
==== Front
Mater Today Bio
Mater Today Bio
Materials Today Bio
2590-0064
Elsevier

S2590-0064(24)00300-4
10.1016/j.mtbio.2024.101239
101239
Full Length Article
Polymeric PD1/PDL1 bispecific antibody enhances immune checkpoint blockade therapy
Xue Fuxin a
Ren Xitong b
Kong Chaoying b
Wang Jianfeng a
Liu Linlin liulinl@jlu.edu.cn
a∗
Hu Junli hujl100@nenu.edu.cn
c∗∗
Shen Na nshen@ciac.ac.cn
b∗∗∗
Tang Zhaohui b
a Department of Radiation Oncology, China-Japan Union Hospital of Jilin University, Changchun, Jilin, 130033, China
b Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, China
c Key Laboratory of UV-Emitting Materials and Technology, Northeast Normal University, Ministry of Education, Changchun, Jilin, 130024, China
∗ Corresponding author. liulinl@jlu.edu.cn
∗∗ Corresponding author. hujl100@nenu.edu.cn
∗∗∗ Corresponding author. nshen@ciac.ac.cn
14 9 2024
10 2024
14 9 2024
28 10123913 6 2024
5 9 2024
10 9 2024
© 2024 The Authors. Published by Elsevier Ltd.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Immune checkpoint blockade (ICB) therapy, particularly PD1/PDL1 inhibition, has demonstrated success in bolstering durable responses in patients. However, the response rate remains below 30 %. In this study, we developed a polymeric bispecific antibody (BsAb) targeting PD1/PDL1 to enhance ICB therapy. Specifically, poly(L-glutamic acid) (PGLU) was conjugated with a double cyclic Fc binding peptide, Fc-III-4C, through condensation reactions between the -COOH group of PGLU and the -NH2 group of Fc-III-4C. This conjugate was then mixed with αPD1 and αPDL1 monoclonal antibodies (mAbs) in an aqueous solution. Mechanistically, the PD1/PDL1 BsAb (BsAbαPD1+αPDL1) acts as a bridge between tumor cells and CD8+ T cells, continuously activating CD8+ T cells to a greater extent. This leads to significantly suppressed tumor growth and prolonged survival in a mouse model of colon cancer compared to treatment with either a single mAb or a mixture of free mAbs. The tumor suppression rate achieved by the BsAbαPD1+αPDL1 was 90.1 %, with a corresponding survival rate of 83.3 % after 48 days. Thus, this study underscores the effectiveness of the BsAbαPD1+αPDL1 as a synchronizing T cell engager and dual ICBs, offering theoretical guidance for clinical ICB therapy.

Graphical abstract

Graphical abstract: BsAbαPD1+αPDL1) simultaneously binds PD1 on the tumor cell and PDL1 on the CD8+ T cell, engaging both cell types together while reinvigorating the CD8+ T cells, further contributing to tumor cell death.Image 1

Keywords

T cell engager
Bispecific antibody
PD1/PDL1 blockage
Cancer immunotherapy
Fc binding peptide
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pmc1 Introduction

In recent decades, immunotherapies, including checkpoint inhibitors, adoptive cell transfer, bispecific antibodies (BsAbs), monoclonal antibodies (mAbs), and vaccines, have emerged as efficient and highly specific treatments to combat cancer by enhancing the patient's immune response [1,2]. Notably, blocking coinhibitory immune checkpoints, such as the programmed cell death protein 1 (PD1; also known as CD279) antibody and its ligand PDL1 (also known as B7-H1 or CD274) antibody, has revolutionized cancer treatment by revitalizing T cells [3]. PD1, an inhibitory receptor found in T lymphocytes, interacts with its ligand PDL1, which is expressed in certain types of tumors [[4], [5], [6]]. Targeting the PD1/PDL1 coinhibitory pathway with monoclonal antibodies (mAbs) has led to durable tumor inhibition effects and approval for administration in various indications, including colon cancer, lung cancer, and melanoma [[7], [8], [9], [10]]. By 2022, ten PD1 products (nivolumab, pembrolizumab, cemiplimab, sintilimab, camrelizumab, toripalimab, tislelizumab, zimberelimab, prolgolimab, and dostarlimab) and three PDL1 products (atezolizumab, durvalumab, and avelumab) have been approved for different cancer types since 2014 [[11], [12], [13], [14]]. However, the response rates of anti-PD1 antibodies (αPD1) or anti-PDL1 antibodies (αPDL1) are approximately 20–30 % in patients with PDL1-positive tumors, indicating that the majority of cancer patients do not benefit from them [[15], [16], [17]]. Enhancing the response rate and therapeutic efficacy of PD1/PDL1-related therapy is therefore crucial.

Bispecific T cell engagers (BiTEs) are emerging and prominent bispecific antibodies (BsAbs) capable of binding spontaneously to both tumor antigens and T cells, effectively bridging these two entities. This interaction redirects and activates T cells, ultimately resulting in the targeted elimination of tumor cells [18,19]. Currently, several BiTEs are available on the market, including Blinatumomab (CD3 × CD19), Tebentafusp-tebn (CD3 × GP100), Mosunetuzumab (CD3 × CD20), and Teclistamab (CD3 × BCMA) [[20], [21], [22], [23], [24]]. BiTEs represent a significant portion of the BsAb pipeline in preclinical and clinical development, offering promising treatment prospects for various tumor types [25]. However, the primary production methods for BiTEs, such as the hybridoma approach and protein engineering technologies, are complex, involving a trial-and-error process that results in low yield, inconsistent quality, and significant impurities [26]. Given the maturity of monoclonal antibody (mAb) production technology, constructing BiTEs using a linker and well-established mAbs could offer a straightforward and efficient alternative, providing a new avenue for BiTE production.

