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WO2021113648A1 - Molécules d'affinité dirigeant le métabolisme et la polarisation de macrophages et permettant une synergie de la thérapie de blocage de point de contrôle immunitaire - Google Patents

Molécules d'affinité dirigeant le métabolisme et la polarisation de macrophages et permettant une synergie de la thérapie de blocage de point de contrôle immunitaire Download PDF

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WO2021113648A1
WO2021113648A1 PCT/US2020/063328 US2020063328W WO2021113648A1 WO 2021113648 A1 WO2021113648 A1 WO 2021113648A1 US 2020063328 W US2020063328 W US 2020063328W WO 2021113648 A1 WO2021113648 A1 WO 2021113648A1
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cancer
tumor
ccl2
bisccl2
cells
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Rihe Liu
Ying Wang
Karthik TIRUTHANI
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University of North Carolina at Chapel Hill
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • 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.
  • mAb Monoclonal antibodies 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.
  • TAMs tumor-associated macrophages
  • MDSCs myeloid-derived suppressor cells
  • Tregs regulatory T cells
  • liver cancer 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).
  • HCC hepatocellular carcinoma
  • liver metastasis is the second common sites of metastatic spread, in particular for colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC).
  • CRC colorectal cancer
  • PDAC pancreatic ductal adenocarcinoma
  • 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.
  • 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.
  • 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.
  • 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,
  • 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. 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
  • 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).
  • 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. 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).
  • FIGS. 2A-2N show that the dual blockade of CCL2 and CCL5 via LNP
  • 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).
  • 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.
  • FIG. 21 is a schematic of the macrophage depletion and treatment method to elucidate the role of macrophage polarization in priming tumor immunosuppression.
  • FIG. 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.
  • 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.
  • 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. 31 is a treatment scheme for HCC tumors established by hemi-spleen approach.
  • FIG. 31 is a treatment scheme for HCC tumors established by hemi-spleen approach.
  • FIG. 3N is representative flow dot plots of CD3 + CD8 + T cells in HCC tumor tissue.
  • 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. 4E is a treatment scheme for colorectal cancer liver metastasis tumors treated intravenously with various formulations.
  • 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. 5 A is representative flow dot plots of M2 macrophages in tumor cell educated-BMDMs cultured in low-dose or high-dose glucose.
  • FIG. 5C is a graph of the
  • OCR oxygen consumption rate
  • 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.
  • FIGS. 7C and 7D are graphs of the mRNA expression of classic Ml and M2
  • 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
  • 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.
  • 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).
  • 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.
  • 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.
  • FIGS. 11 A-l IB show that BisCCL2/5i mRNA gene delivery therapy synergizes ICB therapy in the CT26-FL3 liver metastasis model.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIGS. 14A-14E show that the single domain antibody that binds both CCL2 and CCL5 blocks their biological activities.
  • 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.
  • 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. 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).
  • 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).
  • ⁇ 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.
  • 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.
  • 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.
  • 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. 16K is a treatment scheme for HCC tumors established by hemi- spleen approach.
  • FIGS. 17A-17H show that a dual blockade of CCL2 and CCL5 sensitized KPC liver metastasis tumor to ICB therapy.
  • FIG. 17E is a treatment scheme for KPC liver metastasis tumors administered intravenously with various formulations (mRNA: 1 mg/kg).
  • 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.
  • 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.
  • 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.
  • FIGS. 22A-22B are graphs of the biodistribution of the luciferase activity after the treatment with Luc mRNA-LNPs.
  • 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.
  • 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
  • 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.
  • 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
  • the mean plasma concentrationtime profile FIG. 26B
  • the BisCCL2/5i mRNA was mainly expressed in the liver tissue and the repeated administration showed comparable protein level.
  • 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 + ).
  • 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,
  • 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.
  • 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.).
  • FIG. 29B 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
  • 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.
  • 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.
  • 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.
  • 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.
  • TAMs Tumor-associated macrophages
  • 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.
  • GPCRs G-protein coupled receptors
  • 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.
  • MDSCs myeloid-derived suppressor cells
  • Tregs regulatory T cells
  • 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.
  • 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.
  • Antibody fragment 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.
  • “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.
  • “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.
  • 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.
  • 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.
  • a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.
  • 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.
  • polypeptide and “protein,” are used interchangeably 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.
  • 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.
  • 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.
  • 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.
  • non-mammals include, but are not limited to, birds, fish and the like.
  • the mammal is a human.
  • treat 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.
  • 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.
  • the present disclosure relates to isolated single domain antibodies that specifically bind both CCL2 and CCL5 or CCL2.
  • the isolated 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.
  • 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.
  • 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).
  • the isolated single domain antibody, antibody fragment or derivative thereof specifically binds CCL2.
  • 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.
  • 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).
  • 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.
  • the polynucleotide is an mRNA.
  • compositions comprising a single domain antibody or a polynucleotide as described herein in Section 2.
  • the pharmaceutical composition further comprises one or more additional cancer immunotherapy agents.
  • 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, TI ⁇ 3, 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).
  • Immune checkpoint inhibitors may reduce the function of the immune checkpoint protein or result in a full blockade.
  • the checkpoint inhibitor is a PD-1 ligand inhibitor or PD-1 inhibitor.
  • the checkpoint inhibitor may be those disclosed in WO 2017/053170, the contents of which are herein incorporated herein by reference.
  • compositions and formulations may include pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carrier means a nontoxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • 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 sa
  • compositions 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.
  • 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.
  • 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 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.
  • the non-viral nanoparticle is a lipid nanoparticle.
  • the polynucleotide, the nucleic acid encoding a checkpoint inhibitor, or a combination thereof is encapsulated in the lipid nanoparticle.
