Soluble CD80 oral delivery by recombinant Lactococcus suppresses tumor growth by enhancing antitumor immunity

Abstract CD80 is an important co‐stimulatory molecule that participates in the immune response. Soluble CD80 can induce T cell activation and overcome PDL1‐mediated immune suppression. In this study, we aimed to construct recombinant Lactococcus lactis for oral delivery of the soluble CD80 (hsCD80) protein or the fusion protein containing the cholera toxin B subunit (CTB) and hsCD80 (CTB‐hsCD80) under the control of the nisin‐inducible expression system. The recombinant L. lactis expressed and secreted hsCD80 or CTB‐hsCD80 fusion proteins after induction by nisin in vitro and in the enteric cavity. Additionally, the CTB‐hsCD80 fusion protein showed uptake by intestinal epithelial cells, was cleaved by the furin protease, and was released as free hsCD80 protein into the blood circulation. Orally administered hsCD80 and CTB‐hsCD80 containing L. lactis increased the proportion of activated T cells in the spleen and intestinal epithelium, inhibited tumor growth, and prolonged the survival of tumor‐bearing mice. The hsCD80‐containing L. lactis showed greater therapeutic effects on primary colonic adenoma in APCmin/− mice and completely suppressed tumor growth. Further, recombinant CTB‐hsCD80 in L. lactis was more efficient than hsCD80‐containing bacteria in inhibiting the growth of xenografted colon cancer and melanoma cells. hsCD80 engineered probiotics may serve as a promising new approach for antitumor immunotherapy, especially for colorectal cancer.