Although most established BiTEs contain a fragment recognizing and activating CD3 on T cells, there is currently no CD3 monoclonal antibody available on the market for anti-tumor therapy. However, PD1 and PDL1 monoclonal antibodies (mAbs) are more accessible. PD1/PDL1 bispecific antibody (BsAbαPD1+αPDL1) can bind to specific antigen epitopes on PD1-expressing T cells and highly expressed PDL1 on tumor cells, fostering the cytotoxic effect of T cells on PDL1-positive tumor cells. In this study, polymer-multiple Fc binding peptide conjugate (PGLU-Fc-III-4C) was synthesized by condensing the side –COOH of poly(L-glutamic acid) (PGLU) with the –NH2 of Fc-III-4C (a double cyclic peptide [[27], [28]]). BsAb was then prepared by mixing PGLU-Fc-III-4C with αPD1 and αPDL1 mAbs (Scheme 1). The resulting BsAbαPD1+αPDL1 acted as bridges between tumor cells and CD8+ T cells, persistently activating CD8+ T cells to a greater extent than a solution of free mixed mAbs. The preparation of BsAbαPD1+αPDL1, its physiochemical properties, activation of CD8+ T cells in vitro, subsequent induction of tumor cell death, tumor suppression, and the mechanism of BsAbαPD1+αPDL1 were all verified. This strategy offers a novel and effective BsAbαPD1+αPDL1, significantly enhancing the therapeutic efficacy of immune checkpoint blockade (ICB) therapies.Scheme 1 BsAbαPD1+αPDL1 simultaneously binds PD1 on the tumor cell and PDL1 on the CD8+ T cell, engaging both cell types together while reinvigorating the CD8+ T cells, further contributing to tumor cell death.

Scheme 1

2 Material and methods

2.1 Materials

Fc-III-4C (amino acid sequence: NH2-Cys-Asp-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Cys-COOH, two disulfide bonds form between Cys1-Cys4 and Cys2-Cys3) was procured from GL Biochem Ltd. (Shanghai, China). γ-Benzyl-L-glutamate-N-carboxyanhydride (BLG-NCA) was obtained from Chengdu Enlai Biological Technology Co., Ltd. (Chengdu, China), purified by recrystallization from ethyl acetate, and vacuum-dried at room temperature before use. N,N-dimethylformamide (DMF) was acquired from Shanghai Haohong Scientific Co., Ltd. (Shanghai, China), dried over CaH2 for 72 h, and distilled under reduced pressure prior to use. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and N-Hydroxy succinimide (NHS) were also sourced from Shanghai Haohong Scientific Co., Ltd. (Shanghai, China). Cy5-NH2 was purchased from Wuhan Duofluor Inc. (Wuhan, China). Additionally, prestained Color Protein Molecular Weight Marker, Coomassie Blue Fast Staining Solution and BeyoGel™ Plus Precast PAGE Gel were purchased from Beyotime Biotechnology (Shanghai, China).

Anti-mouse PD1 antibody (αPD1, Rat IgG1, κ; Catalog: BE0146), anti-mouse PDL1 antibody (αPDL1, Rat IgG2b, κ; Catalog: BE0101) were procured from Bio X Cell (West Lebanon, NH, USA). Mouse IgG (Catalog: SP031) was obtained from Solarbio Science & Technology (Beijing, China). Fetal bovine serum (FBS) was sourced from Zhejiang Tianhang Biotechnology Co., Ltd., China. (Zhejiang, China). Penicillin and streptomycin were acquired from Huabei Pharmaceutical (Shijiazhuang, China). ELISA kits were purchased from Anoric.

2.2 Cells and animals

Murine colon cancer cell line MC38 was procured from Shanghai Bogoo Biotechnology Co. Ltd. (Shanghai, China). CD8+ T cells for in vitro experiments were isolated from murine spleens. Spleens were meticulously removed, washed thrice with sterile PBS on ice, and gently fragmented using a syringe end before being filtered through 250-mesh nylon strainers. Red blood cells were lysed using Ammonium-Chloride-Potassium (ACK) Lysing Buffer (BioLegend). Subsequently, splenocytes were suspended in magnetic-activated cell sorting (MACS) buffer and CD8+ T cells were isolated using a CD8a (Ly2) microbeads Isolation Kit (Miltenyi Biotec.). For T cell stimulation, the isolated CD8+ T cells were incubated with plate-bound anti-CD3 antibodies (5 μg/mL) and soluble anti-CD28 antibodies (5 μg/mL) for 48 h. The cells were cultured in RPMI 1640 medium with high glucose, supplemented with 10 % FBS, 1 % penicillin and 1 % streptomycin, and incubated at 37 °C in an atmosphere of 5 % CO2 and 95 % air (approximately 20 % O2 equivalent).

Female C57BL/6 mice were sourced from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The experimental animal procedures adhered to the guidelines outlined in the “Guide for Care and Use of Laboratory Animals” and received approval from the Animal Welfare and Ethics Committee of the Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. The reference number for this approval is [CIAC-2023-0141].

