WO2023225613A2 - Combination anti-epn1 and anti-pd-l1 antibody therapies - Google Patents

Combination anti-epn1 and anti-pd-l1 antibody therapies Download PDF

Info

Publication number
WO2023225613A2
WO2023225613A2 PCT/US2023/067195 US2023067195W WO2023225613A2 WO 2023225613 A2 WO2023225613 A2 WO 2023225613A2 US 2023067195 W US2023067195 W US 2023067195W WO 2023225613 A2 WO2023225613 A2 WO 2023225613A2
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
specific antibody
epn1
seq
acid sequence
Prior art date
Application number
PCT/US2023/067195
Other languages
French (fr)
Other versions
WO2023225613A3 (en
Inventor
Matthew K. Robinson
Michael John MORIN
John P. Dowling
Fang Shen
Original Assignee
Immunome, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Immunome, Inc. filed Critical Immunome, Inc.
Publication of WO2023225613A2 publication Critical patent/WO2023225613A2/en
Publication of WO2023225613A3 publication Critical patent/WO2023225613A3/en

Links

Classifications

    • 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
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • 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
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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

  • Those antigens can result either from genetic changes within the tumor that lead to mutated proteins or aberrant presentation, of otherwise normal, proteins to the immune system.
  • Aberrant presentation may occur through processes that include, but are not limited to, ectopic expression of neonatal proteins, over ⁇ expression of proteins to a high level, mis ⁇ localization of intracellular proteins to the cell surface, or lysis of cells.
  • Aberrant glycosylation of proteins which may occur because of changes Patent Application Attorney Docket No. 172.0016 ⁇ WO00 in the expression of enzymes, such as, but not limited to, glycosyltransferases, can also result in generation of nonself ⁇ antigens that are recognized by the humoral immune system.
  • Antibodies which bind selectively to disease ⁇ related proteins, including proteins related to cancer, have proven successful at modulating the functions of their target proteins in ways that lead to therapeutic efficacy.
  • the ability of the human immune system to mount antibody responses against mutated, or otherwise aberrant, proteins suggests that patients’ immune responses may include antibodies that are capable of recognizing, and modulating the function of, critical tumor ⁇ drivers.
  • the tumor microenvironment is essential for tumor cells to evade detection and elimination by the immune system. This is achieved through several mechanisms including the recruitment of suppressive immune cells, expression of immune checkpoint molecules like PD ⁇ L1, PD ⁇ 1, LAG ⁇ 3, and CTLA ⁇ 4 and the presence of immunosuppressive cytokines.
  • PD ⁇ 1 is an immune checkpoint receptor expressed on the surface of immune effector cells, including CD8+ T cells.
  • Programmed Death Ligand ⁇ 1 is normally expressed on antigen presenting cells, such as dendritic cells. Binding of PD ⁇ L1 to Programmed cell death protein 1 (PD ⁇ 1) leads to T cell inactivation and inhibition of cytokine secretion.
  • Tumor cells can induce PD ⁇ L1 expression, leading to inactivation of T cells within the tumor microenvironment, and a dampening of anti ⁇ tumor immunity.
  • Antibodies specific for either PD ⁇ 1 or PD ⁇ L1 can block the PD ⁇ 1 ⁇ mediated inhibitory signaling pathway, which, in turn, can result in increased levels of T cell activation. Indeed, both anti ⁇ PD1 and anti ⁇ PD ⁇ L1 antibodies have demonstrated significant clinical benefit in a number of different cancer indications.
  • This disclosure describes methods, compositions, and kits of an invention for treating cancer by targeting the cell membrane ⁇ localized adaptor protein, Epsin ⁇ 1 (EPN1), in combination with targeting one or more immune checkpoint proteins.
  • Epsin ⁇ 1 Epsin ⁇ 1
  • the cancer therapy method combines targeting EPN1 with targeting programmed cell death ⁇ ligand 1 (PD ⁇ L1), while in another preferred embodiment of the invention, the cancer therapy method combines targeting EPN1 with targeting programmed cell death receptor (PD ⁇ 1).
  • a therapy that combines targeting EPN1 with targeting one or more immune checkpoint proteins may administer EPN1 ⁇ specific and immune checkpoint protein targeting agents in separately ⁇ administered pharmaceutical compositions, respectively, or administer EPN1 ⁇ specific and immune checkpoint protein targeting agents by administering a single pharmaceutical composition that contains EPN1 ⁇ specific and immune checkpoint protein targeting agents.
  • a step of administering EPN1 ⁇ targeting and immune checkpoint protein targeting agents separately, according to the invention, is inclusive of administering EPN1 ⁇ specific agents concurrently, simultaneously, essentially simultaneously, or sequentially with one or more separately ⁇ administered immune checkpoint protein targeting agents.
  • EPN1 targeting agents and immune checkpoint targeting agents are typically, but not exclusively, antibodies specific for EPN1 and an immune checkpoint protein, respectively.
  • the method combines the administration of a pharmaceutical composition containing an anti ⁇ EPN1 antibodies to a subject, in need thereof, with the administration of a pharmaceutical composition containing one or more immune checkpoint protein specific antibodies.
  • administration of the pharmaceutical composition containing an anti ⁇ EPN1 antibodies to a subject in need thereof may be separate from the administration of the administration of the pharmaceutical composition containing one or more immune checkpoint protein specific antibodies.
  • the pharmaceutical composition containing an anti ⁇ EPN1 antibodies may be administered to a subject in need thereof concurrently, simultaneously, essentially simultaneously, or sequentially with the pharmaceutical composition containing one or more antibodies specific for one or more immune checkpoint proteins.
  • a single pharmaceutical composition containing anti ⁇ EPN1 antibodies and one or more antibodies specific for one or more immune checkpoint proteins are administered to a subject in need thereof.
  • EPN1 ⁇ specific antibodies of the invention are typically monospecific for EPN1.
  • the EPN1 ⁇ specific antibody contains: A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 1; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 2.
  • VHC variable heavy chain
  • VLC variable light chain
  • the EPN1 ⁇ specific antibody contains at least one of the following complementary determining region (CDR) amino acid sequences: a CDR ⁇ H1 amino acid sequence of SEQ ID NO: 3; a CDR ⁇ H2 amino acid sequence of SEQ ID NO: 4; a CDR ⁇ H3 amino acid sequence of SEQ ID NO: 5; a CDR ⁇ L1 amino acid sequence of SEQ ID NO: 6; a CDR ⁇ L2 amino acid sequence of SEQ ID NO: 7; and a CDR ⁇ L3 amino acid sequence of SEQ ID NO: 8.
  • CDR complementary determining region
  • the PD ⁇ L1 ⁇ specific antibody contains: A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 9; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 10.
  • VHC variable heavy chain
  • VLC variable light chain
  • the PD ⁇ L1 ⁇ specific antibody contains at least one of the following complementary determining region (CDR) amino acid sequences: a CDR ⁇ H1 amino acid sequence of SEQ ID NO: 11; a CDR ⁇ H2 amino acid sequence of SEQ ID NO: 12; a CDR ⁇ H3 amino acid sequence of SEQ ID NO: 13; a CDR ⁇ L1 amino acid sequence of SEQ ID NO: 14; a CDR ⁇ L2 amino acid sequence of SEQ ID NO: 15; and a CDR ⁇ L3 amino acid sequence of SEQ ID NO: 16.
  • CDR complementary determining region
  • the PD ⁇ L1 ⁇ specific antibody contains: A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 17; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 18.
  • the PD ⁇ L1 ⁇ specific antibody contains: A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 19; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 20.
  • the PD ⁇ L1 ⁇ specific antibody contains: A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 21; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 22.
  • the PD1 ⁇ specific antibody contains: A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 23; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 24.
  • the PD1 ⁇ specific antibody contains: A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 25; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 26.
  • VHC variable heavy chain
  • VLC variable light chain
  • the PD1 ⁇ specific antibody contains: A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 27; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 28.
  • VHC variable heavy chain
  • VLC variable light chain
  • Fig. 1 is a graph of mean B16F10 tumor volumes of mice harboring tumors over a 10 ⁇ day period in which the mice were administered 12.5 mg/kg of Atezolizumab (anti ⁇ PD ⁇ L1), 12.5 mg/kg IMM20059 (anti ⁇ EPN1), or a combination of 12.5 mg/kg each of Atezolizumab and IMM20059 on days 1, 4 and 7. Control mice were administered 25 mg/kg hIgG1 on the same days.
  • Fig. 2 depicts the individual and mean B16F10 tumor volumes at day 7 of the study described in Fig. 1.
  • Fig. 3. contains graphs that show the tumor volumes for each mouse over the 10 ⁇ day period that is described for Fig.
  • Fig. 4. demonstrates EPN1 is overexpressed in multiple cancer types.
  • Fig. 4A is a graph of RNA expression levels of EPN1 in various types of tumor vs normal tissues based upon analyses of data from the TCGA database.
  • Fig. 4B is publicly available immunohistochemical staining of various tumor types to evaluate EPN1 protein expression.
  • Fig. 5. demonstrates IMM20059 binds to the surface of cancer, but not normal, cell lines.
  • Fig. 5A is a scatter plot showing the level of binding to cancer cells relative to normal cells derived from various tissues.
  • FIG. 5B is a heatmap showing selective binding by flow cytometry of IMM20059 to various cancer cell lines derived from various tissues.
  • Fig. 6 presents Surface Plasmon Resonance (SPR) data showing sub ⁇ nanomolar binding of IMM20059 to recombinant human EPN1 protein.
  • FIG. 8A is a heat map of detectable intratumoral cytokines in tumor lysates prepared from tumors harvested at Day 9 in a B16.F10 syngeneic model comparing treatments with isotype controls, Atezolizumab, IMM20059, and Atezolizumab + IMM20059. Lysates were multiplexed for 32 cytokines. Color scale represents fold ⁇ change of cytokine concentrations in each sample over mean cytokine concentration in Isotype control group (Luminex). [0029] Fig. 8B shows scatter plots of the intratumoral cytokines MIP ⁇ 1 ⁇ , MIP ⁇ 1 ⁇ , and RANTES detected in the Day 9 individual lysates described in the summary of Fig. 8.
  • FIG. 9 is a line graph of mean B16.F10 tumor volumes over a 9 day period in mice dosed with Isotype control (30 mg/kg or 90 mg/kg), Atezolizumab (5 mg/kg, 15 mg/kg, 45 mg/kg), IMM20059 (5 mg/kg, 15 mg/kg, 45 mg/kg), or a combination of Atezolizumab and IMM2005 (5 mg/kg, 15 mg/kg, 45 mg/kg of each test agent) every 3 days.
  • the error bars represent standard error of tumor volumes in specific group.
  • Treatment of IMM20059 alone (45 mg/kg) or Atezolizumab + IMM20059 (45 mg/kg of each test agent) showed trends of inhibition of tumor growth compared to isotype control treatment. [0031] Fig.
  • Fig. 10 is a scatter plot depicting the individual B16.F10 tumor volumes at Day 9 in mice from different treatment groups described in Fig. 9.
  • the error bars represent standard error of tumor volumes in specific group.
  • Treatment of IMM20059 alone (45 mg/kg) or Atezolizumab + IMM20059 (45 mg/kg of each test agent) showed trends of inhibition of tumor growth compared to isotype control treatment.
  • Fig. 11 depicts scatter plots of tumor volumes from individual animals on the final day (Day 11) of the study described in Fig. 9 and the terminal exposures of the indicated treatments in the plasma as measured by hIgG1 concentration.
  • the "r’ represents correlation coefficient and “p” represents false discovery rate determined by Pearson correlation analysis.
  • Fig. 12 is a line plot depicting that IMM20059 or positive control, but not Fc null IMM20059, bind Fc ⁇ RIIIa in vitro in concentration ⁇ dependent manner. The error bars represent standard deviation of normalized relative luminescence at indicated concentration.
  • FIG. 13 contains scatter plots depicting live cell counts on day 5 in bone marrow (Fig. 13A), thymus (Fig. 13B), lymph node (Fig.
  • Fig. 14A is bar chart depicting the frequency of positive surface staining of IMM20059 on different normal human cells at 667 nM , 67 nM and 6.7 nM in flow cytometry analysis. Error bars represent standard deviation of biological duplicates of IMM20059 were performed within the same experiment .
  • IMM20059 showed no significant staining on the surface of eight (8) different normal human cells.
  • HRE human renal epithelial cells
  • NHBE normal human bronchial epithelial cells
  • NHEK normal human epidermal keratinocytes
  • NHDF normal human dermal fibroblast cells
  • HRCE human renal cortical epithelial cells
  • HPrEC human prostate epithelial cells
  • HMEC human mammary epithelial cells
  • SKMC human skeletal muscle cells.
  • Fig. 14B is a bar chart depicting the frequencies of positive surface staining of an isotype control antibody for the staining of IMM20059 shown in Fig. 14A.
  • Fig. 14B is a bar chart depicting the frequencies of positive surface staining of an isotype control antibody for the staining of IMM20059 shown in Fig. 14A.
  • This disclosure describes methods, compositions and kits used for treating various types of cancer by combining therapies that target Epsin ⁇ 1 (EPN1) and target one or more immune checkpoint proteins.
  • EPN1 Epsin ⁇ 1
  • some methods, compositions, and kits relate to administering an Epn1 targeting agent to a subject in need thereof in combination with administering a targeting agent that targets a Patent Application Attorney Docket No. 172.0016 ⁇ WO00 component of the programmed cell death axis.
  • the targeted component of the programmed cell death axis is programmed cell death receptor (PD ⁇ 1) or programmed cell death ⁇ ligand 1 (PD ⁇ L1).
  • a therapy according to the invention that combines targeting EPN1 with targeting one or more immune checkpoint proteins may administer EPN1 ⁇ specific and immune checkpoint protein targeting agents in separately ⁇ administered pharmaceutical compositions, respectively, or administer EPN1 ⁇ specific and immune checkpoint protein targeting agents by administering a single pharmaceutical composition that contains EPN1 ⁇ specific and immune checkpoint protein targeting agents.
  • EPN1 targeting agents and immune checkpoint protein targeting agents are known in the art. Briefly, an EPN1 targeting agent specifically binds EPN1 and blocks binding of EPN1 with an EPN1 ligand (e.g., an immune checkpoint protein). Similarly, an immune checkpoint protein targeting agent specifically binds an immune checkpoint protein and blocks binding of the immune checkpoint protein to EPN1. EPN1 targeting agents and immune checkpoint targeting agents are typically, but not exclusively, antibodies specific for EPN1 and an immune checkpoint protein, respectively.
  • the method combines the administration of a pharmaceutical composition containing an anti ⁇ EPN1 antibodies to a subject, in need thereof, with the administration of a pharmaceutical composition containing one or more immune checkpoint protein specific antibodies.
  • administration of the pharmaceutical composition containing an anti ⁇ EPN1 antibodies to a subject in need thereof may be separate from the administration of the administration of the pharmaceutical composition containing one or more immune checkpoint protein specific antibodies.
  • the pharmaceutical composition containing an anti ⁇ EPN1 antibodies may be administered to a subject in need thereof concurrently, simultaneously, essentially simultaneously, or sequentially with the pharmaceutical composition containing one or more antibodies specific for one or more immune checkpoint proteins.
  • a single pharmaceutical composition containing anti ⁇ EPN1 antibodies and one or more antibodies specific for one or more immune checkpoint proteins are administered to a subject in need thereof.
  • EPN1 ⁇ specific antibodies and the PD ⁇ L1 ⁇ specific or PD ⁇ 1 ⁇ specific antibodies are each administered separately.
  • the EPN1 ⁇ specific antibodies and either the PD ⁇ L1 ⁇ specific or PD ⁇ 1 ⁇ specific antibodies may be administered concurrently, simultaneously, essentially simultaneously, or sequentially.
  • a single pharmaceutical composition that contains the EPN1 ⁇ specific antibodies and either the PD ⁇ L1 ⁇ specific or PD ⁇ 1 ⁇ specific antibodies may be administered to a subject in need thereof.
  • the EPN1 ⁇ specific antibodies and either the PD ⁇ L1 ⁇ specific or PD ⁇ 1 ⁇ specific antibodies may be formulated into separate, respective pharmaceutical compositions, may be formulated in a single pharmaceutical composition.
