THERAPY

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/944,849, filed on December 6, 2019, the contents of which is incorporated herein by reference.

GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant Number CA157738 awarded by National Institutes of Health. The government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

[0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 11,746 Byte ASCII (Text) file named " 38170-601_ST25.TXT," created on December 4, 2020.

FIELD

[0004] The present disclosure relates to polypeptides which bind CCL2 (C-C motif chemokine ligand 2) or both CCL2 and CCL5 (C-C motif chemokine ligand 5), polynucleotides encoding polypeptides which bind CCL2 or both CCL2 and CCL5, and compositions and methods of use thereof.

BACKGROUND

[0005] Monoclonal antibodies (mAb) that block the immune checkpoint signaling pathways have shown favorable therapeutic efficacies across various cancer types. However, only a minority of cancer patients within each subtype respond to the immune checkpoint blockade (ICB) therapy, indicating the existence of other important factors co-shaping the anti-tumor immunity. ICB- resistant tumors are primed by a high density of immunosuppressive cells such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) with little T cell infiltration in the tumor microenvironment (TME), a characteristic that emerged as a major barrier to the effectiveness ofICB therapy.

[0006] As the first extraintestinal organ, the liver is constantly at the risk of attack from various harmful substances such as bacterial endotoxins and virus infection, making it be encircled by immunosuppressive microenvironment and particularly attractive for malignancy development and metastasis. Liver cancer is not only the second most deadly cancer, but also the only cancer that has an increased incidence and mortality in the US. As reported, a majority of macrophages were found in the peritumor and intratumor tissues (38.6%) of hepatocellular carcinoma (HCC) patients and the cell density of FOXP3 ⁺ Tregs in tumor tissues was much higher than that in normal liver tissues (3.9% vs. 0.3%; P<0.0001). Furthermore, compared to normal liver tissue, the frequency of MDSCs in an HCC tumor was significantly increased and correlated with tumor stage, size and burden. In addition to the primary HCC, liver is the second common sites of metastatic spread, in particular for colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC). About 60-70% of CRC patients ultimately develop liver metastasis with a five-year survival rate less than 12%. More than 50% of PDAC patients have liver metastasis at the time of diagnosis and such metastasis is associated with a horrible prognosis. Unfortunately, the clinical trials ofICB (pembrolizumab) therapy failed in treating advanced HCC.

SUMMARY

[0007] Disclosed herein is an isolated single domain antibody, a fragment or derivative thereof, that specifically binds both CCL2 and CCL5, an isolated single domain antibody, or a fragment or derivative thereof, that specifically binds to CCL2, and polynucleotides comprising a nucleic acid encoding the isolated single domain antibodies that specifically binds both CCL2 and CCL5 or CCL2.

[0008] Also disclosed herein are pharmaceutical compositions comprising any of the isolated single domain antibodies that specifically binds both CCL2 and CCL5 or CCL2 or the polynucleotide comprising a nucleic acid encoding the isolated single domain antibodies that specifically binds both CCL2 and CCL5 or CCL2.

[0009] Further disclosed are method of treating cancer and inhibiting the growth or survival of a cancer cells. The methods may comprise administering to a subject in need thereof an effective amount of the isolated antibody or a fragment or derivative thereof that specifically binds both CCL2 and CCL5 or CCL2 or a polynucleotide or pharmaceutical composition thereof. The methods may comprise contacting cells with an effective amount of the isolated antibody or a fragment or derivative thereof that specifically binds both CCL2 and CCL5 or CCL2 or a polynucleotide or pharmaceutical composition thereof.

[0010] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS [0011] FIGS. 1A-1K show the identification of the top-ranked monocytes-related genes that are associated with HCC cancer progression and immunosuppression, and the characterization of an evolved bispecific single domain antibody against CCL2 and CCL5. FIG. 1 A is a heat map showing monocyte-related gene signatures between HCC-free sites (Adjacent) and tumor sites (HCC) in liver cancer patients. Columns represent 197 patient samples from gene expression omnibus (GEO) database; rows represent monocytes-related genes. Values represent the log2 ratio over control (gene expression in adjacent samples). FIG. IB is representative immunofluorescent images of CCL2 and CCL5 staining in the human HCC tumor tissues and non-HCC liver tissues. Higher magnification images are shown under each panel (Scale bars,

100 pm). FIGS. 1C and ID are graphs of the mRNA expression of classic Ml (FIG. 1C) and M2 (FIG. 1 D) markers in Hepal -6 tumor cell-educated BMDMs in different treated groups, n = 3 biologically independent samples; ns means no significance; two-tailed Student’s t-test. FIGS.

IE and IF are Kaplan-Meier survival curves of HCC tumor-bearing mice treated with CCL2 and CCL5 neutralizing antibodies (a-CCL2: 200 pg/mouse, i.p; a-CCL5: 100 pg/mouse, i.p), respectively, using 30% weight loss as the endpoint criteria. Each line represents one survival curve for each group of five mice; Log-rank (Mantel-Cox) test FIG. 1G is a graph demonstrating dual specificity of BisCCL2/5i by flow cytometry. FIG. 1H is showing the binding affinity of BisCCL2/5i to mCCL2, mCCL5 or other related chemokine family members, measured by MST. FIG. II is a graph of the i n vitro inhibition of chemotaxis of monocytes via BisCCL2/5i. FIGS. 1J-1K are graphs of the mRNA expression of Ml and M2 markers in Hepal-6 tumor cell- educated BMDMs after treatment with BisCCL2/5i protein or LPS. n = 3 biologically independent samples; two-tailed Student’s t-test. Results are presented as mean (SD). [0012] FIGS. 2A-2N show that the dual blockade of CCL2 and CCL5 via LNP-mediated mKNA delivery of BisCCL2/5i polarizes macrophage Ml phenotype and reprograms the immunosuppressive TME. FIG. 2A is a schematic of the mRNA-loaded LNPs. FIG. 2B is a graph of the size distribution of mRNA-loaded LNPs. The experiment was conducted independently three times with similar results. Representative conventional transmission electron micrographs of mRNA-loaded LNPs (Insert). FIG. 2C is a graph of the expression of BisCCL2/5i protein in the serum at different time points after intravenous injection with LNP- formulated BisCCL2/5i mRNA. n=3 biologically independent samples. FIGS. 2D and 2E are graphs of the mRNA expression of classic Ml and M2 markers, respectively, in the HCC tumor tissues after systemic administration of formulated LNPs as a dose corresponding to 20 pg mRNA. Each data point is an individual sample (n=3). FIGS. 2F-2G are graphs of the percentage of Ml -phenotype and M2-phenotype macrophages in the HCC tumor across treatments measured by flow cytometry. n=3 biologically independent samples. FIG. 2H is a graph of the ratio of M1/M2 macrophages in the HCC tumor after different treatments. n=3 biologically independent samples. FIG. 21 is a schematic of the macrophage depletion and treatment method to elucidate the role of macrophage polarization in priming tumor immunosuppression. FIG. 2J is a graph of the percentage of macrophages in the HCC tumor tissue with (W) or without (W/O) depletion. n=3 biologically independent samples. FIGS. 2K and 2L are graphs of the number of Tregs and MDSCs, respectively, in the HCC tumor tissue after various treatments. Each data point is an individual sample (n=3); ns means no significance. FIG. 2M is a graph of the percentage of macrophages 5 days after depletion in different treated groups. n=3 biologically independent samples. FIG. 2N is a graph of the cytotoxic CD8 ⁺ T cell infiltration in the tumor sites after various treatments. Each data point is an individual sample (n=3). Significant differences were assessed using two-tailed Student’s t-test Results are presented as mean (SD). [0013] FIGS. 3A-3P show the dual blockade of CCL2 and CCL5 sensitizes HCC tumors to ICB therapy. FIG. 3A is an intratumoral administration scheme for mice with an established subcutaneous HCC tumor. Mice with about 100 mm ³ subcutaneous tumors were administered BisCCL2/5i mRNA-LNPs plus PD-Li mRNA-LNPs, PD-Li mRNA-LNPs alone, BisCCL2/5i mRNA-LNPs alone, or PBS three times. FIG. 3B is a graph of the average tumor volumes in various treated groups. n=5 biologically independent samples; P = 0.0077, P=0.0078, and P = 0.001 denote the significance levels of BisCCL2/5i mRNA-LNPs, PD-Li mRNA-LNPs, and BisCCL2/5i mKNA-LNPs + PD-Li mRNA-LNPs treated mice, respectively, versus those treated with PBS as the control (two-way ANOVA with multiple comparisons). FIG. 3C is spider plots of individual tumor growth curves. The experiment was conducted two times independently with similar results. FIG. 3D is Kaplan-Meier survival curves of mice treated with indicated formulations using a 1,500 mm ³ tumor volume as the endpoint criteria. Each line represents one survival curve for each group of 6 mice; Log-rank (Mantel-Cox) test. FIG. 3E is representative images of the tumors 7 d post final administration. FIG. 3F is a treatment scheme for orthotopic HCC tumor-bearing mice treated intravenously with various formulations. FIG. 3G is a graph of average tumor weight of orthotopic HCC tumors for different treatments. n=5 biologically independent sample; two-tailed Student’s t-test. FIG. 3H is Kaplan-Meier survival analysis using a 30% weight loss as the endpoint criteria. n=5; Log-rank (Mantel-Cox) test FIG. 31 is a treatment scheme for HCC tumors established by hemi-spleen approach. FIG. 3J is a graph of the average tumor weight of different treated groups. n=6 biologically independent samples; two- tailed Student’s t-test. FIG. 3K is a Kaplan-Meier survival curve in different treated groups. n=8 biologically independent samples; Log-rank (Mantel-Cox) test. FIG. 3L is a graph of mRNA expression of IFN-γ in HCC tumors across different treatments. n=4 biologically independent samples; two-tailed Student’s t-test. FIG. 3M is a graph of mRNA expression of TGF-β in tumor tissue after systemic administration of formulated LNPs. Each data point is an individual sample (n=4); two-tailed Student’s t-test. FIG. 3N is representative flow dot plots of CD3 ⁺ CD8 ⁺ T cells in HCC tumor tissue. FIG. 30 is a graph of the percentage of CD3 ⁺ CD8 ⁺ T cells in tumor tissue 4 days after final treatment with different formulations measured by flow cytometry. n=3 biologically independent samples; two-tailed Student’s t-test. FIG. 3P is Kaplan-Meier survival analysis of HCC tumor bearing-mice (for CD4 ⁺ or CD8 ⁺ T-cell depletion and BisCCL2/5i plus PD-Li treatments) n=5 biologically independent samples; Log-rank (Mantel-Cox) test. Results are presented as mean (SD).

[0014] FIGS. 4A-4H show that the dual blockade of CCL2 and CCL5 sensitizes other tumors to ICB therapy. FIG. 4A is an intratumoral administration scheme for mice with an established subcutaneous CT26 colorectal tumor. Mice with about 100 mm ³ subcutaneous tumors were administered different formulations three times. FIG. 4B is representative images of the tumors 10 d after final administration. FIG. 4C is spider plots of individual tumor growth curves and FIG. 4D is Kaplan-Meier survival curves of intratumoral treated mice. n=5 biologically independent samples; Log-rank (Mantel-Cox) test. FIG. 4E is a treatment scheme for colorectal cancer liver metastasis tumors treated intravenously with various formulations. FIG. 4F is a graph of tumor growth burden and FIG. 4G is Kaplan-Meier survival curves in tumor-bearing mice treated with indicated formulations. n=5 per group; in s, two-way ANOVA with multiple comparisons; in t, Log-rank (Mantel-Cox) test FIG. 4H is representative in vivo bioluminescence imaging of mice bearing CT26-FL3 liver metastasis receiving various treatments. Results are presented as mean (SD).

[0015] FIGS. 5A-5G show that the dual blockade of CCL2 and CCL5 induces a shift in macrophage metabolism that regulates the macrophage polarity. FIG. 5 A is representative flow dot plots of M2 macrophages in tumor cell educated-BMDMs cultured in low-dose or high-dose glucose. FIG. 5B is a graph of the percentage of M2 macrophages in BMDMs measured by flow cytometry. n=3 biologically independent samples. FIG. 5C is a graph of the qRT-PCR analysis for glycolysis-related gene expression. n=4 biologically independent samples. FIG. 5D is a graph of the qRT-PCR analysis for gene expression of fatty acid oxidation and synthesis in BMDMs. n=4 biologically independent samples. FIG 5E is a graph of the profiles of extracellular acidification rate (ECAR) of tumor cell-educated BMDMs pretreated with BisCCL2/5i protein for 24 h and 48 h, upon sequential administration with glucose (25 mM), oligomycin (1.5 mM), and 2-DG (50 mM); measured as mpH per minute. n=4 biologically independent samples. FIG. 5F is a graph of the basal ECAR of BMDMs 48 h post treatments. n=4 per group. FIG. 5G is a graph of the profiles of oxygen consumption rate (OCR) of Hepal-6 tumor cell-educated BMDMs pretreated with BisCCL2/5i protein for 24 h and 48 h, upon the administration with oligomycin (25 mM); measured as picomoles of O2 per minute. n=4 per group; two-tailed Student’s t-test. Results are presented as mean (SD).

[0016] FIG. 6 is a schematic overview of BisCCL2/5i-mediated reprograming of the tumor microenvironment and synergy of the ICB immunotherapy. Liver malignancies are resistant to ICB therapy due to the enrichment of immunosuppressive cells, in which abundant TAMs are tumor-supportive M2-phenotype. Both CCL2 and CCL5 cooperatively induce the M2 polarization of macrophage, prime the immunosuppressive microenvironment, and render liver cancers resistant to ICB therapy. The BisCCL2/5i niRNA was delivered and transiently expressed in the tumor sites to bind and inhibit both murine CCL2 and CCL5, thereby reprograming the immunosuppressive TME and increasing the T cell infiltration. The combination of BisCCL2/5i with PD-Li immune checkpoint blockade therapy results in a synergistic antitumor response.

[0017] FIGS. 7A-7B show the mRNA expression of CCL2 and CCL5, respectively, in Hepal-6 tumor cell-educated BMDMs treated with siRNA against CCL2, CCL5, or control, respectively, n = 3 per group. FIGS. 7C and 7D are graphs of the mRNA expression of classic Ml and M2 markers, respectively, in Hepal-6 tumor cell-educated RAW 264.7 cells in different treated groups, n = 3 biological independent samples. FIGS. 7E is a graph of the ratio of M1/M2 macrophages in Hepal-6 tumor cell-educated RAW 264.7 cells. n=4 biologically independent replicates. FIG. 7F is a graph of the ratio of M1/M2 macrophages in Hepal-6 tumor cell- educated BMDMs. n = 3 per group. FIG. 7G is a graph of the ratio of M1ZM2 macrophages in Hepal-6 tumor cell-educated BMDMs after BiCCL2/5i protein or LPS treatments. n= 3 biologically independent samples. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P<0.0001, ns means no significance; two-tailed Student’s t-test; results are presented as mean (SD).