2.3 Synthesis of PGLU

PGLU was synthesized by ring-opening polymerization of BLG-NCA, followed by deprotection. Briefly, BLG-NCA (10.0 g, 38 mmol) was dissolved in 100 mL of anhydrous DMF under a nitrogen atmosphere. Then, 1-hexylamine (24.0 mg, 0.2375 mmol), dissolved in 1.8 mL of anhydrous DMF, was added to initiate polymerization, which proceeded at 30 °C for 3 days. Subsequently, acetic anhydride (242 mg, 2.375 mmol) and triethylamine (240 mg, 2.375 mmol) were introduced, and the reaction continued for an additional 24 h at 30 °C. The resulting mixture was precipitated with an excess of ether to obtain poly(γ-benzyl L-glutamate) (PBLG) as a white solid. PBLG was next dissolved in 100 mL dichloroacetic acid, and 40 mL HBr/acetic acid (33 wt%) was added to facilitate deprotection. The solution was stirred at 30 °C for 1 h and subsequently precipitated with an excess of ether. The precipitate underwent dialysis against distilled water, and was subsequently lyophilized to yield PGLU with an 87 % yield.

2.4 Synthesis of PGLU-Fc-III-4C

Under anhydrous conditions, PGLU (10 mg, 0.3 μmol) and N,N′-disuccinimidyl carbonate (10.24 mg, 0.04 mmol) were dissolved completely in 10 mL DMF. Next, triethylamine (15 mg, 0.15 mol) was added to the mixture. The solution was then incubated at 37 °C for 48 h. Subsequently, Fc-III-4C (16 mg, 0.009 mmol) and an additional amount of triethylamine (10.2 mg, 0.1 mmol) were added. After another 2 h of incubation at 37 °C, the mixture solution was dialyzed sequentially against DMF and water. Finally, the solution was freeze-dried, resulting in the production of light-yellow powder with a yield of 42 %.

2.5 Synthesis of NPαPD1+NPαPDL1 and BsAbαPD1+αPDL1

For the preparation of NPαPD1, a mixture comprising αPD1 and PGLU-Fc-III-4C was formulated at a precise mass ratio of 18.75:1, and a specified amount of PBS (0.01 M, pH 7.4) was added to dissolve the mixture. For NPαPD1, the mixture of αPD1 and PGLU-Fc-III-4C was prepared at a mass ratio of 18.75:1, too. NPαPD1+NPαPDL1 was achieved by mixing NPαPD1 and NPαPD1 in equal quantities. Each antibody in the solution was at a concentration of 1.0 mg/mL. Subsequently, the solution was incubated at 4 °C for 48 h. With the same preparation method, the mixture of αPD1, αPDL1, and PGLU-Fc-III-4C was prepared at a mass ratio of 18.75:18.75:1 The resulting products underwent direct measurements of molecular weight and size. To confirm whether PGLU-Fc-III-4C was linked to both monoclonal antibodies simultaneously, the solution was mixed with FITC-labeled anti-mouse PD1 antibody and PE/Cy7-labeled anti-mouse CD274 antibody to label αPD1 and αPDL1, respectively. The dual-color fluorescence of the solution was assessed using flow cytometry (BD FACS Celesta Multicolor Flow Cytometer, China).

2.6 Characterizations

The chemical structures of PGLU and PGLU-Fc-III-4C were analyzed using 1H nuclear magnetic resonance spectroscopy (1H NMR, Bruker, AV-300) with NaOD/D2O as the solvent. Gel Permeation Chromatography (Waters GPC 3 system, Waters Ultra hydrogel Linear Column) was utilized to determine the molecular weight distribution and dispersion of PGLU, PGLU-Fc-III-4C, BsAbαPD1&αPDL1. Polyethylene glycol was employed as standards, with 0.2 M phosphate buffer (pH 7.4) as the eluting solvent. This test was conducted under 35 °C with the flow rate was 0.5 mL/min. The concentration of the sample is 2.0 mg/mL. All size measurements were performed with a Nano-Particle Analyzer (Malvern, Zetasizer Nano ZS, Shanghai Sibaiji Instrument System Co., LTD). TEM imaging was conducted using a JEOL JEM-1011 transmission electron microscope, located in Tokyo, Japan, operating at an accelerating voltage of 100 kV. 10 μL aqueous solution of nanoparticles (0.2 mg/mL) was dripped onto the copper net and TEM was performed after the water evaporated and dried. The molecular weight of αPD1, αPDL1 and BsAbαPD1+αPDL1 were determined using asymmetric flow field-flow fractionation coupled with muti-angle laser light scattering (AF4-MALLS), conducted on a Wyatt Eclipse DualTec system (Dernbach, Germany). The system was equipped with a DAWN HELEOS-II (Santa Barbara, CA, USA), both operating at a wavelength of 658 nm. For these measurements, the refractive index increment of the samples in the eluent was assumed to be dn/dc = 0.185 mL/g. Data acquisition and analysis were performed using Wyatt Astra software.

2.7 Stability

PGLU-Fc-III-4C-IgG was synthesized following a previously published protocol. The mixture of IgG/PE-Cy7 and PGLU-Fc-III-4C was prepared at a mass ratio of 37.5:1, with an IgG/PE-Cy7 concentration of 37.5 μg/mL containing sodium azide (NaN3, 1 μg/mL) in phosphate-buffered saline (PBS, 0.01 M, pH 7.4). The PGLU-Fc-III-4C-IgG/PE-Cy7 complex was incubated in human serum at a concentration of 9.5 mg/mL for 0 h, 8 h, 24 h, 48 h, and 72 h to assess its stability. The proportion of IgG/PE-Cy7 positive particles was analyzed using flow cytometry (BD FACS Celesta) to determine the stability of the complex over time.

