CXCR5-expressing AIDS-NHL cells. Over subsequent days, specificity declined, likely due to the diffusion of released immunotoxin within the system.

Fig. 5. Assessment of tumor-targeting efficiency of the addition of CXCL13 to the proimmunotoxin.

(A) Targeted elimination of 2F7-BR44 cells by native ch128.1Av/b-SO6, n(ch128.1Av/b-SO6), or n(ch128.1Av/b-SO6)-CXCL13 (4.6 μg/mL) was assessed in a dual-chamber co-culture system, where 2F7-BR44 and Jurkat cells were cultured in separate wells but connected by a diffusion chamber. Samples were tested in triplicates. Data are presented as means ± S.D., with significance determined using the Student’s t-test with Welch’s correction. ns: not significant, ** p < 0.01. (B) A microfluidic platform was used to simulate the mechanical environment of blood vessels to study the tumor-targeting efficacy of proimmunotoxins on 2F7-BR44 cell spheroids. The light microscopic image shows one tumor spheroid captured inside the microfluidic chip wells. (C) FITC-labeled BSA and n(BSA)-CXCL13 were introduced at a flow rate at 15 μL/min into the microfluidics cultured with targeted tumor 2F7-BR44 spheroids (mCherry+) or non-targeted Jurkat T spheroids (labeled with Hoechst-33342 dye), respectively to identify cells. Binding was analyzed using a fluorescent microscope. (D) Representative bioluminescence and fluorescence images show improved tumor targeting of n(BSA)-CXCL13 compared to BSA in the xenograft AIDS-NHL mouse model. NSG mice were inoculated i.v. via the tail vein with 10⁶ 2F7-BR44 cells (n = 3). 1 mg/kg BSA or n(BSA)-CXCL13 was administrated in the mice at Week 2 post-tumor xenograft. Bioluminescent imaging was used to monitor tumor location, while fluorescent imaging was employed for detection of BSA or n(BSA)-CXCL13 24 hrs post-injection.

It is of interest to assess the specific binding of n(ch128.1Av/b-SO6)-CXCL13 to 2F7-BR44 cells. To avoid cytotoxicity, we used BSA and a microfluidic platform designed to simulate the mechanical environment of fluid dynamics within the TME (Fig. 5B, Supplemental Fig. S8A). By modeling laminar flow characteristic of the TME, especially in interstitial regions and abnormal vasculature, this system enables physiologically relevant evaluation of drug delivery [62, 63]. The microfluidic chip design allowed for precise flow rate control and the maintenance of a stable microenvironment for the spheroids (Supplemental Fig. S8B). 2F7-BR44 tumor spheroids were introduced into the microfluidic platform and settled into the wells. After 24 hrs of flow stabilization, BSA or n(BSA)-CXCL13 were introduced at a steady flow rate of 15 μL/min, mimicking bloodstream flow dynamics. COMSOL Multiphysics simulations confirmed that the platform successfully replicated targeted nanoparticle interactions under stable laminar flow, effectively mimicking blood vessel conditions (Supplemental Fig. S8C–E). To prevent spheroid disruption due to immunotoxin cytotoxicity and enable direct evaluation using imaging, FITC-labeled BSA and n(BSA)-CXCL13 were used to demonstrate the specific binding in the microfluidic platform with tumor spheroids of 2F7-BR44 (mCherry+) and Jurkat (Hoechst 33342), respectively (Fig. 5C). Facilitated by the interaction between CXCL13 and CXCR5, n(BSA)-CXCL13 exhibited strong binding to 2F7-BR44 spheroids but not to Jurkat spheroids at 24 hrs post-injection. The tumor-targeting ability of n(BSA)-CXCL13 was evaluated in xenograft AIDS-NHL mice at two weeks post-tumor xenograft (Fig. 5D). At 24 hrs post-injection, bioluminescent imaging was used to monitor tumor location, while fluorescent imaging of BSA or n(BSA)-CXCL13 labeled with Cy5.5 was performed using an excitation filter (660–690 nm) to minimize potential signal overlap from mCherry in 2F7-BR44 cells. The fluorescent signal from BSA was undetectable, whereas n(BSA)-CXCL13 exhibited strong retention in the tumor region, highlighting its superior tumor-targeting capability.