[0018] FIGS. 8A-8E show Luc mRNA-LNP in vivo transfection. The luciferase was injected (i.p.) 6 hours post LNP-Luc mRNA administration (i.v.), and the Luc bioluminescence signal was observed by IVIS imaging. Representative IVIS images (FIG. 8A) and quantification of luciferase activity in different organs measured by Luciferase assay kit (FIG. 8B). n=3 biologically independent samples. FIG. 8C is a graph of the verification of the biological activity of delivered BiCCL2/5i mRNA. The percentage of M2 macrophages in a BMDM and Hepal-6 cell co-cultured system with the addition of BiCCL2/5i mRNA-LNPs. The BiCCL2/5i mRNA delivery system suppressed the M2 polarization. n=4 biologically independent samples. FIG. 8D is a graph of the expression of BiCCL2/5i protein in different organs 6 h after intravenous injection with LNP-formulated mRNA. n=3 biologically independent samples. FIG. 8E is an image of the cellular localization of LNPs (labeled with Cy5.5, red) in Hepal-6-GFP/Luc (GFP, green) HCC-bearing mice 24 h post systemic injection. **P < 0.01; two-tailed Student’s t-test; results are presented as mean (SD).

[0019] FIGS. 9A-9D show the characterization of the self-assembled trimeric PD-Li. FIG. 9A is a schematic of self-assembled trimeric PD-Li from a fusion protein between the PD-1 extracellular domain and a CMP1 trimerization domain. FIG. 9B shows the expressed trimeric PD-Li using 293T cells under reducing condition (Lanes: different concentration of PD-Li protein). FIG. 9C are curves of the binding of trimeric PD-Li at different concentrations to immobilized PD-L1 measured by Octet. The binding affinity was estimated at -870 pM. FIG. 9D is a graph of nm over time.

[0020] FIGS. 10A-10H show the systemic toxicity and immune-related adverse response in Hepal-6 bearing mice after various treatments. FIGS. 10A-10F are graphs of the complete blood count and blood chemistry analysis of Hepal-6 tumor-bearing mice from various therapeutic groups, as indicated, 4 days after the last treatment. FIG. 10G is a graph of the body weight changes of Hepal-6 bearing mice from various therapeutic groups throughout the treatment period. FIG. 10H is a graph of the flow cytometry analysis of Thl7 cells in the spleen of Hepal- 6 tumor-bearing mice 4 days post various therapeutic groups. n=3 biologically independent samples; results are presented as mean (SD). No significant difference in combination therapy versus mock or monotherapy; two-tailed Student’s t-test

[0021] FIGS. 11 A-l IB show that BisCCL2/5i mRNA gene delivery therapy synergizes ICB therapy in the CT26-FL3 liver metastasis model. The infiltration of CD3 ⁺ CD8 ⁺ T cells into the CT26-FL3 liver metastatic lesion after PBS or BisCCL25i plus ICB treatment groups analyzed by flow cytometry (FIG. 11 A) and immunofluorescence staining using anti-CD3 (red) and DAPI (blue) (FIG. 11B).

[0022] FIGS. 12A-12F show gating strategies used for flow cytometry analysis of CD4~ T cells (CD3 ⁺ CD4 ⁺ ), CD8 ⁺ T cells (CD3 ⁺ CD8 ⁺ ), macrophages (CD 1 lb+F4/O"), MDSCs (CD1 lb ⁺ Gr- 1*), and Tregs (CD3 ⁺ CD4 ⁺ CD25 ⁺ FOXP3 ⁺ ), respectively, in the HCC tumor tissue.

[0023] FIGS. 13A-13G show the identification of the top-ranked monocytes-related genes that are associated with HCC cancer progression. FIG. 13A is a volcano plot showing fold changes for genes differentially expressed between HCC-free sites (Adjacent) and tumor sites (HCC) in the diseased samples from liver cancer patients. FIG. 13B-Upper panel is representative IHC staining images of CCL2 and CCL5 (10x) and its regional magnification (40x) in the human liver cancer tissues and paired adjacent non-tumor liver tissues. FIG. 13B-Lower panel is representative scores of IHC staining. Positive staining is indicated by brown color. FIGS. 13C- 13D are staining score analyses of CCL2 (FIG. 13C) and CCL5 (FIG. 13DS) expression in tumor samples from 9 HCC patients; data were analyzed by unpaired two-tailed Student’s t-test. FIGS. 13E-13F are graphs of mRNA expression of classic Ml (FIG. 13E) and M2 (FIG. 13F) markers in BMDMs 24 hr post the addition of the conditioned medium from Hepal-6 tumor cells. The Hepal-6 cells were pre-treated with the siRNA against CCL2, CCL5, or control, respectively. n = 9 biologically independent samples; data were analyzed by one-way ANOVA and Tukey’s multiple comparisons test. FIGS. 13G is Kaplan-Meier survival curves of HCC tumor-bearing mice treated with PBS, BMS-813160 (25 mg/kg/day, i.p., 5 doses, 1 day apart), CCL2 neutralizing antibody alone (a-CCL2: 10 mg/kg, i.p., 3 doses, 3 days apart), CCL5 neutralizing antibody alone (a-CCL5: 5 mg/kg, i.p., 3 doses, 3 days apart), and a-CCL2 plus a-CCL5 antibodies, using 30% weight loss as the endpoint criteria. Each line represents one survival curve for each group of ten mice; Log-rank (Mantel-Cox) test. Data are represented as the mean±s.d.

[0024] FIGS. 14A-14E show that the single domain antibody that binds both CCL2 and CCL5 blocks their biological activities. FIG. 14 A is a graph of in vitro inhibition of chemotaxis of monocytes in the Transwell assays. The cells were stained with crystal violet and counted 4 hr after the treatment with different concentrations of BisCCL2/5i protein. IC ₅₀ was determined by fitting the number of migrated cells vs the protein concentration using a 4 parameter logistic regression model (n = 3 biologically independent samples). The experiment was performed three times independently with similar results. FTSG. 14B-14C are graphs of mRNA expression of Ml and M2 markers in BMDMs 24 hr after the treatment with PBS, BisCCL2/5i protein, and LPS, respectively, n = 9 biologically independent samples; data were analyzed by one-way ANOVA and Tukey’s multiple comparisons test. FIGS. 14D and 14E show M2 macrophages sorted from IL4 stimulated BMDMs and stained with F4/80, CD1 lb, and CD206 16 hr post the incubation with PBS, BisCCL2/5i protein, and LPS. The representative flow dots (FIG. 14D) and FACS quantification (FIG. 14E) showed BisCCL2/5i and LPS promoted Ml polarization of macrophages (n = 5 biologically independent samples; data were analyzed by one-way ANOVA and Tukey’s multiple comparisons test). Data are represented as the mean± s.d.

[0025] FIGS. 15A-15J show that dual blockade of CCL2 and CCL5 via LNP-mediated mRNA delivery of BisCCL2/5i polarizes macrophage Ml phenotype and reduces the immunosuppression in the TME. FIG. 15A is an image of Luc mRNA-LNPs in vivo transfection after repeated administration. The luciferase was injected intraperitoneally into the mice 6 hr post the administration of Luc mRNA-LNPs (i.v.), and the luc bioluminescence signal after each administration was measured by IVIS imaging, n = 3 biologically independent samples. FIG.

15B is the quantification of mCherry-positive cells expressed in murine orthotopic HCC tumor tissue 6 hr after injection of mCheriy mRNA-LNPs (mCherry mRNA: 0.5 mg/kg). mRNA is mainly expressed in monocytes (CD45 ⁺ CD1 lb ⁺ ) and tumor cells (Hepal-6-GFP ⁺ ) (n = 8 biologically independent mice per group). FIG. 15C and 15D are graphs of mKNA expression of classic Ml (FIG. 15C) and M2 (FIG. 15D) markers in the HCC tumor tissues 48 hr after systemic administration of formulated LNPs as a dose corresponding to 1 mg/kg mRNA (Mock, HcRed mRNA). Each data point is an individual sample (n = 9); one-way ANOVA and Tukey’s multiple comparisons test. FIGS. 15E-15J show the change of the immunocellular composition in the HCC TME 48 hr following Mock mRNA-LNPs and BisCCL2/5i mRNA-LNPs treatments (mRNA: 1 mg/kg), measured by flow cytometry (n = 4 biologically independent samples; unpaired two-tailed Student’s t-test; the experiment was conducted three times independently with similar results). FIGS. 15E and 15F show the representative flow dots of Ml- and M2- phenotype macrophages (FIG. 15E) and the ratio of M1/M2 (FIG. 15F). FIGS. 15G-15H are graph of the percentage and cell counts of total macrophages (FIG. 1SG) and their M2 subtype (FIG. 15H). FIGS. 15I-15J are graphs of the ratio of CD8 ⁺ T (FIG. 151) and Tregs (FIG. 15J). ΜΦ, macrophages (CD45 ⁺ CDllb ⁺ CDllc ^' Ly6C ^' Ly6G ^' F4/80 ⁺ ); M2, M2-phenotype macrophages (CD206 ⁺ ); CD8 ⁺ T (CD45 ⁺ CD3 ⁺ CD8 ⁺ ); Treg (CD45 ⁺ CD25 ⁺ CD4 ⁺ Foxp3 ⁺ ). Data are represented as the mean ± s.d.

[0026] FIGS. 16A-16N show that the dual blockade of CCL2 and CCL5 sensitizes HCC tumors to ICB therapy. FIG. 16A is a treatment scheme for orthotopic HCC tumor-bearing mice with the indicated formulations. HCC tumor-bearing mice were administered with indicated mRNA- LNPs (mRNA: 1 mg/kg, 3 days apart, 3 doses, i.v.) or a-CCL2 plus a-CCL5 antibodies (a- CCL2: 10 mg/kg; a-CCL5: 5 mg/kg; 3 days apart, 3 doses, i.p.). FIG. 16B is Kaplan-Meier survival analysis using a 30% weight loss as the endpoint criteria. Each line represents one survival curve for each group of ten mice; Log-rank (Mantel-Cox) test. FIG. 16C is an intratumoral administration scheme for mice with an established subcutaneous HCC tumor. Mice with about 100 mm ³ subcutaneous tumors were administered BisCCL2/5i mRNA-LNPs plus PD- Li mRNA-LNPs, PD-Li mRNA-LNPs alone, BisCCL2/5i mRNA-LNPs alone, or Mock (HcRed) mRNA-LNPs (mRNA: 1 mg/kg) three times, 3 days apart. FIG. 16D is a graph of average tumor volume from three independent experiments, n = 12 biologically independent samples; P = 0.0152, P = 0.1429, and P < 0.001 denote the significance levels of BisCCL2/5i mRNA-LNPs, PD-Li mRNA-LNPs, and BisCCL2/5i mRNA-LNPs + PD-Li mRNA-LNPs treated mice, respectively, versus those treated with Mock mRNA-LNPs as the control (two-way ANOVA with multiple comparisons). FIG. 16E is spider plots of individual tumor growth curves (n = 12 biologically independent samples in each group). FIG. 16F is Kaplan-Meier survival curves of mice treated with indicated formulations using a 1,500 mm ³ tumor volume as the endpoint criteria. Each line represents one survival curve for each group of 15 mice; Log-rank (Mantel- Cox) test. FIG. 16G is representative images of the tumors 7 d post final administration. FIG.

16H is a treatment scheme for orthotopic HCC tumor-bearing mice treated intravenously with the indicated formulations. FIG. 161 is a graph of average tumor weight from three independent experiments in orthotopic HCC tumors, n = 13 biologically independent sample; one-way ANOVA and Tukey’s multiple comparisons test. FIG. 16J is Kaplan-Meier survival analysis using a 30% weight loss as the endpoint criteria, n = 13 biologically independent samples; Log- rank (Mantel-Cox) test FIG. 16K is a treatment scheme for HCC tumors established by hemi- spleen approach. FIG. 16L is a graph of average tumor weight of different treated groups, n = 12 biologically independent samples; unpaired two-tailed Student’s t-test. FIG. 16M is Kaplan- Meier survival curves in different treated groups, n = 14 biologically independent samples; Log- rank (Mantel-Cox) test FIG. 16N is Kaplan-Meier survival analysis of HCC tumor bearing-mice (for CD4~ or CD8 ⁺ T-cell depletion and BisCCL2/5i plus PD-Li treatments), n = 11 biologically independent samples; Log-rank (Mantel-Cox) test. Data are represented as the mean±s.d.

[0027] FIGS. 17A-17H show that a dual blockade of CCL2 and CCL5 sensitized KPC liver metastasis tumor to ICB therapy. FIGS. 17A-17D are graphs showing the change of the immunocellular composition in the KPC liver metastatic TME 48 hr following Mock mRNA and BisCCL2/5i mRNA-LNPs treatment (mRNA: 1 mg/kg, i.v.), measured by flow cytometry (n = 6 biologically independent samples; unpaired two-tailed Student’s t-test). FIG. 17E is a treatment scheme for KPC liver metastasis tumors administered intravenously with various formulations (mRNA: 1 mg/kg). FIGS. 17F-17G are in vivo bioluminescence imaging (FIG. 17F) and tumor growth burden (FIG. 17G) of mice bearing KPC liver metastasis receiving various treatments (n = 7 biologically independent samples; two-way ANOVA with multiple comparisons). The experiments were conducted twice independently with similar results. FIG. 17H is spider plots of individual tumor growth curves (n = 7 biologically independent samples in each group). Data are represented as the mean ± s.d.

[0028] FIGS. 18A-18B are graphs of the qRT-PCR analysis for glycolysis-related (FIG. 18 A) and fatty acid oxidation-related (FIG. 18B) gene expression in BMDMs 24 hr post the indicated treatment, n = 6 biologically independent samples; one-way ANOVA and Tukey’s multiple comparisons test The experiment was performed two times independently with similar results. The experiment was performed two times independently with similar results. Data are represented as the mean ± s.d.