2.8 Non-denaturing polyacrylamide gel electrophoresis

PGLU-Fc-III-4C-IgG was synthesized according to a previously established protocol. IgG and PGLU-Fc-III-4C were combined at a mass ratio of 37.5:1, with an IgG concentration of 1.0 mg/mL, and then incubated in PBS (0.01 M, pH 7.4) at 4 °C for 48 h. Subsequently, loading buffer was added, and the samples were boiled at 100 °C for 10 min. Non-denaturing polyacrylamide gel electrophoresis (PAGE) was conducted using an electrophoresis apparatus, followed by Coomassie Brilliant blue staining for 10 min. The gel was then shaken overnight in distilled water at 4 °C to remove excess stain, and visualization was performed using a gel imaging analyzer (Beijing Junyi Huaxin Technology Co., LTD., JY04S-3C, Beijing, China).

2.9 In vitro immune activation capability of BsAbαPD1+αPDL1

MC38 cells were seeded at a density of 5 × 103 cells per well in 96-well plates and incubated overnight. 5 × 104 stimulated CD8+ T cells were then added into each well and co-cultured with MC38 cells in 150 μL RPMI 1640 for another 24 h. 50 μL fresh RPMI 1640 medium containing mAbs, free mixed mAbs (αPD1+αPDL1), free mixed NPs (NPαPD1+NPαPDL1) or BsAbαPD1&αPDL1 was then added into wells. The concentration of mAbs was 20 μg/mL, and the BsAbαPD1+αPDL1 had an equimolar of total antibodies to mAbs. After incubation at 37 °C and 5 % CO2 for 6 h, cells were imaged using an optical microscope (Olympus, Japan). For another 48 h, tumor cells were analyzed by CCK-8 assay and supernatants were subjected to IFN-γ, perforin, and granzyme B analysis by ELISA kit according to the manufacturer's instructions.

2.10 In vivo anti-tumor study

MC38 tumor models were established by subcutaneous injecting 1.0 × 106 MC38 cells to the right flank of C57BL/6 mice (female, 6 weeks old, 20 g). When tumor volumes reached around 80–100 mm3, the mice were divided into 6 groups randomly with 6 mice per group, and treated with: PBS, αPD1, αPDL1, FreeαPD1+αPDL1, NPαPD1+NPαPDL1 and BsAbαPD1+αPDL1. All treatments are performed by intravenous (i.v.) injection with 200 μL PBS as solvent on day 0, 2, 4, 7.

Long and short tumor diameters were measured using a caliper, and the body weight was measured and recorded every 2 days. The volumes of the tumor were calculated using the formula for mouse tumor calculation: V = (a × b2)/2, where “a” represents the longest diameter of the tumor and “b” is the shortest diameter. The Tumor inhibition rate (TSR) was determined by the following formula: TSR (%) = [(Vc - Vt)/Vc] × 100 %, with Vc being the average tumor volume of the control group, and Vt the average tumor volume of the treatment group (PBS). The survival rate of the mice was monitored every other day. The mice with tumors larger than 2000 mm3 were considered dead.

To establish a bilateral tumor model, When the primary tumor on the right reached approximately 100 mm3 and the secondary tumors on the left (as the secondary tumor) reached approximately 50 mm3, mice were randomly divided into six groups: PBS, αPD1, αPDL1, FreeαPD1+αPDL1, NPαPD1+NPαPDL1 and BsAbαPD1+αPDL1. All treatments are performed by intravenous (i.v.) injection with 200 μL PBS as solvent on day 0, 2, 4, 7.

2.11 Biodistribution and pharmacokinetics analyses

Under anhydrous conditions, PGLU (10 mg) and N,N′-disuccinimidyl carbonate (10.24 mg, 0.04 mmol) were dissolved completely in 10 mL DMF. Next, triethylamine (15 mg, 0.15 mol) was added to the mixture. The solution was then incubated at 37 °C for 48 h. Fc-III-4C (16 mg, 0.009 mmol) was added for another 2 h at 37 °C in dark. Subsequently, Cy5-NH2 (3 mg, 0.005 mmol) was added for another 12 h at a room temperature in dark. This solution was transferred into a dialysis bag (MWCO = 5000 Da, Mengyimei Biotechnology Co., LTD, Beijing, China) and dialyzed against water for 72 h. After freeze-drying, a blue solid, Cy5-labeled PGLU (PGLU/Cy5) was obtained with a yield of 96.6 %. Next, PGLU-Fc-III-4C/Cy5, NPαPD1+NPαPDL1/Cy5 and BsAbαPD1+αPDL1/Cy5 were prepared according to the previous method.

MC38 tumor models were established via the subcutaneous injection of 1.0 × 106 MC38 cells to the right flank of C57BL/6 mice (female, 6 weeks old, 20 g). When the tumor had reached approximately 300–500 mm3, mice were randomly allocated into three groups of 3 mice per group and treated with: Cy5-labed PGLU-Fc-III-4C-IgG, NPαPD1+NPαPDL1 and BsAbαPD1+αPDL1 by intravenous (i.v.) with 200 μL PBS as solvent. 24 h after post-administration, the tumors and normal organs were harvested and the biodistribution of nanoparticles was visualized using an IVIS Lumina LT Seres III in vivo imaging system.

The pharmacokinetics of IgG, PGLU-Fc-III-4C-IgG and BsAbαPD1+αPDL1 were assessed by the fluorescence intensity of Cy5 in the serum at various time points after the intravenous injection of different drugs into rats (n = 3).