  • 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).
  • lipids include, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides).
  • phosphatidyl compounds e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
  • 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.
  • 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.
  • the effective amount ranged from 0.25 mg/kg to 8 mg/kg, dosed over 3-4 doses, every 3-5 days.
  • 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
  • the cancer is selected from the group consisting of liver cancer, pancreatic cancer, gastrointestinal cancer, lung cancer, ovarian cancer, eye cancer, and combinations thereof.
  • 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.
  • 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.
  • the administration is intratumoral, peripheral to the tumor, or systemic.
  • the methods may further comprise a combination treatment with other agents.
  • 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.
  • 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.
  • FBS fetal bovine serum
  • 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.
  • mice Animal experiments were carried out in accordance with approved protocols.
  • HCC liver cancer and KPC liver metastasis model male C57BL/6J mice (5-6 weeks old) were purchased from the Jackson Laboratory (stock #000664).
  • female BALB/cJ mice (6-8 weeks) were purchased from the Jackson Laboratory (stock #000651).
  • 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.
  • 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
  • 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
  • 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.
  • PBS A PBS + 0.1% BSA
  • VH sequences that bispecifically bind both mCCL2 and mCCL5 were used as the parental sequence for error-prone PCR using the GeneMorph ⁇ 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.
  • 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.
  • 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 ⁇ L 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 HisPurTM Ni- NTA Resin (Thermo Fisher). The purified proteins were analyzed on 4-12% SDS-PAGE gel (Invitrogen) with Coomassie G-250 (Bio-Rad) stain.
  • 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.
  • 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.
  • MST microscale thermophoresis
  • purified BisCCL2/5i was first fluorescently labeled by using REDtris-NTA dye according to standard manufacturer protocols.
  • Bio-layer Interferometry (BLI) analyses of the interaction between recombinant PD-Li and PD-LI were performed on forteBIO Octet QK system.
  • Ni-NTA or SAX biosensors Pall forteBIO Corp
  • 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.
  • 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 (MEGAscriptTM T7 Transcription Kit, Thermo Fisher).
  • 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).
  • 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.
  • the mouse was anesthetized and the upper abdomen was opened for tumor cell inoculation.
  • the murine Hepal -6 cells (2*10 6 to 5x1 ⁇ 6 cells/mouse) are inoculated at the subcapsular region of the left lobes of the liver.
  • 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.
  • 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 ⁇ L) 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.
  • 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.
  • CD8 + T cells or CD4 + T cells
  • CD8 + T cells or CD4 + T cells
  • 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.
  • RT-PCR Quantitative reverse transcription polymerase chain reaction
  • 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.
  • RIP A radio-immunoprecipitation
  • BCA bicinchoninic acid
  • ELISA 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.
  • HRP horseradish peroxidase
  • G-21040 goat anti-mouse IgG
  • Invitrogen horseradish peroxidase
  • 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/DEADTM Fixable Near-IR dye (Thermo fisher, LI 0119). The single cells were stained with fluorescently conjugated antibodies according to the protocol of manufacturer.
  • 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).
  • BMDMs were incubated with BisCCL2/5i and seeded into XFe 96-well plates (Agilent) at 2x10 5 per well.
  • samples were washed and incubated in Seahorse media (Agilent) with 1 mM glutamine (Agilent).
  • 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.
  • TAMs most differentiated from monocytes, form a major component of the immune cells recruited to TME during cancer progression.
  • 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).
  • 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.
  • CXCL12 and CD274 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.
  • BMDMs murine bone-marrow-derived macrophages
  • 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).
  • 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).
  • IL interleukin
  • Arg-1 arginase-1
  • M2 markers CD206
  • Ml macrophages express high levels of IL12 and inducible nitric oxide synthase (iNOS) (Ml markers).
  • iNOS inducible nitric oxide synthase
  • CCL2 and CCL5 cognate receptors may also account for CCL2- and CCL5-driven chemotaxis, leading to the unsatisfactory antitumor effect of BMS-813160.
  • CCL2 and CCL5 were simultaneously blocked using a combination of two neutralizing antibodies.
  • a unique single domain antibody that binds both CCL2 and CCL5 and blocks their biological activities
  • 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).
  • 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).
  • MST microscale thermophoresis
  • 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).
  • 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).
  • 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.
  • the LNPs were mainly internalized by Hepal-6 tumor cells (GFP + ) and monocytes (GDI lb " ').
  • 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).
  • 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.
  • 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).
  • CMP-1 cartilage matrix protein- 1
  • 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.
  • 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.
  • 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).
  • mice bearing diffused ( ⁇ 5 d) tumor FIG. 31 and 16K.
  • CD4 + and CD8 + T cells were 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.
  • the level of IFN- ⁇ , 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- ⁇ 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).
  • irAEs immunotherapy-related adverse events
  • 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).
  • 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.
  • CPTla carnitine palmitoyltransferase la
  • LCAD long chain acyl-CoA dehydrogenase
  • HADHa long-chain L-3-hydroxyacyl-coenzyme A dehydrogenase a
  • 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 ⁇ -oxidation (FAO) in macrophages.
  • FOS fatty acid synthase
  • 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).
  • the basal ECAR of macrophages had a 12-fold increase in the BisCCL2/5i treated group compared to the control group (FIG. 5F).
  • BisCCL2/5i reduced the basal OCR of macrophages, signifying lower rates of oxidative phosphorylation (FIG. 5G).
  • CPTla carnitine palmitoyltransferase la
  • LCAD Long chain acyl-CoA dehydrogenase
  • ACOX-1 straight-chain acyl-CoA oxidase- 1
  • FEO reduced fatty acid ⁇ -oxidation
  • 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.
  • 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.
  • Clause 3 An isolated single domain antibody, or a fragment or derivative thereof, that specifically binds to CCL2.
  • 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.
  • Clause 5 A polynucleotide comprising a nucleic acid encoding the isolated single domain antibody of any of clauses 1-4.
  • 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.
  • Clause 8 The pharmaceutical composition of clause 7, further comprising a checkpoint inhibitor or a nucleic acid encoding a checkpoint inhibitor.
  • Clause 10 The pharmaceutical composition of any of clauses 7-9, further comprising a viral nanoparticle or a non-viral nanoparticle.
  • Clause 11 The pharmaceutical composition of clause 10, wherein the non-viral nanoparticle is a lipid nanoparticle.
  • 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.
  • 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.
  • Clause 14 The method of clause 13, wherein the cancer comprises a solid tumor.
  • 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.
  • 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.
  • 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.
  • Clause 18 The method of any of clauses 13-17, wherein the administration is intratumoral, peripheral to the tumor, or systemic.
  • Clause 19 The method of any of clauses 13-18, further comprising administering to the subject an effective amount of a conventional cancer treatment.
  • 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.
  • 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.
  • Clause 22 The method of clause 21, wherein the contacting is performed in vitro in cells or tissues.
  • Clause 23 The method of clause 22, wherein the cells or tissues comprise human cells or tissues.