[0029] FIGS. 19A-19D are graphs showing CCL2 and CCL5 expression was positively correlated with M2 macrophage-associated gene expression in the HCC tumor site in the diseased tissues from liver cancer patients. FIGS. 19A-19B are graphs of the comparison of CCL2 expression with MRC1 (FIG. 19 A) orILlO (FIG. 19B) gene expression. FIGS. 19C-19D are graphs of the comparison of CCL5 expression with MRC1 (FIG. 19C) or IL10 (FIG. 19D) gene expression. Plotted is the expression pattern (normalized log2 intensity). Pearson’s correlation (R) values and P-values are indicated within each graph. The human data were downloaded from International Cancer Genome Consortium (ICGC) database under the accession code LIRI-JP.

[0030] FIGS. 20A-20D are graphs showing the mRNA expression in Hepal-6 tumor cell- educated macrophages treated with siKNA against CCL2, CCL5, or control, respectively. FIGS. 20A-20B are graphs of the mRNA expression of CCL2 (FIG. 20A) and CCL5 (FIG. 20B) in Hepal -6 cells 24 hr after the transfection with indicated siRNA. n = 6 biologically independent samples; data were analyzed by one-way ANOVA and Tukey’s multiple comparisons test. FIGS. 20C-20D are graphs of the mRNA expression of classic Ml (FIG. 20C) and M2 (FIG. 20D) markers in RAW 264.7 cells 24 hr post the addition of the conditioned medium from tumor cells that were pre-treated with the indicated siRNA. n = 3 biological independent samples; data were analyzed by one-way ANOVA and Tukey’s multiple comparisons test. Data are represented as the mean ± s.d.

[0031] FIG. 21 is a graph of the in vitro inhibition of chemotaxis of monocytes in the Transwell assays. The RAW 264.7 cells were stained with crystal violet and counted at 4 hr after the treatment with different concentration of anti-mCCL2 or anti-mCCL5 neutralizing antibodies. IC ₅₀ was determined by fitting the number of migrated cells vs the protein concentration using a 4 parameter logistic regression model (n = 3 biologically independent samples). The experiment was performed three times independently with similar results. Data are represented as the mean ± s.d. [0032] FIGS. 22A-22B are graphs of the biodistribution of the luciferase activity after the treatment with Luc mRNA-LNPs. FIG. 22A is a graph of the quantification of luciferase activity in the liver tissue at different time points after the injection of Luc mRNA-LNPs (mKNA: 0.5 mg/kg, i.v.), measured by luciferase assay kit. n = 6 biologically independent samples. FIG. 22B is a graph of the quantification of luciferase activity in different organs 6 hr after each administration of Luc mRNA-LNPs (mRNA: 0.5 mg/kg, i.v., 3 days apart), n = 3 biologically independent samples. The repeat administration showed comparable protein level. Data are represented as the mean ± s.d.

[0033] FIGS. 23A-23C are images of the cellular localization of mCherry mRNA-LNPs in Hepal -6-GFP/Luc (GFP, green) HCC tumor tissue 6 hr post systemic injection. LNPs were labeled with Cy5.5 (white), mCherry expression (red), cell nuclei (DAPI, blue). FIG. 23 A is representative confocal laser scanning microscopy (CLSM) images under 20* magnification in different channels. FIG. 23B is a representative image under 40* magnification and its regional magnification in the tumor site. The arrows indicate the location of the expressed mCherry protein and LNPs in the tumor cells. FIG. 23C is representative images and its regional magnification in the tumor site and non-tumor site.

[0034] FIGS. 24A-24C are flow cytometric analysis of the transfected cell types after the injection of mCherry mRNA-LNPs. FIG. 24A shows the gating strategy of the transfected cells (mCherry ^" *) 6 hr post injection of mCherry mRNA-LNPs. mCherry mRNA was mainly delivered and expressed in the monocytes (CD1 lb ⁺ ) and the tumor cells (GFP*) and the quantification was shown in FIG. 15B. FIGS. 24B and 24C are the gating strategy and the quantification, respectively, of the cell uptake of LNPs (PE-Cy7 ⁺ ) 2 hr post injection of mRNA-LNPs. LNPs were mainly internalized in monocytes (CD1 lb ⁺ ) and tumor cells (GFP ⁺ ), consistent with the location of the protein product encoded by mCherry mRNA. Data are represented as the mean ± s.d.

[0035] FIG. 25 is representative IHC staining images of CCL2 and CCL5 in mouse liver tissue, as well as in tissues from murine Hepal -6 HCC tumor, KPC liver metastasis, and CT26 liver metastasis. Positive staining is indicated by brown color.

[0036] FIGS. 26A-26C show in vivo expression and biological activity evaluation of BisCCL2/5i in the mRNA-LNP delivery system. FIGS. 26A-26B show the plasma concentration of BisCCL2/5i protein at different time points after the administration of BisCCL2/5i mRNA-LNPs (1 mg/kg; i.v.). Pharmacokinetic parameters (FIGS. 26A) and the mean plasma concentrationtime profile (FIG. 26B) following single-dose administration, n = 6 biologically independent samples. FIG. 26C are graphs of BisCCL2/5i expression in different organs 6 hr after each administration of BisCCL2/5i mRNA-LNPs (mRNA: 1 mg/kg, i.v., 3 days apart), n = 6 biologically independent samples. The BisCCL2/5i mRNA was mainly expressed in the liver tissue and the repeated administration showed comparable protein level.

[0037] FIGS. 27A-27F show the change of the immunocellular composition in the HCC TME 48 hr after the different treatments, measured by flow cytometry and the percentage of g-MDSC (FIG. 27A), m-MDSC (FIG. 27B), NK (FIG. 27C), CD4 ⁺ T (FIG. 27D), GDI lb ⁺ DC (FIG. 27E), and CD8 ⁺ DC (FIG. 27F) following Mock mRNA-LNPs or BisCCL2/5i mRNA-LNPs treatments (mRNA: 1 mg/kg), n = 4 biologically independent samples; unpaired two-tailed Student’s t-test; the experiment was conducted three times independently with similar results. Data are represented as the mean ± s.d. m-MDSC, monocytic MDSC (CD45 ⁺ CD11 b ⁺ Ly6C ^hi Ly6G ^low ); g- MDSC, granulocytic MDSC (CD45 ⁺ CD1 lb"Ly6G ^hi Ly6C ^low ); CD4 ⁺ T (CD45 ⁺ CD3 ⁺ CD4 ⁺ ); NK ⁺ (CD45 ⁺ NK1.1 ⁺ ); DCs (CD45 ⁺ CDllc ⁺ CDl lb ⁺ /CD8 ⁺ ).

[0038] FIGS. 28A-28D show the change of the immunocellular composition in the KPC liver metastatic TME 48 hr following Mock (HcRed) mRNA-LNPs or BisCCL2/5i mRNA-LNPs treatment (mRNA: 1 mg/kg, i.v.). FIGS. 28A-28C are graphs of the percentage of m-MDSC,

NK, and CD4 ⁺ T cells, respectively, measured by flow cytometry (n = 6 biologically independent samples; unpaired two-tailed Student’s t-test). The experiment was conducted two times independently with similar results. FIG. 28D is representative immunofluorescence staining images using anti-CD3 antibody (red) and DAPI (blue) in the KPC liver metastatic TME. Data are represented as the mean ± s.d.

[0039] FIG. 29A is a representative immunofluorescence staining images using anti-CD3 antibody (red) and DAPI (blue) in the CT26 liver metastatic TME 48 hr following Mock mRNA- LNPs or BisCCL2/5i mRNA-LNPs treatment (mRNA: 1 mg/kg, i.v.). FIGS. 29B and 29C are graphs of the percentage of macrophage (FIG. 29B) and its M2-phenotype (FIG. 29C) 48 hr after indicated treatments, measured by flow cytometry (n = 6 biologically independent samples; unpaired two-tailed Student’s t-test). The experiment was conducted two times independently with similar results. [0040] FIG. 30 shows the effect of BisCCL2/5i on macrophage polarization in the presence or absence of glucose. M2-phenotype macrophages sorted from BMDMs were incubated with PBS or recombinant BisCCL2/5i protein for 16 hr, followed by measuring the polarization via flow cytometry. Representative flow dot plots (left) and the quantification (right) of M2-phenotype macrophages ((+) Glu: 80 mM glucose; (-) Glu: 0 mM glucose), n = 6 biologically independent samples; one-way ANOVA and Tukey’s multiple comparisons test. The experiment was performed three times independently with similar results. Data are represented as the mean± s.d. [0041] FIG. 31 shows the gating strategies used for flow cytometry analysis of macrophages (CD45 ⁺ CD11 b ⁺ Ly6C ^" Ly6G ^" F4/80 ⁺ ), M2-phenotype macrophages (CD206 ⁺ gated in macrophages), g-MDSCs (CD45 ⁺ CD11 b ⁺ Ly6G ^hi Ly6C ^low ), and m-MDSCs (CD45 ⁺ CD1 lb ⁺ Ly6C ^hl Ly6G ^,low ) in the HCC tumor tissue.

[0042] FIG. 32 shows the gating strategies used for flow cytometry analysis of DCs (CD45 ⁺ CD1 lc ⁺ CD8 ⁺ ), CD8 ⁺ DCs (CD45 ⁺ CD1 lc ⁺ CD8 ⁺ ), and CD1 lb ⁺ DCs (CD45 ⁺ CD1 lc ⁺ CDl lb ⁺ ) in the HCC tumor tissue.

[0043] FIG. 33 shows the gating strategies used for flow cytometry analysis of CD4~ T cells (CD45 ⁺ CD3 ⁺ CD4 ⁺ ), CD8 ⁺ T cells (CD45 ⁺ CD3 ⁺ CD8 ⁺ ), NK cells (CD45 ⁺ NK1. V), and Tregs (CD45 ⁺ CD4 ⁺ CD25 ⁺ FOXP3 ⁺ ) in the HCC tumor tissue.

DETAILED DESCRIPTION

[0044] Tumor-associated macrophages (TAMs) have been identified as a main switch to induce tumor immune suppression via M2 subtype and promote resistance to ICB during cancer progression. As described herein, it was found that tumor-derived C-C Motif Chemokine Ligands CCL2 and CCL5 are associated with HCC progression. Both CCL2 and CCL5 are chemoattractants of monocytes and act through G-protein coupled receptors (GPCRs) to evoke their respective biological responses. The receptors for CCL2 are CCR2 and CCR4, whereas those that use CCL5 as a ligand include CCR1, CCR3, and CCR5. Previous clinical trials used small molecular antagonists to block either CCR2 or CCR5 for cancer therapy. However, the therapeutic effects have not been fully realized in clinical studies. Herein it is shown that downregulation of either CCL2 or CCL5 alone in macrophages was not enough to suppress the tumor-supportive M2 macrophage and improve the immunosuppression. [0045] Herein, a single domain antibody was designed that tightly binds and potently inhibits both CCL2 and CCL5 in a bispecific manner (BisCCL2/5i). The mRNA encoding BisCCL2/5i was delivered to the primary or metastatic cancerous liver malignancy specifically using clinically validated liver-homing lipid nanoparticles (LNPs) to restrict the therapeutic activity only in the diseased organ. The dual blockade of CCL2 and CCL5 signaling via BisCCL2/5i mRNA-LNPs led to metabolic reprogramming of the macrophage, including glycolysis, fatty acid oxidation and lipid synthesis, which subsequently triggered the Ml polarization of macrophage in the tumor sites. The M2 to Ml macrophage phenotype transition further suppressed the recruitment of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), facilitating the infiltration of T cells into tumor sites. The combination of BisCCL2/5i with ICB provided a synergistic anti-tumor immune response that inhibited local tumors as well as orthotopic HCC and CRC liver metastatic tumors. This dual CCL2/CCL5 targeting and ICB combination therapy are safe due to the low dosage administration via the LNPs-mRNA delivery system. The strategy disclosed herein would be clinically amenable and has the great potential to inflame the TME of other cancer types and synergistically enhance the therapeutic efficacy of the ICB therapy.

[0046] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.

1. Definitions

[0047] The terms “comprise(s) ,” “include^),” “having,” “has, " " can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. [0048] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0049] “Antibody fragment” as used herein refers to a portion of an intact antibody comprising the antigen-binding site or variable region. The portion does not include the constant heavy chain domains (i.e., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody.

[0050] “Derivative” of an antibody as used herein may refer to an antibody having one or more modifications to its amino acid sequence when compared to a genuine or parent antibody and exhibit a modified domain structure. The derivative may still be able to adopt the typical domain configuration found in native antibodies, as well as an amino acid sequence, which is able to bind to targets (antigens) with specificity. Typical examples of antibody derivatives are antibodies coupled to other polypeptides, rearranged antibody domains, or fragments of antibodies. The derivative may also comprise at least one further compound, e.g., a protein domain, said protein domain being linked by covalent or non-covalent bonds. The linkage can be based on genetic fusion according to the methods known in the art. The additional domain present in the fusion protein comprising the antibody may preferably be linked by a flexible linker, advantageously a peptide linker, wherein said peptide linker comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of the further protein domain and the N-terminal end of the antibody or vice versa. The antibody may be linked to an effector molecule having a conformation suitable for biological activity or selective binding to a solid support, a biologically active substance (e.g., a cytokine or growth hormone), a chemical agent, a peptide, a protein, or a drug, for example. [0051] “Identical” or “identity,” as used herein in the context of two or more polypeptide or polynucleotide sequences, can mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of the single sequence are included in the denominator but not the numerator of the calculation.

[0052] “Polynucleotide” or “oligonucleotide” or “nucleic acid," as used herein, means at least two nucleotides covalently linked together. The polynucleotide may be DNA, both genomic and cDNA, RNA, or a hybrid, where the polynucleotide may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods. Polynucleotides may be single- or double-stranded or may contain portions of both double stranded and single stranded sequence. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.

[0053] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The proteins may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain. The terms “polypeptide”, and “protein,” are used interchangeably herein.

[0054] As used herein, the term "preventing" refers to partially or completely delaying onset of a disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular disease, disorder, and/or condition; partially or completely delaying progression from a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.

[0055] As used herein, the terms "providing", "administering," "introducing," are used interchangeably herein and refer to the placement of the compounds and/or compositions of the present disclosure into a subject by a method or route which results in at least partial localization of the compound and/or composition to a desired site. The compound and/or compositions can be administered by any appropriate route which results in delivery to a desired location in the subject.

[0056] A "subject" or "patient" may be human or non-human and may include, for example, animal strains or species used as "model systems" for research purposes. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compounds and/or compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like. In one aspect of the methods provided herein, the mammal is a human.