2.12 Immune analysis

Flow cytometry assay was performed to analyze immune cells infiltrated in tumor. The subcutaneous MC38 tumor model, the grouping of tumor-bearing mice, and the way of drug administration are the same as those in vitro antitumor experiments. On day 9 in the tumor inhibition experiment of treatment, the mice were sacrificed, and both the tumor and spleen were collected. These tissues were gently grinded and strained through 250-μm nylon strainers to produce single cell suspensions. The single-cell suspensions were then washed with PBS (0.01 M, pH 7.4) supplemented with 2 % FBS. Subsequently, the cells were stained with a panel of fluorescent dyes conjugated to anti-mouse antibodies specific for CD3, CD4 and CD8 markers, followed by fixation with 4 % paraformaldehyde. The presence and distribution of T lymphocytes within the tumor and spleen tissues were detected and analyzed using flow cytometry (BD FACS Celesta).

2.13 Cytokine analysis

Peripheral blood was collected from mice 48 h after treat by different drugs and centrifuged at 1000×g at 4 °C for 15 min within 30 min of collection. The concentrations of the cytokines (IFN-γ, TNF-α and IL-12) in serum were then measured using ELISA kit according to the manufacturer's instructions.

2.14 Hematoxylin and eosin (H&E) staining

MC38 tumor models were established by subcutaneous injection of 1.0 × 106 MC38 cells to the right flank of C57BL/6 mice (female, 6 weeks old, 20 g). Mice bearing a unilateral MC38 tumor were treated with PBS, NPαPD1+NPαPDL1 and BsAbαPD1+αPDL1 (n = 3 per group) administered by intravenous (i.v.) injection with 200 μL PBS as solvent on days 0, 2, 4, and 7. Mice were euthanized on day 13, and major organs including heart, liver, spleen, lung and kidney were excised. Those organs were then fixed in 4 % buffered paraformaldehyde overnight and subsequently embedded in paraffin. Tissue sections of 5 μm thickness were prepared and stained with hematoxylin and eosin for histopathological analysis, which was conducted under a light microscope (Nikon TE2000U).

2.15 Systemic toxicity

Groups of three mice each were intravenously administered with αPD1, αPDL1, FreeαPD1+αPDL1, NPαPD1+NPαPDL1 and BsAbαPD1+αPDL1. Nine days post-injection, blood samples were collected and centrifuged at 3000 rmp for 5 min to isolate plasma. The plasma was then analyzed using an automated biochemical analyzer to measure liver and kidney function parameters such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), blood urea nitrogen (BUN), creatinine (CRE) and uric acid (UA).

2.16 Statistical analysis

All experiments were conducted with a minimum of two repetitions, and the results are presented as mean values ± standard deviation (SD). Statistical analysis for multiple group comparisons was performed using one-way ANOVA followed by Tukey's multiple comparison. Unpaired student's t-test was used for statistical analysis in experiments with two groups of data. “ns” means no significance. Survival differences were determined using Kaplan-Meier analysis, with the overall significance determined by og-rank test. ∗p < 0.05 was considered statistically significant, while ∗∗p < 0.01 and ∗∗∗p < 0.001 were considered highly and extremely levels of significant, respectively. p values were annotated on the figures. The figure legend detail the number of animals included in each study.

3 Results and discussion

3.1 Synthesis and characterization of the carrier polymer PGLU-Fc-III-4C

The carrier polymer contains a backbone of PGLU and multiple grafts of Fc-III-4C peptide. PGLU is a biocompatible and biodegradable polymer, with various downstream being tested in clinical trials. Fc-III-4C is an artificially synthesized 15-amino acid double cyclic peptide mimicking the Z-domain of Protein A, and it binds to the IgG Fc region with high affinity (dissociation constant (KD): KD = 2.45 nM to human IgG and 12.3 nM for mouse IgG) [29,30]. The PGLU, PGLU-Fc-III-4C were similarly synthesized as our previous works [[31], [32], [33]]. PGLU was synthesized via the ring-opening polymerization of γ-benzyl-L-glutamate N-carboxyanhydride, which was subsequently followed by the removal of the γ-benzyl protecting groups [31,34]. The Fc-III-4Cs were attached to PGLU molecular chains by a condensation reaction between the terminal amino groups and the side carboxyl groups of PGLU (Fig. S1). The PGLU-Fc-III-4C contained a chain of 160 L-glutamic acid residues as depicted in Figure S2. Sulfur element analysis revealed a 3.9 % sulfur content in PGLU-Fc-III-4C, with the side chains of L-glutamic acid (Glu) present as sodium salts (Table S2). This data coupled with the integration of areas and structural hydrogen numbers of δ 6.69–7.73 ppm (h, i) in the 1H NMR spectrum (Fig. S2), confirmed that each PGLU-Fc-III-4C molecule contained an average of 13.6 Fc-III-4C grafts.