Leveraging the superior tumor-targeting capability of the CXCL13 conjugated proimmunotoxin, a dose of 1 mg/kg, 10-fold higher than the safe dose of ch128.1Av/b-SO6, was evaluated for therapeutic efficacy in xenograft AIDS-NHL mice. Xenografted mice were treated with PBS, ch128.1Av/b-SO6, n(ch128.1Av/b-SO6), or n(ch128.1Av/b-SO6)-CXCL13 via i.v. injection on Days 7 and 21 post-tumor xenograft. Longitudinal antitumor effects were monitored weekly using in vivo bioluminescent imaging, with all animals euthanized on Day 35 for tissue analysis (Fig. 6A). Treatment with n(ch128.1Av/b-SO6)-CXCL13 resulted in significantly greater control of lymphoma burden compared to other groups, as measured by BLIs in a defined region of interest (ROI) (Fig. 6B). Total body or head-region BLIs were quantitively compared among the four groups (Fig. 6C). Both n(ch128.1Av/b-SO6) and n(ch128.1Av/b-SO6)-CXCL13 significantly reduced BLIs at multiple time points compared to PBS and ch128.1Av/b-SO6. Notably, n(ch128.1Av/b-SO6)-CXCL13 exhibited the lowest tumor burden in both systemic and CNS at the end point of the experiment. Kaplan-Meier survival analysis further highlighted the therapeutic benefits of CXCL13 ligand conjugation. Mice treated with n(ch128.1Av/b-SO6)-CXCL13 exhibited 100% survival throughout the experiment up to Day 35, indicating superior survival benefits (Fig. 6D). Tumor burden in major metastatic organs, including the brain, BM, and kidneys, was further quantified by assessing the percentages of mCherry+ 2F7-BR44 cells within total tissue cells using flow cytometry (Fig. 6E). Different from the insufficient efficacy of ch128.1Av/b-SO6, both n(ch128.1Av/b-SO6) and n(ch128.1Av/b-SO6)-CXCL13 significantly reduced tumor burden. The reduction was particularly pronounced in the n(ch128.1Av/b-SO6)-CXCL13-treated group, attributable to its enhanced tumor-targeting capability. Moreover, whereas ch128.1Av/b-SO6 treatment was associated with increased liver enzyme levels, both n(ch128.1Av/b-SO6) and n(ch128.1Av/b-SO6)-CXCL13 showed liver enzyme levels comparable to PBS controls, suggesting minimal liver toxicity with the pro-immunotoxin formulations (Fig. 6F). The therapeutic efficacy of n(ch128.1Av/b-SO6) or n(ch128.1Av/b-SO6)-CXCL13 was evaluated again in another independent test in the xenograft murine model with 2F7-BR44 cells. Consistently, the administration of n(ch128.1Av/b-SO6)-CXCL13 achieved a significant extension in mouse survival compared to the other treatments including n(ch128.1Av/b-SO6) as shown in Supplemental Fig. S9. These findings underscore the potential of n(ch128.1Av/b-SO6)-CXCL13 as a therapeutic agent for disseminated AIDS-NHL.

Fig. 6. Evaluation of therapeutic efficacy of a CXCL13-targeted proimmunotoxin the xenograft AIDS-NHL mouse model.