  • the EPN1 ⁇ specific antibodies and either the PD ⁇ L1 ⁇ specific or PD ⁇ 1 ⁇ specific antibodies are administered in separate or a single pharmaceutical compositions may be a choice made by a practitioner based on the stage and type of cancer, the condition of the patient.
  • EPN1 ⁇ specific antibodies and either the PD ⁇ L1 ⁇ specific or PD ⁇ 1 ⁇ specific antibodies may be based on the same or similar factors.
  • Combinations of EPN1 antibody and either PD ⁇ L1 antibody or PD ⁇ 1 antibody therapies may treat cancer by slowing or inhibiting growth or metastasis of primary tumors in a subject in need thereof.
  • the effect of the combining anti ⁇ EPN1 and either the anti ⁇ PD ⁇ L1 or anti ⁇ PD ⁇ 1 therapies on the growth or metastasis of tumors is synergistic.
  • some methods of the invention reduce or limit the volume of one or more cancerous tumors in a subject afflicted by cancer, to no more than 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% of the volume (i.e., the size) of the tumor an oncologist or other cancer treatment practitioner would expect if the subject had been treated with anti ⁇ EPN1, anti ⁇ PD ⁇ L1, or anti ⁇ PD ⁇ 1 antibodies.
  • the therapeutic effect of a combination therapy of the invention is greater in comparison to an expected therapeutic effect produced by administering anti ⁇ EPN1 antibodies without administering either anti ⁇ PD ⁇ L1 or anti ⁇ PD ⁇ 1 antibodies during the course of a treatment.
  • a reduction in the volume of one or more primary tumors or at metastatic sites in a subject treated by a method of the invention is an indicator of tumor regression and clearance.
  • a method of the invention for treating cancer in a subject by combining anti ⁇ EPN1 antibody therapy with either anti ⁇ PD ⁇ L1 antibody therapy or anti ⁇ PD ⁇ 1 antibody therapy reduces the volume of one or more tumors in the subject by greater percentage of reduction as compared to the reduction of one or more tumors in a subject who received i) anti ⁇ EPN1 antibody therapy, but not anti ⁇ PD ⁇ L1 antibody therapy, or anti ⁇ PD ⁇ 1 antibody therapy; or ii) anti ⁇ PD ⁇ L1 antibody therapy or anti ⁇ PD ⁇ 1 antibody therapy, but not anti ⁇ EPN1 antibody therapy.
  • Patent Application Attorney Docket No. 172.0016 ⁇ WO00 [0045] Accordingly, as stated above, a method of the invention in which a cancer is treated in a subject by combining the administration of IMM20059, or other EPN1 ⁇ specific antibodies, with the administration of Atezolizumab, or other PD ⁇ L1 ⁇ specific antibodies, the observed reduction in volume of one or more tumors in the subject is synergistic in comparison to the observed or expected reduction in the volume of one or more tumors as a consequence an additive effect of administering IMM20059, or other EPN1 ⁇ specific antibodies with Atezolizumab, or other PD ⁇ L1 ⁇ specific antibodies. Similarly.
  • a method of the invention in which a cancer is treated in a subject by combining the administration of IMM20059, or other EPN1 ⁇ specific antibodies, with the administration of PD ⁇ 1 ⁇ specific antibodies, the observed reduction in volume of one or more tumors in the subject is synergistic in comparison to the observed or expected reduction in the volume of one or more tumors as a consequence an additive effect of administering IMM20059, or other EPN1 ⁇ specific antibodies with PD ⁇ 1 ⁇ specific antibodies.
  • the tumor may be, but is not limited to classification as lung squamous carcinoma, lung adenocarcinoma, breast cancer, prostate cancer, uterine cancer, cervical cancer, thyroid cancer, melanoma, gastroesophageal cancer, bladder cancer, or hepatocellular carcinoma.
  • an indicator of an increased probability of tumor regression and clearance is the detection of one or more chemokines in the intratumoral environment of one or more tumors in a subject who received anti ⁇ EPN1 antibody therapy in combination with anti ⁇ EPN1 antibody therapy with either anti ⁇ PD ⁇ L1 antibody therapy or anti ⁇ PD ⁇ 1 antibody therapy.
  • one or more chemokines are detected at a higher level in the intratumoral environment of one or more tumors in a subject who received anti ⁇ EPN1 antibody therapy in combination with anti ⁇ EPN1 antibody therapy with either anti ⁇ PD ⁇ L1 antibody therapy or anti ⁇ PD ⁇ 1 antibody therapy, as compared to the detected levels of the same one or more chemokines in the intratumoral environment of one or more tumors in a subject who received: i) anti ⁇ EPN1 antibody therapy, but not anti ⁇ PD ⁇ L1 antibody therapy, or anti ⁇ PD ⁇ 1 antibody therapy; or ii) anti ⁇ PD ⁇ L1 antibody therapy or anti ⁇ PD ⁇ 1 antibody therapy, but not anti ⁇ EPN1 antibody therapy.
  • an additive or synergistic effect of combining the administration of IMM20059, or other EPN1 ⁇ specific antibodies, with the administration of Atezolizumab, or other PD ⁇ L1 ⁇ specific antibodies is the detection of higher levels of one or more chemokines in the intratumoral environment of one or more tumors in the subject in comparison to the observed or expected detected levels of one or more tumors as a consequence an additive effect of receiving: i) IMM20059 therapy or other anti ⁇ EPN1 antibody therapy, but not anti ⁇ PD ⁇ L1 antibody therapy or anti ⁇ PD ⁇ 1 antibody therapy; or ii) anti ⁇ PD ⁇ L1 antibody therapy or anti ⁇ PD ⁇ 1 antibody therapy, but not IMM20059 therapy or other anti ⁇ EPN1 Patent Application Attorney Docket No.
  • the levels of one or more of the chemokines MIP ⁇ 1 ⁇ , MIP ⁇ 1 ⁇ , and RANTES detected in the intratumoral environment of one or more tumors are increased as a consequence of a synergistic effect of receiving anti ⁇ EPN1 antibody therapy in combination with anti ⁇ EPN1 antibody therapy with either anti ⁇ PD ⁇ L1 antibody therapy or anti ⁇ PD ⁇ 1 antibody therapy.
  • the detection of higher levels of one or more chemokines in the intratumoral environment is an indicator of an increased probability of tumor regression and clearance.
  • the tumor may be, but is not limited to classification as lung squamous carcinoma, lung adenocarcinoma, breast cancer, prostate cancer, uterine cancer, cervical cancer, thyroid cancer, melanoma, gastroesophageal cancer, bladder cancer, or hepatocellular carcinoma.
  • EPN1 ⁇ specific antibodies and immune checkpoint protein specific antibodies reduce suppression of immune cell ⁇ mediated anti ⁇ tumor activity.
  • one or both the EPN1 specific antibodies and the PD ⁇ L1 specific or PD ⁇ 1 specific antibodies reduce suppression of immune cell ⁇ mediated anti ⁇ tumor activity by targeting EPN1 and PD ⁇ L1 ⁇ mediated signaling on tumor ⁇ cell derived exosomes.
  • suppression of immune cell ⁇ mediated anti ⁇ tumor activity is mediated by CD8 + suppressor T cells.
  • EPN1 ⁇ specific antibodies bind EPN1 that is expressed on the surface of tumor cells, thereby effecting the death of the cancer cell.
  • This disclosure also does not limit the types of cancers that are treated by methods of the invention.
  • anti ⁇ EPN1 and either anti ⁇ PD ⁇ L1 or anti ⁇ PD ⁇ 1 antibodies function together synergistically to slow or inhibit the growth or metastasis of one or more of lung squamous carcinoma, lung adenocarcinoma, breast cancer, prostate cancer, uterine cancer, cervical cancer, thyroid cancer, melanoma, gastroesophageal cancer, bladder cancer, or hepatocellular carcinoma.
  • Subjects treated by methods of the invention are typically human subjects that have been diagnosed with cancer or are otherwise suspected as being afflicted by cancer.
  • Therapeutically effective Patent Application Attorney Docket No. 172.0016 ⁇ WO00 amounts of anti ⁇ EPN1 and either anti ⁇ PD ⁇ L1 or PD ⁇ 1 antibodies administered to a subject according to the invention will depend upon the severity of the cancer, and the general state of the patient’s health.
  • a therapeutically effective amount of the foregoing antibodies provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by a clinician or other qualified professional.
  • therapeutically effective amounts of the anti ⁇ EPN1 and either the anti ⁇ PD ⁇ L1 or anti ⁇ PD ⁇ 1 antibodies are dosage amounts that, when administered to a subject in need thereof, are sufficient to inhibit growth, or metastasis of cancer cells, or to inhibit a sign or a symptom of the cancer.
  • the administered therapeutic dosage amounts of the anti ⁇ EPN1 antibody and either the anti ⁇ PD ⁇ L1 or anti ⁇ PD ⁇ 1 antibody are equivalent.
  • the administered therapeutic dosage amount of the anti ⁇ EPN1 antibody is greater than the administered therapeutic dosage amount of either the anti ⁇ PD ⁇ L1 or anti ⁇ PD ⁇ 1 antibody.
  • the administered therapeutic dosage amount of the anti ⁇ EPN1 antibody is less than the administered therapeutic dosage amount of either the anti ⁇ PD ⁇ L1 antibody or anti ⁇ PD ⁇ 1.
  • This disclosure does not limit minimum or maximum dosage amounts of either the EPN1 ⁇ specific, PD ⁇ L1 ⁇ specific, or PD ⁇ 1 ⁇ specific antibodies that are administered to a subject in need thereof.
  • the administered dosage amount of an EPN1 ⁇ specific antibody is from 0.2 mg/kg to 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 105 mg/kg, 110 mg/kg, or any amount therein.
  • the foregoing antibody dosage amounts correlate to intravenously administering a pharmaceutical composition, containing from 800 ⁇ 1200 mg of PD ⁇ L1 ⁇ specific antibodies and/or 800 ⁇ 1200 mg of PD ⁇ 1 specific antibodies, to a human subject every two weeks(Q2W) or every three weeks (Q3W).
  • a pharmaceutical composition comprising EPN1 ⁇ specific antibodies may be intravenously administered, either as a single administered dose, or in a series of administered doses, at 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, or less than 1 week before or after the first or last administration of PD ⁇ L1 and/or PD1 specific antibodies.
  • the dosage amount of EPN1 ⁇ specific antibody in a pharmaceutical composition administered in one or more of the Patent Application Attorney Docket No. 172.0016 ⁇ WO00 above ⁇ methods may contain, but is not limited to, containing 200 mg – 1800 mg of EPN1 ⁇ specific antibodies administered to a human subject every week (Q1W), every two weeks (Q2W), every three weeks (Q3W), or every 4 weeks (Q4W).
  • the foregoing antibody dosage amounts correlate to intravenously administering from 1500 ⁇ 1680 mg of a PD ⁇ L1 ⁇ specific antibody to a human subject every three(Q3W) or every four weeks (Q4W).
  • a pharmaceutical composition comprising EPN1 ⁇ specific antibodies may be intravenously administered, either as a single administered dose, or in a series of administered doses, at 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, or less than 1 week before or after the first or last administration of PD ⁇ L1 ⁇ specific antibodies.
  • the dosage amount of EPN1 ⁇ specific antibody in a pharmaceutical composition administered in one or more of the above ⁇ methods may contain, but is not limited to, containing 200 mg – 1800 mg of EPN1 ⁇ specific antibodies administered to a human subject every week (Q1W), every two weeks (Q2W), every three weeks (Q3W), or every 4 weeks (Q4W).
  • initial loading dosage amounts of EPN1 ⁇ specific and/or PD ⁇ L1 ⁇ specific antibodies are administered, with subsequent, maintenance doses being administered at lower dosage levels.
  • a lower weekly maintenance dose may be administered to the subject for the subsequent 4, 5, 6, 7, or 8 weeks.
  • the foregoing antibody dosage amounts correlate to intravenously administering from 200 ⁇ 400 mg of a PD ⁇ 1 ⁇ specific antibody to a human subject every three (Q3W) or every six weeks (Q6W).
  • a pharmaceutical composition comprising EPN1 ⁇ specific antibodies may be intravenously administered, either as a single administered dose, or in a series of administered doses, at 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, or less than 1 week before or after the first or last administration of PD ⁇ 1 ⁇ specific antibodies.
  • the dosage amount of EPN1 ⁇ specific antibody in a pharmaceutical composition administered in one or more of the above ⁇ methods may contain, but is not limited to, containing 200 mg – 1800 mg of EPN1 ⁇ specific antibodies administered to a human subject every week (Q1W), every two weeks (Q2W), every three weeks (Q3W), or every 4 weeks (Q4W).
  • the foregoing antibody dosage amounts correlate to intravenously administering either 3 mg/Kg or 240 mg of a PD ⁇ 1 ⁇ specific antibody to a human subject every two (Q2W), or 480 mg every four weeks (Q4W).
  • a pharmaceutical composition comprising EPN1 ⁇ specific antibodies may be intravenously administered, either as a single administered Patent Application Attorney Docket No. 172.0016 ⁇ WO00 dose, or in a series of administered doses, at 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, or less than 1 week before or after the first or last administration of PD ⁇ 1 ⁇ specific antibodies.
  • the dosage amount of EPN1 ⁇ specific antibody in a pharmaceutical composition administered in one or more of the above ⁇ methods may contain, but is not limited to, containing 200 mg – 1800 mg of EPN1 ⁇ specific antibodies administered to a human subject every week (Q1W), every two weeks (Q2W), every three weeks (Q3W), or every 4 weeks (Q4W).
  • the foregoing antibody dosage amounts correlate to intravenously administering 350 mg of a PD ⁇ 1 ⁇ specific antibody to a human subject every three (Q3W).
  • a pharmaceutical composition comprising EPN1 ⁇ specific antibodies may be intravenously administered, either as a single administered dose, or in a series of administered doses, at 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, or less than 1 week before or after the first or last administration of PD ⁇ 1 ⁇ specific antibodies.
  • the dosage amount of EPN1 ⁇ specific antibody in a pharmaceutical composition administered in one or more of the above ⁇ methods may contain, but is not limited to, containing 200 mg – 1800 mg of EPN1 ⁇ specific antibodies administered to a human subject every week (Q1W), every two weeks (Q2W), every three weeks (Q3W), or every 4 weeks (Q4W).
  • EPN1 ⁇ specific and/or PD ⁇ L1 ⁇ specific or PD ⁇ 1 ⁇ specific antibodies that are administered in methods of the invention are formulated into compositions. More particularly, the antibodies may be formulated for systemic administration, or local administration, such as intra ⁇ tumor administration. For example, EPN ⁇ specific and/or PD ⁇ L1 ⁇ specific or PD ⁇ 1 ⁇ specific antibodies may be formulated for parenteral administration, such as intravenous administration.
  • the compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating clinician to achieve the desired outcome.
  • Antibody compositions according to the invention may also be controlled release formulations. Controlled release parenteral formulations, for example, can be made as implants, or oily injections.
  • Particulate systems including microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles, may also be used to deliver EPN1 ⁇ specific and/or PD ⁇ L1 ⁇ specific or PD ⁇ 1 ⁇ specific antibodies.
  • Microcapsules as referred to herein, contain EPN1 ⁇ specific and/or PD ⁇ L1 ⁇ specific or PD ⁇ 1 ⁇ specific antibodies in central core components. In microspheres, EPN1 ⁇ specific and/or PD ⁇ L1 ⁇ specific or PD ⁇ 1 ⁇ specific antibodies are dispersed throughout the particle. Particles, microspheres, and Patent Application Attorney Docket No.
  • 172.0016 ⁇ WO00 microcapsules smaller than about 1 ⁇ m are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively.
  • Administration of the EPN ⁇ specific and/or PD ⁇ L1 ⁇ specific or PD ⁇ 1 ⁇ specific antibodies may also be accompanied by administration of other anti ⁇ cancer agents or therapeutic treatments, such as surgical resection of a tumor. Any suitable anti ⁇ cancer agent can be administered in combination with the antibodies disclosed herein.
  • anti ⁇ cancer agents include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, anti ⁇ metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti ⁇ survival agents, biological response modifiers, anti ⁇ hormones (e.g., anti ⁇ androgens) and anti ⁇ angiogenesis agents.
  • chemotherapeutic agents such as, for example, mitotic inhibitors, alkylating agents, anti ⁇ metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti ⁇ survival agents, biological response modifiers, anti ⁇ hormones (e.g., anti ⁇ androgens) and anti ⁇ angiogenesis agents.
  • Other anti ⁇ cancer treatments include radiation therapy and other antibodies that specifically target cancer cells.
  • a basic antibody structure includes two heavy (H) and two light (L) polypeptide chains, each of which contains a constant region and
  • immunoglobulin light chains which are termed lambda (“ ⁇ ”) and kappa (“ ⁇ ")
  • immunoglobulin heavy chain classes also known as isotypes, which determine functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.
  • V H variable heavy
  • V L variable light
  • a full ⁇ length heavy chain also has three constant domains (CH1, CH2, CH3).
  • V H and V L regions contain "framework" regions interrupted by three hypervariable regions, called complementarity ⁇ determining regions ("CDRs").
  • CDRs complementarity ⁇ determining regions