| INTRODUCTION
Spontaneous antitumor immunity is ordinarily repressed by the immune checkpoint pathway. Malignant tumors usually express various immune checkpoint molecules to escape immune surveillance by interacting with the checkpoint receptors expressed on immune cells, and this is the main obstacle for tumor immunotherapy. 1 PD-1 is a well-known checkpoint receptor that belongs to the immunoglobulin gene superfamily. It is expressed on the surfaces of activated T cells, B cells, natural killer cells, and dendritic cells (DCs). PD-1 ligand 1 (PDL1, also called B7-H1 or CD274) and PDL2 (also called B7-DC or CD273) are expressed either constitutively or in response to exposure to interferon γ (IFN-γ) by many tumor cells. 2 The interaction of PD-1 with PDL1 or PDL2 results in immune tolerance by inducing T cell apoptosis, decreasing the killing capacity of effector T cells (Te), and inhibiting T cell activation. 3 Recently, checkpoint inhibitors have been identified as promising antitumor drugs, as these can restore T cell activity by blocking the interaction between PD-1 and PDL1 or PDL2.
Humanized antibodies for PD-1 or PDL1, such as nivolumab, pembrolizumab, or atezolizumab, have shown promising clinical efficacy for the treatment of melanoma, colon cancer, lung cancer, and several other malignant tumors. [4][5][6][7] However, recent clinical data indicate that only 17-28% of patients with advanced cancers showed complete or partial remission following PD-1 or PDL1 antibody treatment. The majority of patients do not benefit from checkpoint inhibitor therapy. [4][5][6][7] The main reason for this is the high degree of heterogeneity of malignant tumors. One tumor cell usually expresses several inhibitory molecules and generates immune tolerance through multiple mechanisms, and different types of tumors have different immunosuppression mechanisms. 8 Therefore, most malignant tumors require combination therapy with multiple drugs or alternative routes to achieve curative results.
Humanized antibodies are expensive for low-and middle-income families, especially when continuous treatment is required. Moreover, these drugs are administered via injection, which may cause side effects such as allergy, infection, and local vascular stimulation in patients. 9,10 Therefore, it is necessary to explore new strategies to overcome immune suppression and to identify novel checkpoint inhibitors.
CD80 is an immune co-stimulatory molecule, which plays an important role in antitumor and anti-viral immunity. 11 CD80, also known as B7-1, was first cloned from active B cells, and is a type 1 transmembrane protein of the immunoglobulin superfamily. Molecules homologous to CD80 include PDL1 and PDL2. 12 Full activation of T cells requires that the main signal is recognized by the T-cell receptor and antigen peptide/major histocompatibility complex, and the auxiliary signals driven by co-stimulatory molecules, such as CD80 and CD86, which are present on tumor or antigen-presenting cells (APCs). The engagement of CD80 with its positive receptor CD28 enhances T cell proliferation and IL-2 secretion. 13 In contrast, CD80 interaction with the negative regulatory receptor cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibits the early activation of T cells. [14][15][16] However, co-stimulatory molecules like CD80, which are crucial for T cell activation, are often poorly expressed, whereas immunosuppressive molecules such as PDL1 and PDL2 are highly expressed on tumor and microenvironment cells. 17 Therefore, low expression of CD80 may predict a poor prognosis in tumor patients. 18 CD80 has been mainly used as an immunoadjuvant molecule in tumor or pathogenic microorganism vaccines to excite antigenspecific T cells by transfection or co-expression with antigens in vector cells. 19 Tumor vaccines transfected with the CD80 gene restored tumor-specific T cell activation and reversed PDL1-mediated immunosuppression. Further, CD80 co-expression inhibits the expression of PDL1 on the tumor cell surface. 20 Recently, recombinant soluble CD80 (sCD80) containing the extracellular domains alone was used for tumor treatment because the binding affinity of CD80 to PDL1 was similar to its affinity with CD28. 3,21 The sCD80-Fc fusion protein (sCD80 fused with an IgG Fc domain) showed greater therapeutic efficacy in preventing PD1: PDL1-mediated suppression and restored T cell activation compared to treatment with monoclonal antibodies (mAb) against either PD1 or PDL1. sCD80-Fc overcame PDL1-mediated suppression in human or mouse tumor cells, facilitating T cell activation by binding to PDL1 to inhibit PDL1:PD1 interaction and by co-stimulation via CD28. 22,23 Furthermore, sCD80-Fc was able to prime T cells via the CD28 and non-CD28 pathways in CD28-knockout mice, in which CD80:CD28 binding was blocked. 24 Therefore, sCD80 may serve as a promising antitumor factor as it primes active antitumor immunity by providing a co-stimulatory signal and restores T cell activity by blocking the binding of PDL-1 to PD-1. 25 It was reported that sCD80-Fc delivered via intraperitoneal injection has a superior antitumor effect to PD-1 antibodies in mice. 26 Production of recombinant sCD80 protein using traditional in vitro expression methods has several disadvantages, such as high production costs and inconvenient administration. Therefore, it is necessary to explore new ways to produce checkpoint inhibitors.
The gastrointestinal tract is a suitable and economical bioreactor for biological drug generation and oral delivery. It is estimated that the total amount of biochemical metabolism in the gut equals the amount of liver metabolism. 27 Lactic acid bacteria (LAB) and other probiotics are favorable carriers for the oral administration of immunotherapeutic drugs. In recent years, the use of live bacterial oral vaccine preparations or gene therapy using genetically engineered probiotics has been increasing. Probiotics are used to express pathogenic microorganism antigens, such as the rotavirus antigen and tetanus toxin C. 28,29 Bifidobacteria expressing cytosine deaminase have been used with 5-fluorocytosine for tumor therapy via intravenous injection. 30 In recent years, our group has used recombinant bifidobacteria to express several exogenous genes, such as oxyntomodulin, thymosin alpha, IL-10, and tumstatin, for disease treatment in mice, resulting in positive curative effects. [31][32][33][34][35] Importantly, the intestinal epithelium is involved in mucosal immunity besides serving as the main organ of nutrition and drug absorption, and may be useful for effective immunotherapy. Receptor-mediated endocytosis is usually used to help recombinant proteins cross the intestinal mucosal barrier. Ligands, such as transferrin, M cell-specific ligand, and cholera toxin B subunit (CTB), can bind to the receptors on the intestinal epithelium, thus aiding in the transportation of recombinant proteins across the barrier. CTB is a regulatory subunit of cholera toxin without intestinal toxicity, and the CTB receptor ganglioside M1 is highly expressed in the intestinal epithelium. Therefore, CTB is often used to guide the target protein across the intestinal mucosal barrier through receptor-mediated endocytosis. [35][36][37] In this study, we aimed to establish recombinant Lactococcus lactis carrying the human soluble CD80 (hsCD80) gene for colorectal cancer therapy. Additionally, recombinant L. lactis carrying the CTB-hsCD80 fusion gene was also established to facilitate the free absorption of hsCD80 protein into blood circulation through the F I G U R E 1 Identification and expression of Lactococcus lactis transformants in vitro. (a) Schematic of hsCD80 gene structure and plasmid construction. The hsCD80 gene or cholera toxin B (CTB)-hsCD80 fusion gene with the SPK1 signal sequence was inserted into the Escherichia coli-L. lactis shuttle vector pLAN at the NcoI and XbaI restriction sites. The food-grade recombinant vectors pLN-hsCD80 and pLN-CTB-hsCD80 were obtained by excision of the ampicillin resistance (AmpR) gene with the SalI restriction enzyme. (b) Agarose gel electrophoresis analysis of plasmids extracted from L-vector, L-hsCD80, and L-CTB-hsCD80. (c) The expression of hsCD80 and CTB-hsCD80 recombinant proteins was analyzed via western blotting. The proteins were detected with anti-Flag, anti-CD80, or anti-CTB antibodies, respectively, after being induced by 2.0 ng/ml nisin for 6 h. intestinal mucosal barrier by CTB-mediated endocytosis for tumor therapy in other tissues. 38,39 The hsCD80-transformed L. lactis showed therapeutic effects in primary colonic adenoma in APC min/À mice, whereas the CTB-hsCD80-transformed L. lactis had pronounced effects on the growth of subcutaneous xenografts and melanoma lung metastases.
F I G U R E 2 Uptake of the recombinant proteins expressed from Lactococcus lactis transformants was analyzed in intestinal epithelial cells in vitro. (a) The localization of recombinant proteins in normal human NCM460 intestinal epithelial cells, colon cancer HT29, and mouse colon cancer CT26 cells was evaluated via immunofluorescence analysis in vitro. Several cholera toxin B (CTB)-hsCD80 proteins (green fluorescence) showed uptake into the cytoplasm. Green fluorescence indicates hsCD80 detected with anti-Flag Ab. Blue fluorescence indicates the DAPIstained nucleus. (b) The recombinant CTB-hsCD80 protein was digested and released as free hsCD80 after co-incubation with colon cancer HT29 cells. (c) Scatterplot of CD3 + IFN-γ + T cells analyzed using flow cytometry. The accumulation area of CD3 + IFN-γ + cells is indicated with an arrow. (d) Percentage histogram of CD3 + , CD3 + CD4 + , CD3 + CD8 + , and CD3 + IFN-γ + cells. (e) Localization of the recombinant protein in the colon was visualized by immunofluorescence with anti-Flag Ab. Recombinant protein (red fluorescence) was observed in the submucosal vascular area (white arrows). (f) The distribution of the recombinant hsCD80 protein in the intestinal contents, and the intestinal, muscle, cardiac, liver, and stomach tissues were detected via western blotting using anti-Flag Ab. (g) The concentration of hsCD80 in the peripheral blood was detected using the hsCD80 ELISA kit. The data are representative of six independent experiments. Error bars indicate the mean ± SD. **p < 0.01.   Free hsCD80 protein was detected in the intestine, stomach, liver, spleen, heart, and muscle tissues, as well as in the intestinal contents in the L-CTB-hsCD80 and L-hsCD80 groups by western blotting. However, the hsCD80 protein was detected in the intestinal content, intestine, and heart of the L-hsCD80 group alone. These results indicate that fusion expression with CTB enables hsCD80 to cross the intestinal mucosal barrier, enter the circulatory system, and reach various tissues and organs (Figure 2f,g).   Supplementary Figures 1-3), which was higher than that in the L-vector and L-CTB-hsCD80 groups, respectively ( p < 0.05).