(A) Treatment scheme in the xenograft AIDS-NHL mouse model. NSG mice engrafted with 10⁶ 2F7-BR44 cells i.v. via tail vein injection were treated i.v. via the tail vein with PBS, or with 1 mg/kg of ch128.1Av/b-SO6, n(ch128.1Av/b-SO6), n(ch128.1Av/b-SO6)-CXCL13 on Days 7 and 21 post-tumor xenograft (n = 6). Dosages were normalized to ch128.1Av antibody fusion protein content. (B) Longitudinal tumor progression was monitored weekly using bioluminescent imaging. Boxes with a red X indicate deceased mice. (C) Tumor burden was quantified based on bioluminescence signals detected through imaging. No statistical analysis was performed at Wk 4 due to unequal animal numbers across groups. (D) Kaplan-Meier plot shows survival of all treatment groups after cell inoculation. Statistical significance was calculated using log-rank test compared to the PBS control group. * p < 0.05, *** p < 0.001, **** p < 0.0001. (E) Tumor burden in major metastatic sites (brain, BM, and kidneys) was quantified by flow cytometry as the percentage of mCherry+ 2F7-BR44 cells within the total tissue cell population. (F) Liver toxicity was assessed by measuring the plasma levels of ALT, AST, and ALP using ELISA assays. Data in panels C, E, F are presented as means ± S.D., with significance calculated with Student’s t-test with Welch’s correction. ns: not significant, * p < 0.05, ** p < 0.01.

Discussion

AIDS-NHL in people living with HIV tends to manifest earlier in life and with more aggressive, advanced-stage disease, frequently involving extranodal sites such as the CNS [2–7]. An important challenge in designing effective therapies for NHL lies in achieving effective delivery to the CNS and targeting of malignant cells while minimizing off-target toxicity. Elevated human TfR1 is associated with tumorigenesis, cancer progression, and poor prognosis [12, 14, 15, 64], including solid cancers such as esophageal squamous cell carcinoma [65, 66], breast cancer [67, 68], ovarian cancer [69], lung cancer [70], cervical cancer [71], bladder cancer [72], osteosarcoma [73], pancreatic cancer [74–76], cholangiocarcinoma [77], renal cell carcinoma [78], hepatocellular carcinoma [79–81], adrenal cortical carcinoma [82], and malignancies of the CNS such as glioblastomas [83, 84] as well as hematopoietic malignancies such as acute lymphoblastic leukemia (ALL) [85, 86], chronic lymphocytic leukemia (CLL) [16], and NHL [16–18]. Notably, AIDS-NHL expresses even higher TfR1 mRNA levels compared to NHL cells from non-infected individuals [19, 20]. However, it is well known that not all tumor types respond equally to anti-TfR1 antibodies and that even in those that respond there is the possibility that resistance may develop [15]. Thus, it is of interest to explore the use of antibodies, and their derivatives bound to antitumor agents to be delivered into cancer cells by RME [13, 37]. Immunotoxins containing anti-TfR1 antibodies function independently of the immune system by directly delivering toxins into malignant cells, ensuring effectiveness even in the presence of a dysfunctional immune system. Moreover, immunotoxins bypass tumor resistance to antibody-mediated mechanisms by directly causing cancer cell death through the action of the toxin. Therefore, immunotoxins offer a promising antitumor therapy for cancer, including AIDS-NHL.