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • sequences of the framework regions of different light or heavy chains are relatively conserved within a species and serve to position and align the CDRs in three ⁇ dimensional space.
  • the three CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N ⁇ terminus, and are often identified by the chain in which the particular CDR is located. Accordingly, heavy chain CDRs are designated H ⁇ CDR1, H ⁇ CDR2, and H ⁇ CDR3; likewise, light chain CDRs are designated L ⁇ CDR1, L ⁇ CDR2, and L ⁇ CDR3.
  • An antigen ⁇ binding fragment, one constant and one variable domain of each of the heavy and the light chain is referred to as an Fab fragment.
  • An F(ab)' 2 fragment contains two Fab fragments and can be generated by cleaving an immunoglobulin molecule below its hinge region.
  • EPN1 ⁇ specific antibodies of the invention are typically monospecific for EPN1. However, this disclosure does not limit the EPN1 ⁇ specific antibodies that may be administered in methods of the invention, as described above.
  • the EPN1 ⁇ specific antibody contains: A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 1; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 2.
  • VHC variable heavy chain
  • VLC variable light chain
  • the EPN1 ⁇ specific antibody contains at least one of the following complementary determining region (CDR) amino acid sequences: a CDR ⁇ H1 amino acid sequence of SEQ ID NO: 3; a CDR ⁇ H2 amino acid sequence of SEQ ID NO: 4; a CDR ⁇ H3 amino acid sequence of SEQ ID NO: 5; a CDR ⁇ L1 amino acid sequence of SEQ ID NO: 6; a CDR ⁇ L2 amino acid sequence of SEQ ID NO: 7; and a CDR ⁇ L3 amino acid sequence of SEQ ID NO: 8.
  • CDR complementary determining region
  • the EPN1 ⁇ specific antibodies of the invention are typically monospecific for EPN1, PD ⁇ L1 ⁇ specific antibodies of the invention are also typically monospecific for PD ⁇ L1, though this disclosure does not limit the EPN1 ⁇ specific antibodies that may be administered in methods of the invention.
  • the PD ⁇ L1 ⁇ specific antibody contains: A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 9; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 10.
  • the PD ⁇ L1 ⁇ specific antibody contains at least one of the following complementary determining region (CDR) amino acid sequences: a CDR ⁇ H1 amino acid sequence of SEQ ID NO: 11; a CDR ⁇ H2 amino acid sequence of SEQ ID NO: 12; a CDR ⁇ H3 amino acid sequence of SEQ ID NO: 13; a CDR ⁇ L1 amino acid sequence of SEQ ID NO: 14; a CDR ⁇ L2 amino acid sequence of SEQ ID NO: 15; and a CDR ⁇ L3 amino acid sequence of SEQ ID NO: 16.
  • CDR complementary determining region
  • the PD ⁇ L1 ⁇ specific antibody contains: A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 17; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 18.
  • the PD ⁇ L1 ⁇ specific antibody contains: A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 19; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 20.
  • the PD ⁇ L1 ⁇ specific antibody contains: A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 21; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 22.
  • VHC variable heavy chain
  • VLC variable light chain
  • the PD ⁇ 1 ⁇ specific antibody contains: A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 23; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 24.
  • the PD ⁇ 1 ⁇ specific antibody contains: A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 25; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 26.
  • the PD ⁇ 1 ⁇ specific antibody contains: A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 27; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 28.
  • VHC variable heavy chain
  • VLC variable light chain
  • the EPN1 ⁇ specific, PD ⁇ L1 ⁇ specific, and PD ⁇ 1 ⁇ specific antibodies of the invention are monoclonal antibodies, meaning an antibody is produced by a single clonal B ⁇ lymphocyte population, a clonal hybridoma cell population, or a clonal population of cells into which the genes of a single antibody, or portions thereof, have been transfected.
  • Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody ⁇ forming cells from a fusion of myeloma cells with immune lymphocyte cells.
  • Monoclonal antibodies according to the invention are also typically humanized monoclonal antibodies.
  • a “human” antibody also called a “fully human” antibody, according to the invention, is an antibody that includes human framework regions and CDRs from a human immunoglobulin.
  • the framework and the CDRs of an antibody are from the same originating human heavy chain, or human light chain amino acid sequence, or both.
  • the framework regions may originate from one human antibody, and be engineered to include CDRs from a different human antibody.
  • “Humanizing substitutions” are amino acid substitutions in which the amino acid residue present at a particular position in the VH or VL domain of an antibody, such as an IL ⁇ 38 antibody, is replaced with an amino acid residue which occurs at an equivalent position in a reference human VH or VL domain.
  • the reference human VH or VL domain may be a VH or VL domain encoded by Patent Application Attorney Docket No. 172.0016 ⁇ WO00 the human germline. Humanizing substitutions may be made in the framework regions and/or the CDRs of the antibodies, defined herein.
  • a "humanized variant” is a variant antibody of the invention, which contains one or more "humanizing substitutions" relative to a reference antibody, wherein a portion of the reference antibody (e.g., the VH domain and/or the VL domain or parts thereof containing at least one CDR) has an amino acid derived from a non ⁇ human species, and the "humanizing substitutions" occur within the amino acid sequence derived from a non ⁇ human species.
  • EPN1 ⁇ specific, PD ⁇ L1 ⁇ specific, and PD ⁇ 1 ⁇ specific antibodies of the invention may also be "antigen ⁇ binding fragments".
  • An antigen ⁇ binding fragment refers to a polypeptide fragment of an immunoglobulin or antibody that binds antigen or competes with intact antibody (i.e., with the intact antibody from which they were derived) for antigen binding (i.e., specific binding to IL ⁇ 38).
  • fragment of an antibody molecule includes antigen ⁇ binding fragments of antibodies, for example, an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single chain antibody (scFv), a F(ab')2 fragment, a Fab fragment, an Fd fragment, an Fv fragment, and a single domain antibody fragment (Dab). Fragments can be obtained, e.g., via chemical or enzymatic treatment of an intact or complete antibody or antibody chain or by recombinant means.
  • immunoglobulin variants that are considered antibodies according to the invention include single ⁇ domain antibodies (such as VH domain antibodies), Fab fragments, Fab' fragments, F(ab)' 2 fragments, single chain Fv proteins ("scFv”), and disulfide stabilized Fv proteins ("dsFv").
  • a VH single ⁇ domain antibody is an immunoglobulin fragment consisting of a heavy chain variable domain.
  • An Fab fragment contains a monovalent antigen ⁇ binding immunoglobulin fragment, which can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain.
  • a Fab' fragment also contains a monovalent antigen ⁇ binding immunoglobulin fragment, which can be produced by digestion of whole antibody with the enzyme pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained per immunoglobulin molecule.
  • a (Fab') 2 fragment is a dimer of two Fab' fragments, that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction, so Fab' monomers remain held together by two disulfide bonds.
  • a Fv fragment is a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains.
  • a single chain (“sc”) antibody such as scFv fragment
  • scFv fragment is a genetically engineered molecule containing the V L region of a light chain, the V H region of a heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
  • a dimer of a single chain antibody such as a scFV 2 antibody, is a dimer of a scFV, and may also be known as a "miniantibody”.
  • a Patent Application Attorney Docket No. 172.0016 ⁇ WO00 dsFvs variant also contains a V L region of an immunoglobulin and a V H region, but the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
  • EPN1 ⁇ specific, PD ⁇ L1 ⁇ specific, and PD ⁇ 1 ⁇ specific antibodies of the invention may also possess a "tagged" immunoglobulin CH3 domain to facilitate detection of the biologic against a background of endogenous antibodies.
  • a tagged CH3 domain is a heterogenous antibody epitope that has been incorporated into one or more of the AB, EF, or CD structural loops of a human IgG ⁇ derived CH3 domain.
  • CH3 tags are preferably incorporated into the structural context of an IgG1 subclass antibody, other human IgG subclasses, including IgG2, IgG3, and IgG4, are also available according to the invention.
  • Epitope ⁇ tagged CH3 domains also referred to as "CH3 scaffolds" can be incorporated into any antibody of the invention having a heavy chain constant region, generally in the form of an immunoglobulin Fc portion. Examples of CH3 scaffold tags, and methods for incorporating them into antibodies are disclosed in International Patent Application Publication No.
  • EPN1 ⁇ specific, PD ⁇ L1 ⁇ specific, and/or PD ⁇ 1 ⁇ specific antibodies of the invention may be packaged into a kit for administering the antibodies to a subject for the purpose of treating cancer.
  • kits include suitable storage containers, such as, ampules, vials, and tubes, for EPN1 ⁇ specific, PD ⁇ L1 ⁇ specific, and/or PD ⁇ 1 ⁇ specific antibody compositions and other included reagents, such as buffers and balanced salt solutions, for use in administering the compositions to subjects.
  • the compositions and other reagents may be present in the kits in any convenient form, such as, in a solution or in a powder form.
  • the kits may further include instructions for using the components of the kit.
  • Example 1 ⁇ Efficacy study in the B16.F10 syngeneic model A B16F10 melanoma model in C57BL/6 female mice was used to evaluate the impact of IMM20059 and Atezolizumab – alone and in combination – on melanoma tumor growth. To generate the model for these studies, B16.F10 cells were thawed and cultured according to manufacturer’s instruction, washed, counted, and resuspended in PBS, and subcutaneously injected in the right rear flank of C57BL/6 female mice. Injected cell suspension containing 2.5 x 10 5 cells/100 ⁇ l. The tumors of mice harboring Patent Application Attorney Docket No.
  • EPN1 overexpression is associated with certain cancers
  • Overexpression of EPN1 RNA is observed in several different cancer types.
  • EPN1 RNA expression across various tumor types was assessed using the TCGA database (Fig. ⁇ 4A).
  • Multiple cancers including breast, lung squamous, lung adenocarcinoma, uterus, cervical, prostate, thyroid, bladder, and liver, were shown to overexpress EPN1 as compared to normal tissues from the same organ. Cancers, including colorectal, glioblastoma, and kidney were shown to have lower expression of EPN1 as compared to normal tissue, publicly available immunohistochemistry data demonstrates expression of EPN1 protein in various types of cancer (Fig. 4B) [0081] Example 3.
  • IMM20059 binds the surface of cancer cells as compared to normal cells. IMM20059 was assessed for binding to the surface of additional cancer cell lines as well as normal cell lines by flow cytometry (Fig. 5). Cell lines represent several different cancer indications and normal tissues. Data are represented as scatter plots (Fig. 5A) and heatmap (Fig. 5B). Flow cytometry analysis on expanded normal human cells (Fig. 14) and cancer cell lines (Fig. 15) are presented as bar charts. [0082] Example 4. – IMM20059 binds recombinant human EPN1 protein with sub ⁇ nanomolar affinity.
  • Binding of IMM20059 to recombinant human EPN1 protein was quantified by surface plasmon resonance (SPR) performed at 25 ⁇ C using a BiacoreTM 2000 optical biosensor with a BiacoreTM Protein A sensor chip.
  • IMM20059 was diluted in SPR running buffer (10 mM HEPES, pH7.4, 150 mM NaCl, 0.005% Tween ⁇ 20, 0.2% bovine serum albumin) and captured at 2 densities ( ⁇ 200 and ⁇ 450 RU).
  • EPN1 was diluted in SPR running buffer to a concentration of 33 nM and a 3 ⁇ fold dilution series was run across the chip.
  • Example 5 IMM20059 binds to the surface of live B16.F10 cells IMM20059 binding to the surface of live mouse B16.F10 melanoma cells was detected by fluorescently labeled anti ⁇ human IgG1 secondary antibody and assessed by flow cytometry. Concentration ⁇ dependent binding of IMM20059 to B16.F10 cells was observed with an EC 50 of 7.5 nM (Fig. 7). Patent Application Attorney Docket No. 172.0016 ⁇ WO00 [0084] Example 6. — Combination of IMM20059 and Atezolizumab results in the significant upregulation of intratumoral chemokines.
  • B16.F10 tumors from Example 1 were harvested on Day 9 and flash frozen. To generate tumor homogenates, frozen tumors were placed in 2 mL Bead Ruptor Prefilled Bead Tubes (Omni International Catalog # 19 ⁇ 628) with lysis buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, and 0.5% NP ⁇ 40. A bead mill homogenizer (Bead Ruptor Elite, Omni International Catalog # 19 ⁇ 042E) was used to perform two 30 second bead beating cycles. Tubes were spun at >10,000 x g for 10 minutes and the supernatant was transferred to a fresh tube. Total protein concentration was measured using the Pierce BCA Protein Assay kit (ThermoFischerTM Scientific Catalog # 23225).
  • Luminex xMAP technology was used for multiplexed quantification of 32 Mouse cytokines, chemokines, and growth factors.
  • the multiplexing analysis was performed using the LuminexTM 200 system (Luminex®, Austin, TX, USA) by Eve Technologies Corp. (Calgary, Alberta). Thirty ⁇ two markers were simultaneously measured in the samples using Eve Technologies' Mouse Cytokine 31 ⁇ Plex Discovery Assay® (MilliporeSigmaTM, Burlington, Massachusetts, USA) according to the manufacturer's protocol.
  • the 32 ⁇ plex consisted of Eotaxin, G ⁇ CSF, GM ⁇ CSF, IFN ⁇ , IL ⁇ 1 ⁇ , IL ⁇ 1 ⁇ , IL ⁇ 2, IL ⁇ 3, IL ⁇ 4, IL ⁇ 5, IL ⁇ 6, IL ⁇ 7, IL ⁇ 9, IL ⁇ 10, IL ⁇ 12 (p40), IL ⁇ 12 (p70), IL ⁇ 13, IL ⁇ 15, IL ⁇ 17, IP ⁇ 10, KC, LIF, LIX, MCP ⁇ 1, M ⁇ CSF, MIG, MIP ⁇ 1 ⁇ , MIP ⁇ 1 ⁇ , MIP ⁇ 2, RANTES, TNF ⁇ , and VEGF. Assay sensitivities of these markers range from 0.3 ⁇ 30.6 pg/mL for the 32 ⁇ plex.
  • B16.F10 cells were thawed and cultured according to manufacturer’s instruction, washed, counted, and resuspended in PBS.
  • Cell suspension containing 2.5 x 10 5 cells/100 ⁇ l was subcutaneously injected in the right rear flank of C57BL/6 female mice. Tumor volumes were measured with a caliper. When the majority of the tumors reached 50 ⁇ 150 mm 3 , the mice were randomized to begin the study on Day 0.
  • mice On Days 1, 4, 7, and 10, the mice were dosed intraperitoneally (IP) with either Isotype control (30 mg/kg or 90 mg/kg), IMM20059 (5 mg/kg, 15 mg/kg, 45 mg/kg), Atezolizumab (5 mg/kg, 15 mg/kg, 45 mg/kg), or a combination of IMM20059 and Atezolizumab (each dosed at 5 mg/kg, 15 mg/kg, 45 mg/kg.
  • Tumor Patent Application Attorney Docket No. 172.0016 ⁇ WO00 volume and body weight were measured three times per week up to Day 11. Mean tumor volumes were plotted up to Day 9, the final day all mice were on study (Fig. 9).
  • Tumor volumes were measured across the groups on Day 9 and plotted as individual and mean tumor volumes (Fig. 10). The study was concluded on Day 11, 24 hours after the final dosing, and blood was collected. Exposure of antibodies in the plasma in each treatment group were assessed using the Human Therapeutic IgG1 ELISA Kit (Cayman Chemical, catalog number 500910) following instructions from the manufacturer. Terminal plasma exposure and terminal tumor volume on Day 11 were plotted. Pearson correlation analysis was performed to assess a positive or negative correlation between these variables.
  • Example 8 Effects of IMM20059 on lymphocytes numbers in naive female C57BL/6 mice Fc null IMM20059 was generated by introducing the following mutations in Fc region of IMM20059: L234A, L235A, P329G, and N297A. To confirm these mutations abrogated FcR binding, LumitTM Fc ⁇ RIIIA (V158) binding immunoassay kit (PromegaTM, catalog # CS3041A04) were used according to manufacturer’s instruction (Fig. 12).
  • lymphoid organs bone marrow, thymus, lymph node, and spleen
  • total and live cell numbers were counted on a Nexcelom Cellometer using acridine orange/propidium iodide (AOPI) dye.
  • AOPI acridine orange/propidium iodide
  • cells stained with Live/Dead Near IR Fixable dye InvitrogenTM
  • the total number of cells were counted by running cells on the Attune NxT Flow Cytometer (InvitrogenTM) with 123count eBeads Counting Beads (InvitrogenTM).
  • the frequency of live cells and immune cell subsets are enumerated using the manufacturer’s instructions and live cells are plotted (Fig. 13).
  • samples were stained with lineage ⁇ specific, fluorochrome ⁇ conjugated antibodies to identify populations including T cells (CD4, CD8), B cells (B220, IgM, IgD), NK cells (NK1.1) and myeloid cells (Gr1, CD11b, CD11c). Samples were run on the Attune NxT Flow Cytometer and the percentage of each population was multiplied by the number of total live cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