[0057] As used herein, "treat," "treating" and the like means a slowing, stopping, prevention or reversing of progression of a disease, disorder and/or condition when provided a compound and/or composition described herein to an appropriate control subject. The term also means a reversing of the progression of such a disease to a point of eliminating or greatly reducing the cell proliferation. As such, "treating" means an application or administration of the compounds and/or compositions described herein to a subject, where the subject has a disease, disorder and/or condition or a symptom of a disease, disorder and/or condition, or is exhibiting symptoms that indicate high risk for disease, disorder and/or condition where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, affect, or prevent the disease or symptoms of the disease, disorder and/or condition.

[0058] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0059] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. 2. Monospecific and Bispecific Antibody from the selection

[0060] The present disclosure relates to isolated single domain antibodies that specifically bind both CCL2 and CCL5 or CCL2.

[0061] In some embodiments, the isolated single domain antibody, an antibody fragment or derivative thereof that specifically binds both CCL2 and CCL5. In some embodiments, the single domain antibody comprises an amino acid sequence with at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% amino acid sequence identity to any of SEQ ID NOs: 1-3. In select embodiments, the single domain antibody comprises an amino acid sequence with at least 70% amino acid sequence identity to any of SEQ ID NO: 1-3. Complementary determining regions (CDRs) are underlined and based on Kabat numbering. [0062] SEQ ID NO: 1 Q [0063] SEQ ID NO:2

OVOLLESGGGLVOPGGSLRLSCAASGYDVSYENMAWVRRAPGKGLEWVSTINTDD R Q [0064] SEQ ID NO:3

[0065] In another aspect, the disclosure relates to a single domain antibody, antibody fragment or derivative thereof having at least one of the following sets of CDRs: (1) YENIA (SEQ ID NO: 8), K (SEQ ID NO:9), and VDVSPVQAVDEALRF (SEQ ID NO: 10); (2) (SEQ ID NO: 11) and VDVSPVQAVDEALRF (SEQ ID NO: 10); or (3) YENMA (SEQ ID NO: 12), G (SEQ ID NO: 13) and V (SEQ ID NO: 14).

[0066] In some embodiments, the isolated single domain antibody, antibody fragment or derivative thereof specifically binds CCL2. In some embedments, the single domain antibody comprises an amino acid sequence with at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% amino acid sequence identity to any of SEQ ID NOs: 4-7. CDRs are underlined and based on Kabat numbering.

[0067] SEQ ID NO:4 L Q [0068] SEQ ID NO:5 [0069] SEQ ID NO:6

O V OLLES GGGL V QPGGSLRLS C AAS GYNHTKDMGWVRO APGKGLEWV S SIEDGGG [0070] SEQ ID NO:7

[0071] In another aspect, the disclosure relates to a single domain antibody, antibody fragment or derivative thereof having at least one of the following sets of CDRs: (1) YENMA (SEQ ID NO.12), (SEQ ID NO: 13) and V Q (SEQ ID NO: 10); (2) DKFMS (SEQ ID NO: 15), ( ID NO: 16), and GQGELDSPLSY (SEQ ID NO: 17); (3) TKDMG (SEQ ID NO: 18), G (SEQ ID NO: 19) and LVDHEDSMTS (SEQ ID NO:20); or (4) (SEQ ID NO:21), (SEQ ID NO:22) and RDQGLNYGSLFDY (SEQ ID NO:23).

[0072] The present disclosure also relates to polynucleotides comprising a nucleic acid encoding the isolated single domain antibodies that specifically binds both CCL2 and CCL5 or CCL2. In some embodiments, the polynucleotide is an mRNA.

3. Pharmaceutical Compositions

[0073] The present disclosure relates to pharmaceutical compositions comprising a single domain antibody or a polynucleotide as described herein in Section 2.

[0074] In some embodiments, the pharmaceutical composition further comprises one or more additional cancer immunotherapy agents. In some aspects, the cancer immunotherapy agent is a checkpoint inhibitor or a nucleic acid encoding a checkpoint inhibitor.

Immune checkpoint proteins are well-known in the art as components of the immune system which provide inhibitory signals to its components in order to balance immune reactions. Known immune checkpoint proteins include, but are not limited to, CTLA-4, PD1 and its ligands PD-L1 and PD-L2 and in addition LAG-3, BTLA, B7H3, B7H4, TIΜ3, and MR Immune checkpoint proteins are described in the art (see, for example, Pardoll, 2012 Nature Rev. Cancer 12: 252-264, incorporated herein by reference in its entirety).

[0075] Immune checkpoint inhibitors may reduce the function of the immune checkpoint protein or result in a full blockade. In some embodiments, the checkpoint inhibitor is a PD-1 ligand inhibitor or PD-1 inhibitor. In select embodiments, the checkpoint inhibitor may be those disclosed in WO 2017/053170, the contents of which are herein incorporated herein by reference.

[0076] The pharmaceutical compositions and formulations may include pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier,” as used herein, means a nontoxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, com starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0077] The route by which the disclosed compounds are administered and the form of the composition will dictate the type of carrier to be used. The composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis). Techniques and formulations may generally be found in “Remington's Pharmaceutical Sciences,” (Meade Publishing Co., Easton, Pa.). Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage.

[0078] The pharmaceutical compositions may further comprise a viral nanoparticle or a non- viral nanoparticle. Non-viral nanoparticles include those which are not based upon a virus or protein components derived from a virus. For example, non-viral nanoparticles include, but are not limited to those using lipids, polymers, ceramic-based nanomaterials, metal nanoparticles, e.g., gold nanoparticles, and silica-based nanoparticles. Viral nanoparticles (VNPs) are virus- based nanoparticle formulations. VNPs can be bacteriophages, plant or animal viruses, and they can be infectious or non-infectious. VNPs, as used herein include virus-like particles (VLPs). Virus-like particles refer to a structure resembling a virus particle, but which has been demonstrated to be non-pathogenic. In general, virus-like particles lack at least part of the viral genome. Also, virus-like particles can often be produced in large quantities by heterologous expression and can be easily purified. A virus-like particle in accordance with the invention may contain nucleic acid distinct from their genome.

[0079] In some embodiments, the non-viral nanoparticle is a lipid nanoparticle. In some embodiments, the polynucleotide, the nucleic acid encoding a checkpoint inhibitor, or a combination thereof is encapsulated in the lipid nanoparticle. As used herein, the phrase "lipid nanoparticle" refers to a transfer vehicle comprising one or more lipids (e.g., cationic lipids, non- cationic lipids, ionizable cationic lipids, and PEG-modified lipids). Examples of suitable lipids include, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides).

4. Methods of Use

[0080] The present disclosure relates to methods of treating cancer and inhibiting the growth or survival of a cancer cells. The methods of treating cancer may comprise administering to a subject in need thereof an effective amount of the isolated antibody or a fragment or derivative thereof that specifically binds both CCL2 and CCL5 or CCL2, as described herein in Section 2, or a polynucleotide, also described herein in Section 2, or pharmaceutical composition thereof, as described herein in Section 3. The methods of inhibiting the growth or survival of cancer cells may comprise contacting cells with an effective amount of the isolated antibody or a fragment or derivative thereof that specifically binds both CCL2 and CCL5 or CCL2, as described herein in Section 2, or a polynucleotide, also described herein in Section 2, or pharmaceutical composition thereof, as described herein in Section 3.

[0081] An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. An effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of a compound of the invention (e.g., a compound of formula (I)) are outweighed by the therapeutically beneficial effects. In some embodiments, the effective amount ranged from 0.25 mg/kg to 8 mg/kg, dosed over 3-4 doses, every 3-5 days.

[0082] The methods can be used with any cancer cell or in a subject having any type of cancer, for example those described by the National Cancer Institute. Exemplary cancers may include the following: digestive/gastrointestinal cancers such as anal cancer; bile duct cancer; extrahepatic bile duct cancer; appendix cancer; carcinoid tumor, gastrointestinal cancer; colon cancer; colorectal cancer including childhood colorectal cancer; esophageal cancer including childhood esophageal cancer; gallbladder cancer; gastric (stomach) cancer including childhood gastric (stomach) cancer; hepatocellular (liver) cancer including adult (primary) hepatocellular (liver) cancer and childhood (primary) hepatocellular (liver) cancer; pancreatic cancer including childhood pancreatic cancer; sarcoma, rhabdomyosarcoma; islet cell pancreatic cancer; rectal cancer; and small intestine cancer; endocrine cancers such as islet cell carcinoma (endocrine pancreas); adrenocortical carcinoma including childhood adrenocortical carcinoma; gastrointestinal carcinoid tumor; parathyroid cancer; pheochromocytoma; pituitary tumor; thyroid cancer including childhood thyroid cancer; childhood multiple endocrine neoplasia syndrome; and childhood carcinoid tumor; eye cancers such as intraocular melanoma or uveal melanoma; and retinoblastoma; musculoskeletal cancers such as Ewing's family of tumors; osteosarcoma/malignant fibrous histiocytoma of the bone; childhood rhabdomyosarcoma; soft tissue sarcoma including adult and childhood soft tissue sarcoma; clear cell sarcoma of tendon sheaths; and uterine sarcoma; breast cancer such as breast cancer including childhood and male breast cancer and breast cancer in pregnancy; neurologic cancers such as childhood brain stem glioma; brain tumor; childhood cerebellar astrocytoma; childhood cerebral astrocytoma/malignant glioma; childhood ependymoma; childhood medulloblastoma; childhood pineal and supratentorial primitive neuroectodermal tumors; childhood visual pathway and hypothalamic glioma; other childhood brain cancers; adrenocortical carcinoma; central nervous system lymphoma, primary; childhood cerebellar astrocytoma; neuroblastoma; craniopharyngioma; spinal cord tumors; central nervous system atypical teratoid/rhabdoid tumor; central nervous system embryonal tumors; and childhood supratentorial primitive neuroectodermal tumors and pituitary tumor; genitourinary cancers such as bladder cancer including childhood bladder cancer; renal cell (kidney) cancer; ovarian cancer including childhood ovarian cancer; ovarian epithelial cancer; ovarian low malignant potential tumor; penile cancer; prostate cancer; renal cell cancer including childhood renal cell cancer; renal pelvis and ureter, transitional cell cancer; testicular cancer; urethral cancer; vaginal cancer; vulvar cancer; cervical cancer; Wilms tumor and other childhood kidney tumors; endometrial cancer; and gestational trophoblastic tumor; Germ cell cancers such as childhood extracranial germ cell tumor; extragonadal germ cell tumor; ovarian germ cell tumor; head and neck cancers such as lip and oral cavity cancer; oral cancer including childhood oral cancer; hypopharyngeal cancer; laryngeal cancer including childhood laryngeal cancer; metastatic squamous neck cancer with occult primary; mouth cancer; nasal cavity and paranasal sinus cancer; nasopharyngeal cancer including childhood nasopharyngeal cancer; oropharyngeal cancer; parathyroid cancer; pharyngeal cancer; salivary gland cancer including childhood salivary gland cancer; throat cancer; and thyroid cancer; hematologic/blood cell cancers such as a leukemia (e.g., acute lymphoblastic leukemia including adult and childhood acute lymphoblastic leukemia; acute myeloid leukemia including adult and childhood acute myeloid leukemia; chronic lymphocytic leukemia; chronic myelogenous leukemia; and hairy cell leukemia); a lymphoma (e.g., AIDS- related lymphoma; cutaneous T cell lymphoma; Hodgkin's lymphoma including adult and childhood Hodgkin's lymphoma and Hodgkin's lymphoma during pregnancy; non-Hodgkin's lymphoma including adult and childhood non-Hodgkin's lymphoma and non-Hodgkin's lymphoma during pregnancy; mycosis fungoides; Sezary syndrome; Waldenstrom's macroglobulinemia; and primary central nervous system lymphoma); and other hematologic cancers (e.g., chronic myeloproliferative disorders; multiple myeloma/plasma cell neoplasm; myelodysplastic syndromes; and myelodysplastic/myeloproliferative disorders); lvrng cancer such as non-small cell lung cancer; and small cell lung cancer; respiratory cancers such as adult malignant mesothelioma; childhood malignant mesothelioma; malignant thymoma; childhood thymoma; thymic carcinoma; bronchial adenomas/carcinoids including childhood bronchial adenomas/carcinoids; pleuropulmonary blastema; non-small cell lung cancer; and small cell lung cancer; skin cancers such as Kaposi's sarcoma; Merkel cell carcinoma; melanoma; and childhood skin cancer; AIDS-related malignancies; other childhood cancers, unusual cancers of childhood and cancers of unknown primary site; and metastases of the aforementioned cancers.

[0083] In some embodiments, the cancer is selected from the group consisting of liver cancer, pancreatic cancer, gastrointestinal cancer, lung cancer, ovarian cancer, eye cancer, and combinations thereof. In select embodiments, the cancer comprises hepatocellular carcinoma, colorectal cancer, gastric cancer, pancreatic ductal adenocarcinoma, lung cancer, ovarian cancer, eye cancer, cholangiocarcinoma (bile duct cancer), or a combination thereof. In certain embodiments, the cancer comprises liver metastasis of hepatocellular carcinoma, colorectal cancer, gastric cancer, pancreatic ductal adenocarcinoma, lung cancer, ovarian cancer, uveal melanoma, cholangiocarcinoma, or a combination thereof.

[0084] In some embodiments, the administration is intratumoral, peripheral to the tumor, or systemic.

[0085] The methods may further comprise a combination treatment with other agents. In some embodiments, the methods further comprise administering to the subject an effective amount of a conventional cancer treatment. Conventional treatments are well-known to those in the art and include, but are not limited to, chemotherapy, radiotherapy, immunotherapy, proton therapy, photodynamic therapy, and surgery.

