Leukemia2021ResearchDrug Delivery

Nanoparticle T-cell engagers as a modular platform for cancer immunotherapy

Kinan Alhallak, Jennifer Sun, Katherine Wasden, Nicole Guenthner, Julie O'neal, Barbara Muz, Justin King, Daniel Kohnen, Ravi Vij, Samuel Achilefu, John F. Dipersio, Abdel Kareem Azab29 September 2026DOI 10.1038/s41375-021-01127-2

Summary

T-cell-based immunotherapies such as CAR-T cells and bispecific T-cell engagers (BiTEs) have shown promise but have significant limitations: (1) poor pharmacokinetics requiring continuous infusion (BiTE half-life ~2 h), and (2) single-antigen targeting leading to antigen-loss tumor escape and relapse. A modular nanoparticle platform that addresses both limitations—extending half-life and enabling multispecific targeting—could improve efficacy. ### Nanoparticle Characterization & Pharmacokinetics | Parameter | Result | |---------------|------------| | Liposome size | ~100 nm | | Half-life (non-PEGylated nanoBiTE) | ~36 h | | Half-life (PEGylated nanoBiTE) |.

Keywords

T cellsCancer immunotherapyNanoparticlesBiodistributionLiposomesCAR-T cellsNanocarriers

Purpose: T-cell-based immunotherapies such as CAR-T cells and bispecific T-cell engagers (BiTEs) have shown promise but have significant limitations: (1) poor pharmacokinetics requiring continuous infusion (BiTE half-life ~2 h), and (2) single-antigen targeting leading to antigen-loss tumor escape and relapse. A modular nanoparticle platform that addresses both limitations—extending half-life and enabling multispecific targeting—could improve efficacy and patient quality of life.

Hypothesis: Liposomal nanoparticles decorated with anti-CD3 antibodies (targeting T cells) and one or more antibodies targeting cancer antigens will: (1) have a prolonged half-life (~60 h) enabling once-weekly administration; (2) function as bispecific T-cell engagers (nanoBiTEs) to redirect T cells to kill cancer cells; (3) when decorated with multiple antibodies against multiple cancer antigens (nanoMuTEs), prevent antigen-loss tumor escape; and (4) show superior efficacy compared to single-antigen targeting in Waldenström macroglobulinemia (WM) and multiple myeloma (MM) models.

Aims: 1. Develop and characterize liposomal nanoparticles decorated with anti-CD3 and anti-cancer antigen antibodies (nanoBiTEs) with PEGylation for extended half-life 2. Evaluate pharmacokinetics of nanoBiTEs compared to conventional BiTEs 3. Test therapeutic efficacy of CD20/CD3 nanoBiTEs in Waldenström macroglobulinemia (WM) models in vitro and in vivo 4. Develop and validate multi-antigen targeting nanoparticles (nanoMuTEs) targeting BCMA, CS1, and CD38 for multiple myeloma 5. Demonstrate that nanoMuTEs prevent antigen-loss tumor escape compared to single-antigen nanoBiTEs 6. Compare in vivo efficacy of nanoBiTEs vs. nanoMuTEs in MM xenograft models

Delivery system:

Component: Nanoparticle Type; Description: Liposomes (large unilamellar vesicles); extruded through 100 nm membranes

Component: Lipid Composition; Description: DPPC : Cholesterol : DSPE-PEG2000 = 65:30:5 (mass ratio)

Component: PEGylation; Description: DSPE-PEG2000 incorporated for stealth properties and extended circulation

Component: Surface Functionalization; Description: Streptavidin conjugated to liposome amine groups; biotinylated antibodies attached via streptavidin-biotin

Component: Antibody Density; Description: ~1 anti-CD3 + ~1 anti-cancer antibody per liposome (optimized; increasing density did not improve binding)

Component: Size/Zeta Potential; Description: ~100 nm (Supplementary Table 2)

Component: Cancer Targets (WM); Description: CD20 (highly expressed on WM cells)

