Development and Evaluation of a Bispecific Single-Chain Antibody Targeting PSMA and CD3 to Enhance T-Cell-Mediated Lysis

Abstract

Prostate cancer is among the most prevalent malignancies in men worldwide and is associated with poor outcomes in advanced stages. Conventional therapies, including androgen deprivation and chemotherapy, provide limited benefit and are often accompanied by significant toxicity. This study aimed to evaluate the efficacy and immunological impact of a novel bispecific single-chain antibody targeting prostate-specific membrane antigen (PSMA) and CD3 in preclinical models. A bispecific single-chain variable fragment (scFv) antibody was engineered to simultaneously bind PSMA on tumor cells and CD3 on T cells. The construct was expressed in mammalian cell lines, and its purity, specificity, and binding affinity were characterized. In vivo efficacy was assessed in murine xenograft models of prostate cancer, with treatment groups receiving human T cells with or without the bispecific antibody. Tumor growth, survival, and immunological responses were evaluated using flow cytometry, immunohistochemistry, and histopathological analysis. The bispecific antibody demonstrated high purity (mean 97.7%), strong binding affinity to PSMA (Kd 0.23 nM) and CD3 (Kd 0.30 nM), and specificity >99%. In vivo, treatment significantly reduced tumor growth (75.45 ± 3.52 mm³ vs. 543.39 ± 44.35 mm³ in controls at Day 28; Student’s t-test, t(28) = 40.52, p < .001) and prolonged survival (59.00 ± 0.84 vs. 31.00 ± 0.84 days; Log-rank test, p < .001). Enhanced T-cell activation, infiltration, and IFN-γ release were observed, accompanied by increased tumor necrosis and apoptosis. The PSMA-CD3 bispecific antibody effectively redirected T-cell cytotoxicity against prostate cancer cells, resulting in robust antitumor activity and survival benefit. These findings support further translational development of bispecific immunotherapies for advanced prostate cancer.

Keywords

prostate cancer PSMA CD3 bispecific antibody immunotherapy T-cell activation

Introduction

Prostate cancer is the most frequently diagnosed malignancy among men in industrialized nations and represents the second leading cause of cancer-related mortality worldwide (Wang et al., 2022). The disease is often clinically silent until advanced stages, contributing to delayed diagnosis and poor patient outcomes (Barsouk et al., 2020).

Initially, prostate cancer growth is androgen-dependent, and androgen deprivation therapy (ADT) remains the standard first-line treatment (de Bono et al., 2011).

Nearly all patients eventually develop resistance, progressing to an androgen-independent stage characterized by aggressive behavior and limited therapeutic options (Teo et al., 2019). Standard treatments for advanced disease, including anti-androgens and chemotherapy, provide only transient disease control and are associated with significant toxicity (Scher et al., 2012).

The identification of tumor-associated antigens has enabled the development of targeted therapies. Among these, prostate-specific membrane antigen (PSMA), a 100-kDa transmembrane glycoprotein with enzymatic activities including folate hydrolase and NAALADase, has emerged as a particularly promising target (Liu et al., 1997). PSMA is highly expressed in prostate cancer cells, retains its orientation in malignant tissue, undergoes endocytosis, and traffics through lysosomal compartments, making it suitable for both active immunization and passive immunotherapy (Bander et al., 2005; Drake, 2010; Virgolini et al., 2018).

Bispecific antibodies (BsAbs) represent a novel class of immunotherapeutics capable of simultaneously engaging tumor-associated antigens and immune effector cells (Klein et al., 2024; Labrijn et al., 2019). By redirecting T-cell cytotoxicity toward malignant cells, BsAbs have demonstrated potent antitumor activity in hematologic malignancies and are increasingly being explored in solid tumors (Cheng et al., 2024; Falchi et al., 2023; Heitmann et al., 2021; Huehls et al., 2015).

The present study aimed to evaluate the efficacy, safety, and immunological impact of a novel bispecific single-chain antibody targeting PSMA and CD3 in preclinical models of prostate cancer.