This invention relates to treating cancer by targeting the cell membrane-localized adaptor protein, Epsin-1 (EPN1), in combination with targeting one or more immune checkpoint proteins, including targeting programmed cell death-ligand 1 (PD-L1) and/or targeting Programmed cell death protein 1 (PD-1)

Description

Patent Application  Attorney Docket No. 172.0016‐WO00    COMBINATION ANTI‐EPN1 AND ANTI‐PD‐L1 ANTIBODY THERAPIES  Cross Reference to Related Applications  [0001]     This application claims priority to U.S. Provisional Application No. 63/380,457, filed on October  21, 2022; U.S. Provisional Application No. 63/368,583, filed on July 15, 2022; and U.S. Provisional  Application No. 63/343,282, filed on May 18, 2022; the disclosures of which are all incorporated by  reference.  Sequence Listing  [0002]     The Sequence Listing, submitted herewith through Patent Center as an eXtensible Markup  Language file (2023‐05‐16_Sequence_listing_172.0016‐WO00.xml; created on May 16, 2023; 27,474  bytes), is incorporated by reference.  Background  [0003]     The human adaptive immune system responds through both cellular (T cell) and humoral (B  cell) processes.  The humoral response results in selection and clonal amplification of B cells that express  surface bound immunoglobulin (Ig) molecules capable of binding to antigens.  The processes of somatic  hypermutation and class switching take place concordant with the clonal amplification.  Together these  processes lead to secreted antibodies that have been affinity matured against a target antigen and  contain a constant domain belonging to one of the five general classes, also called isotypes, (M, D, A, G,  or E).  Each class of antibody (IgM, IgD, IgA, IgG, and IgE) interact in distinct ways with the cellular  immune system.  Hallmarks of antibodies that have been affinity matured against a target antigen can  include: 1) nucleotide, and subsequent amino acid, changes relative to the germline gene, 2) high  binding affinity for the target antigen, 3) binding selectivity for the target antigen as compared to other  proteins.  [0004]     It is well understood that oncology patients can mount an immune response against tumor  antigens.  Those antigens can result either from genetic changes within the tumor that lead to mutated  proteins or aberrant presentation, of otherwise normal, proteins to the immune system.  Aberrant  presentation may occur through processes that include, but are not limited to, ectopic expression of  neonatal proteins, over‐expression of proteins to a high level, mis‐localization of intracellular proteins to  the cell surface, or lysis of cells.  Aberrant glycosylation of proteins, which may occur because of changes  Patent Application  Attorney Docket No. 172.0016‐WO00    in the expression of enzymes, such as, but not limited to, glycosyltransferases, can also result in  generation of nonself‐antigens that are recognized by the humoral immune system.  [0005]     Antibodies, which bind selectively to disease‐related proteins, including proteins related to  cancer, have proven successful at modulating the functions of their target proteins in ways that lead to  therapeutic efficacy.  The ability of the human immune system to mount antibody responses against  mutated, or otherwise aberrant, proteins suggests that patients’ immune responses may include  antibodies that are capable of recognizing, and modulating the function of, critical tumor‐drivers.  [0006]     The tumor microenvironment is essential for tumor cells to evade detection and elimination by  the immune system.  This is achieved through several mechanisms including the recruitment of  suppressive immune cells, expression of immune checkpoint molecules like PD‐L1, PD‐1, LAG‐3, and  CTLA‐4  and the presence of immunosuppressive cytokines.  Therefore, some immune‐oncology  therapies aim to target key molecules that are responsible for the immunosuppressive barriers within  the tumor microenvironment.  Successful immune‐oncology therapies can lead to the infiltration of  cytotoxic T cells and NK cells as well as an upregulation of Th1 cytokines and the induction of a  successful anti‐tumor response.   [0007]     Programmed Death‐1 (PD‐1) is an immune checkpoint receptor expressed on the surface of  immune effector cells, including CD8+ T cells.  Programmed Death Ligand‐1 (PD‐L1) is normally  expressed on antigen presenting cells, such as dendritic cells.  Binding of PD‐L1 to Programmed cell  death protein 1 (PD‐1) leads to T cell inactivation and inhibition of cytokine secretion.  Tumor cells can  induce PD‐L1 expression, leading to inactivation of T cells within the tumor microenvironment, and a  dampening of anti‐tumor immunity.  Antibodies specific for either PD‐1 or PD‐L1 can block the PD‐1‐ mediated inhibitory signaling pathway, which, in turn, can result in increased levels of T cell activation.   Indeed, both anti‐PD1 and anti‐PD‐L1 antibodies have demonstrated significant clinical benefit in a  number of different cancer indications.   Summary of the Invention  [0008]     This disclosure describes methods, compositions, and kits of an invention for treating cancer  by targeting the cell membrane‐localized adaptor protein, Epsin‐1 (EPN1), in combination with targeting  one or more immune checkpoint proteins.  For example, in a preferred embodiment of the invention,  the cancer therapy method combines targeting EPN1 with targeting programmed cell death‐ligand 1  (PD‐L1), while in another preferred embodiment of the invention, the cancer therapy method combines  targeting EPN1 with targeting programmed cell death receptor (PD‐1).    Patent Application  Attorney Docket No. 172.0016‐WO00    [0009]     According to the invention, a therapy that combines targeting EPN1 with targeting one or  more immune checkpoint proteins, may administer EPN1‐specific and immune checkpoint protein  targeting agents in separately‐administered pharmaceutical compositions, respectively, or administer  EPN1‐specific and immune checkpoint protein targeting agents by administering a single pharmaceutical  composition that contains EPN1‐specific and immune checkpoint protein targeting agents.  A step of  administering EPN1‐targeting and immune checkpoint protein targeting agents separately, according to  the invention, is inclusive of administering EPN1‐specific agents concurrently, simultaneously, essentially  simultaneously, or sequentially with one or more separately‐administered immune checkpoint protein  targeting agents.   [0010]     EPN1 targeting agents and immune checkpoint targeting agents are typically, but not  exclusively, antibodies specific for EPN1 and an immune checkpoint protein, respectively.  For example,  in some methods of the invention for treating cancer, the method combines the administration of a  pharmaceutical composition containing an anti‐EPN1 antibodies to a subject, in need thereof, with the  administration of a pharmaceutical composition containing one or more immune checkpoint protein  specific antibodies.  In such methods, administration of the pharmaceutical composition containing an  anti‐EPN1 antibodies to a subject in need thereof may be separate from the administration of the  administration of the pharmaceutical composition containing one or more immune checkpoint protein  specific antibodies.  For example, the pharmaceutical composition containing an anti‐EPN1 antibodies  may be administered to a subject in need thereof concurrently, simultaneously, essentially  simultaneously, or sequentially with the pharmaceutical composition containing one or more antibodies  specific for one or more immune checkpoint proteins.  [0011]     Alternatively, in other methods of the invention, a single pharmaceutical composition  containing anti‐EPN1 antibodies and one or more antibodies specific for one or more immune  checkpoint proteins are administered to a subject in need thereof.  [0012]     In various embodiments, methods of the invention treat lung squamous carcinoma, lung  adenocarcinoma, breast cancer, prostate cancer, uterine cancer, cervical cancer, thyroid cancer,  melanoma, gastroesophageal cancer, bladder cancer, or hepatocellular carcinoma.  [0013]     EPN1‐specific antibodies of the invention are typically monospecific for EPN1.  In some  methods of the invention, the EPN1‐specific antibody contains: A) a variable heavy chain (VHC) that  comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID  NO: 1; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 2.  In the same or different methods of the  Patent Application  Attorney Docket No. 172.0016‐WO00    invention, the EPN1‐specific antibody contains at least one of the following complementary determining  region (CDR) amino acid sequences: a CDR‐H1 amino acid sequence of SEQ ID NO: 3; a CDR‐H2 amino  acid sequence of SEQ ID NO: 4; a CDR‐H3 amino acid sequence of SEQ ID NO: 5; a CDR‐L1 amino acid  sequence of SEQ ID NO: 6; a CDR‐L2 amino acid sequence of SEQ ID NO: 7; and a CDR‐L3 amino acid  sequence of SEQ ID NO: 8.  [0014]     Like EPN1 antibodies of the invention, PD‐L1‐specific antibodies of the invention are also  typically monospecific.  In some methods of the invention, the PD‐L1‐specific antibody contains: A) a  variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the  amino acid sequence of SEQ ID NO: 9; and B) a variable light chain (VLC) that comprises an amino acid  that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 10.  In the same or  different methods of the invention, the PD‐L1‐specific antibody contains at least one of the following  complementary determining region (CDR) amino acid sequences: a CDR‐H1 amino acid sequence of SEQ  ID NO: 11; a CDR‐H2 amino acid sequence of SEQ ID NO: 12; a CDR‐H3 amino acid sequence of SEQ ID  NO: 13; a CDR‐L1 amino acid sequence of SEQ ID NO: 14; a CDR‐L2 amino acid sequence of SEQ ID NO:  15; and a CDR‐L3 amino acid sequence of SEQ ID NO: 16.  [0015]     In another method of the invention, the PD‐L1‐specific antibody contains: A) a variable heavy  chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid  sequence of SEQ ID NO: 17; and B) a variable light chain (VLC) that comprises an amino acid that shares  at least 90% homology with the amino acid sequence of SEQ ID NO: 18.  [0016]     In yet another method of the invention, the PD‐L1‐specific antibody contains: A) a variable  heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid  sequence of SEQ ID NO: 19; and B) a variable light chain (VLC) that comprises an amino acid that shares  at least 90% homology with the amino acid sequence of SEQ ID NO: 20.  [0017]     In yet another method of the invention, the PD‐L1‐specific antibody contains: A) a variable  heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid  sequence of SEQ ID NO: 21; and B) a variable light chain (VLC) that comprises an amino acid that shares  at least 90% homology with the amino acid sequence of SEQ ID NO: 22.  [0018]     In yet another method of the invention, the PD1‐specific antibody contains: A) a variable heavy  chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid  sequence of SEQ ID NO: 23; and B) a variable light chain (VLC) that comprises an amino acid that shares  at least 90% homology with the amino acid sequence of SEQ ID NO: 24.  Patent Application  Attorney Docket No. 172.0016‐WO00    [0019]     In yet another method of the invention, the PD1‐specific antibody contains: A) a variable heavy  chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid  sequence of SEQ ID NO: 25; and B) a variable light chain (VLC) that comprises an amino acid that shares  at least 90% homology with the amino acid sequence of SEQ ID NO: 26.  [0020]     In yet another method of the invention, the PD1‐specific antibody contains: A) a variable heavy  chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid  sequence of SEQ ID NO: 27; and B) a variable light chain (VLC) that comprises an amino acid that shares  at least 90% homology with the amino acid sequence of SEQ ID NO: 28.  Brief Description of the Figures  [0021]     Fig. 1 is a graph of mean B16F10 tumor volumes of mice harboring tumors over a 10‐day  period in which the mice were administered 12.5 mg/kg of Atezolizumab (anti‐PD‐L1), 12.5 mg/kg  IMM20059 (anti‐EPN1), or a combination of 12.5 mg/kg each of Atezolizumab and IMM20059 on days 1,  4 and 7. Control mice were administered 25 mg/kg hIgG1 on the same days.  [0022]     Fig. 2 depicts the individual and mean B16F10 tumor volumes at day 7 of the study described  in Fig. 1.  [0023]     Fig. 3. contains graphs that show the tumor volumes for each mouse over the 10‐day period  that is described for Fig. 1 for groups administered Atezolizumab, IMM20059, (Atezolizumab and  IMM20059), and hIgG1, respectively.  [0024] Fig. 4. demonstrates EPN1 is overexpressed in multiple cancer types. Fig. 4A is a graph of RNA  expression levels of EPN1 in various types of tumor vs normal tissues based upon analyses of data from  the TCGA database. Fig. 4B is publicly available immunohistochemical staining of various tumor types to  evaluate EPN1 protein expression.   [0025]     Fig. 5. demonstrates IMM20059 binds to the surface of cancer, but not normal, cell lines.  Fig. 5A is a scatter plot showing the level of binding to cancer cells relative to normal cells derived from  various tissues. Fig. 5B is a heatmap showing selective binding by flow cytometry of IMM20059 to  various cancer cell lines derived from various tissues.  [0026]     Fig. 6 presents Surface Plasmon Resonance (SPR) data showing sub‐nanomolar binding of  IMM20059 to recombinant human EPN1 protein.  [0027]     Fig. 7 is a binding curve showing concentration‐dependent binding of IMM20059 to the surface  of live B16.F10 melanoma cells (EC50 = 7.5 nM).    Patent Application  Attorney Docket No. 172.0016‐WO00    [0028]     Fig. 8A is a heat map of detectable intratumoral cytokines in tumor lysates prepared from  tumors harvested at Day 9 in a B16.F10 syngeneic model comparing treatments with isotype controls,  Atezolizumab, IMM20059, and Atezolizumab + IMM20059. Lysates were multiplexed for 32 cytokines.  Color scale represents fold‐change of cytokine concentrations in each sample over mean cytokine  concentration in Isotype control group (Luminex).   [0029]     Fig. 8B shows scatter plots of the intratumoral cytokines MIP‐1 ^, MIP‐1 ^, and RANTES  detected in the Day 9 individual lysates described in the summary of Fig. 8. These cytokines in tumors  harvested from animals treated with Atezolizumab + IMM20059 was statistically significant higher than  those in Isotype control group by one‐way ANOVA with Dunnett’s post‐hoc test. * p < 0.05; ***p <  0.001.  [0030]     Fig. 9 is a line graph of mean B16.F10 tumor volumes over a 9 day period  in mice dosed with  Isotype control (30 mg/kg or 90 mg/kg), Atezolizumab (5 mg/kg, 15 mg/kg, 45 mg/kg), IMM20059 (5  mg/kg, 15 mg/kg, 45 mg/kg), or a combination of Atezolizumab and IMM2005 (5 mg/kg, 15 mg/kg, 45  mg/kg of each test agent) every 3 days. The error bars represent standard error of tumor volumes in  specific group. Treatment of IMM20059 alone (45 mg/kg) or Atezolizumab + IMM20059 (45 mg/kg of  each test agent) showed trends of inhibition of tumor growth compared to isotype control treatment.  [0031]     Fig. 10 is a scatter plot depicting the individual B16.F10 tumor volumes at Day 9 in mice from  different treatment groups described in Fig. 9. The error bars represent standard error of tumor  volumes in specific group. Treatment of IMM20059 alone (45 mg/kg) or Atezolizumab + IMM20059 (45  mg/kg of each test agent) showed trends of inhibition of tumor growth compared to isotype control  treatment.  [0032]     Fig. 11 depicts scatter plots of tumor volumes from individual animals on the final day (Day 11)  of the study described in Fig. 9 and the terminal exposures of the indicated treatments in the plasma as  measured by hIgG1 concentration. The "r’ represents correlation coefficient and “p” represents false  discovery rate determined by Pearson correlation analysis.  It demonstrates a significant negative  correlation between tumor volumes and concentrations of IMM20059 or a combination of IMM20059  and Atezolizumab in terminal plasma.  [0033]     Fig. 12 is a line plot depicting that IMM20059 or positive control, but not Fc null IMM20059,  bind FcγRIIIa in vitro in concentration‐dependent manner.  The error bars represent standard deviation  of normalized relative luminescence at indicated concentration. IMM20059 (triangle) and positive  control (open circle) showed comparable binding to FcγRIIIa (V158 isoform) and displaced FcγRIIIa  binding to IgG tracer in the Lumit™ FcγRIIIa Binding Immunoassay (Promega™), while Fc null IMM20059  Patent Application  Attorney Docket No. 172.0016‐WO00    (diamond) showed no effects. This data suggests that IMM20059 has intact FcγRIIIa binding function and  Fc null IMM20059 lost binding to FcγRIIIa.  [0034]     Fig. 13 contains scatter plots depicting live cell counts on day 5 in bone marrow (Fig. 13A),  thymus (Fig. 13B), lymph node (Fig. 13C), spleen (Fig. 13D), and blood (Fig. 13E) of naïve C57BL/6 mice  after treatment with IMM20059, Fc null IMM20059, or isotype control at 45 mg/Kg intraperitoneally on  day 1 and day 4. Each dot represents the number of live cells in a specific organ from one mouse. The  error bar represents standard error. The numbers on top of horizontal lines are p values from One‐Way  ANOVA analysis with Dunnett post‐hoc comparison using IgG1 isotype as the control group. Both  IMM20059 and Fc null IMM20059 showed no significant changes in the numbers of live cells in vivo.   There were also no significant differences in the number of T cells, B cells, NK cells, or myeloid cells  between groups in all organs tested as determined by flow cytometry with lineage‐specific markers  (data not shown).  This suggests that the efficacy of IMM20059 in B16.F10 model (Fig. 1‐3 and Fig. 8‐11)  is independent of immune cell depletion.    [0035]     Fig. 14A is bar chart depicting the frequency of positive surface staining of IMM20059 on  different normal human cells at 667 nM , 67 nM and 6.7 nM in flow cytometry analysis. Error bars  represent standard deviation of biological duplicates of IMM20059 were performed within the same  experiment . IMM20059 showed no significant staining on the surface of eight (8) different normal  human cells. HRE: human renal epithelial cells; NHBE: normal human bronchial epithelial cells; NHEK:  normal human epidermal keratinocytes; NHDF: normal human dermal fibroblast cells; HRCE: human  renal cortical epithelial cells; HPrEC: human prostate epithelial cells; HMEC: human mammary epithelial  cells; SKMC: human skeletal muscle cells.  [0036]     Fig. 14B is a bar chart depicting the frequencies of positive surface staining of an isotype  control antibody for the staining of IMM20059 shown in Fig. 14A.  [0037]     Fig. 15 is a bar chart of relative mean florescent intensity (MFI) of surface staining of  IMM20059 on eleven (11) cancer cell lines at 667 nM, 67 nM, and 6.7 nM concentrations. IMM20059  showed concentration‐dependent binding to 10 out of 11 cancer cell lines tested.   Detailed Description  [0038]     This disclosure describes methods, compositions and kits used for treating various types of  cancer by combining therapies that target Epsin‐1 (EPN1) and target one or more immune checkpoint  proteins.  For example, some methods, compositions, and kits relate to administering an Epn1 targeting  agent to a subject in need thereof in combination with administering a targeting agent that targets a  Patent Application  Attorney Docket No. 172.0016‐WO00    component of the programmed cell death axis.  More particularly, in such methods of the invention, the  targeted component of the programmed cell death axis is programmed cell death receptor (PD‐1) or  programmed cell death‐ligand 1 (PD‐L1).    [0039]     A therapy according to the invention that combines targeting EPN1 with targeting one or more  immune checkpoint proteins, may administer EPN1‐specific and immune checkpoint protein targeting  agents in separately‐administered pharmaceutical compositions, respectively, or administer EPN1‐ specific and immune checkpoint protein targeting agents by administering a single pharmaceutical  composition that contains EPN1‐specific and immune checkpoint protein targeting agents.   Administering EPN1‐targeting and immune checkpoint protein targeting agents separately means EPN1‐ specific agents may be administered concurrently, simultaneously, essentially simultaneously, or  sequentially with one or more separately‐administered immune checkpoint protein targeting agents.   [0040]     EPN1 targeting agents and immune checkpoint protein targeting agents are known in the art.   Briefly, an EPN1 targeting agent specifically binds EPN1 and blocks binding of EPN1 with an EPN1 ligand  (e.g., an immune checkpoint protein).  Similarly, an immune checkpoint protein targeting agent  specifically binds an immune checkpoint protein and blocks binding of the immune checkpoint protein  to EPN1.  EPN1 targeting agents and immune checkpoint targeting agents are typically, but not  exclusively, antibodies specific for EPN1 and an immune checkpoint protein, respectively.  For example,  in some methods of the invention for treating cancer, the method combines the administration of a  pharmaceutical composition containing an anti‐EPN1 antibodies to a subject, in need thereof, with the  administration of a pharmaceutical composition containing one or more immune checkpoint protein  specific antibodies.  In such methods, administration of the pharmaceutical composition containing an  anti‐EPN1 antibodies to a subject in need thereof may be separate from the administration of the  administration of the pharmaceutical composition containing one or more immune checkpoint protein  specific antibodies.  For example, the pharmaceutical composition containing an anti‐EPN1 antibodies  may be administered to a subject in need thereof concurrently, simultaneously, essentially  simultaneously, or sequentially with the pharmaceutical composition containing one or more antibodies  specific for one or more immune checkpoint proteins.  [0041]     Alternatively, in other methods of the invention, a single pharmaceutical composition  containing anti‐EPN1 antibodies and one or more antibodies specific for one or more immune  checkpoint proteins are administered to a subject in need thereof.  [0042]     In some methods of treatment according to the invention, EPN1‐specific antibodies and the  PD‐L1‐specific or PD‐1‐specific antibodies are each administered separately.  In such methods of the  Patent Application  Attorney Docket No. 172.0016‐WO00    invention, the EPN1‐specific antibodies and either the PD‐L1‐specific or PD‐1‐specific antibodies may be  administered concurrently, simultaneously, essentially simultaneously, or sequentially.  Alternatively, in  other methods of the invention, a single pharmaceutical composition that contains the EPN1‐specific  antibodies and either the PD‐L1‐specific or PD‐1‐specific antibodies may be administered to a subject in  need thereof.  Accordingly, in some methods of the invention, the EPN1‐specific antibodies and either  the PD‐L1‐specific or PD‐1‐specific antibodies may be formulated into separate, respective  pharmaceutical compositions, may be formulated in a single pharmaceutical composition.  Whether the  EPN1‐specific antibodies and either the PD‐L1‐specific or PD‐1‐specific antibodies are administered in  separate or a single pharmaceutical compositions may be a choice made by a practitioner based on the  stage and type of cancer, the condition of the patient.  Likewise, the order of administration of the  EPN1‐specific antibodies and either the PD‐L1‐specific or PD‐1‐specific antibodies, as well as the number  of repetitions of administration of each antibody during a treatment protocol, may be based on the  same or similar factors.  [0043]     Combinations of EPN1 antibody and either PD‐L1 antibody or PD‐1 antibody therapies may  treat cancer by slowing or inhibiting growth or metastasis of primary tumors in a subject in need  thereof.  Notably, the effect of the combining anti‐EPN1 and either the anti‐PD‐L1 or anti‐PD‐1 therapies  on the growth or metastasis of tumors is synergistic. For example, some methods of the invention  reduce or limit the volume of one or more cancerous tumors in a subject afflicted by cancer, to no more  than 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% of the  volume (i.e., the size) of the tumor an oncologist or other cancer treatment practitioner would expect if  the subject had been treated with anti‐EPN1, anti‐PD‐L1, or anti‐PD‐1 antibodies.  In other words, the  therapeutic effect of a combination therapy of the invention is greater in comparison to an expected  therapeutic effect produced by administering anti‐EPN1 antibodies without administering either anti‐ PD‐L1 or anti‐PD‐1 antibodies during the course of a treatment.    [0044]     In some methods of the invention, a reduction in the volume of one or more primary tumors  or at metastatic sites in a subject treated by a method of the invention, is an indicator of tumor  regression and clearance.  In other words, a method of the invention for treating cancer in a subject by  combining anti‐EPN1 antibody therapy with either anti‐PD‐L1 antibody therapy or anti‐PD‐1 antibody  therapy, reduces the volume of one or more tumors in the subject by greater percentage of reduction as  compared to the reduction of one or more tumors in a subject who received i) anti‐EPN1 antibody  therapy, but not anti‐PD‐L1 antibody therapy, or anti‐PD‐1 antibody therapy; or ii) anti‐PD‐L1 antibody  therapy or anti‐PD‐1 antibody therapy, but not anti‐EPN1 antibody therapy.   Patent Application  Attorney Docket No. 172.0016‐WO00    [0045]     Accordingly, as stated above, a method of the invention in which a cancer is treated in a  subject by combining the administration of IMM20059, or other EPN1‐specific antibodies, with the  administration of Atezolizumab, or other PD‐L1‐specific antibodies, the observed reduction in volume of  one or more tumors in the subject is synergistic in comparison to the observed or expected reduction in  the volume of one or more tumors as a consequence an additive effect of administering IMM20059, or  other EPN1‐specific antibodies with Atezolizumab, or other PD‐L1‐specific antibodies.  Similarly. a  method of the invention in which a cancer is treated in a subject by combining the administration of  IMM20059, or other EPN1‐specific antibodies, with the administration of PD‐1‐specific antibodies, the  observed reduction in volume of one or more tumors in the subject is synergistic in comparison to the  observed or expected reduction in the volume of one or more tumors as a consequence an additive  effect of administering IMM20059, or other EPN1‐specific antibodies with PD‐1‐specific antibodies.  In  such methods of the invention, the tumor may be, but is not limited to classification as lung squamous  carcinoma, lung adenocarcinoma, breast cancer, prostate cancer, uterine cancer, cervical cancer, thyroid  cancer, melanoma, gastroesophageal cancer, bladder cancer, or hepatocellular carcinoma.  [0046]     In some methods of the invention, an indicator of an increased probability of tumor regression  and clearance is the detection of one or more chemokines in the intratumoral environment of one or  more tumors in a subject who received anti‐EPN1 antibody therapy in combination with anti‐EPN1  antibody therapy with either anti‐PD‐L1 antibody therapy or anti‐PD‐1 antibody therapy.  In the same or  other methods of the invention, one or more chemokines are detected at a higher level in the  intratumoral environment of one or more tumors in a subject who received anti‐EPN1 antibody therapy  in combination with anti‐EPN1 antibody therapy with either anti‐PD‐L1 antibody therapy or anti‐PD‐1  antibody therapy, as compared to the detected levels of the same one or more chemokines in the  intratumoral environment of one or more tumors in a subject who received: i) anti‐EPN1 antibody  therapy, but not anti‐PD‐L1 antibody therapy, or anti‐PD‐1 antibody therapy; or ii) anti‐PD‐L1 antibody  therapy or anti‐PD‐1 antibody therapy, but not anti‐EPN1 antibody therapy.   [0047]     Accordingly, an additive or synergistic effect of combining the administration of IMM20059, or  other EPN1‐specific antibodies, with the administration of Atezolizumab, or other PD‐L1‐specific  antibodies, is the detection of higher levels of one or more chemokines in the intratumoral environment  of one or more tumors in the subject in comparison to the observed or expected detected levels of one  or more tumors as a consequence an additive effect of receiving: i) IMM20059 therapy or other anti‐ EPN1 antibody therapy, but not anti‐PD‐L1 antibody therapy or anti‐PD‐1 antibody therapy; or ii) anti‐ PD‐L1 antibody therapy or anti‐PD‐1 antibody therapy, but not IMM20059 therapy or other anti‐EPN1  Patent Application  Attorney Docket No. 172.0016‐WO00    antibody therapy.  In some methods of the invention, the levels of one or more of the chemokines MIP‐ 1α, MIP‐1β, and RANTES detected in the intratumoral environment of one or more tumors are increased  as a consequence of a synergistic effect of receiving anti‐EPN1 antibody therapy in combination with  anti‐EPN1 antibody therapy with either anti‐PD‐L1 antibody therapy or anti‐PD‐1 antibody therapy.  [0048]     As described above, in some methods of the invention, the detection of higher levels of one or  more chemokines in the intratumoral environment is an indicator of an increased probability of tumor  regression and clearance.  Thus, it follows that, in some methods on the invention, increased levels of  one or more intratumoral chemokines may be used to predict the probability of success of the  treatment.  In other words, an increased level of one or more intratumoral chemokines in a subject who  was treated by a method of the invention, may be used as a marker of a higher probability of success of  the treatment.  In such methods of the invention, the tumor may be, but is not limited to classification  as lung squamous carcinoma, lung adenocarcinoma, breast cancer, prostate cancer, uterine cancer,  cervical cancer, thyroid cancer, melanoma, gastroesophageal cancer, bladder cancer, or hepatocellular  carcinoma.  [0049]     This disclosure does not place limits on mechanisms of action mediated by methods of the  invention to treat cancer by limiting or reducing the growth or formation.  EPN1‐specific antibodies and  immune checkpoint protein specific antibodies, such as, for example, PD‐L1‐specific antibodies and or  PD‐1‐specific antibodies reduce suppression of immune cell‐mediated anti‐tumor activity.  In some  methods of the invention, one or both the EPN1 specific antibodies and the PD‐L1 specific or PD‐1  specific antibodies reduce suppression of immune cell‐mediated anti‐tumor activity by targeting EPN1  and PD‐L1‐mediated signaling on tumor‐cell derived exosomes.  In some of those methods, suppression  of immune cell‐mediated anti‐tumor activity is mediated by CD8+ suppressor T cells.  Alternatively, in the  same or other methods of the invention, at least some EPN1‐specific antibodies bind EPN1 that is  expressed on the surface of tumor cells, thereby effecting the death of the cancer cell.   [0050]     This disclosure also does not limit the types of cancers that are treated by methods of the  invention.  In some methods of the invention, anti‐EPN1 and either anti‐PD‐L1 or anti‐PD‐1 antibodies  function together synergistically to slow or inhibit the growth or metastasis of one or more of lung  squamous carcinoma, lung adenocarcinoma, breast cancer, prostate cancer, uterine cancer, cervical  cancer, thyroid cancer, melanoma, gastroesophageal cancer, bladder cancer, or hepatocellular  carcinoma.  [0051]     Subjects treated by methods of the invention are typically human subjects that have been  diagnosed with cancer or are otherwise suspected as being afflicted by cancer.  Therapeutically effective  Patent Application  Attorney Docket No. 172.0016‐WO00    amounts of anti‐EPN1 and either anti‐PD‐L1 or PD‐1 antibodies administered to a subject according to  the invention will depend upon the severity of the cancer, and the general state of the patient’s health.   A therapeutically effective amount of the foregoing antibodies provides either subjective relief of a  symptom(s) or an objectively identifiable improvement as noted by a clinician or other qualified  professional.  