| CTB-hsCD80 fusion protein crosses the intestinal mucosa and releases hsCD80 into the blood circulation in vivo
The number of CD8 + IFNγ + cells in the L-CTB-hsCD80 group showed no significant differences compared with the L-vector and saline groups. On the contrary, the number of CD11b + Gr-1 + cells in the three tissues in the L-hsCD80 group was significantly decreased ( Figure 3g, Supplementary Figures 4-6), which was lower than that in the L-vector and L-CTB-hsCD80 groups, respectively. The numbers of CD11b + Gr-1 + cells in intestinal adenoma and Peyer's patches in the L-CTB-hsCD80 group were slightly lower than that in the L-vector and saline groups. However, there was no significant difference between the three groups.    The results of flow cytometry analysis in CT26 xenograft-bearing mice showed that the proportion of CD3 + CD4 + , CD3 + CD8α + , or IFNγ + CD3 + T cell subsets in the spleen in the L-hsCD80 group was higher than that of the L-vector group. Furthermore, the proportion of CD3 + CD4 + , CD3 + CD8α + , or IFN-γ + CD3 + T cell subsets in the L-CTB-hsCD80 group was significantly higher than that of the L-vector or L-hsCD80 group. Similar results were obtained in B16F10 xenograft-bearing mice (Figure 8c,d).
The CD8 + and IFN-γ + infiltrating lymphocytes within the local tumor microenvironment were identified via immunofluorescence analysis using anti-CD8 (green) and anti-IFN-γ Ab (red) to ascertain whether the hsCD80-transformed L. lactis promotes T lymphocyte activation and antitumor immunity. In Figure 8e, the number of CD8 and IFN-gamma double-positive cells in the L-CTB-hsCD80 group increased, and the expression levels of CD8 and IFN-gamma in most lymphocytes increased slightly (exhibiting higher fluorescence intensity). However, the variance between different activated lymphocytes was large after L-hsCD80 and L-CTB-hsCD80 treatment. (Figure 8e), and most CD8 + cells coincided with IFN-γ + cells. Over 65% of CD8 + cells had high IFN-γ + expression in the LL-sCD80 group, which was significantly higher than that of the L-vector group (46.14%). Furthermore, the percentage of CD8 + T cells with high IFN-γ + expression in the LL-CTB-sCD80 group (85.56%) was significantly higher than that in the LL-sCD80 and L-vector groups. Additionally, the percentage of IFN-γ + CD8 + cells relative to the total cells in the LL-CTB-sCD80 group (29.84%) was significantly higher than that in the sCD80 (12.12%) and L-vector (6.87%) groups (Figure 8f  reduced, resistance to PD-1 Ab was reversed, and better curative effects were obtained when the tumor vaccine was administered before the PD-1 blocking Ab. 44 Reportedly, soluble CD80 (sCD80) evokes antitumor immunity through CD28 and non-CD28 pathways and restores T cell activity by blocking PD-1: PDL1 binding, exhibiting an improved antitumor effect than PD-1 and PD-L1 Abs. 23,24 Considering the high cost and inconvenience of recombinant