Component: Cancer Targets (MM); Description: BCMA, CS1 (SLAMF7), CD38 (single or multiplexed)

Component: T Cell Target; Description: CD3 (pan-T cell marker)

Component: nanoBiTE Formats; Description: CD20/CD3 (WM); BCMA/CD3, CS1/CD3, CD38/CD3 (MM; single-antigen)

Component: nanoMuTE Format; Description: BCMA/CS1/CD38/CD3 (triple-antigen targeting)

Component: Half-Life (PEGylated); Description: ~60 hours (vs. ~36 h non-PEGylated; vs. ~2 h conventional BiTE)

Component: Dosing Schedule; Description: Once weekly (IV bolus)

Approach:

Parameter: Cell Lines; Details: • WM: BCWM.1, MWCL.1 • MM: H929, MM.1S, RPMI-8226 • Primary patient MM cells (CD138⁺)

Parameter: In Vitro Model; Details: 3D Tissue-Engineered Bone Marrow (3DTEBM): cross-linked fibrinogen from patient BM supernatant; includes tumor cells, T cells, and accessory cells; 4-day culture

Parameter: In Vitro Assays; Details: • Binding (flow cytometry; DiO-labeled liposomes) • T-cell-mediated killing (counting beads; DiO⁺ tumor cells) • T-cell activation (CD69 on CD4⁺/CD8⁺ T cells) • Cytokine secretion (RayBiotech array) • Antigen loss modeling (blocking antibodies + flow cytometry)

Parameter: In Vivo WM Model; Details: NSG mice; IV injection of BCWM.1 cells (2×10⁶); human T cells (5×10⁶) day 7; treatment IV weekly (0.5 mg/mouse); IVIS bioluminescence

Parameter: In Vivo MM Model; Details: NSG mice; IV injection of MM.1S-luc cells (2×10⁶); human T cells day 7; treatment IV weekly; survival monitored

Parameter: Pharmacokinetics; Details: DiD-labeled nanoparticles; blood sampling at 0.25, 6, 24, 48, 72, 96 h; fluorescence measurement; polynomial regression

Parameter: Biodistribution; Details: DiD-labeled nanoparticles + calcein violet-labeled T cells; organs harvested at 24 h; flow cytometry

Parameter: Controls; Details: Isotype/CD3 nanoparticles (non-targeting); no T cell controls

Parameter: Sample Sizes; Details: In vitro: n=3-4; In vivo: n=7 per group

Parameter: Statistical Tests; Details: Student's t-test; one-way/two-way ANOVA; log-rank test (survival); P < 0.05 significant

Key methods:

Analysis Category: Nanoparticle Characterization; Methods: Zetasizer (size, zeta potential, PDI); fluorescence spectrophotometry (pharmacokinetics)

Analysis Category: Antigen Expression; Methods: Flow cytometry (APC-conjugated antibodies); RMFI (relative mean fluorescence intensity); % positive cells

Analysis Category: Liposome Binding; Methods: DiO-labeled liposomes; flow cytometry; MFI of DiO

Analysis Category: Cell Survival/Killing; Methods: DiO-labeled tumor cells + counting beads; flow cytometry; normalized to beads

Analysis Category: T Cell Activation; Methods: Flow cytometry: CD3-PE, CD4-FITC, CD8-Violet, CD69-APC; % CD69⁺ within CD4⁺/CD8⁺ subsets

Analysis Category: Cytokine Secretion; Methods: Human Cytokine Array Q1 (RayBiotech); InnoScan 710 scanner

Analysis Category: Antigen Loss Modeling; Methods: Pre-treatment with blocking antibodies (anti-BCMA, anti-CS1, anti-CD38); then nanoBiTE/nanoMuTE binding measured

Analysis Category: In Vivo Imaging; Methods: IVIS 50 bioluminescence (D-luciferin i.p.); Living Image 2.6 software

Analysis Category: Gene Expression Analysis; Methods: Affymetrix U133 Plus 2.0 (600 MM patients); Python analysis