Despite the above-mentioned advantages, the clinical application of immunotoxins is limited by issues of off-target toxicity and immunogenicity. Moreover, for ch128.1Av/b-SO6, rapid clearance driven by the presence of avidin shortens its circulating half-life and limits its antitumor efficacy. In this study, we developed a proimmunotoxin nanodrug platform using ch128.1Av/b-SO6, an immunotoxin targeting TfR1 for the treatment of AIDS-NHL, including primary and metastatic brain disease. Our strategy mitigated safety and efficacy concerns by encapsulating ch128.1Av/b-SO6 within a zwitterionic polymeric nanocapsule, synthesized using MPC and an MMP-2-responsive crosslinker. Compared to native ch128.1Av/b-SO6, proimmunotoxin nanodrugs, both n(ch128.1Av/b-SO6) and n(ch128.1Av/b-SO6)-CXCL13, improve the antitumor efficacy. MPC-based zwitterionic nanocapsules offer multiple advantages, including protection from potential immunogenicity and prolonged half-life of encapsulated therapeutics [51–53, 87]. The zwitterionic MPC polymers form a strong hydration layer due to their positively and negatively charged functional groups, creating steric hindrance that prevents protein adsorption and immune cell recognition [87, 88]. Furthermore, the zwitterionic shell reduces cellular uptake by immune cells, such as macrophages and dendritic cells, thereby limiting antigen presentation and adaptive immune responses [51–53]. Moreover, the choline analogue structure on MPC-based nanocapsules facilitate transcytosis of the BBB and tumor infiltration through binding to ChTs on the endothelial cells of the BBB [51–53] and cancer cells [89]. The enzyme-responsive peptide crosslinkers ensure targeted therapeutic release, activating in the tumor microenvironment [55, 90]. The enhanced tumor penetration of nanocapsules is driven by a combination of zwitterionic surface properties, optimal nanoscale size, choline analogue modification, and controlled release behavior. The zwitterionic PMPC shell forms a dense hydration layer that minimizes nonspecific interactions with extracellular matrix (ECM) components, enabling low-fouling, stealth-like diffusion through the tumor interstitium. This effect is further supported by the nanocapsule’s small hydrodynamic size (~25 nm), which facilitates interstitial transport through tight ECM pores. Simultaneously, the choline analogue of PMPC promotes active uptake via ChTs, which are upregulated in tumor cells to meet increased demands for phospholipid biosynthesis, particularly in the hypoxic inner layers of spheroids where expression of ChTs is elevated [55, 58, 59]. Some nanocapsules remain structurally intact during interstitial transport and cellular uptake due to their MMP-2-sensitive controlled release design. Following ChT-mediated internalization, these nanocapsules may undergo exocytosis and be subsequently uptaken by adjacent cells, facilitating a transcytosis-like mechanism that supports the tumor penetration and the BBB crossing. Together, the combination of passive stealth diffusion and active transcellular transport enables efficient traversal of multilayered tumor spheroids. In contrast, penetration across the BBB relies more heavily on transcellular movement due to the minimal interstitial space between tightly packed endothelial cells. The encapsulated ch128.1Av/b-SO6 is further optimized by conjugating tumor-targeting ligands, redirecting them to specific cells. Together, these features significantly improve the potency and specificity of the proimmunotoxin nanodrug. The biosafety profile of n(ch128.1Av/b-SO6) was significantly improved compared to the native immunotoxin. Encapsulation effectively mitigated the systemic toxicity of ch128.1Av/b-SO6, as evidenced by the absence of liver toxicity in treated mice. These findings suggest that the proimmunotoxin strategy could potentially overcome the limitations of traditional immunotoxin therapies, providing a safer and more effective treatment option for NHL, particularly in patients with primary or metastatic brain tumors.

An important limitation of present in vivo studies is that the full extent of potential toxicity of the proimmunotoxin nanodrug cannot be adequately addressed given the lack of cross-reactivity of the antibody with mouse TfR1 [30]. Therefore, additional studies in relevant animal models are required to properly evaluate potential toxicities of the present strategy. However, the results of this study suggest that n(ch128.1Av/b-SO6)-CXCL13, along with other constructs based on this proimmunotoxin nanodrug strategy, warrant further investigation as potential therapeutics for AIDS-NHL and potentially other malignancies.

There are several key areas for further exploration. First, it is essential to optimize the formulation of n(ch128.1Av/b-SO6) for clinical application, particularly in scaling up production and ensuring stability since nanocapsule production has only been achieved in small volume so far and potential problems such as gelling could occur during preparation in large quantity in free radical polymerization [91]. Additionally, the versatility of this nanocapsule platform suggests potential applications beyond NHL, particularly in treating other cancers with brain metastases. The microfluidic platform developed in this study offers a valuable tool for screening and optimizing these nanodrug formulations in a controlled environment that mimics in vivo conditions. Future studies should also explore the integration of n(ch128.1Av/b-SO6) with other treatment modalities to enhance therapeutic outcomes.

In conclusion, the development of proimmunotoxin nanodrugs using a nanoencapsulation strategy represents a significant advancement in targeted cancer therapy. This platform not only offers a promising treatment option for AIDS-NHL but also opens new avenues for treating NHL in the general population as well as other malignancies with and without CNS involvement. Continued research and development will be crucial in translating this innovative therapy from preclinical to clinical transition, with the potential to significantly improve patient outcomes.

Data and materials availability:

All data required to evaluate the conclusion in this paper are present in the main text or the Supplementary Materials. Raw data may be requested from the authors if needed.