| Vector construction
The scheme of the food-grade expression vector construction is shown in Figure 1

| Gene transformation and recombinant bacterial identification
The recombinant plasmids pLN-hsCD80, pLN-CTB-hsCD80, or empty vector pLN were transformed into L. lactis NZ3900 by electroporation using a Gene Pulser and Pulse Controller apparatus (Bio-Rad, USA) at 2.5 kV, 25 μF, and 200 Ω as described previously. 30 The transformed L. lactis were selected using Elliker medium with lactose as the sole carbon source. Positive colonies were identified by PCR, restriction enzyme digestion, and DNA sequencing. The recombinant L. lactis transformed with pLN, pLN-hsCD80, or pLN-CTB-hsCD80 was subsequently referred to as L-vector, L-hsCD80, and L-CTB-hsCD80, respectively.

| Recombinant protein expression analysis in vitro
The transformed L. lactis were grown in Elliker medium and cultured until the OD 600 reached 0.6. Recombinant protein expression was  The positive control group was treated with 1 μg/ml ionomycin + 100 ng/ml PMA or 1 μg/ml anti-PDL1 mAb. The proportion of CD3 + CD4 + , CD3 + CD8 + , and CD3 + IFNγ + T cell subsets in the lower chambers was identified via flow cytometry.

| Protein expression and secretion in vivo
The mice were subsequently randomly classified into five groups (n = 30, six in each group). Mice were administered with 2 Â 10 8 recombinant L. lactis live bacteria intragastrically and 20 ng/ml nisin daily for 1 week. Western blotting and immunohistochemistry were used to analyze the content and distribution of the Flag label-free hsCD80 protein in the heart, liver, spleen, kidney, stomach, intestine, intestinal contents, muscle, and fat. Analysis of the receptor-mediated endocytosis effect of CTB was also performed.