5. Examples

Materials and Methods

[0086] Cell lines The metastatic murine CT26-FL3 colorectal cancer cell line stably expressing RFP/Luc were cultured in complete DMEM medium with 10% fetal bovine serum (FBS) and lpg/mL puromycin. The CT26 wild type colorectal cancer, Hepal-6 wild type liver cancer, and HEK 293 T cell lines were purchased from ATCC and cultured in complete DMEM medium with 10% fetal bovine serum (FBS). Hepal-6 cells were stably transfected with the vector carrying the GFP, firefly luciferase, and the puromycin resistance gene (Hepal-6-GFP-Luc) and maintained in complete DMEM medium with 10% fetal bovine serum (FBS) and 1 pg/mL puromycin. The primary mouse pancreatic cancer cell line was derived from the spontaneous KPC mouse model (LSL-Kras G12D/+; LSL-Trp53R172H/+; Pdx-l-Cre, synergetic to C57BL/6 strain) and was provided by Dr. Serguei Kozlov from the Center for Advanced Preclinical Research, Frederick National Laboratory for Cancer Research (NCI). KPC cells were then stably transfected with the vector carrying the GFP, firefly luciferase, and the puromycin resistance gene (KPC- GFP-Luc) and cultured in complete DMEMZF-12 medium with 10% fetal bovine serum (FBS) and 1 pg/mL puromycin. All cells were grown at 37 °C in a humidified atmosphere (5% CCh) and 95% humidity.

[0087] Mice Animal experiments were carried out in accordance with approved protocols. For the HCC liver cancer and KPC liver metastasis model, male C57BL/6J mice (5-6 weeks old) were purchased from the Jackson Laboratory (stock #000664). For the colorectal cancer and colorectal cancer liver metastasis model, female BALB/cJ mice (6-8 weeks) were purchased from the Jackson Laboratory (stock #000651).

[0088] Selection of CCL2- and CCL5-binding bispecific single domain antibody using Yeast Surface Display The initial VH domain library was constructed by PCR-amplifying a human VH domain library sequences followed by homologous recombination by transformation of the purified PCR product into EBY100 yeast cells along with linearized pCTCON2 vector through electroporation. The diversity of the library was determined to be around 10 ⁸ by plating serial dilutions of the library on selective plates. Yeast cells were grown in SDCAA (20 g/L dextrose, 5 g/L Casamino acids, 6.7 g/L yeast nitrogen base, 5.40 g/L Na2HP04, 7.45 g/L NaJfcPCU) to expand and maintain the library and in SGCAA (20 g/L galactose, 5 g/L Casamino acids, 6.7 g/L yeast nitrogen base, 5.40 g/L Na2HPC>4, 7.45 g/L NaEbP04) to induce protein expression. The yeast displaying VH were first incubated with target-free streptavidin-coated magnetic Dynabeads for 1 h at 4°C, to deplete clones that bound bare Dynabeads or streptavidin. Unbound yeast cells were collected and used for the magnetic-bead positive selection by incubating with biotinylated mouse CCL2 (-200 pmol) immobilized on Dynabeads for 1 h at 4°C. The beads with attached cells were isolated and washed with 1 mL PBS A (PBS + 0.1% BSA) at room temperature 3-5 times, followed by transferring to SD-CAA for growth. After one round of magnetic-bead enrichment, three rounds of FACS sorting selection were performed by using decreased amount of mCCL2, with concentration changed from 500 nM to 10 nM. Each round of FACS sorting was performed simultaneously with an anti-c-myc chicken antibody (Life technologies) to quantify surface protein expression and appropriate amount of biotinylated mCCL2. The secondary antibodies used were Streptavidin-PE and goat-anti-chicken conjugated to Alexa 647 (Life technologies). Yeast cells with the highest binding and expression ratio were selected. The selection was performed until a homogenous population was obtained and no further enrichment was possible. The collected yeast cells were grown on SD-CAA and the corresponding plasmid DNAs were extracted for sequencing.

[0089] To develop VH sequences that bispecifically bind both mCCL2 and mCCL5, a mCCL2- binding VH sequence that showed weak binding with CCL5 was used as the parental sequence for error-prone PCR using the GeneMorph Π Random Mutagenesis Kit. The mutated VH genes were co-transformed with linearized plasmid vector to produce intact plasmid via homologous recombination. The mutagenized yeast population was sorted once each on magnetic beads using biotinylated mCCL5 or mCCL2 as the target in a sequentially manner, followed by three rounds of FACS sorting selection using mCCL2 or mCCL5, sequentially, at 25 nM, 10 nM, and 1 nM, respectively.

[0090] BisCCL2/5i and PD-Li protein generation The expression vectors encoding BisCCL2/5i and PD-Li were generated by inserting synthesized DNAs into the pCDNAS.l vector. For protein expression, HEK 293T cells were cultured until 80% confluence and transfected with the expression vector using lipofectamine 3000 according to the manufacturer's instructions. The transfection complex was made with 24 pg plasmid, 20 μL lipofectamine 3000, and 2 mL Opti- MEM to each T75 flask. The supernatant was collected 3 days, 6 days, and 9 days post transfection and stored at 4 °C until purification. The protein was purified by using HisPur™ Ni- NTA Resin (Thermo Fisher). The purified proteins were analyzed on 4-12% SDS-PAGE gel (Invitrogen) with Coomassie G-250 (Bio-Rad) stain.

[0091] Affinity measurement and specificity analysis The target-binding of individual clones of interest was assayed by flow cytometry using yeast cells displaying the corresponding VH sequence that were grown and induced as for the selection. Washed cells were suspended in PBSA containing a biotinylated chemokine of interest over a range of concentrations and incubated at 22 °C for a sufficient time to reach close to complete equilibrium. After incubation, cells were washed and analyzed by flow cytometry. Fluorophore signal was compared with both unlabeled and non-displaying cells. The relative binding was estimated by subtracting the background signal from the unlabeled control and normalized to the saturated signal at high concentrations. The dissociation constant was determined as the concentration corresponding to half-maximal binding.

[0092] To more accurately measure the target-binding affinity and specificity of the BisCCL2/5i, the ORF sequence encoding BisCCL2/5i with a C-terminal His-tag was cloned into pRS314 vector (pRS314-4M5.3 was a gift from Dane Wittrup (Addgene plasmid #45830) for secreted expression in yeast. The affinities of purified BisCCL2/5i to a panel of related chemokines, including mCCL2, mCCL5, hCCL2, hCCL5, hCCLll, mCCL3, mCCL4, mCCL7, mCCL8, and mCCL19, were accessed with microscale thermophoresis (MST), respectively. In brief, purified BisCCL2/5i was first fluorescently labeled by using REDtris-NTA dye according to standard manufacturer protocols. Ten μL of the labeled protein (50 nM) was then applied to 10 μL (10 pM initial concentration) of serially 2-fold diluted chemokine of interest using a PBST buffer (PBS with 0.05% Tween 20). The resulting samples were subsequently loaded into 16 standard treated capillaries, samples excited by Red LED at 50% power and the thermophoresis of each sample was measured at 40% MST power on Monolith NT.115 (NanoTemper Technologie, Munich, Germany).

[0093] Bio-layer Interferometry (BLI) analyses of the interaction between recombinant PD-Li and PD-LI were performed on forteBIO Octet QK system. To measure the interaction between PD-LI with the trimeric PD-Li inhibitor. Ni-NTA or SAX biosensors (Pall forteBIO Corp) were used to immobilize PD-LI tagged with His or biotin, respectively. Purified trimeric PD-Li was prepared in the assay buffer (1 xPBS, 0.002% Tween 20, pH 7.4) and applied to a 96-well microplate in column arrangement. Different concentrations of PD-Li (0 - 400 nM) were used to test the binding affinity. All data were acquired and analyzed with forteBIO Data Acquisition 6.4 software. Data processing was normalized by the reference biosensors, applying Savitzky-Golay filtering, and fitting binding curves.

[0094] In vitro suppression of cell migration via BisCCL2/5i Cells were seeded on the transwell plates (24 well, 5 pm pore size) at a density of 5 x10 ⁵ cells/mL in a serum-free medium (RPMI1640). One well was exposed to serum-free medium, while all other groups were exposed to serum-free medium containing an appropriate amount of chemokine of interest in the bottom tray. Migration was measured in cells either in the presence/absence of purified BisCCL2/5i (2,

4, 10, 20, and 40 nM) or a control. Assays were performed at 37 °C in a 5% CO2 environment for 4 h and the migrated cells were counted using a hemacytometer. The number of migrated cells was fit to the BisCCL2/5i concentration using a 4-parameter logistic regression model. The experiment was repeated in triplicate and IC ₅₀ was determined by fitting the number of migrated cells to the protein concentration using a 4-parameter logistic regression model.

[0095] BisCCL2/5i and PD-Li mRNA synthesis and LNP preparation The expression vectors encoding BisCCL2/5i and PD-Li were generated by inserting the ORF into a pUC57 vector preconstructed and optimized with 80 As the 3’-UTR for in vitro transcription. The T7 promoter- AG mutation was generated by using the Q5 site-directed mutagenesis kit according to the manufacturer’s instructions. All plasmids were sequenced to confirm the sequence accuracy. mRNAs were transcribed in vitro by T7 RNA polymerase-mediated transcription according to the manufacturer’s instructions (MEGAscript™ T7 Transcription Kit, Thermo Fisher). CleanCap AG (TriLink) was used as a capping reagent that results in a Cap 1 structure at the 5’ end of in vitro transcribed mRNA. The resulting mRNAs were highly purified by using MEGAclear™ Transcription Clean-Up Kit (Thermo Fisher) and characterized prior to being packed in LNPs. LNP formulations were prepared using a previously described method (Miao, L. etal. Nature Biotechnology, 1-12 (2019). The particles were diluted in PBS (pH7.4) and characterized using a Malvern Nano ZS (Westborough, MA) to measure the particle size distribution. For transmission electron microscopy, particles were stained with a 2% phosphotungstic acid (PTA) for 30 s and imaged on a 200 kV JEOL 1230 transmission Electron Microscope.

[0096] Generation of tumor cell-educated macrophages BMDMs were derived by isolating bone marrow from adult male C57BL/6 mice for 7 d at 37 °C 5% CO2 in DMEM medium with 10% heat deactivated FBS and 20% L929 conditioned medium under non-adherent conditions using Ultra Low Adherence Flasks (Coming).

[0097] For tumor cell-education macrophages, the conditioned medium was collected from Hepal-6 liver cancer cells incubated with different treated groups for 24 h, and then added to BMDMs or Raw 264.7 cells for 24-48 h. Following incubation, the flow cytometry or qRT-PCR was performed to measure the amounts of Ml and M2 macrophages and the gene expression of Ml and M2 markers. [0098] In vivo tumor model and treatment For orthotopic HCC tumor models, the mouse was anesthetized and the upper abdomen was opened for tumor cell inoculation. The murine Hepal -6 cells (2*10 ⁶ to 5x1ο ⁶ cells/mouse) are inoculated at the subcapsular region of the left lobes of the liver. To establish orthotopic hemi-spleen HCC tumor model and CT26 CRC liver metastasis model, the mice were anaesthetized, and 1 cm vertical incision was made under rib cage on the left side of the mouse near approximate spleen area. The spleen was exposed and tied in half using suture. Murine CT26-FL3 cells (3x10 ⁵ cells/mouse) or Hepal -6 cells (1 xlO ⁶ cells/mouse) were inoculated directly into spleen, followed by resecting the inoculated portion of spleen. The rest of the spleen was returned before suturing. For subcutaneous tumor model, 1 xlO ⁶ CT26 wild type cells or 3x 10 ⁶ Hepal -6 wild type cells, respectively, were inoculated subcutaneously into mice to generate the local tumor. For all the different mouse models, mice were randomly assigned to treatment groups. The progression of tumor growth (with Luc) was monitored by an IVIS® Kinetics Optical System (Perkin Elmer, CA) after intraperitoneal (i.p.) injection of D- luciferin (10 mg/mL, 100 μL) to mice. The growth of subcutaneous tumors was followed by directly measuring the tumor size using an electronic caliper. The tumor volume was calculated by (LXW ² )/2, where L is the largest diameter measurement of the tumor and W is the shorter perpendicular tumor measurement. For survival studies, mice were euthanized when tumors reached -1500 mm ³ or mice lost more than 30% of their weight.

[0099] Depletion of macrophages, CD8 ⁺ T cells, or CD4 ^÷ T cells To elucidate the role of macrophage polarization in the activity of other immunosuppressive cells, the clodronate liposome (Liposoma BV, 10 mL/kg) was used to deplete the macrophages in tumor-bearing mice. The liposome was injected intravenously every 3 days (3 times in total), beginning 1 day before therapy. Depletion of macrophages was confirmed by flow cytometry. To evaluate which immune cells are required to confer the observed antitumor effect, specific cell subsets (CD8 ⁺ T cells, or CD4 ⁺ T cells) were depleted by administering a depleting antibody intraperitoneally every 3 days (4 times in total), beginning 1 day before therapy. The antibodies used for T-cell depletion were anti-mouse CD8a (clone 53-6.7) and anti-mouse CD4 (clone GK1.5), respectively. The polycolonal Rat IgG antibody was used as a control. All antibodies were purchased from BioXCell, and 100 pg of antibody was used. Depletion of CD8 ⁺ T cells, or CD4 ⁺ T cells was confirmed by flow cytometry. [0100] Quantitative reverse transcription polymerase chain reaction (RT-PCR) assay Cells and tumor tissues were homogenized using QIAshredder (Qiagen), followed by RNA extraction using the RNeasy Plus Mini Kit with gDNA Eliminator (Qiagen). Complementary DNA (cDNA) was synthesized using iScript Reverse Transcription Supermix (Bio-Rad) and amplified with the TaqManTM Gene Expression Master Mix. qRT-PCR was performed using the 7500 Real-Time PCR System and data were analyzed with the 7500 Software. All the mouse-specific primers are listed in Table 2. Expression was normalized to internal control GAPDH.

[0101] ELISA assay The BisCCL2/5i protein (engineered with a C-terminal E-tag) expression in the different organs and serum was measured by epitope tag (E-tag) sandwich ELISA. Tissue samples were prepared using a radio-immunoprecipitation (RIP A) buffer containing protease inhibitor cocktail mix and 0.5 M EDTA. The cell lysates were centrifuged, and the supernatant proteins were collected. A bicinchoninic acid (BCA) kit was used to measure the protein concentration according to the manufacturer’s protocols. ELISA assay was performed according to the manufacturer’s protocols after appropriate titration. For ELISA, flat-bottomed 96-well plates (ThermoFisher Scientific) were precoated with anti-E-tag antibody (Abeam, ab3397) at a concentration of 2 pg/mL per well in 100 mM carbonate buffer (pH 9.6) at 4 °C overnight. Then the 5% BSA in PBST was used to block the non-specific binding. The samples from liver tissue and serum were diluted 50 times in PBST buffer and added to the wells, while the samples from other organs were added to wells without dilution. After incubation at RT for 2 hr, the horseradish peroxidase (HRP)-conj ugated goat anti-mouse IgG (G-21040, Invitrogen) was used at a dilution of 1 : 2000 in the PBST buffer with 5% BSA. After incubation at RT for 1 hr, the HRP substrates were added and the optical densities were determined at a wavelength of 450 nm in the plate reader (Bio-Rad).