Method

Ethical Considerations

The study protocol was approved by the Institutional Ethics Committee/Institutional Review Board (IRB) of the Oncology Teaching Hospital (Reference No: 000898761, Date: 15/10/2024). Human T cells used in this study were isolated from peripheral blood samples. Written informed consent was obtained from all participants prior to blood collection, in accordance with the Declaration of Helsinki

Antibody Design and Construction

A bispecific single-chain variable fragment (scFv) antibody was engineered to simultaneously target PSMA on tumor cells and CD3 on T cells. The construct was generated by fusing the variable heavy (VH) and variable light (VL) domains of a PSMA-specific scFv (clone J591) with the VH and VL domains of a CD3-specific scFv (clone OKT3). A flexible glycine-serine linker [(G₄S)₃] was incorporated to maintain conformational stability and enable dual binding. The sequence was codon-optimized for mammalian expression and verified by Sanger sequencing.

Molecular Cloning and Expression

The bispecific construct was cloned into a mammalian expression vector under the Cytomegalovirus (CMV) promoter using restriction digestion and Gibson assembly. HEK293 cells achieved ~70% transfection efficiency, yielding ~50 mg/L antibody, while Chinese Hamster Ovary (CHO) cells provided stable long-term expression. Antibody purity was assessed by high-performance liquid chromatography (HPLC), and binding specificity was confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR).

In Vivo Studies

Murine xenograft models were established using Lymph Node Carcinoma of the Prostate (LNCaP) prostate cancer cells. Mice were divided into three groups:

Control group: LNCaP cells only.

T-cell group: LNCaP cells + human T cells (no antibody).

Treatment group: LNCaP cells + human T cells + PSMA-CD3 bispecific antibody.

Tumor growth was monitored twice weekly using calipers, with volumes calculated as (length × width²)/2. Treatment commenced when tumors reached ~200 mm³. Survival was tracked for up to 12 weeks, and Kaplan–Meier curves were generated.

Flow Cytometry

Peripheral blood and tumor-infiltrating lymphocytes were isolated at defined time points. Cells were stained with fluorochrome-conjugated antibodies against CD3, CD4, CD8, CD69, CD25, and FoxP3. Data were acquired using a BD FACSCanto II cytometer and analyzed with FlowJo software. Activated T cells (CD69+), effector subsets (CD4+, CD8+), and regulatory T cells (CD25+FoxP3+) were quantified.

Immunohistochemistry

Tumor tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned at 4 µm. Sections underwent antigen retrieval in citrate buffer (pH 6.0), blocking with 3% hydrogen peroxide and 5% bovine serum albumin, and incubation with primary antibodies against CD3 (T-cell infiltration), Ki-67 (proliferation), and cleaved caspase-3 (apoptosis). Horseradish peroxidase–conjugated secondary antibodies and 3,3’-Diaminobenzidine (DAB) substrate were used for visualization, followed by hematoxylin counterstaining. Quantification was performed by two blinded pathologists.

Results

Antibody Characterization

The BsAb demonstrated high purity (mean 97.7%), strong binding affinity to PSMA (Kd 0.23 nM) and CD3 (Kd 0.30 nM), and specificity >99% (Table 1, Figure 1).

Table 1.

Antibody Characterization Parameters (Purity, Binding Affinity, Specificity, Concentration).

Parameter	N	Minimum	Maximum	Mean	Std. Deviation
Purity (%)	10	96.5	98.8	97.770	0.80
PSMA binding affinity (Kd, nM)	10	0.20	0.26	0.2310	0.019
CD3 binding affinity (Kd, nM)	10	0.27	0.33	0.3000	0.01
Specificity (%)	10	98.5	99.4	99.010	0.28
Concentration (µg/mL)	10	150	200	174.50	15.88

Figure 1.

Antibody Characterization.

In Vitro Cytotoxicity

LDH assays revealed significantly enhanced cytotoxicity in the presence of the BsAb. At effector-to-target ratios of 5:1 and 10:1, cell lysis reached 66.25 ± 2.75% and 79.00 ± 1.82%, respectively, compared to 21.50 ± 1.29% in the absence of antibody (one-way analysis of variance [ANOVA], F(2, 27) = 2181.74, p < .001) (Table 2, Figure 2).

Table 2.