For example, therapeutically effective amounts of the anti‐EPN1 and either the anti‐PD‐L1  or anti‐PD‐1 antibodies are dosage amounts that, when administered to a subject in need thereof, are  sufficient to inhibit growth, or metastasis of cancer cells, or to inhibit a sign or a symptom of the cancer.   [0052]     In some methods of the invention, the administered therapeutic dosage amounts of the anti‐ EPN1 antibody and either the anti‐PD‐L1 or anti‐PD‐1 antibody are equivalent.  Whereas, in other  methods of the invention, the administered therapeutic dosage amount of the anti‐EPN1 antibody is  greater than the administered therapeutic dosage amount of either the anti‐PD‐L1 or anti‐PD‐1  antibody.  In yet other methods of the invention, the administered therapeutic dosage amount of the  anti‐EPN1 antibody is less than the administered therapeutic dosage amount of either the anti‐PD‐L1  antibody or anti‐PD‐1.   [0053]     This disclosure does not limit minimum or maximum dosage amounts of either the EPN1‐ specific, PD‐L1‐specific, or PD‐1‐specific antibodies that are administered to a subject in need thereof. In  some methods of the invention, the administered dosage amount of an EPN1‐specific antibody is from  0.2 mg/kg to 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg,  50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg,  95 mg/kg, 100 mg/kg, 105 mg/kg, 110 mg/kg, or any amount therein.  Likewise, the administered  dosage amount of either a PD‐L1‐specific or PD‐1‐specific antibody is from 0.2 mg/kg to 10 mg/kg, 15  mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg,  65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 105 mg/kg, 110  mg/kg, or any amount therein, wherein mg/kg = (mg antibody/kg body weight).  [0054]     In some methods of the invention, the foregoing antibody dosage amounts correlate to  intravenously administering a pharmaceutical composition, containing from 800‐1200 mg of PD‐L1‐ specific antibodies and/or 800‐1200 mg of PD‐1 specific antibodies, to a human subject every two  weeks(Q2W) or every three weeks (Q3W).  In such methods, a pharmaceutical composition comprising  EPN1‐specific antibodies may be intravenously administered, either as a single administered dose, or in  a series of administered doses, at 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, or less than 1 week  before or after the first or last administration of PD‐L1 and/or PD1 specific antibodies.  The dosage  amount of EPN1‐specific antibody in a pharmaceutical composition administered in one or more of the  Patent Application  Attorney Docket No. 172.0016‐WO00    above‐methods may contain, but is not limited to, containing 200 mg – 1800 mg of EPN1‐specific  antibodies administered to a human subject every week (Q1W), every two weeks (Q2W), every three  weeks (Q3W), or every 4 weeks (Q4W).   [0055]     In alternative methods of the invention, the foregoing antibody dosage amounts correlate to  intravenously administering from 1500‐1680 mg of a PD‐L1‐specific antibody to a human subject every  three(Q3W) or every four weeks (Q4W).  In such methods, a pharmaceutical composition comprising  EPN1‐specific antibodies may be intravenously administered, either as a single administered dose, or in  a series of administered doses, at 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, or less than 1 week  before or after the first or last administration of PD‐L1‐specific antibodies.  The dosage amount of EPN1‐ specific antibody in a pharmaceutical composition administered in one or more of the above‐methods  may contain, but is not limited to, containing 200 mg – 1800 mg of EPN1‐specific antibodies  administered to a human subject every week (Q1W), every two weeks (Q2W), every three weeks (Q3W),  or every 4 weeks (Q4W).  [0056]     In some methods of the invention, initial loading dosage amounts of EPN1‐specific and/or  PD‐L1‐specific antibodies are administered, with subsequent, maintenance doses being administered at  lower dosage levels.  For example, after administering initial loading doses of EPN1‐specific and/or PD‐ L1‐specific antibodies to a subject, a lower weekly maintenance dose may be administered to the  subject for the subsequent 4, 5, 6, 7, or 8 weeks.  [0057]     In alternative methods of the invention, the foregoing antibody dosage amounts correlate to  intravenously administering from 200 ‐ 400 mg of a PD‐1‐specific antibody to a human subject every  three (Q3W) or every six weeks (Q6W). In such methods, a pharmaceutical composition comprising  EPN1‐specific antibodies may be intravenously administered, either as a single administered dose, or in  a series of administered doses, at 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, or less than 1 week  before or after the first or last administration of PD‐1‐specific antibodies. The dosage amount of EPN1‐ specific antibody in a pharmaceutical composition administered in one or more of the above‐methods  may contain, but is not limited to, containing 200 mg – 1800 mg of EPN1‐specific antibodies  administered to a human subject every week (Q1W), every two weeks (Q2W), every three weeks (Q3W),  or every 4 weeks (Q4W).  [0058]     In alternative methods of the invention, the foregoing antibody dosage amounts correlate to  intravenously administering either 3 mg/Kg or 240 mg of a PD‐1‐specific antibody to a human subject  every two (Q2W), or 480 mg every four weeks (Q4W). In such methods, a pharmaceutical composition  comprising EPN1‐specific antibodies may be intravenously administered, either as a single administered  Patent Application  Attorney Docket No. 172.0016‐WO00    dose, or in a series of administered doses, at 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, or less than 1  week before or after the first or last administration of PD‐1‐specific antibodies.  The dosage amount of  EPN1‐specific antibody in a pharmaceutical composition administered in one or more of the above‐ methods may contain, but is not limited to, containing 200 mg – 1800 mg of EPN1‐specific antibodies  administered to a human subject every week (Q1W), every two weeks (Q2W), every three weeks (Q3W),  or every 4 weeks (Q4W).  [0059]     In alternative methods of the invention, the foregoing antibody dosage amounts correlate to  intravenously administering 350 mg of a PD‐1‐specific antibody to a human subject every three (Q3W).  In such methods, a pharmaceutical composition comprising EPN1‐specific antibodies may be  intravenously administered, either as a single administered dose, or in a series of administered doses, at  5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, or less than 1 week before or after the first or last  administration of PD‐1‐specific antibodies.  The dosage amount of EPN1‐specific antibody in a  pharmaceutical composition administered in one or more of the above‐methods may contain, but is not  limited to, containing 200 mg – 1800 mg of EPN1‐specific antibodies administered to a human subject  every week (Q1W), every two weeks (Q2W), every three weeks (Q3W), or every 4 weeks (Q4W).  [0060]     EPN1‐specific and/or PD‐L1‐specific or PD‐1‐specific antibodies that are administered in  methods of the invention are formulated into compositions.  More particularly, the antibodies may be  formulated for systemic administration, or local administration, such as intra‐tumor administration.  For  example, EPN‐specific and/or PD‐L1‐specific or PD‐1‐specific antibodies may be formulated for  parenteral administration, such as intravenous administration.  The compositions can be prepared in  unit dosage forms for administration to a subject. The amount and timing of administration are at the  discretion of the treating clinician to achieve the desired outcome.  Options for administering the EPN1‐ specific and/or PD‐L1‐specific or PD‐1‐specific antibodies include, but are not limited to, slow infusion  and via an intravenous push or bolus.  Prior to being administered, the antibodies may be provided in  lyophilized form, and rehydrated in a sterile solution to a desired concentration before administration.    [0061]     Antibody compositions according to the invention may also be controlled release formulations.  Controlled release parenteral formulations, for example, can be made as implants, or oily injections.  Particulate systems, including microspheres, microparticles, microcapsules, nanocapsules, nanospheres,  and nanoparticles, may also be used to deliver EPN1‐specific and/or PD‐L1‐specific or PD‐1‐specific  antibodies.  Microcapsules, as referred to herein, contain EPN1‐specific and/or PD‐L1‐specific or PD‐1‐ specific antibodies in central core components.  In microspheres, EPN1‐specific and/or PD‐L1‐specific or  PD‐1‐specific antibodies are dispersed throughout the particle. Particles, microspheres, and  Patent Application  Attorney Docket No. 172.0016‐WO00    microcapsules smaller than about 1 µm are generally referred to as nanoparticles, nanospheres, and  nanocapsules, respectively.  [0062]     Administration of the EPN‐specific and/or PD‐L1‐specific or PD‐1‐specific antibodies may also  be accompanied by administration of other anti‐cancer agents or therapeutic treatments, such as  surgical resection of a tumor.  Any suitable anti‐cancer agent can be administered in combination with  the antibodies disclosed herein. Exemplary anti‐cancer agents include, but are not limited to,  chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, anti‐metabolites,  intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase  inhibitors, anti‐survival agents, biological response modifiers, anti‐hormones (e.g., anti‐androgens) and  anti‐angiogenesis agents.  Other anti‐cancer treatments include radiation therapy and other antibodies  that specifically target cancer cells.  [0063]     A basic antibody structure includes two heavy (H) and two light (L) polypeptide chains, each of  which contains a constant region and a variable region and are interconnected by disulfide bonds. In  humans, there are two types of immunoglobulin light chains, which are termed lambda ("λ") and kappa  ("κ"), and five main immunoglobulin heavy chain classes, also known as isotypes, which determine  functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.  Together, a variable heavy ("VH")  region and a variable light ("VL") region form a fragment variable "Fv" that is responsible for the specific  binding of the antibody to its antigen.  A full‐length heavy chain also has three constant domains (CH1,  CH2, CH3).  The constant regions of the Abs may mediate the binding of the immunoglobulin to host  tissues or factors, including various cells of the immune system (e.g., effector cells) and the first  component (C1q) of the classical complement system.  [0064]     VH and VL regions contain "framework" regions interrupted by three hypervariable regions,  called complementarity‐determining regions ("CDRs").  The CDRs are primarily responsible for binding to  an epitope of an antigen.  The sequences of the framework regions of different light or heavy chains are  relatively conserved within a species and serve to position and align the CDRs in three‐dimensional  space. The three CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered  sequentially starting from the N‐terminus, and are often identified by the chain in which the particular  CDR is located.  Accordingly, heavy chain CDRs are designated H‐CDR1, H‐CDR2, and H‐CDR3; likewise,  light chain CDRs are designated L‐CDR1, L‐CDR2, and L‐CDR3.  An antigen‐binding fragment, one  constant and one variable domain of each of the heavy and the light chain is referred to as an Fab  fragment.  An F(ab)'2 fragment contains two Fab fragments and can be generated by cleaving an  immunoglobulin molecule below its hinge region.  Patent Application  Attorney Docket No. 172.0016‐WO00    [0065]     EPN1‐specific antibodies of the invention are typically monospecific for EPN1. However, this  disclosure does not limit the EPN1‐specific antibodies that may be administered in methods of the  invention, as described above. In some methods of the invention, the EPN1‐specific antibody contains:   A) a variable heavy chain (VHC) that comprises an amino acid that shares at least 90% homology with  the amino acid sequence of SEQ ID NO: 1; and B) a variable light chain (VLC) that comprises an amino  acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO: 2.  In the same or  different method of the invention, the EPN1‐specific antibody contains at least one of the following  complementary determining region (CDR) amino acid sequences: a CDR‐H1 amino acid sequence of SEQ  ID NO: 3; a CDR‐H2 amino acid sequence of SEQ ID NO: 4; a CDR‐H3 amino acid sequence of SEQ ID NO:  5; a CDR‐L1 amino acid sequence of SEQ ID NO: 6; a CDR‐L2 amino acid sequence of SEQ ID NO: 7; and a  CDR‐L3 amino acid sequence of SEQ ID NO: 8.  [0066]     As the EPN1‐specific antibodies of the invention are typically monospecific for EPN1, PD‐L1‐ specific antibodies of the invention are also typically monospecific for PD‐L1, though this disclosure does  not limit the EPN1‐specific antibodies that may be administered in methods of the invention. In some  methods of the invention, the PD‐L1‐specific antibody contains: A) a variable heavy chain (VHC) that  comprises an amino acid that shares at least 90% homology with the amino acid sequence of SEQ ID NO:  9; and B) a variable light chain (VLC) that comprises an amino acid that shares at least 90% homology  with the amino acid sequence of SEQ ID NO: 10.  In the same or different method of the invention, the  PD‐L1‐specific antibody contains at least one of the following complementary determining region (CDR)  amino acid sequences: a CDR‐H1 amino acid sequence of SEQ ID NO: 11; a CDR‐H2 amino acid sequence  of SEQ ID NO: 12; a CDR‐H3 amino acid sequence of SEQ ID NO: 13; a CDR‐L1 amino acid sequence of  SEQ ID NO: 14; a CDR‐L2 amino acid sequence of SEQ ID NO: 15; and a CDR‐L3 amino acid sequence of  SEQ ID NO: 16.  [0067]     In other methods of the invention, the PD‐L1‐specific antibody contains: A) a variable heavy  chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid  sequence of SEQ ID NO: 17; and B) a variable light chain (VLC) that comprises an amino acid that shares  at least 90% homology with the amino acid sequence of SEQ ID NO: 18.  [0068]     In other methods of the invention, the PD‐L1‐specific antibody contains: A) a variable heavy  chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid  sequence of SEQ ID NO: 19; and B) a variable light chain (VLC) that comprises an amino acid that shares  at least 90% homology with the amino acid sequence of SEQ ID NO: 20.  Patent Application  Attorney Docket No. 172.0016‐WO00    [0069]     In other methods of the invention, the PD‐L1‐specific antibody contains: A) a variable heavy  chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid  sequence of SEQ ID NO: 21; and B) a variable light chain (VLC) that comprises an amino acid that shares  at least 90% homology with the amino acid sequence of SEQ ID NO: 22.  [0070]     In other methods of the invention, the PD‐1‐specific antibody contains: A) a variable heavy  chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid  sequence of SEQ ID NO: 23; and B) a variable light chain (VLC) that comprises an amino acid that shares  at least 90% homology with the amino acid sequence of SEQ ID NO: 24.  [0071]     In other methods of the invention, the PD‐1‐specific antibody contains: A) a variable heavy  chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid  sequence of SEQ ID NO: 25; and B) a variable light chain (VLC) that comprises an amino acid that shares  at least 90% homology with the amino acid sequence of SEQ ID NO: 26.  [0072]     In other methods of the invention, the PD‐1‐specific antibody contains: A) a variable heavy  chain (VHC) that comprises an amino acid that shares at least 90% homology with the amino acid  sequence of SEQ ID NO: 27; and B) a variable light chain (VLC) that comprises an amino acid that shares  at least 90% homology with the amino acid sequence of SEQ ID NO: 28.  [0073]     The EPN1‐specific, PD‐L1‐specific, and PD‐1‐specific antibodies of the invention are monoclonal  antibodies, meaning an antibody is produced by a single clonal B‐lymphocyte population, a clonal  hybridoma cell population, or a clonal population of cells into which the genes of a single antibody, or  portions thereof, have been transfected.  Monoclonal antibodies are produced by methods known to  those of skill in the art, for instance by making hybrid antibody‐forming cells from a fusion of myeloma  cells with immune lymphocyte cells.  [0074]     Monoclonal antibodies according to the invention are also typically humanized monoclonal  antibodies. More specifically, a "human" antibody, also called a "fully human" antibody, according to the  invention, is an antibody that includes human framework regions and CDRs from a human  immunoglobulin.  For example, the framework and the CDRs of an antibody are from the same  originating human heavy chain, or human light chain amino acid sequence, or both.  Alternatively, the  framework regions may originate from one human antibody, and be engineered to include CDRs from a  different human antibody. "Humanizing substitutions" are amino acid substitutions in which the amino  acid residue present at a particular position in the VH or VL domain of an antibody, such as an IL‐38  antibody, is replaced with an amino acid residue which occurs at an equivalent position in a reference  human VH or VL domain.  The reference human VH or VL domain may be a VH or VL domain encoded by  Patent Application  Attorney Docket No. 172.0016‐WO00    the human germline.  Humanizing substitutions may be made in the framework regions and/or the CDRs  of the antibodies, defined herein. A "humanized variant" is a variant antibody of the invention, which  contains one or more "humanizing substitutions" relative to a reference antibody, wherein a portion of  the reference antibody (e.g., the VH domain and/or the VL domain or parts thereof containing at least  one CDR) has an amino acid derived from a non‐human species, and the "humanizing substitutions"  occur within the amino acid sequence derived from a non‐human species.  [0075]     EPN1‐specific, PD‐L1‐specific, and PD‐1‐specific antibodies of the invention may also be  "antigen‐binding fragments".  An antigen‐binding fragment refers to a polypeptide fragment of an  immunoglobulin or antibody that binds antigen or competes with intact antibody (i.e., with the intact  antibody from which they were derived) for antigen binding (i.e., specific binding to IL‐38).  As used  herein, the term "fragment" of an antibody molecule includes antigen‐binding fragments of antibodies,  for example, an antibody light chain variable domain (VL), an antibody heavy chain variable domain  (VH), a single chain antibody (scFv), a F(ab')2 fragment, a Fab fragment, an Fd fragment, an Fv fragment,  and a single domain antibody fragment (Dab).  Fragments can be obtained, e.g., via chemical or  enzymatic treatment of an intact or complete antibody or antibody chain or by recombinant means.  Examples of immunoglobulin variants that are considered antibodies according to the invention include  single‐domain antibodies (such as VH domain antibodies), Fab fragments, Fab' fragments, F(ab)'2  fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv").  A VH single‐ domain antibody is an immunoglobulin fragment consisting of a heavy chain variable domain. An Fab  fragment contains a monovalent antigen‐binding immunoglobulin fragment, which can be produced by  digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one  heavy chain.  Similarly, a Fab' fragment also contains a monovalent antigen‐binding immunoglobulin  fragment, which can be produced by digestion of whole antibody with the enzyme pepsin, followed by  reduction, to yield an intact light chain and a portion of the heavy chain.  Two Fab' fragments are  obtained per immunoglobulin molecule.  A (Fab')2 fragment is a dimer of two Fab' fragments, that can  be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction, so Fab'  monomers remain held together by two disulfide bonds.  A Fv fragment is a genetically engineered  fragment containing the variable region of the light chain and the variable region of the heavy chain  expressed as two chains. A single chain ("sc") antibody, such as scFv fragment, is a genetically  engineered molecule containing the VL region of a light chain, the VH region of a heavy chain, linked by a  suitable polypeptide linker as a genetically fused single chain molecule. A dimer of a single chain  antibody, such as a scFV2 antibody, is a dimer of a scFV, and may also be known as a "miniantibody".  A  Patent Application  Attorney Docket No. 172.0016‐WO00    dsFvs variant also contains a VL region of an immunoglobulin and a VH region, but the chains have been  mutated to introduce a disulfide bond to stabilize the association of the chains.  [0076]     One of skill in the art will realize that conservative variants of the antibodies can be produced.   Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv  fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between  the VH and the VL regions, and will retain the charge characteristics of the residues to preserve the low pI  and low toxicity of the molecules.   [0077]     EPN1‐specific, PD‐L1‐specific, and PD‐1‐specific antibodies of the invention may also possess a  "tagged" immunoglobulin CH3 domain to facilitate detection of the biologic against a background of  endogenous antibodies.  More particularly, a tagged CH3 domain is a heterogenous antibody epitope  that has been incorporated into one or more of the AB, EF, or CD structural loops of a human IgG‐ derived CH3 domain.  CH3 tags are preferably incorporated into the structural context of an IgG1  subclass antibody, other human IgG subclasses, including IgG2, IgG3, and IgG4, are also available  according to the invention.  Epitope‐tagged CH3 domains, also referred to as "CH3 scaffolds" can be  incorporated into any antibody of the invention having a heavy chain constant region, generally in the  form of an immunoglobulin Fc portion.  Examples of CH3 scaffold tags, and methods for incorporating  them into antibodies are disclosed in International Patent Application Publication No. WO 2019/222575.   [0078]     EPN1‐specific, PD‐L1‐specific, and/or PD‐1‐specific antibodies of the invention may be  packaged into a kit for administering the antibodies to a subject for the purpose of treating cancer. Such  a kits include suitable storage containers, such as, ampules, vials, and tubes, for EPN1‐specific, PD‐L1‐ specific, and/or PD‐1‐specific antibody compositions and other included reagents, such as buffers and  balanced salt solutions, for use in administering the compositions to subjects.  The compositions and  other reagents may be present in the kits in any convenient form, such as, in a solution or in a powder  form.  The kits may further include instructions for using the components of the kit.    Examples  [0079]     Example 1. ‐ Efficacy study in the B16.F10 syngeneic model  A B16F10 melanoma model in C57BL/6 female mice was used to evaluate the impact of IMM20059 and  Atezolizumab – alone and in combination – on melanoma tumor growth.  To generate the model for  these studies, B16.F10 cells were thawed and cultured according to manufacturer’s instruction, washed,  counted, and resuspended in PBS, and subcutaneously injected in the right rear flank of C57BL/6 female  mice.  Injected cell suspension containing 2.5 x 105 cells/100 µl.  The tumors of mice harboring  Patent Application  Attorney Docket No. 172.0016‐WO00    established B16F10 tumors were measured with a caliper 2‐3 times per week. When the majority of  tumors reached 50‐150 mm3, the mice were randomized to begin the study on Day 0.  On Days 1, 4,  and 7, the mice were dosed intraperitoneally (IP) with either 25 mg/kg Isotype control, 12.5 mg/kg  IMM20059, 12.5 mg/kg Atezolizumab, or 12.5 mg/kg each of IMM20059 and Atezolizumab.  Tumor  volume and body weight were measured three days per week and the study was concluded on Day 9.   Tumor volumes were measured across the groups on Day 7 and compared using one‐way ANOVA with  Dunnett’s post‐hoc test.  [0080]     Example 2. ‐ EPN1 overexpression is associated with certain cancers   Overexpression of EPN1 RNA is observed in several different cancer types. EPN1 RNA expression across  various tumor types was assessed using the TCGA database (Fig.^4A).  Multiple cancers, including breast,  lung squamous, lung adenocarcinoma, uterus, cervical, prostate, thyroid, bladder, and liver, were shown  to overexpress EPN1 as compared to normal tissues from the same organ.   Cancers, including  colorectal, glioblastoma, and kidney were shown to have lower expression of EPN1 as compared to  normal tissue, publicly available immunohistochemistry data demonstrates expression of EPN1 protein  in various types of cancer (Fig. 4B)   [0081]     Example 3. – IMM20059 binds the surface of cancer cells as compared to normal cells.   IMM20059 was assessed for binding to the surface of additional cancer cell lines as well as normal cell  lines by flow cytometry (Fig. 5). Cell lines represent several different cancer indications and normal  tissues. Data are represented as scatter plots (Fig. 5A) and heatmap (Fig. 5B). Flow cytometry analysis on  expanded normal human cells (Fig. 14) and cancer cell lines (Fig. 15) are presented as bar charts.   [0082]     Example 4. – IMM20059 binds recombinant human EPN1 protein with sub‐nanomolar affinity.  Binding of IMM20059 to recombinant human EPN1 protein (Origene™, catalog number TP307099) was  quantified by surface plasmon resonance (SPR) performed at 25 ^C using a Biacore™ 2000 optical  biosensor with a Biacore™ Protein A sensor chip.  IMM20059 was diluted in SPR running buffer (10 mM  HEPES, pH7.4, 150 mM NaCl, 0.005% Tween‐20, 0.2% bovine serum albumin) and captured at 2 densities  (~200 and ~450 RU). EPN1 was diluted in SPR running buffer to a concentration of 33 nM and a 3‐fold  dilution series was run across the chip.  Data were collected at 25  ^C and were fit to a 1:1 interaction  model yielding the rate constants depicted in Fig. 6.   [0083]     Example 5. – IMM20059 binds to the surface of live B16.F10 cells  IMM20059 binding to the surface of live mouse B16.F10 melanoma cells was detected by fluorescently  labeled anti‐human IgG1 secondary antibody and assessed by flow cytometry.  Concentration‐ dependent binding of IMM20059 to B16.F10 cells was observed with an EC50 of 7.5 nM (Fig. 7).  Patent Application  Attorney Docket No. 172.0016‐WO00    [0084]     Example 6. – Combination of IMM20059 and Atezolizumab results in the significant  upregulation of intratumoral chemokines.  B16.F10 tumors from Example 1 were harvested on Day 9 and flash frozen.  To generate tumor  homogenates, frozen tumors were placed in 2 mL Bead Ruptor Prefilled Bead Tubes (Omni International  Catalog # 19‐628) with lysis buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, and 0.5% NP‐40.  A bead  mill homogenizer (Bead Ruptor Elite, Omni International Catalog # 19‐042E) was used to perform two 30  second bead beating cycles.  Tubes were spun at >10,000 x g for 10 minutes and the supernatant was  transferred to a fresh tube.  Total protein concentration was measured using the Pierce BCA Protein  Assay kit (ThermoFischer™ Scientific Catalog # 23225). All tumor lysates were diluted in lysis buffer and  normalized to 5 mg/mL of total protein.  Luminex xMAP technology was used for multiplexed  quantification of 32 Mouse cytokines, chemokines, and growth factors.  The multiplexing analysis was  performed using the Luminex™ 200 system (Luminex®, Austin, TX, USA) by Eve Technologies Corp.  (Calgary, Alberta). Thirty‐two markers were simultaneously measured in the samples using Eve  Technologies' Mouse Cytokine 31‐Plex Discovery Assay® (MilliporeSigma™, Burlington,  Massachusetts, USA) according to the manufacturer's protocol.  The 32‐plex consisted of Eotaxin, G‐CSF,  GM‐CSF, IFNγ, IL‐1α, IL‐1β, IL‐2, IL‐3, IL‐4, IL‐5, IL‐6, IL‐7, IL‐9, IL‐10, IL‐12 (p40), IL‐12 (p70), IL‐13, IL‐15,  IL‐17, IP‐10, KC, LIF, LIX, MCP‐1, M‐CSF, MIG, MIP‐1α, MIP‐1β, MIP‐2,  RANTES, TNFα, and VEGF.  Assay  sensitivities of these markers range from 0.3‐ 30.6 pg/mL for the 32‐plex.  Individual analyte sensitivity  values are available in the MilliporeSigma™ MILLIPLEX® MAP protocol.  Detectable markers are plotted  as a heat map normalized to the isotype control group (Fig. 8A).  Tumors treated with the combination  of IMM20059 and Atezolizumab had a significant increase in MIP‐1 ^, MIP‐1 ^, RANTES, and MIG  compared with the isotype control group (Fig. 8B).  [0085]     Example 7 – Efficacy study in the B16.F10 model  A B16.F10 melanoma model was used to evaluate the efficacy of IMM20059 and Atezolizumab  treatments alone and in combination on tumor growth.  To generate the model for these studies,  B16.F10 cells were thawed and cultured according to manufacturer’s instruction, washed, counted, and  resuspended in PBS. Cell suspension containing 2.5 x 105 cells/100 µl was subcutaneously injected in the  right rear flank of C57BL/6 female mice.  Tumor volumes were measured with a caliper. When the  majority of the tumors reached 50‐150 mm3, the mice were randomized to begin the study on Day 0.   On Days 1, 4, 7, and 10, the mice were dosed intraperitoneally (IP) with either Isotype control (30 mg/kg  or 90 mg/kg), IMM20059 (5 mg/kg, 15 mg/kg, 45 mg/kg), Atezolizumab (5 mg/kg, 15 mg/kg, 45 mg/kg),  or a combination of IMM20059 and Atezolizumab (each dosed at 5 mg/kg, 15 mg/kg, 45 mg/kg.  Tumor  Patent Application  Attorney Docket No. 172.0016‐WO00    volume and body weight (data not shown) were measured three times per week up to Day 11.  Mean  tumor volumes were plotted up to Day 9, the final day all mice were on study (Fig. 9).  Tumor volumes  were measured across the groups on Day 9 and plotted as individual and mean tumor volumes (Fig. 10).   The study was concluded on Day 11, 24 hours after the final dosing, and blood was collected.  Exposure  of antibodies in the plasma in each treatment group were assessed using the Human Therapeutic IgG1  ELISA Kit (Cayman Chemical, catalog number 500910) following instructions from the manufacturer.   Terminal plasma exposure and terminal tumor volume on Day 11 were plotted. Pearson correlation  analysis was performed to assess a positive or negative correlation between these variables.  [0086]     Example 8 – Effects of IMM20059 on lymphocytes numbers in naive female C57BL/6 mice  Fc null IMM20059 was generated by introducing the following mutations in Fc region of IMM20059:  L234A, L235A, P329G, and N297A. To confirm these mutations abrogated FcR binding, Lumit™ FcγRIIIA  (V158) binding immunoassay kit (Promega™, catalog # CS3041A04) were used according to  manufacturer’s instruction (Fig. 12).  [0087]     To assess the effects of IMM20059 on lymphocytes numbers in vivo, IMM20059, Fc null  IMM20059 and isotype control (human IgG1) antibodies were dosed intraperitoneally in naive female  C57BL/6 (n=8) at 7‐8 weeks old at 45 mg/kg on day 1 and day 5.  On day 5, bone marrow, thymus, lymph  node, spleen, and blood were collected.  Tissues are processed into single cell suspension and red blood  cells were lysed. Cell suspension was washed and filtered through a 50‐micron cell strainer.  For  lymphoid organs (bone marrow, thymus, lymph node, and spleen), total and live cell numbers were  counted on a Nexcelom Cellometer using acridine orange/propidium iodide (AOPI) dye.  For blood  samples, cells stained with Live/Dead Near IR Fixable dye (Invitrogen™) and fixed. The total number of  cells were counted by running cells on the Attune NxT Flow Cytometer (Invitrogen™) with 123count  eBeads Counting Beads (Invitrogen™).  The frequency of live cells and immune cell subsets are  enumerated using  the manufacturer’s instructions and live cells are plotted (Fig. 13).  To calculate  numbers of immune cell subsets, samples were stained with lineage‐specific, fluorochrome‐conjugated  antibodies to identify populations including T cells (CD4, CD8), B cells (B220, IgM, IgD), NK cells (NK1.1)  and myeloid cells (Gr1, CD11b, CD11c).  Samples were run on the Attune NxT Flow Cytometer and the  percentage of each population was multiplied by the number of total live cells.    