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Abstract

La présente invention concerne des polypeptides qui se lient à CCL2-2 ou à la fois à CCL2 et CCL5 et des polynucléotides codant des polypeptides qui se lient à CCL2 ou à la fois à CCL2 et CCL5. La présente invention concerne en outre des compositions comprenant les polypeptides et les polynucléotides, et des procédés d'utilisation associés. L'invention concerne un anticorps à domaine unique isolé, un fragment ou un dérivé de celui-ci, qui se lie spécifiquement à la fois à CCL2 et CCL5, un anticorps à domaine unique isolé, ou un fragment ou dérivé de celui-ci, qui se lie spécifiquement à CCL2, et des polynucléotides comprenant un acide nucléique codant les anticorps à domaine unique isolés qui se lient spécifiquement à la fois à CCL2 et CCL5 ou à CCL2.
PCT/US2020/063328 2019-12-06 2020-12-04 Molécules d'affinité dirigeant le métabolisme et la polarisation de macrophages et permettant une synergie de la thérapie de blocage de point de contrôle immunitaire Ceased WO2021113648A1 (fr)

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CN119220679A (zh) * 2024-10-10 2024-12-31 浙江大学医学院附属第二医院 Dicer及其调控miRNA作为标志物在制备诊断、预防和/或治疗肿瘤肝转移的产品中的应用

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023122062A1 (fr) * 2021-12-21 2023-06-29 Cornell University Procédés d'administration améliorée d'acide nucléique
CN119220679A (zh) * 2024-10-10 2024-12-31 浙江大学医学院附属第二医院 Dicer及其调控miRNA作为标志物在制备诊断、预防和/或治疗肿瘤肝转移的产品中的应用

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