[0102] Flow cytometry Tumor tissues were harvested and digested with collagenase type I (200 U/mL, Invitrogen), collagenase type IV (200 U/mL, Invitrogen), and DNAase I (100 pg/mL, Invitrogen) in 2% FBS at 37 °C for 40-50 min to generate a single-cell suspension. After the cells were treated with ACK buffer, samples were diluted to 1 xlO ⁶ cells/mL for staining with LIVE/DEAD™ Fixable Near-IR dye (Thermo fisher, LI 0119). The single cells were stained with fluorescently conjugated antibodies according to the protocol of manufacturer. For intracellular staining, cells were permeabilized in Cytofix/Cytoperm solution (BD Biosciences) for 15 min. For the in vitro flow cytometry assay, cells were collected and stained with fluorescently conjugated antibodies. All antibodies were diluted after optimization. All the experiments were conducted in triplicate. Flow cytometry was performed on a BD LSRFortessa (BD Biosciences). All antibodies were purchased from BioLegend or BD Biosciences (Table 1). All the data were analyzed via FlowJo software (TreeStar, Ashland, OR) and singlets were gated on using FSC-H versus FSC-A.

[0103] Confocal microscopy Paraffin-embedded liver tissues from patients with hepatocellular carcinoma were provided by the Department of Hepatology at HwaMei Hospital of University of Chinese Academy of Sciences and approved by the ethics committee. Liver samples were collected during hepatic resection surgery and patient's identity of pathological specimens remained anonymous.

[0104] To assess the distribution of the LNPs, the DOPE-Cy5.5 was used to prepare the LNPs and the tumor cells were labeled with GFP. Tumor tissues for frozen sections were resected and placed in 4% paraformaldehyde (PFA) overnight at 4 °C. The resulting tissues were dehydrated with 15% and 30% sucrose solution, successively. Tissues were snap frozen in O.C.T. (Fisher Scientific) 10 pm thickness. The slices were mounted with Prolong® Diamond Antifade Mountant with DAPI (ThermoFisher Scientific) and imaged under a confocal laser scanning microscope (CLSM, Zeiss LSM 700).

[0105] For immunofluorescence staining, the sections were washed with PBS, blocked in 5 % goat serum at room temperature for 1 h and incubated with primaiy antibody overnight at 4 °C. The sections were washed and stained with fluorescent secondary antibody at R.T for 1 h. The cell nuclear was stained with DAPI and the slices were imaged under CLSM.

[0106] ECAR and OCR measurements BMDMs were incubated with BisCCL2/5i and seeded into XFe 96-well plates (Agilent) at 2x10 ⁵ per well. Before ECAR measurements, samples were washed and incubated in Seahorse media (Agilent) with 1 mM glutamine (Agilent). Before OCR measurements, samples were washed and incubated in Seahorse media (Agilent) with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose. The Glycolysis Stress Test Kit and Mito Stress Test Kit were prepared according to the manufacturer instructions.

[0107] Blood chemistry analysis and H&E staining After the final treatments, whole blood was obtained from the mice and the serum was collected. The concentrations of liver enzymes (aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) and the kidney function (blood urea nitrogen (BUN) and creatinine (CRE)) in the blood were measured across treatment groups. Blood cell counts including white blood cell (WBC) and red blood cell (RBC) counts were measured with whole blood analysis. All the measurements were performed by UNC histology facility at UNC Chapel Hill.

[0108] Statistical analysis Data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed by two-tailed Student’s t-test. Log-rank Mantel-Cox test was used for survival curves. Two-way ANOVA with multiple comparisons was used for comparison between multiple groups in tumor growth curve. All statistical analyses were done using GraphPad Prism

7.0.

[0109] Biodistribution and mRNA transfection analysis A time course biodistribution of the luciferase activity was performed. After the injection of Luc mRNA-LNPs, the luciferase activity at different time points was determined via Luciferase Assay System (Promega, USA) according to the manufacturer’s protocols. To determine the luciferase mRNA transfection in vivo, 6 hr after each injection of the mRNA-LNPs, mice were injected intraperitoneally with D-luciferin (10 mg/ml, 100 μL). The mice were anesthetized with 2.5% isoflurane in oxygen and imaged 5 min after the injection with AMI optical imaging system (Spectral Instruments Imaging, AZ). The repeated administration (in total 3 dosage) was performed to determine the luciferase expression after each injection. Luciferase activity on the different organs was quantified using the Luciferase Assay System.

[0110] To determine the biodistribution of mRNA-LNPs in the Hepal-6-GFP+ HCC tumor tissue, theDOPE-Cy5.5 (confocal microscope imaging) or DOPE-PE-Cy7 (flow cytometric analysis) was used to label LNPs and the mCherry mRNA (TriLink) was encapsulated into LNPs. HCC tumor-bearing mice were sacrificed 6 hr post the injection and the HCC tumor tissue was collected, followed by different performance. The biodistribution of LNPs and the mCherry expression were determined by the confocal laser scanning microscope. Moreover, the mCherry expression was also analyzed by flow cytometry. In addition, due to the rapid endosome escape, the HCC tumor-bearing mice were sacrificed 2 hr post the injection of LNP-PE-Cy7 for flow cytometric analysis to determine their internalization in different cell types.

[0111] For BisCCL2/5i protein expression in vivo, the serum was collected at different time points post the single injection of BisCCL2/5i mRNA-LNPs and the protein concentration was determined via ELISA assay. All pharmacokinetic and pharmacodynamic parameters including maximum concentration (Cmax), half-life (tl/2), area under the curve (AUC0-336hr), clearance and terminal slopes (λΖ) were calculated with Phoenix WinNonlin 7.0 (Certara, Princeton, NJ, USA). The repeated administration was also performed to determine the protein expression in different organs 6 hr after each injection.

Table 1. Antibodies

Table 2. Primers

Example 1

Identification of top candidate monocytes-associated genes that drive the M2 macrophage polarization in HCC

[0112] TAMs, most differentiated from monocytes, form a major component of the immune cells recruited to TME during cancer progression. In an attempt to assess the top candidates of monocyte-associated genes that prime the immunosuppression in the liver malignancy, the gene expression profiles (data extracted from the Gene Expression Omnibus database under the accession number GSE22058) of HCC liver tumor lesions and their matched adjacent normal liver samples from 197 patients were first analyzed (FIG. 1A and 13 A). Comparison of the gene expression profiles of monocyte attractants showed profound changes within the tumor environment (Fig. 13 A). Among the monocyte attractants, both CCL2 and CCL5 chemokines were significantly correlated with HCC cancer progression and were significantly upregulated (Log2 fold change >1.5, PO.OOOl, FDRcO.05) in the HCC tumor sites. Not only CCL2 and CCL5 were highly expressed in the tumor sites, but they drove TAMs accumulation. In contrast, no obvious difference was found between HCC malignant and normal liver tissues in CXCL5, CXCL10, and CSF2, which are known to promote Ml -phenotype polarization of macrophages. Two other monocyte-associated genes, CXCL12 and CD274 (PD-L1), were also significantly increased in HCC tumor sites compared to HCC-free adjacent sites. CXCL12, was also upregulated at HCC tumor sites compared to HCC-free adjacent sites (Log2 fold change >5.0, P<0.0001, FDR<0.05), supporting the potential target on CXCL12 signaling pathway. Consistent with the gene signature analysis, up-regulation of CCL2 and CCL5 was observed via immunofluorescence in HCC tumor tissues relative to normal liver tissues (FIG. IB). CCL2 and CCL5 appear to be the two top-ranked genes that trigger the tumor-infiltrating monocytes in liver cancer patients. In these human cases, the importance of monocyte recruitment and differentiation into M2-phenotype macrophages was also implicated in HCC cancer development.

[0113] To assess the potential impact of CCL2 and CCL5 on TAMs, gene expression correlation analysis in the human HCC tumor sites was performed. As shown in FIG. 19, gene expression of CCL2 and CCL5 was positively correlated with the expression of M2 -phenotype macrophage markers (MRC1 and IL10), suggesting the indispensable role of CCL2 and CCL5 in the M2 polarization of TAMs during HCC progression.

[0114] To evaluate the crosstalk between TAMs and tumor cells mediated by tumor-derived CCL2 and CCL5 in liver cancer progression, murine bone-marrow-derived macrophages (BMDMs) were exposed to the culture medium of tumor cells to mimic the interactions in TME. CCL2 and CCL5 were knocked down in murine Hepal -6 cells, a mouse HCC cell line with similar pathological features to human HCC, and then co-cultured the cancer cells with BMDMs to track the macrophage polarization in the presence or absence of CCL2 and CCL5 secreted microenvironment. Stable siRNA-mediated knockdown of CCL2 and CCL5 was confirmed by quantitative PCR (qRT-PCR) (FIGS. 7A-7B and 20A-20B).

[0115] Macrophages have functional plasticity, with the capacity to polarize their phenotype in response to differently educated microenvironment M2 macrophages produce large amounts of interleukin (IL) 10, arginase-1 (Arg-1), and CD206 (M2 markers), whereas Ml macrophages express high levels of IL12 and inducible nitric oxide synthase (iNOS) (Ml markers). qRT-PCR analyses confirmed that silencing either CCL2 or CCL5 suppressed the gene expression of M2 markers and increased the expression of Ml markers to some extent. However, compared to the mono depletions, the combination of CCL2 and CCL5 silencing was most effective in priming the macrophage toward the Ml phenotype (FIGS. 1C-1D and 13E-13F). Consistent with the finding from BMDMs, the co-culture of RAW 264.7 cells, a murine macrophage cell line, with tumor cells showed significant inhibition of M2 polarization (FIGS. 7C-7D and 20C-D). In both cell lines, the ratio of M1/M2 significantly increased with combined silencing of CCL2 and CCL5 (FIGS. 7E and 7F). Consistent with the analysis using tumor samples from human HCC patients, high levels of CCL2 and CCL5 were detected via IHC staining in murine orthotopic HCC tumor tissues compared to normal liver tissues (FIG. 25).

[0116] The anti-tumor efficacy when CCL2 or CCL5 alone was inhibited by using a neutralizing antibody or a CCR2/CCR5 dual antagonist (BMS-813160) in orthotopic HCC tumor-bearing mice was further investigated (FIG. 13G). As shown in FIGS. IE and IF, blockade of either CCL2 or CCL5 alone delayed tumor growth to some extent. However, such mono-blockade didn’t result in significant prolonged survival. Moreover, dual blockade of CCR2 and CCR5 via BMS-813160 showed negligible survival benefit, which might be caused by poor pharmacokinetic profile of small molecule through systemic administration. The existence of other CCL2 and CCL5 cognate receptors (e.g., CCR1 and CCR4) may also account for CCL2- and CCL5-driven chemotaxis, leading to the unsatisfactory antitumor effect of BMS-813160. In contrast, a significant survival benefit was observed when CCL2 and CCL5 were simultaneously blocked using a combination of two neutralizing antibodies. These results support that both CCL2 and CCL5 play roles in mediating the polarization of TAMs and driving the immunosuppressive response, and single blockade of CCL2 or CCL5 only confers limited survival benefit in the HCC tumor model.

Example 2

A unique single domain antibody that binds both CCL2 and CCL5 and blocks their biological activities

[0117] While two different affinity molecules can be genetically fused to achieve bispecificity, the development, production, and the delivery of such bispecific antibodies could be complicated. To address the difficulty in designing an affinity molecule that would bind both CCL2 and CCL5 at critical regions involved in interaction with their receptors with less than 50% homology, directed molecular evolution for a dual mCCL2 and mCCL5 inhibitor from a single domain antibody library (VH) displayed on yeast cell surface (~10 ⁸ variants) was performed. From the primary selections, VH sequences that tightly bound either CCL2 or CCL5 were successfully identified. Interestingly, one CCL2-binding VH sequence showed weak interaction with mCCL5. A sub-library based on this VH sequence via error-prone PCR was generated and used for the secondary selection in which mCCL2 and mCCL5 were used as the target in a sequential manner. Three more rounds of yeast surface display selection followed by optimization resulted in a unique single domain antibody that recognizes both mCCL2 and mCCL5 with desired bispecificity (BisCCL2/5i).

[0118] Unlike other bispecific affinity molecules typically composed of two moieties with different specificity, the resulting dual inhibitor BisCCL2/5i was composed of only one single domain (-135 amino acids) that could be encoded by an mRNA with less than 500-nucleotide for efficient gene delivery. BisCCL2/5i was shown to bind both mCCL2 and mCCL5 (FIG. 1G). The binding affinity and specificity of BisCCL2/5i to a panel of related chemokines were measured through microscale thermophoresis (MST). The evolved BisCCL2/5i was found to have a binding affinity of -11.5 nM and -9.4 nM for CCL2 and CCL5, respectively (FIG. 1H). Moreover, BisCCL2/5i is at least 100x more selective compared to other chemokines tested. An initial migration assay showed BisCCL2/5i potently inhibited CCL2- or CCL5-mediated migration of monocytes with an initial IC ₅₀ around 5.1 nM and 4.1 nM, respectively (FIG. II). Subsequent migration inhibition assays showed that BisCCL2/5i potently inhibited CCL2- or CCL5-mediated migration of macrophages, with IC ₅₀ values of approximately 4.0 nM and 2.6 nM, respectively (FIG. 14A), similar to those of anti-CCL2 and anti-CCL5 neutralizing antibodies (a-CCL2: IC ₅₀ 1.2 nM; a-CCL5: IC ₅₀ 2.2 nM, FIG. 21).

[0119] The treatment of BMDMs with BisCCL2/5i significantly increased the gene expression of Ml markers while suppressing the expression of M2-related genes (FIGS. 1 J- IK and 14B- 14C). The ratio of M1/M2 macrophages also increased after BisCCL2/5i treatment (FIG. 7G). Flow cytometric analysis confirmed a significant decline in the percentage of M2-phenotype macrophages after BisCCL2/5i and LPS (a classic Ml inducer) treatment, revealing a shift of macrophage polarization toward the Ml subtype (FIGS. 14D-14E). These results demonstrated that BisCCL2/5i can simultaneously block CCL2 and CCL5 signaling and promote macrophage polarization toward the cancer-inhibitory Ml phenotype.