LDH Assay Results: Percentage of Cell Lysis at Different Effector-to-Target Ratios.

Groups	% Cell Lysis (LDH Assay)
No Antibody	21.50 ± 1.29
5:1	66.25 ± 2.75^a
10:1	79.00 ± 1.82^a,b

Note. Each value represents the mean ± SD (standard deviation) of 10 animals.

Significant result as compared to No Antibody. ^bSignificant result as compared to 5:1.

Figure 2.

In Vitro Cytotoxicity: LDH Assay Showing Percentage of Cell Lysis at Different Effector-To-Target Ratios (No Antibody, 5:1, 10:1).

Immunological Activation

Flow cytometry demonstrated increased CD69+ T-cell activation (63.00 ± 2.58% at 5:1, 71.25 ± 2.20% at 10:1 vs. 12.50 ± 1.29% without antibody; one-way ANOVA, F(2, 27) = 2306.10, p < .001) (Table 3, Figure 3). IFN-γ release was markedly elevated (1235.00 ± 50.66 pg/mL at 5:1, 1512.50 ± 29.86 pg/mL at 10:1 vs. 302.50 ± 9.57 pg/mL without antibody; one-way ANOVA, F(2, 27) = 3395.64, p < .001) (Tables 4, 5, Figure 4).

Table 3.

Percentage of CD69+ T Cells at Different Effector-to-Target Ratios.

Groups	CD69+ T Cells (%)
No Antibody	12.50 ± 1.29
5:1	63.00 ± 2.58^a
10:1	71.25 ± 2.2^b

Note. Each value represents the mean ± SD (standard deviation) of 10 animals.

Significant result as compared to No Antibody. ^bSignificant result as compared to 5:1.

Figure 3.

T-Cell Activation: Percentage of CD69+ T Cells at Different Effector-To-Target Ratios (No Antibody, 5:1, 10:1).

Table 4.

IFN-γ Release (pg/mL) at Different Effector-to-Target Ratios.

Groups	IFN-γ Release (pg/mL)
No Antibody	302.50 ± 9.57
5:1	1235.00 ± 50.66^a
10:1	1512.50 ± 29.86^{a, b}

Note. Each value represents the mean ± SD (standard deviation) of 10 animals.

Significant result as compared to No Antibody. ^bSignificant result as compared to 5:1.

Table 5.

Correlation Analysis Between Cell Lysis, CD69+ T Cells, and IFN-γ Release.

Parameters		CD69+ T Cells (%)	IFN-γ Release (pg/mL)
% Cell lysis (LDH assay)	r	.996**	.999**
% Cell lysis (LDH assay)	p	.001	.001
CD69+ T cells (%)	r		.995**
CD69+ T cells (%)	p		.001

**p < 0.01.

Figure 4.

IFN-γ Release: Concentration of IFN-γ (pg/mL) at Different Effector-To-Target Ratios (No Antibody, 5:1, 10:1).

Tumor Growth and Survival

Treatment significantly reduced tumor growth (75.45 ± 3.52 mm³ vs. 543.39 ± 44.35 mm³ in controls at Day 28; Student’s t-test, t(28) = 40.52, p < .001) (Table 6, Figure 5). Survival was prolonged in the treatment group (59.00 ± 0.84 days vs. 31.00 ± 0.84 days in controls; Log-rank test, p < .001). The T-cell-only group demonstrated intermediate survival (41 days) (Table 7, Figure 6).

Table 6.

Tumor Growth Monitoring (mm³) in Control, T-Cell Only, and Treatment Groups.

Groups	Control (n = 15)	Treatment Group (n = 15)	t-test (p Value)
Groups	Mean ± SD	Mean ± SD	t-test (p Value)
Day 0 (mm³)	0.00 ± 0.00	0.00 ± 0.00	-
Day 7 (mm³)	12.75 ± 2.62	4.11 ± 1.14	11.68 (0.001*)
Day 14 (mm³)	88.05 ± 24.74	23.79 ± 3.20	9.45 (0.001*)
Day 21 (mm³)	251.05 ± 21.02	41.4 ± 1.56	38.23 (0.001*)
Day 28 (mm³)	543.39 ± 44.35	75.45 ± 3.52	40.52 (0.001*)

Note. Control Group: Mice received no antibody treatment. Treatment Group: Mice received the bispecific antibody targeting PSMA and CD3, along with human T cells to simulate T-cell engagement. *p < 0.05.