3.2 Bispecific antibody (BsAbαPD1+αPDL1)

Tumor cells frequently overexpress these immune checkpoint proteins as a strategy to evade detection and destruction by the host immune system, effectively suppressing T-cell-mediated tumor rejection [[35], [36], [37], [38]]. Preclinical and clinical evidence has demonstrated that antibody blockade of immune checkpoints can significantly augment enhance antitumor immunity, with combined administration proven to intensify T-cell activation in the context of immunotherapy [39,40]. They were selected as two representative mAbs for BsAbαPD1+αPDL1 preparation with PGLU-Fc-III-4C. A 1:1 αPD1 to αPDL1 molar ratio was taken. The PGLU-Fc-III-4C was incubated with FreeαPD1+αPDL1 in PBS (Fig. 1A). As shown by Fig. 1D, DLS results showed that free αPD1 and αPDL1 had an average hydrodynamic diameter of 11.3 ± 0.8 nm and 13.5 ± 0.6 nm, respectively. The FreeαPD1+αPDL1 show a hydrodynamic diameter of 19.3 ± 3.4 nm, while the PGLU-Fc-III-4C's hydrodynamic diameter is 43.8 ± 1.7 nm. The BsAbαPD1+αPDL1 exhibited a substantially enhanced diameter of 112.3 ± 1.9 nm which without purification. The diameters determined by DLS experiments was in agreement with those observed via transmission electron microscopy (TEM) for BsAbαPD1+αPDL1 with an average diameter of 88.3 ± 15.9 nm as shown in Fig. 1B. In contrast, PGLU-Fc-III-4C-IgG exhibited a profile that negated the need for purification, which was corroborated by nondenaturing polyacrylamide gel electrophoresis, followed by Coomassie blue staining, as depicted in Figure S3. As illustrated in Fig. 1E and Table S1, the elution time of BsAbαPD1+αPDL1 was earlier than that of PGLU-Fc-III-4C and PGLU, suggesting that the BsAbαPD1+αPDL1 possesses a significantly larger mass compared to PGLU-Fc-III-4C and PGLU.Fig. 1 Synthesis and characterization of BsAbαPD1+αPDL1. (A) Schematic representation and preparation of BsAbαPD1+αPDL1. (B) Transmission electron microscopy (TEM) images of BsAbαPD1+αPDL1 (Scale bar: 1 μm). (C) Molecular weight of αPD1, αPDL1 and BsAbαPD1+αPDL1, as measured by asymmetric flow field-flow fractionation coupled with multiangle laser light scattering. (D) Particle size of PGLU-Fc-III-4C and BsAbαPD1+αPDL1, as detected by DLS. Average diameters for each sample are shown. (E) The molecular weights of PGLU, PGLU-Fc-III-4C, BsAbαPD1+αPDL1 were measured with Gel Permeation Chromatography (GPC). (F) Flow cytometric analysis detecting FITC labeled anti-PD1 and PE-Cy7 labeled anti-PDL1 antibodies conjugated into a BsAbαPD1+αPDL1.

Fig. 1

The expression of PDL1 on MC38 was analyzed by flow cytometry (Fig. S4). The mixture of αPD1 and αPDL1 was labeled with FITC and PE-Cy7, respectively, and then subjected to two-color fluorescence detection with flow cytometry. Nearly all particles of BsAbαPD1+αPDL1 (93.9 %) and PGLU-Fc-III-4C (97.5 %) were respectively categorized into double-positive and double-negative gates, indicating of the dual fluorescent labeling on PGLU-Fc-III-4C and the effective construction of BsAbαPD1+αPDL1 (Fig. 1F). Based on the average weight mass (Mw) determined by static light scattering detector (Fig. 1C), and the inputted Mw ratio of αPD1+αPDL1 and PGLU-Fc-III-4C is 37.5:1. It is theoretically deduced that the 1470 kDa BsAbαPD1+αPDL1 consisted of 42.4 kDa from PGLU-Fc-III-4C and 1427.6 kDa from antibodies (αPD1+αPDL1). Assuming the Mw of αPD1 and αPDL1 to be 247.7 kDa and 159.7 kDa, respectively. Each BsAbαPD1+αPDL1 were bound an average of 7.0 total antibodies at the 1:1 of the αPD1: αPDL1 input molar ratio. These results demonstrate the high binding ability of the Fc-III-4C grafts as well as the multivalent characteristics of the obtained BsAbαPD1+αPDL1.

3.3 The T cell-activation ability of BsAbαPD1+αPDL1

The stability of PGLU-Fc-III-4C-IgG was monitored over a period of 72 h in IgG-stimulated blood environment (9.6 g/L IgG), and it exhibited no significant alterations. The percentage of IgG-PE-Cy7 conjugated PGLU-Fc-III-4C was maintain a level above 91.0 % (Fig. 2A–B). This finding demonstrated that BsAbαPD1+αPDL1 could be stable in mice for about three days.Fig. 2 In vitro stability testing, tumor inhibition and CD8+ T cell activation activity of BsAbαPD1+αPDL1. (A) Schematic representation of the experimental of stability testing. (B) Flow cytometry analysis detecting the stimulative stability of PGLU-Fc-III-4C-hIgG-PE-Cy7 in human blood in 72 h, in which the hIgG was 9.5 g/L. PGLU-Fc-III-4C without hIgG-PE-Cy7 was used as a control for gating. (C) Schematic representation of the experimental process assessing in vitro activity of BsAbαPD1+αPDL1 in a co-culture of MC38 cells and CD8+ T cells. (D–F) IFN-γ, perforin, and granzyme B ELISA results from supernatants of co-cultured MC38 cells and CD8+ T cells at 24 h after treatment. (G) CCK-8 assay detecting different drugs induced anti-tumor effect of CD8+ T cells.

Fig. 2

MC38 tumor cells were incubated with CD8+ T cells in medium containing FreeαPD1+αPDL1, NPαPD1+NPαPDL1 or BsAbαPD1+αPDL1. Then cytokine production was subsequently analyzed 24 h after coincubation. When compared with equivalent dosage of non-conjugated mix mAbs (FreeαPD1+αPDL1), the BsAbαPD1+αPDL1 stimulated significantly higher level of interferon-gamma (IFN-γ), perforin and granzyme B (Fig. 2D–F). These cytokines are crucial for mediating the immune response of cytotoxic T lymphocyte [41,42]. Moreover, the FreeαPD1+αPDL1 only kill ∼33 % MC38 cancer cells, the NPαPD1+NPαPDL1 could kill ∼46 % MC38 cells, while the BsAbαPD1+αPDL1 killed ∼90 % MC38 cells (Fig. 2G). These results demonstrate the BsAbαPD1+αPDL1 has superior T-cell activation ability and tumor cell killing ability than the free antibody mixture.