Analysis Category: Biodistribution; Methods: Flow cytometry of organs (bone marrow, spleen, blood, liver, lung)

Key results: ### Nanoparticle Characterization & Pharmacokinetics

Parameter: Liposome size; Result: ~100 nm

Parameter: Half-life (non-PEGylated nanoBiTE); Result: ~36 h

Parameter: Half-life (PEGylated nanoBiTE); Result: ~60 h

Parameter: Conventional BiTE half-life; Result: ~2 h

Parameter: Antibody density optimization; Result: ~1 antibody per liposome (optimal)

Parameter: CD20/CD3 nanoBiTE binding to WM cells; Result: ~50× greater than isotype/CD3

Waldenström Macroglobulinemia (CD20:

Parameter: WM cell CD20 expression; Result: ~90% of cells; high intensity

Parameter: WM killing (3DTEBM, 4 days); Result: 60-70% (vs. ~0% isotype/CD3)

Parameter: CD4⁺ T cell activation (CD69); Result: Significantly increased vs. isotype

Parameter: CD8⁺ T cell activation (CD69); Result: Higher than CD4⁺

Parameter: Cytokines increased; Result: IL-2, IL-6, IL-10, TNF-α, IFN-γ

Parameter: In vivo tumor cure; Result: 100% survival (>60 days)

Parameter: In vivo tumor progression; Result: Complete eradication by day 35

Multiple Myeloma Antigen Expression (600 Patients + Cell Lines):

Antigen: BCMA; Gene Expression Variability: High heterogeneity; Surface Protein Variability: Variable across cell lines/patients

Antigen: CS1; Gene Expression Variability: High heterogeneity; Surface Protein Variability: Variable across cell lines/patients

Antigen: CD38; Gene Expression Variability: High heterogeneity; Surface Protein Variability: Variable across cell lines/patients

nanoBiTE vs. nanoMuTE Binding to MM Cells:

Formulation: BCMA/CD3; Binding (vs. isotype/CD3): Significant

Formulation: CS1/CD3; Binding (vs. isotype/CD3): Significant

Formulation: CD38/CD3; Binding (vs. isotype/CD3): Significant

Formulation: BCMA/CS1/CD38/CD3 (nanoMuTE); Binding (vs. isotype/CD3): Highest binding

T-Cell-Mediated Killing (MM, 3DTEBM):

Formulation: Isotype/CD3; Killing: ~0%; vs. isotype/CD3: -

Formulation: BCMA/CD3; Killing: Significant; vs. isotype/CD3: P < 0.05

Formulation: CS1/CD3; Killing: Significant; vs. isotype/CD3: P < 0.05

Formulation: CD38/CD3; Killing: Significant; vs. isotype/CD3: P < 0.05

Formulation: nanoMuTE (triple); Killing: Highest killing; vs. isotype/CD3: P < 0.05 vs. each nanoBiTE

Antigen Escape Prevention:

Treatment: BCMA/CD3 nanoBiTE; Effect on Antigen Expression: Decreased BCMA on remaining cells; Interpretation: Antigen-low clones survive/emerge

Treatment: CS1/CD3 nanoBiTE; Effect on Antigen Expression: Decreased CS1 on remaining cells; Interpretation: Antigen-low clones survive/emerge

Treatment: CD38/CD3 nanoBiTE; Effect on Antigen Expression: Decreased CD38 on remaining cells; Interpretation: Antigen-low clones survive/emerge

Treatment: nanoMuTE (triple); Effect on Antigen Expression: No decrease in any antigen; Interpretation: Prevents antigen escape

Treatment: Blocking one antigen (nanoMuTE); Effect on Antigen Expression: Binding maintained via other antigens; Interpretation: Multi-targeting compensates

Treatment: Blocking all three antigens (nanoMuTE); Effect on Antigen Expression: Binding decreased; Interpretation: Confirms mechanism

In Vivo MM Efficacy (MM.1S Xenograft):

Treatment: Isotype/CD3; Tumor Progression: Rapid progression; Survival: All dead by day 40