Figure 5.

Tumor Growth Monitoring: Tumor Volume (mm³) in Control, T-Cell Only, and Treatment Groups Over 28 Days.

Table 7.

Survival Duration (Days) in Control, T-Cell Only, and Treatment Groups.

Groups	Control (n = 15)	Treatment Group (n = 15)	t-test (p Value)
Groups	Mean ± SD	Mean ± SD	t-test (p Value)
Survival (days)	31.0± 0.84	59.00± 0.84	(0.001*)

Note. Control group: Mice received no antibody treatment. Treatment group: Mice received the bispecific antibody targeting PSMA and CD3, along with human T cells to simulate T-cell engagement. *p < 0.05.

Figure 6.

Kaplan–Meier Survival Curves: Survival Analysis of Control, T-Cell Only, and Treatment Groups.

Immunological Response

The treatment group exhibited increased CD4+(12.93 ± 1.28% vs. 4.40 ± 1.28%; Student’s t-test, t(28) = 19.42, p < .001) and CD8+ (17.27 ± 1.48% vs. 7.13 ± 1.18%; Student’s t-test, t(28) = 20.63, p < .001) T-cell populations, with reduced regulatory T cells (4.00 ± 0.75% vs. 10.47 ± 1.06%; Student’s t-test, t(28) = 19.23, p < .001) (Table 8, Figure 7). Tumor infiltration was significantly higher (138.33 ± 11.28 vs. 38.00 ± 7.51 cells/mm²; Student’s t-test, p < .001) (Table 9, Figure 8).

Table 8.

Immunological Response Assessment (CD4+, CD8+, Tregs) in Control, T-Cell Only, and Treatment Groups.

Groups	Control (n = 15)	Treatment Group (n = 15)	t-test (p Value)
Groups	Mean ± SD	Mean ± SD	t-test (p Value)
CD4+ T cells (%)	4.40 ± 1.28	12.93 ± 1.28	19.42 (.001*)
CD8+ T cells (%)	7.13 ± 1.18	17.27 ± 1.48	20.63 (.001*)
Regulatory T cells (Tregs) (%)	10.47 ± 1.06	4.00 ± 0.75	19.23 (.001*)

**p < 0.05.

Figure 7.

Immunological Response: Percentages of CD4+, CD8+, and Regulatory T Cells in Control, T-Cell Only, and Treatment Groups.

Table 9.

Tumor Infiltration Analysis (cells/mm²).

Groups	Control (n = 15)	Treatment Group (n = 15)	t-test (p Value)
Groups	Mean ± SD	Mean ± SD	t-test (p Value)
T-cell infiltration (cells/mm²)	38.00± 7.51	138.33± 11.28	(.001*)

**p < 0.05.

Figure 8.

Tumor Infiltration: Quantification of T-Cell Infiltration (Cells/mm²) in Control, T-Cell Only, and Treatment Groups.

Histopathology

Treated tumors showed increased mean necrosis area (49.27 ± 1.48% vs. 19.40 ± 1.19%; Student’s t-test, t(28) = 73.27, p < .001) and apoptosis (39.40 ± 1.12% vs. 11.00 ± 0.84%; Student’s t-test, t(28) = 78.33, p < .001) (Table 10, Figure 9). Regarding histopathological scores, moderate dysplasia was observed in 10 out of 15 treated animals (66.7%) compared to 0 (0.0%) in controls (Chi-square test, χ²(1) = 17.69, p < .001). Severe dysplasia was present in 5 treated animals (33.3%) versus 8 controls (53.3%), and necrosis was predominant in 0 treated animals (0.0%) versus 7 controls (46.7%) (Table 11, Figure 10). (Tables 12, 13, Figure 11)

Table 10.

Necrosis and Apoptosis Percentages in Control, T-Cell Only, and Treatment Groups.