Example 3

Dual Inhibition of CCL2 and CCL5 Reprograms the Tumor Immunosuppression

[0120] To target both CCL2 and CCL5 for Ml macrophage polarization while minimizing the systemic off-target side-effects, transient delivery of mRNA was performed encoding BisCCL2/5i via clinically validated lipid nanoparticles (LNPs) based on ionizable Dlin-MC3- DMA in a diseased organ specific manner in mouse models with liver malignance (FIGS. 2A and 2B). The mRNA-loaded LNPs with a diameter around 100-120 nm showed a high transfection efficacy in vivo, as demonstrated by using Luc mRNA as a reporter gene. Significantly, the expression of the delivered mRNA was almost exclusively restricted in the liver tissue, appearing much less in other organs (FIGS. 8A-8B, 15 A, and 22), effectively minimizing the side toxicities in non-diseased organs.

[0121] To determine which cell types within the liver tumor were transfected, mCherry mRNA- LNPs were delivered to an orthotopic HCC tumor model in which Hepal-6 tumor cells were stably transfected with a vector carrying GFP. As shown in FIG. 24, the LNPs were mainly internalized by Hepal-6 tumor cells (GFP ⁺ ) and monocytes (GDI lb ^" '). Moreover, the confocal images and flow cytometric results demonstrated that the internalized LNPs were able to transfect cells and induce the expression of mCherry protein (FIGS. 15B, 23 and 24). When mRNA encoding BisCCL2/5i was delivered by LNPs, the corresponding BisCCL2/5i protein showed highest expression in the liver compared with other major organs (FIG. 26C), consistent with the results from use of Luc mRNA.

[0122] To express the therapeutic BisCCL2/5i in vivo, 5 ’-capped and 3’-polyadenylated mRNA was in vitro transcribed and purified, and its biological activities were verified in a co-culture system with BMDMs and Hepal-6 cells (FIG. 8C). In initial studies, systemic injection of mRNA formulated-LNPs in animals resulted in a peak level of BisCCL2/5i protein in the serum after 6 hours with sustained decrease until Day 7 (FIG. 2C). Compared to other organs, BisCCL2/5i protein was almost exclusively expressed in the liver tissue, consistent with the expression of Luc mRNA (FIG. 8D). The cellular localization of LNPs was confirmed via immunofluorescence of mRNA-treated orthotopic HCC tumors (FIG. 8E), indicating the LNPs were efficiently internalized by tumor cells. In additional studies, the maximal protein concentration in plasma (2,086 ng/mL) was observed 6 hr after injection, followed by a decrease yet detectable measurements until day 3 (FIGS. 26A and 26B). The total protein level over time (area under the curve (AUC)) was approximately 34,403 ng½/mL. The decay phase (Az) in serum suggests a distribution process between central blood and peripheral tissues. It has been reported that repeated dosage of mRNA therapy substantially improved survival relative to a single dose in the MC38-R tumor model. Repeated administration of mRNA-LNPs induced comparable protein expression by quantifying the luciferase activity and BisCCL2/5i levels in different organs (FIGS. 22B and 26), supporting the feasibility of long-term treatment with the mRNA therapeutics.

[0123] Antitumor efficacy was further evaluated in the orthotopic HCC tumor model after treatment with either Mock (HcRed) mRNA-LNPs as control, BisCCL2/5i mRNA-LNPs, or combined CCL2- and CCL5-neutralizing antibodies, respectively· As shown in PIGS. 16A-16B, botii BisCCL2/5i and neutralizing antibodies resulted in prolonged survival relative to the Mock group. Notably, the BisCCL2/5i mRNA delivered by LNPs showed a greater survival benefit than neutralizing antibodies, indicating a dear advantage of using the BisCCL2/5i mRNA-loaded LNPs over administration of anti-CCL2 and anti-CCL5 antibodies simultaneously. To study the direct effect of BisCCL2/5i on macrophage polarity, the biomarkers associated with Ml and M2 phenotypes were evaluated via qRT-PGR analysis. Treatment with BisCCL2/5i mRNA-LNPs resulted in the suppression of M2-TAMs polarization with decreased expression of IL-10, Arg-1, and CD206 and increased expression of IL-12 and iNOS (PIGS. 2D-2E, 3C-3D, 15C-15D), respectively·

[0124] The polarization of macrophages was further analyzed by using flow cytometry. After BisCCL2/5i blockade treatment, about 25% of Ml-TAMs infiltrated into the tumors, an amount about 3 times higher than the untreated group, while the percentage of M2-TAMs significantly decreased (FIGS. 2F, 2G, and 15G), indicating the blockage of CCL2 and CCL5 reduced intratumcral trafficking of macrophages. The resulting ratio of M1/M2 within the BisCCL2/5i treatment group increased drastically by 8 times compared with untreated group (FIG. 2H). It is noteworthy that BisCCL2/5i treatment led to more than 50% reduction of M2 fraction in total macrophage population (20.8% vs. 8.0% of M2 fraction in total macrophages in Mock vs. BisCCL2/5i groups) (FIG. 15H) and 4.4-fold increase of M1/M2 ratio (0.3 vs. 1.31 in Mode vs. BisCCL2/5i groups) (FIGS. 15E-15F), suggesting that BisCC12/5i not only inhibited the further infiltration of macrophages, but also drove the polarization of existing M2 macrophage towards Ml subtype. These results indicated that tiie mRNA encoding this novel dual CCL2 and CCL5 inhibitor efficiently promoted the polarization of TAMs from the cancer-promoting M2 phenotype to the cancer-inhibitory Ml phenotype.

[0125] Other immunosuppressive cells including were also suppressed, suggesting that BisCCL2/5i not only disrupted the crosstalk between tumor cells and TAMs but also reprogramed the tumor immunosuppressive microenvironment (FIGS. 2K and 2L). No significant change in monocytic MDSCs (CD45 ⁺ CD11 blLy6C ^hi Ly6G ^low ) was observed between the Mock group and BisCCL2/5i group (FIG. 27B). However, the proportion of granulocytic MDSCs (CD45 ⁺ CDllb ⁺ Ly6G ^hi Ly6C ^low ), a predominate composition of MDSCs in most cancer types was decreased after BisCCL2/5i treatment (FIG. 27 A), indicating the existence of chemotaxis-mediated reduction of g-MDSC by blocking CCL2 and CCL5. The reduced proportion of Tregs (CD45 ⁺ CD25 ⁺ CD4 ⁺ Foxp3 ⁺ ) was also observed in the BisCCL2/5i treated group (FIG. 15H). The suppression of M2-phenotype macrophage polarization, g-MDSCs, and Tregs after BisCCL2/5i treatment indicated improved immunosuppression in the TME, which was confirmed by increased levels of CD8+ T cells (CD45 ⁺ CD3 ⁺ CD8 ⁺ ) and NK (CD45TNK1.1 ^"+" ) cells (FIGS. 15G and 27C). Additionally, Mock LNPs and BisCCL2/5i LNPs showed similar percentages of dendritic cells (DCs) (CDllbDCs and CD8 ⁺ T)Cs) (FIGS. 27E and 27F), suggesting BisCCL2/5i does not promote activation of antigen-presentation. These results demonstrated that BisCCL2/5i efficiently promoted the polarization of TAMs from the cancer- promoting M2 phenotype to the cancer-inhibitory Ml phenotype and shifted the immunocellular composition of TME into antitumor immunity.

[0126] The above findings raised the question whether the M2 to Ml polarization of TAMs affects the activity of other immune cells such as MDSCs and Tregs. To address the question, clodronate liposomes were used to deplete the macrophages in tumor-bearing mice (FIG. 21), as confirmed by flow cytometry analysis (FIG. 2J). The ratio of Tregs in clodronate liposome- treated mice decreased drastically compared to the untreated group (FIG. 2K), indicating the influence of TAMs on Tregs recruitment Significantly, in the macrophage depleted groups, the percentage of Tregs within the BisCCL2/5i treatment showed 7 times lower (0.02%) in comparison with the mock group (0.14%), suggesting that dual blockade of CCL2 and CCL5 may directly inhibit the Treg recruitment. On the other hand, the macrophage depletion did not result in a significant difference of MDSCs when compared to the non-depleted group. In contrast, MDSCs decreased in both BisCCL2/5i-treated groups (with or without depletion), with a more reduced level observed in the depletion group, indicating that BisCCL2/5i also inhibited the attraction of MDSCs into tumor sites (FIG. 2L). In order to explore whether BisCCL2/5i can fundamentally eradicate the differentiation of TAMs from tumor-infiltrating monocytes, the macrophage recovery rate was measured after depletion and BisCCL2/5i-treated mice had a much lower recovery rate than the untreated group, illustrating the fundamental eradication of TAMs differentiation from monocytes (FIG. 2M). The increase of CD8 ⁺ T cells after macrophage depletion confirmed that TAMs are associated with T cell infiltration. Moreover, treatment with BisCCL2/5i resulted in a significant increase of CD8 ⁺ T cells in the mice with macrophage depletion, suggesting that BisCCL2/5i can reprogram the immunosuppressive TME and drive T cell infiltration into tumor sites (FIG. 2N).

Example 4

Dual Blockade of CCL2 and CCL5 Greatly Sensitizes HCC Tumors to ICB Therapy

[0127] To deliver an immune checkpoint inhibitor specifically to the diseased organ, a trimeric PD-1 ligand inhibitor (PD-Li) was developed by fusing the extracellular domain of PD-1 (117 aa) with a small trimerization domain (TMD, 43 aa) from cartilage matrix protein- 1 (CMP-1), an extracellular protein that is highly abundant in both mouse and human cartilages (FIG. 9). The resulting trimeric fusion protein bound both PD-1 ligands with picomolar affinities and effectively blocked the immunosuppressive signaling pathways mediated by PD-1 and its ligands in various syngeneic tumor mouse models. In the current study, the PD-1 ligand inhibitor is encoded in an mRNA of less than 600-nucleotide, a size that can be efficiently encapsulated in LNPs for liver-specific delivery in the same manner as that for the delivery of BisCCL2/5i. The therapeutic efficacy of BisCCL2/5i and PD-Li in treating mice bearing well-established (—15 d) and relatively large subcutaneous Hepal-6 HCC tumors via intratumoral injection (FIGS. 3A-3E and 16C-16G) was investigated. Treatment with BisCCL2/5i mRNA-LNPs or PD-Li mRNA- LNPs alone inhibited the tumor growth and prolonged the survival time relative to the PBS group. However, both monotherapies resulted in only a modest suppression of tumor growth and did not confer a significant survival benefit. Significantly, the combination treatment of BisCCL2/5i mRNA-LNPs and PD-Li mRNA-LNPs resulted in a 27-fold decrease in the tumor volume and substantial increase in the survival time relative to the monotherapies. While treatment with control mRNA-LNPs did not affect tumor growth, approximately 33% (4 out of 12) or 50% (3 out of 6) of mice treated with BisCCL2/5i plus ICB therapy completely rejected tumors, with no evidence of tumor burden at least 80 days after tumor inoculation (FIG. 3D and 16F). These results corroborated the in vitro data described above and demonstrated the effectiveness of BisCCL2/5i-mediated reeducation of TME in sensitizing the tumors for ICB response.

[0128] While localized intratumoral delivery of immunomodulators is emerging as a clinically viable treatment for some cancer types, this administration route is not clinically feasible for most solid tumors, particularly in the setting of advanced metastatic diseases in the liver. Furthermore, orthotopic tumors are much more challenging to cure than subcutaneous tumors since tissues in the orthotopic sites play critical roles in shaping and altering the TME and influencing the ICB response. An orthotopic HCC large tumor model (~15 d) was established to evaluate the therapeutic efficacy of BisCCL2/5i in sensitizing tumor to ICB therapy (FIGS. 3F and 16H). Tumor weight measurements and survival rates showed a modest therapeutic benefit with the monotherapies (FIGS. 3G and 161). Systemic administration of BisCCL2/5i mRNA- LNPs significantly improved the response to ICB, with the Kaplan-Meier survival at 24 or 32 days (PBS), 32 or 37 days (BisCCL2/5i therapy), 34 or 35 days (ICB therapy), and 43 or 49 days (BisCCL2/5i plus ICB combination therapy), respectively, demonstrating the BisCCL2/5i therapy sensitizes the tumors to ICB therapy (FIGS. 3H and 16J).

[0129] From the point of view of clinical treatment, the orthotopic HCC tumor model may not mimic human HCC cancers which are often unresectable and have spread throughout the whole liver. A hemi-spleen approach was adopted that allowed for efficient establishment of uniform and diffused HCC in the liver. Hepal-6 tumor cells were administrated specifically to the liver via a hemi-spleen injection and the treatment was initiated in mice bearing diffused (~5 d) tumor (FIG. 31 and 16K). Notably, about 58% (7 out of 12) to 66% of mice (4 out of 6) administered BisCCL2/5i in combination with PD-Li exhibited complete antitumor responses, without evidence of residual tumor burden at least 50-70 days after the tumor cell inoculation (FIGS. 3J- 3K and 16L-16M). These results demonstrated that the combination therapy can confer a significant survival benefit and tumor eradication in diffused liver cancers. The immune status of TME was analyzed and the BisCCL2/5i treatment increased CD8 ⁺ T cells by approximately 2.5- fold in tumor sites (FIG. 2N), whereas the combination therapy increased infiltrating CD8 ⁺ T cells more efficiently (FIGS. 3N and 30). The CD8 ⁺ T cells showed about 5-fold higher infiltration in the combination treatment group than that in the control group. 'The important involvement of CD4 ⁺ and CD8 ⁺ T cells in the treatments was further verified by a depletion study in which anti-CD4 or anti-CD8 mAbs significantly compromised therapeutic efficacy compared to the IgG control and CDS ^* T cells appeared to play a more important role in antitumor immunity than CD4 ^* T cells (FIGS. 3P and I6N). Additionally, cytokines and chemokines are important mediators in manipulating the tumor immune microenvironment. Indeed, the level of IFN-γ, the lead cytokine in the Th-1 response associated with cytotoxic T- cell killing and improved clinical outcome, was much higher while the release of immunosuppression inducing TGF-β showed 4-fold decrease in the combination therapy than in the monotherapies or mock groups, demonstrating the synergistic antitumor immune responses in the HCC tumor types (FIGS. 3L and 3M).