| Primary colon cancer immunotherapy in vivo
Male APC min/+ mice were fed high-fat diets for 4 weeks to induce the development of primary colonic cancer and were subsequently randomly classified into four groups (n = 24, six in each group). The mice in the L-vector, L-hsCD80, and L-CTB-hsCD80 groups were treated with 0.2 ml of recombinant L. lactis (suspended in saline, 10 9 CFU/ml) containing 10 ng/ml nisin by gavage every alternate day. Mice in the saline group were treated with 0.2 ml of saline by gavage every alternate day. The fecal blood, anal swelling, prolapse, and other physical conditions of the mice were observed constantly. When the control mice became very weak, all animals were sacrificed, and the tumor tissue and the heart, liver, spleen, kidney, stomach, and intestinal contents were removed completely. The size, weight, and quality inhibition rates of the tumors were measured. Each tissue was fixed using 4% paraformaldehyde solution for immunohistochemical and microscopy analysis. Cellular apoptosis was quantified using TUNEL reagent. The proportion of CD3 + , CD3 + CD4 + , CD3 + CD8 + , and CD3 + IFNγ + T cell subsets in splenic lymphocytes was analyzed by flow cytometry. Activated CD8 + IFNγ + T cells and CD11b + Gr-1 + MDSCs in adenoma tissues and intestinal mucosa were detected via immunofluorescence assays. The blank control group (BC) mice were treated with 0.2 ml of saline by gavage every alternate day. When the diameter of tumors in the control group reached 2-3 cm with necrosis, all mice were sacrificed, and the tumor tissue and the heart, liver, spleen, kidney, stomach, and intestinal contents were removed completely. The size, weight, and quality inhibition rates of the tumors were measured. Each tissue was fixed with 4% paraformaldehyde solution for immunohistochemical (CD31, Ki-67), immunofluorescence (double-labeled CD8 + /IFNγ + , double-labeled Flag/Muc 2), and microscopy analysis. Cellular apoptosis was evaluated using TUNEL assays. The proportion of CD3 + , CD3 + CD4 + , CD3 + CD8 + , and CD3 + IFNγ + T cell subsets of spleen lymphocytes was analyzed by flow cytometry.

| Tumor-specific cytotoxicity assay in vitro
To test the tumor-specific cytotoxicity of spleen lymphocytes stimu-

| Immunofluorescence staining
Cells or tissue sections were fixed in 4% paraformaldehyde and methanol for 20 min at room temperature and were subsequently co-incubated with anti-Flag, anti-CD8, anti-IFNγ, anti-MUC2, anti-CD11b, or anti-Gr-1 antibodies, followed by reimaging on the BioStation (Nikon).

| Western blotting
The expression of hsCD80 and CTB-hsCD80 in transformed bacteria was induced using 2.0 ng/ml nisin for 6 h. The proteins in the bacterial extracts were detected by western blotting using Flag mAb (Sigma) against hsCD80 and CTB-hsCD80, and the GAPDH mAb (MC4) (RM2002, Beijing Ray Antibody Biotech) against GAPDH as an internal control. The hsCD80 protein in the intestine and its distribution in the heart, spleen, liver, kidney, stomach, muscle, and fat were evaluated. Protein samples were separated via polyacrylamide gel electrophoresis. Next, proteins were transferred onto a polyvinylidene difluoride membrane (Millipore), which was washed with PBST, blocked in 5% nonfat dry milk, and blotted with the FLAG or GAPDH antibodies.

| Statistical analysis
All data were expressed as the mean ± standard deviation (SD) of each group. The statistical differences between the two groups were analyzed using an unpaired Student's t test (two-tailed). Multiple groups were compared using a one-way analysis of variance (GraphPad Prism 5.0; GraphPad, Bethesda, MD). Any p-values lower than 0.05 were considered statistically significant and were indicated as p < 0.05 (*) or p < 0.01 (**).

AUTHOR CONTRIBUTIONS
Hongying Fan proposed study concepts and designed the research plan.
Xiaojing Meng and Weisen Zeng gave constructive suggestions and reviewed the manuscript. Ziqin Lin, Yanqing Tang, and Zerong Chen organized the data and figures. Xueyan Xu, Xufeng Hou, Junjie Wen, and Zhenhui Chen performed the experiments and statistical analyses.

ACKNOWLEDGMENT
The authors would like to thank Editage (www.editage.cn) for English language editing.

CONFLICT OF INTEREST
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

TRANSLATIONAL IMPACT STATEMENT
CD80 is an important co-stimulatory molecule that induces T cell activation and overcomes PDL1-mediated immune suppression. However, production of recombinant sCD80 protein using traditional methods has a high production costs and inconvenient administration.
We engineered a recombinant strain of L. lactis capable of orally delivering CTB and hsCD80 (CTB-hsCD80), and the CTB-hsCD80-transformed strain having pronounced effects on the growth of subcutaneous xenografts and melanoma lung metastases. Thus, our approach could help patients inhibit tumor with low cost and high efficiency.