3.4 The antitumor ability of BsAbαPD1+αPDL1

As shown in Fig. 3A, optical images illustrated that in the BsAbαPD1+αPDL1 group, CD8+ T cells were mostly found in close proximity to adherent MC38 cells. BsAbαPD1+αPDL1 exhibits superior efficacy to active T-cell and subsequent eliminating tumor cells compared to a mixture of free mAbs.Fig. 3 (A) Optical microscope photographs of the cocultured MC38 and CD8+ T cells at 6 h after treatment with PBS, NPαPD1+NPαPD1 and BsAbαPD1+αPDL1.In vivo biodistribution of Cy5-labeled NPαPD1+NPαPD1 and BsAbαPD1+αPDL1. (B) In vivo fluorescent imaging of drug distribution in mice at 4 h and 24 h post-injection and quantitative analysis of the fluorescence in the tumor (n = 3 mice/group).

Fig. 3

According to the positive results of the cytokine analyses and the optical images in vitro, we tried to evaluate BsAbαPD1+αPDL1 antitumor efficacy in vivo. To assesses BsAbαPD1+αPDL1 antitumor efficacy, imaging experiments were performed in mice. As depicted in Fig. 3B, in vivo imaging showed that BsAbαPD1+αPDL1 exhibited a significantly enhanced distribution compared to those of NPαPD1+NPαPDL1 and BsAbαPD1+αPDL1, corroborating the targeting capabilities of αPDL1. The ex vivo imaging obtained the same results. And the pharmacokinetic results (Fig. S7) showed that BsAbαPD1+αPDL1 showed a considerably prolonged blood circulation time (t1/2 = 20.1 ± 4.4 h) compared to IgG (t1/2 = 10.1 ± 1.7 h), which proving that structure and characteristics of BsAbαPD1+αPDL1 did not affect the circulation time compared with PGLU-Fc-III-4C-IgG (t1/2 = 19.8 ± 1.1 h).

Subsequently, we aimed to determine whether BsAbαPD1+αPDL1 could elicit an enhanced antitumor effect in vivo compared to the mixture of NPs (NPαPD1+NPαPDL1). PDL1 receptors are expressed on MC38 tumor cells (Figure S5). Fig. 4A show the set groups and the corresponding administration methods. Fig. 4B shows the tumor growth rate. In Groups 2 and 3, the administered concentrations of αPD1 and αPDL1 were 2.5 mg/kg, respectively. For Group 4, the concentration of the free antibody mixture was 5.0 mg/kg. In Group 5, the total administered concentration of NPαPD1+NPαPDL1 was 5.27 mg/kg, comprising 5.0 mg/kg of antibodies and 0.27 mg/kg of the PGLU-Fc-III-4C conjugate. The PBS control group show fast tumor growth. The single αPD1, αPDL1, FreeαPD1+αPDL1 and NPαPD1+NPαPDL1 all show relatively slower tumor growth rate, indicating the tumor growth suppression effects of each drug. The BsAbαPD1+αPDL1 shows even improved tumor growth suppression effect than the simple antibody NPs mixture. Specifically, the tumor suppression rate (TSR) of the mixture of two NPs was 72.3 %, while that of the BsAbαPD1+αPDL1 group was 90.1 % on day 16, proving that the BsAbαPD1+αPDL1 effectively boost the efficacy of combination immunotherapy. Fig. 4C shows the fluctuations in body weight observed in mice. At the initial stage of the treatments, the mice in experimental treatment group FreeαPD1+αPDL1 showed slightly body weight loss. After 8 days, the body weight of the treatment group of FreeαPD1+αPDL1 mice returned to normal.Fig. 4 A) Schedule for drug treatment in the tumor inhibition experiment. (B) Tumor growth curve, (C) body weight change, and (D) survival curve for mice after different treatments (n = 6 mice/group). (E) Flow cytometry analyzing CD8+ T cells levels at day 9 in the tumor inhibition experiment. (F) Flow cytometry analyzing CD8+ T cells levels at day 9 in the spleen inhibition experiment (n = 5 mice/group). (G–I) Serum cytokine levels were detected by ELISA assay, including (G) IFN-γ, (H) TNF-α and (I) IL-12 (n = 3 mice/group).

Fig. 4

Fig. 4D shows the survival curve of the mice. In Group BsAbαPD1+αPDL1, the survival time of the mice in is significantly prolonged. Compared with PBS. After 48 days, the STR of the Group BsAbαPD1+αPDL1 is 83.3 %, while the STR of mice in all the other groups is below 35 %. These results prove that the BsAbαPD1+αPDL1 can effectively prolong the survival period of tumor-bearing mice in MC38 model of primary colon cancer. In view of the improved tumor suppressor effect and the well-tolerated condition of mice, the BsAbαPD1+αPDL1 finally significantly prolonged the survival period of mice with lower toxic and side effects. In bilateral tumor model (Fig. S9), both the PD1 group and the PDL1 group demonstrated moderate tumor suppression effects for primary tumors, with a TSR of 42.0 % and 55.6 %, respectively. The TSR of FreeαPD1+αPDL1 and NPαPD1+NPαPDL1 group were 66.6 % and 65.6 %, respectively. Importantly, the group of BsAbαPD1+αPDL1 significantly suppressed primary tumor growth (TSR = 87.0 %). These results indicate that the BsAbαPD1+αPDL1 effectively inhibits primary tumor growth. In line with the observed inhibition of primary tumor growth, BsAbαPD1+αPDL1 group also markedly suppressed secondary tumor growth (TSR = 83.3 %), whereas other treatment groups did not exhibit a substantial suppression of secondary tumor growth (Fig. S9). Furthermore, no significant weight loss was noted in any group of mice during the observation period, underscoring the safety of the treatment regimen (Fig. 4C).