Groups	Control (n = 15)	Treatment Group (n = 15)	t-test (p Value)
Groups	Mean ± SD	Mean ± SD	t-test (p Value)
Necrosis (%)	19.40 ± 1.19	49.27 ± 1.48	73.27 (.001*)
Apoptosis (%)	11.00 ± 0.84	39.40 ± 1.12	78.33 (.001*)

**p < 0.05.

Figure 9.

Histopathological Analysis.

Table 11.

Histopathological Scoring (Moderate Dysplasia, Necrosis Predominance, Severe Dysplasia).

Groups	Control (n = 15)		Treatment Group (n = 15)		χ² (p Value)
Groups	No.	%	No.	%	χ² (p Value)
Moderate dysplasia	0	0.0	10	66.7	17.69 (.001*)
Necrosis predominant	7	46.7	0	0.0
Severe dysplasia	8	53.3	5	33.3

**p < 0.05.

Figure 10.

Histopathological Scoring: Distribution of Moderate Dysplasia, Necrosis Predominance, and Severe Dysplasia in Control, T-Cell Only, and Treatment Groups.

Table 12.

Kaplan–Meier Survival Analysis for Moderate Dysplasia in Treatment Group.

Total N	N of Events	Censored
Total N	N of Events	N	Percent
15	10	5	33.3

**p < 0.05.

Table 13.

Mean And Median Survival Times for Moderate Dysplasia in Treatment Group.^a

Mean^b				Median
Estimate	Std. Error	95% Confidence Interval		Estimate	Std. Error	95% Confidence Interval
Estimate	Std. Error	Lower Bound	Upper Bound	Estimate	Std. Error	Lower Bound	Upper Bound
59.247	0.233	58.789	59.704	60.000	0.471	59.076	60.924

Note. Superscript letters (a, b) indicate statistically significant differences between groups. **p < 0.05.

Figure 11.

Kaplan–Meier Survival Curve: Probability Estimates for Moderate Dysplasia in the Treatment Group.

Discussion

This study demonstrates that a novel BsAb targeting PSMA and CD3 effectively enhances T-cell-mediated cytotoxicity against prostate cancer cells. The construct exhibited high purity and binding specificity, ensuring reliable engagement of both tumor and immune targets. In vivo, the treatment resulted in significant tumor regression and a pronounced survival benefit. Immunological and histopathological analyses confirmed this robust antitumor response, evidenced by increased activation and infiltration of CD4+ and CD8+ T cells, reduced regulatory T-cell populations, elevated IFN-γ release, and enhanced tumor necrosis (49.3%) and apoptosis (39.4%).

These findings align with the growing body of literature emphasizing the potential of T-cell engagers in solid tumors, which have historically presented treatment challenges due to their complex and immunosuppressive microenvironments (Wu et al., 2021). By simultaneously engaging PSMA and CD3, our bispecific construct effectively redirects potent cytotoxic T-cell responses directly to the tumor site (Hummel et al., 2021). Compared to conventional therapies, which frequently result in resistance to androgen deprivation and chemotherapy, this immune-mediated mechanism of action offers a highly targeted approach (Lan et al., 2023). The substantial induction of tumor cell death observed in our models suggests that this strategy maximizes therapeutic efficacy, while its high specificity (>99%) could potentially minimize off-target toxicities.

Despite these promising results, several limitations must be acknowledged. The in vivo experiments relied on a single prostate cancer cell line (LNCaP), which may not fully capture the profound genetic and phenotypic heterogeneity characteristic of advanced human prostate cancer. In addition, although the murine xenograft model successfully incorporated human T cells to demonstrate targeted lysis, it inherently lacks a fully intact human immune system and the full complexity of the tumor microenvironment. Furthermore, the relatively short observation period (28 days) and the specific pharmacokinetic properties typical of scFv constructs limit the comprehensive assessment of long-term efficacy and potential systemic toxicities.

Future research should incorporate diverse patient-derived xenografts (PDX) or humanized mouse models alongside extended safety profiling. Ultimately, addressing these preclinical limitations will be critical for translating this novel therapeutic option into early-phase clinical trials, thereby addressing a critical unmet need in men’s health for advanced prostate cancer.

Footnotes

ORCID iD

Mustafa Abdulkareem Aljaberi

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was self-funded by the authors.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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