Treatment: BCMA/CD3; Tumor Progression: Delayed; Survival: Prolonged vs. isotype

Treatment: CS1/CD3; Tumor Progression: Delayed; Survival: Prolonged vs. isotype

Treatment: CD38/CD3; Tumor Progression: Delayed; Survival: Prolonged vs. isotype

Treatment: nanoMuTE (triple); Tumor Progression: Longest delay; Survival: 100% survival to day 55

T Cell Biodistribution:

Treatment: Isotype/CD3; T Cell Accumulation at Tumor Site (BM): Minimal

Treatment: nanoBiTEs (single antigen); T Cell Accumulation at Tumor Site (BM): Significant

Treatment: nanoMuTE (triple); T Cell Accumulation at Tumor Site (BM): Highest accumulation

Interpretation: The authors conclude that "the nanoBiTE/nanoMuTE platform uses nanotechnology to provide a relatively easy-to-make and off-the-shelf solution to circumvent the major limitations of the current immunotherapy technologies (CAR-T cells and BiTEs)." They emphasize that "the flexibility of the nanoparticle-based immuno-engaging technology provides a general platform with groundbreaking translational potential for developing easy-to-make, specific, and efficacious immunotherapy for cancer in general." The nanoMuTE approach addresses "development of antigen-less clones, causing tumor escape and relapse" by targeting multiple antigens simultaneously. They highlight that the prolonged half-life (60 h) "enables once-a-week administration instead of continuous infusion, dramatically improving the patient's quality of life."

10. Limitations (Explicitly Stated or Evident):

1. Different efficacy between WM and MM: The authors note that "the effect of the CD20/CD3 nanoBiTEs for the treatment of WM was significantly more profound than the nanoBiTEs/nanoMuTEs used for MM; CD20/CD3 cured the WM xenograft murine model, whereas the nanoBiTEs/nanoMuTEs prolonged survival of MM mice by only 10–20 days." This is attributed to higher CD20 expression on WM vs. lower/variable BCMA/CS1/CD38 on MM.

2. In vivo models use humanized T cells in NSG mice: All in vivo studies used immunodeficient NSG mice with adoptively transferred human T cells, not fully immunocompetent mice with endogenous immune systems.

3. No direct comparison to conventional BiTEs in vivo: The study does not compare nanoBiTEs/nanoMuTEs head-to-head with conventional BiTE molecules in the same tumor models.

4. Model antigen systems: The study uses established cell lines and xenografts, not patient-derived tumors or genetically engineered mouse models.

5. Cytokine release syndrome not assessed: While T-cell activation and cytokine secretion were measured in vitro, systemic cytokine release (CRS) in vivo was not evaluated.

6. Antigen escape modeled with blocking antibodies: The study uses blocking antibodies to mimic antigen downregulation, not true genetic loss or long-term selection pressure from treatment.

7. No evaluation of T cell exhaustion: Long-term T cell function, persistence, and exhaustion markers (PD-1, TIM-3) were not assessed in vivo.

8. Antibody orientation and stability: The streptavidin-biotin conjugation method may lead to random antibody orientation; long-term stability of the antibody-liposome conjugate was not extensively characterized.

9. Dose optimization not performed: A single dose (0.5 mg/mouse) was used; dose-response and optimal scheduling were not systematically explored.

10. Potential immunogenicity of streptavidin: The use of streptavidin (a bacterial protein) could elicit immune responses upon repeated administration; this was not assessed.

11. Conflicts of interest: The authors have filed a patent, and one author is founder/owner of Cellatrix LLC and Targeted Therapeutics LLC, which could represent a conflict.

12. No evaluation in solid tumors: The study focuses on hematological malignancies (WM and MM); efficacy in solid tumors with heterogeneous antigen expression and dense stroma was not demonstrated.

Report prepared based on the published Leukemia article. For full experimental details, supplementary figures, and complete references, please refer to the original publication.

Biochemical Pharmacology*, Volume 173, 2020, Article 113648 DOI/