[0130] Systemic side toxicity is a major concern in the anti-cancer therapies. The safety of LNPs-mRNA delivery strategy was confirmed in the orthotopic HCC tumor models. The blood was collected and subjected to a blood panel analysis. No abnormal changes of white blood cell (WBC) and red blood cell (RBC) counts were observed, indicating the lower immunogenicity of LNPs (FIGS. 10A and 10B). The lack of detectable systemic toxicity was further confirmed by the normal liver function (ALT and AST levels) and the kidney function (blood urea nitrogen (BUN) and creatinine (CREAT)) in the blood across all the treatment groups (FIGS. 1 OC-IOF). Moreover, body weight was consistent across all groups tested (FIG. 10G). It should be noted that immunotherapy-related adverse events (irAEs) are the common complication of systemic administration of immunotherapeutics. As reported, Thl7 cells are highly upregulated in inflammatory tissues of autoimmune diseases. Therefore, the ratio of Thl7 cells as the parameter to monitor the irAEs of immunotherapy was measured. No significant upregulation of Thl7 cells was observed in the spleen from the treated groups, indicating the LNP-mRNA local delivery and transient expression system specific to the diseased organ may allow the mitigation of irAEs (FIG. 10H).

Example 5

Dual Blockade of CCL2 and CCL5 Greatly Sensitizes CRC Liver Metastatic Tumors to

ICB Therapy

[0131] To evaluate whether BisCCL2/5i therapy can sensitize other cancer types to ICB therapy, colorectal cancer, one of the leading causes of death for patients with gastrointestinal (GI) malignances, was established subcutaneously using murine CT26 cell line (FIG. 4A). As shown in FIGS. 4B-4C, tumor growth was inhibited after different treatments relative to the PBS group, while the combination of BisCCL2/5i and PD-Li treatment resulted in a significant synergistic antitumor effect. The median survival in the combination treatment was 2-fold prolonged compared to the PBS group (FIG. 4D), indicating the capacity of BisCCL2/5i to enhance the response of CRC to ICB therapy. Clinically, the high recurrence rate in the CRC patients is induced by residual metastases after surgery, and liver metastasis is the major cause of death in patients with CRC.

[0132] The antitumor efficacy of BisCCL2/5i and ICB combination therapy was further evaluated by using murine CT26 CRC liver metastasis tumor model (FIG. 4E), which also shows signatures including high expression levels of CCL2 and CCL5 and enriched macrophages (-30%) within the metastatic lesions (FIGS. 25 and 29B). BisCCL2/5i-mediated immune microenvironment modulation was confirmed by upregulated CD3 ⁺ T cell infiltration, downregulated macrophage accumulation and decreased M2-phenotype inside CT26 liver metastases (FIGS. 29A and 29C). As demonstrated in FIGS. 4F and 4H, the BisCCL2/5i monotherapy exhibited an effect on the inhibition of liver metastasis. However, the combination of BisCCL2/5i with ICB therapy effectively controlled the liver metastatic tumor growth and prolonged the survival time significantly compared to monotherapies (FIG. 4G). The increase of CD3 ⁺ and CD8 ⁺ cells was observed from immunofluorescent images and the CD8 ⁺ T cells showed higher infiltration in the combination treatment than the control group (FIGS. 11 A and 1 IB). These results indicated that CRC tumors and CRC liver metastasis tumors can also be sensitized to ICB therapy by BisCCL2/5i-mediated reeducation of tumor immunosuppression, and this strategy has the potential to become a potent combination immunotherapy in different cancer types with liver metastasis.

[0133] To evaluate whether BisCCL2/5i therapy can sensitize liver metastatic tumors to ICB therapy, pancreatic cancer liver metastasis was established using a KPC-GFP-Luc cell line. Enriched macrophage infiltration (-25%) (FIG. 17A) and upregulation of CCL2 and CCL5 as shown by IHC staining (FIG. 25) in KPC liver metastatic tumors implied the suitability of BisCCL2/5i treatment. Similar to the HCC tumor, BisCCL2/5i treatment decreased M2-polarized macrophage and MDSC populations inside KPC liver metastases (FIG. 17B and 17C), presumably leading to increased intratumoral infiltration of CD3 ⁺ T cells and in particular CDS ⁺ T cells (Fig. 28D) compared to the Mock group. As expected, BisCCL2/5i monotherapy significantly mitigated the progression of liver metastasis, while PD-Li monotherapy showed negligible tumor inhibition compared to the Mock treatment (FIGS. 17F-17H). Relative to monotherapies, the combination of BisCCL2/5i with PD-Li most effectively controlled liver metastatic KPC tumor growth, inducing a complete response in approximately 57% of mice.

Example 6

Dual Blockade of CCL2 and CCL5 Induces a Shift in Macrophage Metabolism that

Regulates die Macrophage Polarity

[0134] Polarized macrophages not only differ in cytokine production and receptor expression but also in metabolic processes. It has been reported that GPCRs are related to the glucose uptake via the regulation of glucose transporters GLUT1 and GLUT4, and the glucose metabolism through phosphorylation of hexokinase 1 (HK-1) and hexokinase 2 (HK-2) to produce glucose-e- phosphate. By analyzing the phenotype ofBMDMs in the presence and absence of glucose, the polarization of macrophages was found to be associated with glucose-mediated energy generation. Without the addition of glucose, BMDMs tended to undergo M2 polarization, while Ml polarization was promoted by the high dose of glucose, suggesting the macrophage polarization could be determined through metabolic pathways in response to the glucose level in the TME (FIGS. 5A-5B and 30). Compared to glucose deprived environment, BisCCL2/5i treatment induced more effective polarization of M2 macrophages towards Ml -phenotype in the presence of high concentration of glucose (1.1% vs. 31.3% of M2 macrophages after treatment with BisCCL2/5i in (+) Glu vs. (-) Glu groups), demonstrating that BisCCL2/5i-mediated Ml- phenotype transition mainly relied on glycolysis. The remaining Ml polarization effect by BisCCL2/5i in glucose-deprived condition suggests the existence of other polarization-associated biological processes that are regulated by BisCCL2/5i

[0135] The contribution of CCL2 and CCL5 to the central metabolic rewiring during alternative activation of macrophages remains elusive. To study the role of CCL2 and CCL5 in regulating the glycolysis of macrophages, the glycolysis-related genes were evaluated via qRT-PCR analysis. The upregulation of GLUT1 expression was observed in BisCCL2/5i-treated BMDMs, which consequently enhanced the glucose uptake. Indeed, metabolic enzymes involved in glucose metabolism, including HK-1, HK-2, lactate dehydrogenase A (LDHA), and pyruvate kinase (PKM), increased significantly in BisCCL2/5i-induced BMDMs (FIG. 5C), suggesting the key role of CCL2 and CCL5 in regulating the glycolysis of BMDMs. Since fatty acid oxidation is the main metabolic program in M2 macrophages, the fatty acid metabolism-related gene expression was assessed in BisCCL2/5i-treated BMDMs (FIG. 5D). The expression of carnitine palmitoyltransferase la (CPTla), a regulatory enzyme that transfers fatty acid to mitochondria prior to β-oxidation, was reduced after BisCCL2/5i treatment, indicating the decreased transport of fatty acid to mitochondria. The long chain acyl-CoA dehydrogenase (LCAD) that modulates β-oxidation of fatty acid also decreased while no significant difference was observed on the long-chain L-3-hydroxyacyl-coenzyme A dehydrogenase a (HADHa). Since fatty acid oxidation is activated upon the accumulation of fatty acids in the mitochondria, straight-chain acyl-CoA oxidase- 1 (ACOX-1), an enzyme responsible for the initial oxidation of long-chain fatty acyl-CoAs was analyzed. A lower expression of ACOX-1 was observed in BisCCL2/5i-induced BMDMs relative to the mock group, indicating BisCCL2/5i could lead to reduced fatty acid β-oxidation (FAO) in macrophages. The expression of fatty acid synthase (FAS) and CD36, two genes involved in de novo lipid biosynthesis and lipid accumulation, was highly increased in BisCCL2/5i-treated BMDMs, suggesting dual blockade of CCL2 and CCL5 resulted in de novo lipid synthesis. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured to explore the impact of BisCCL2/5i on the metabolic state of macrophages. BisCCL2/5i significantly increased ECAR of BMDMs relative to the mock group, indicating an increase in glycolytic flux (FIG. 5E). Drastically, the basal ECAR of macrophages had a 12-fold increase in the BisCCL2/5i treated group compared to the control group (FIG. 5F). However, BisCCL2/5i reduced the basal OCR of macrophages, signifying lower rates of oxidative phosphorylation (FIG. 5G). These data indicated that BisCCL2/5i resulted in enhanced glycolysis and reduced oxidative phosphorylation of macrophages, which predicted the metabolic process of Ml macrophage.

[0136] The expression of glycolysis- and fatty acid metabolism-related genes was measured in BisCCL2/5i-treated BMDMs via qRT-PCR analysis. The upregulation of GLUT1 expression was observed in BisCCL2/5i-treated BMDMs, which consequently enhanced glucose uptake. Indeed, metabolic enzymes involved in glucose metabolism, including HK-1, HK-2, lactate dehydrogenase A (LDHA), and pyruvate kinase (PKM), all increased significantly in BisCCL2/5i-induced BMDMs (FIG. 18A), suggesting a key role of CCL2 and CCL5 in regulating glycolysis in BMDMs. The expression of carnitine palmitoyltransferase la (CPTla), a regulatory enzyme that transfers fatty acid to mitochondria prior to β-oxidation, was reduced after BisCCL2/5i treatment, indicating decreased transport of fatty acids to mitochondria (FIG. 18B). Long chain acyl-CoA dehydrogenase (LCAD), which modulates the β-oxidation of fatty acids, also decreased, while no significant difference in long-chain L-3 -hydroxyacyl-coenzyme A dehydrogenase a (HADHa) was observed. As fatty acid oxidation is activated upon the accumulation of fatty acids in the mitochondria, straight-chain acyl-CoA oxidase- 1 (ACOX-1), an enzyme responsible for the initial oxidation of long-chain fatty acyl-CoAs, was analyzed. Lower expression of ACOX-1 was observed in BisCCL2/5i-induced BMDMs than in the PBS group, indicating that BisCCL2/5i could lead to reduced fatty acid β-oxidation (FAO) in macrophages. The expression of fatty acid synthase (FAS) and CD36, two genes involved in de novo lipid biosynthesis and lipid accumulation, was highly increased in BisCCL2/5i-treated BMDMs, suggesting that dual blockade of CCL2 and CCL5 resulted in de novo lipid synthesis. These data indicated that BisCCL2/5i resulted in enhanced glycolysis and reduced oxidative phosphorylation in macrophages, which was consistent with the metabolic process of Ml macrophages.

[0137] Collectively, dual blockade of CCL2 and CCL5 via BisCCL2/5i enhanced glycolysis and lipid synthesis, reduced oxidative phosphorylation, and FAO metabolic shift, effectively enabling the polarization of cancer-inhibitory Ml macrophages while suppressing the cancer- promoting M2 macrophages.

[0138] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.

[0139] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and may be made without departing from the spirit and scope thereof. [0140] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0141] Clause 1. An isolated single domain antibody, or a fragment or derivative thereof, that specifically binds both CCL2 and CCL5.

[0142] Clause 2. The isolated single domain antibody of clause 2, comprising an amino acid sequence with at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. [0143] Clause 3. An isolated single domain antibody, or a fragment or derivative thereof, that specifically binds to CCL2.

[0144] Clause 4. The isolated single domain antibody of clause 3, comprising an amino acid sequence with at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% amino acid sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0145] Clause 5. A polynucleotide comprising a nucleic acid encoding the isolated single domain antibody of any of clauses 1-4.

[0146] Clause 6. The polynucleotide of clause 5, wherein the polynucleotide is an mRNA.

[0147] Clause 7. A pharmaceutical composition comprising the single domain antibody of any of clauses 1-4, or the polynucleotide of any of clauses 5-6.

[0148] Clause 8. The pharmaceutical composition of clause 7, further comprising a checkpoint inhibitor or a nucleic acid encoding a checkpoint inhibitor.

[0149] Clause 9. The pharmaceutical composition of clause 8, wherein the checkpoint inhibitor is a PD-1 ligand inhibitor or PD-1 inhibitor.

[0150] Clause 10. The pharmaceutical composition of any of clauses 7-9, further comprising a viral nanoparticle or a non-viral nanoparticle.

[0151] Clause 11. The pharmaceutical composition of clause 10, wherein the non-viral nanoparticle is a lipid nanoparticle.

[0152] Clause 12. The pharmaceutical composition of clause 11, wherein the polynucleotide, the nucleic acid encoding a checkpoint inhibitor, or a combination thereof is encapsulated in lipid nanoparticles.

[0153] Clause 13. A method of treating cancer in a subject in need thereof comprising administering an effective amount the isolated antibody or a fragment or derivative thereof of any of clauses 1-4, the polynucleotide of any of clauses 5-6, or the pharmaceutical composition of any of clauses 7-11.

[0154] Clause 14. The method of clause 13, wherein the cancer comprises a solid tumor. [0155] Clause 15. The method of clause 13 or 14, wherein the cancer is selected from the group consisting of liver cancer, pancreatic cancer, gastrointestinal cancer, lung cancer, ovarian cancer, eye cancer, and combinations thereof.

[0156] Clause 16. The method of any of clauses 13-15, wherein the cancer comprises hepatocellular carcinoma, colorectal cancer, gastric cancer, pancreatic ductal adenocarcinoma, lung cancer, ovarian cancer, eye cancer, cholangiocarcinoma, or a combination thereof.

[0157] Clause 17. The method of any of clauses 13-16, wherein the cancer comprises liver metastasis of hepatocellular carcinoma, colorectal cancer, gastric cancer, pancreatic ductal adenocarcinoma, lung cancer, ovarian cancer, uveal melanoma, cholangiocarcinoma, or a combination thereof.

[0158] Clause 18. The method of any of clauses 13-17, wherein the administration is intratumoral, peripheral to the tumor, or systemic.

[0159] Clause 19. The method of any of clauses 13-18, further comprising administering to the subject an effective amount of a conventional cancer treatment.

[0160] Clause 20. The method of any of clauses 13-19, wherein the conventional cancer treatment is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, proton therapy, photodynamic therapy, and surgery.

[0161] Clause 21. A method of inhibiting the growth or survival of a cancer cell comprising contacting a cancer cell with an effective amount the isolated antibody of any of clauses 1-4, the polynucleotide of any of clauses 5-6, or the pharmaceutical composition of any of clauses 7-11. [0162] Clause 22. The method of clause 21, wherein the contacting is performed in vitro in cells or tissues.

[0163] Clause 23. The method of clause 22, wherein the cells or tissues comprise human cells or tissues.