To analyze the tumor growth inhibiting mechanism of BsAbαPD1+αPDL1, we further evaluated the immune cell infiltration and representative immunofluorescence images of tumors and spleens after various treatments. T cells play a role in killing tumors and inhibiting tumor growth in the immune response [43,44]. As depicted in Fig. 4E–F, the up-regulation of CD8+ T cells of Group BsAbαPD1+αPDL1 proved that. The IFN-γ, TNF-α and IL-12's expression levels were also detected with ELISA kits (Fig. 4G–I). These cytokines play important roles in inhibiting tumor growth. The activated CD8+ T cells are capable of secreting IFN-γ and TNF-α, which can inhibit the formation of tumor blood vessels. IL-12, a cytokine regulated by IFN-γ, plays a crucial role in the activation of natural killer (NK) cells. TNF-α can also kill or inhibit the proliferation of tumor cells, and improve the ability of T cells and other killer cells to kill tumor cells [45]. After the treatment with the BsAbαPD1+αPDL1 prepared in the work, the expressions of IFN-γ, TNF-α and IL-12 were all up-regulated, indicating that the BsAbαPD1+αPDL1 can trigger both adaptive and innate immune responses, which probably significantly led to the inhibition of the growth and development of mouse colon tumors and the prolonging of the survival time of tumor-bearing mice.

To further evaluate the therapeutic effect, we performed immunofluorescent labeling of the resected tumor to observe the presence of CD3+ T cells in the tumor tissue. The strongest red fluorescence (CD3+ T and CD8+ T cells) can be seen in the tumors after BsAbαPD1+αPDL1 treatment, indicating that the tumor cells in the treatment group had the most T cells (Fig. 5B–C). H&E sections also observed more bad colored tumor cells (Fig. 5A), demonstrating that large areas of necrosis occurred within the tumor after BsAb treatment, while no significant changes were observed in normal organs [46]. The serum levels of BUN, ALT, AST, AKP, CRE and UA all exhibited similar levels across experimental groups (Fig. S8). This indicating that the treatment of BsAbαPD1+αPDL1 did not adversely impact liver and kidney function. Furthermore, consistent with the results so far, no significant pathological alterations were detected in the heart, liver, spleen, lung and kidney in any of the treatment groups, as detailed in Fig. S6. In tumor, more tumor cell apoptosis was seen in the BsAbαPD1+αPDL1 group compared with the PBS group. The results demonstrated that BsAbαPD1+αPDL1 was safe and effective, with stronger killing effect on tumor cells.Fig. 5 H&E staining of tumors (Scale bar: 400 μm) and representative immunofluorescence images of tumors (Scale bar: 500 μm) after BsAbαPD1+αPDL1 treatment.

Fig. 5

4 Conclusion

In summary, we propose a novel strategy to enhance immune checkpoint blockade therapy using polymeric PD1/PDL1 bispecific antibodies (BsAbαPD1+αPDL1). The desired BsAbαPD1+αPDL1 was constructed by gently mixing a polymer-multiple Fc binding peptides with αPD1 and αPDL1 monoclonal antibodies (mAbs) in an aqueous solution. Furthermore, it acts as an innovative T cell engager, stimulating immune activation, tumor cell elimination, inhibition of tumor growth, and prolonged survival. In vivo imaging and dissection experiments demonstrated that BsAbαPD1+αPDL1 exhibited significantly enhanced distribution compared to NPαPD1+NPαPDL1 and BsAbαPD1+αPDL1, attributed to the targeting ability of αPDL1. H&E staining analysis revealed no significant pathological abnormalities in the major organs of mice treated with BsAbαPD1+αPDL1. The up-regulation of anti-tumor cytokines indicates that BsAbαPD1+αPDL1 can induce the activation of adaptive and innate immunity, likely contributing to the inhibition of mouse colon tumor growth and prolonged survival in tumor-bearing mice. BsAbαPD1+αPDL1 demonstrated favorable therapeutic efficacy and enhanced anti-tumor immune responses compared to free mAbs alone, highlighting its interactions and collaborative functions. This study provides new insights into strategies for enhancing clinical immune checkpoint blockade therapy.

CRediT authorship contribution statement

Fuxin Xue: Writing – original draft, Formal analysis, Data curation. Xitong Ren: Methodology. Chaoying Kong: Methodology. Jianfeng Wang: Methodology. Linlin Liu: Supervision. Junli Hu: Supervision. Na Shen: Conceptualization, Project administration, Supervision, Writing – review & editing, Data validity, Funding acquisition. Zhaohui Tang: Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix B Supplementary data

The following is the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Data availability

Data will be made available on request.

Acknowledgements

This work is financially supported by the the Jilin province, China (20230508102RC ), Ministry of Science and Technology of China (2022YFE0110200 ), 10.13039/501100001809 National Natural Science Foundation of China (52273157 , 52073279 , 52025035 ), as well as the 10.13039/501100004739 Youth Innovation Promotion Association of Chinese Academy of Sciences, China (2022224 ).

Appendix B Supplementary data to this article can be found online at https://doi.org/10.1016/j.mtbio.2024.101239.
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