Claims

Patent Application  Attorney Docket No. 172.0016‐WO00    What is claimed:    1.    A method for treating cancer in a subject in need thereof, the method comprising  administering to the subject a therapeutically effective amount of an Epsin‐1 (EPN1)‐specific antibody 
Patent Application  Attorney Docket No. 172.0016‐WO00    in combination with a therapeutically effective amount of a programmed cell death‐ligand 1 (PD‐L1)‐ specific antibody and/or Programmed cell death protein 1 (PD‐1)‐specific antibody.    2.    The method of claim 1, wherein one or both the EPN1‐specific antibody and the PD‐L1‐specific  antibody and/or the PD‐1‐specific antibody reduce suppression of immune cell‐mediated anti‐tumor  activity.    3.    The method of claim 2, wherein the reduction of suppression of immune cell‐mediated anti‐ tumor activity is achieved by targeting tumor‐cell derived exosomes by one or both the EPN1‐specific   antibody and the PD‐L1‐specific antibody and/or the PD‐1‐specific antibody, wherein one or both the  EPN1‐specific antibody and the PD‐L1‐specific antibody bind EPN1 and PD‐L1, respectively.    4.    The method of claim 2 or 3, wherein suppression of immune cell‐mediated anti‐tumor activity  is mediated by CD8+ suppressor T cells.    5.    The method of claim 1, wherein the cancer in the subject comprises cells that express EPN1,  and the EPN1‐specific antibody effects antitumor activity by binding the EPN1 expressed by the cancer  cells.      6.  The method of claim 5, wherein the cancer in the subject comprises cells that express EPN1, and  wherein the EPN1‐specific antibody effects antitumor activity by binding the EPN1 expressed by the  cancer cells.    7.    The method of any one of claims 1‐6, wherein one or both of the EPN1‐specific antibody and  the PD‐L1‐specific antibody and/or the PD‐1‐specific antibody inhibit tumor growth or metastasis.    8.    The method of any one of claims 1‐7, wherein the cancer is lung squamous carcinoma, lung  adenocarcinoma, breast cancer, prostate cancer, uterine cancer, cervical cancer, thyroid cancer,  melanoma, gastroesophageal cancer, bladder cancer, or hepatocellular carcinoma.  9.  The method of any one of claims 1‐8, wherein the subject in need thereof is human.  10.  The method of any one of claims 1‐9, wherein from 0.2‐110 mg/kg of one or both of the EPN1‐ specific antibody and the PD‐L1‐specific antibody and/or the PD‐1‐specific antibody are administered  to the subject in need thereof by infusion.  Patent Application  Attorney Docket No. 172.0016‐WO00      11.  The method of claim 10, wherein subsequent infusions of from 0.2‐80 mg/kg of one or both of  the EPN1‐specific antibody and the PD‐L1‐specific antibody and/or the PD‐1‐specific antibody are  administered to the subject in need thereof by infusion.    12.  The method of claim 11, wherein the subsequent infusions are performed at least once weekly  for 4, 5, 6, 7, or 8 weeks.    13.  The method of any one of claims 1‐12, wherein the EPN1‐specific antibody comprises:       A)  a variable heavy chain (VHC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 1; and      B)  a variable light chain (VLC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 2.    14.   The method of any one of claims 1‐13, wherein the EPN1‐specific antibody comprises  following complementary determining region (CDR) amino acid sequences:      CDR‐H1, corresponding to SEQ ID NO: 3;      CDR‐H2, corresponding to SEQ ID NO: 4;      CDR‐H3, corresponding to SEQ ID NO: 5;       CDR‐L1, corresponding to SEQ ID NO: 6;      CDR‐L2 corresponding to SEQ ID NO: 7; and       CDR‐L3 corresponding to SEQ ID NO: 8.    15.  The method of any one of claims 1‐14, wherein the PD‐L1‐specific antibody comprises:       A)  a variable heavy chain (VHC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 9; and      B)  a variable light chain (VLC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 10.    16.  The method of any one of claims 1‐15, wherein the PD‐L1‐specific antibody comprises  following complementary determining region (CDR) amino acid sequences:      CDR‐H1, corresponding to SEQ ID NO: 11;      CDR‐H2, corresponding to SEQ ID NO: 12;      CDR‐H3, corresponding to SEQ ID NO: 13;   Patent Application  Attorney Docket No. 172.0016‐WO00        CDR‐L1, corresponding to SEQ ID NO: 14;      CDR‐L2 corresponding to SEQ ID NO: 15; and       CDR‐L3 corresponding to SEQ ID NO: 16.    17.  The method of any one of claims 1‐14, wherein the PD‐L1‐specific antibody comprises:       A)  a variable heavy chain (VHC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 17; and      B)  a variable light chain (VLC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 18.    18.   The method of any one of claims 1‐14, wherein the PD‐L1‐specific antibody comprises:       A)  a variable heavy chain (VHC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 19; and      B)  a variable light chain (VLC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 20.    19.   The method of any one of claims 1‐14, wherein the PD‐L1‐specific antibody comprises:       A)  a variable heavy chain (VHC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 21; and      B)  a variable light chain (VLC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 22.    20.  The method of any one of claims 1‐14, wherein the PD‐1‐specific antibody comprises:       A)  a variable heavy chain (VHC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 23; and      B)  a variable light chain (VLC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 24.    21.  The method of any one of claims 1‐14, wherein the PD‐1‐specific antibody comprises:       A)  a variable heavy chain (VHC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 25; and      B)  a variable light chain (VLC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 26.  Patent Application  Attorney Docket No. 172.0016‐WO00    22.  The method of any one of claims 1‐14, wherein the PD‐1‐specific antibody comprises:       A)  a variable heavy chain (VHC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 27; and      B)  a variable light chain (VLC) that comprises an amino acid that shares at least 90%  homology with the amino acid sequence of SEQ ID NO: 28.    23.  The method of any one of claims 1‐22, wherein one or both of the EPN1‐specific antibody and  the PD‐L1‐specific antibody is a knob‐into‐hole derivative; SEED‐IgG, DEKK mutated Fc, DVD‐Ig,  heteroFc‐scFv, IgG‐scFv, scFv2‐Fc, scDB‐Fc.    24.  The method of any one of claims 1‐22, wherein one or both of the EPN1‐specific antibody and  the PD‐L1‐specific antibody is a tandem scFv, diabody, Fab‐scFv.    25    The method of any one of claims 1‐22, wherein one or both of the EPN1‐specific antibody and  the PD‐L1‐specific antibody is a fully human or humanized antibody.    26.  The method of any one of claims 1‐25, wherein the EPN1‐specific antibody and the PD‐L1‐ specific antibody are administered concurrently.    27.  The method of any one of claims 1‐25, wherein the EPN1‐specific antibody and the PD‐L1‐ specific antibody are administered simultaneously.    28.  The method of claim 27, wherein the EPN1‐specific antibody and the PD‐L1‐specific antibody  are each present in a pharmaceutical formulation, wherein the pharmaceutical formulation is  administered to the subject.    29.  A kit comprising:      (A)  a pharmaceutical formulation comprising an EPN1‐specific antibody and a PD‐L1‐specific  antibody and/or a PD‐1‐specific antibody;      (B)  a suitable storage container;      (C)  a pH buffered solution; and       (D)  instructions for using the kit.     
PCT/US2023/067195 2022-05-18 2023-05-18 Combination anti-epn1 and anti-pd-l1 antibody therapies WO2023225613A2 (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US202263343282P 2022-05-18 2022-05-18
US63/343,282 2022-05-18
US202263368583P 2022-07-15 2022-07-15
US63/368,583 2022-07-15
US202263380457P 2022-10-21 2022-10-21
US63/380,457 2022-10-21

Publications (2)

Publication Number Publication Date
WO2023225613A2 true WO2023225613A2 (en) 2023-11-23
WO2023225613A3 WO2023225613A3 (en) 2023-12-28

Family

ID=88836167

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/067195 WO2023225613A2 (en) 2022-05-18 2023-05-18 Combination anti-epn1 and anti-pd-l1 antibody therapies

Country Status (1)

Country Link
WO (1) WO2023225613A2 (en)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2368113A1 (en) * 2008-12-19 2011-09-28 INSERM (Institut National de la Santé et de la Recherche Médicale) Biomarkers for the diagnosis of renal allograft and kidney status
EP3866851A4 (en) * 2018-10-17 2022-11-02 Immunome, Inc. Exosome-targeting bispecific antibodies

Also Published As

Publication number Publication date
WO2023225613A3 (en) 2023-12-28

Similar Documents

Publication Publication Date Title
EP3344658B1 (en) Antibodies specific to human t-cell immunoglobulin and itim domain (tigit)
CN108368174B (en) FGFR2 inhibitors alone or in combination with immunostimulants for cancer treatment
JP2020090509A (en) Anti-B7-H1 and anti-CTLA-4 antibodies for treating non-small cell lung cancer
EP1960432B1 (en) Anti-ICAM antibody inducing apoptosis.
JP2021521182A (en) Combination therapy of CD73 antagonist and PD-1 / PD-L1 axis antagonist
CN116672445A (en) Connexin (Cx) 43 hemichannel binding antibodies and uses thereof
EP3964525A2 (en) Monoclonal antibody that binds specifically to gitr
CN110312525A (en) The combination and application thereof of T cell redirection multipurpose antibody and immunologic test point regulator
JP2022522775A (en) LILRB4 binding antibody and its usage
US20220154296A1 (en) Use of anti-pd-1 antibody in preparation of medicament for treating solid tumors
US20240052050A1 (en) Multispecific antibodies for the treatment of cancer
JP2022509448A (en) Treatment methods and pharmaceuticals for cancers that are refractory to PD-1 / PD-L1 signal transduction inhibitors
CN112041346A (en) anti-CD 137 antibodies for combination with anti-PD-1 antibodies
Elloumi et al. Monoclonal antibodies as cancer therapeutics
CN112996504A (en) Methods of treating cancer by inhibiting ubiquitin conjugating enzyme E2K (UBE2K)
WO2023225613A2 (en) Combination anti-epn1 and anti-pd-l1 antibody therapies
WO2022177995A1 (en) Anti-lair1 antibodies for the treatment of cancer
KR20220151684A (en) Medicines for the treatment and/or prevention of cancer
CN115551549A (en) Compositions and methods for treating autoimmune diseases and cancer by targeting IGSF8
JP2022506430A (en) Monoclonal antibody that specifically binds to CD20
WO2023165561A1 (en) Anti-cd39 antibodies and use thereof
EP4373522A1 (en) Engineered compositions for bone-targeted therapy
JP2024522172A (en) Humanized anti-CLEC-1A antibodies and antigen-binding fragments thereof and mimetics thereof
AU2022318734A1 (en) Methods for treating acute myeloid leukemia with anti-ilt3 antibodies
CN118139884A (en) Humanized anti-CLEC-1A antibodies and antigen binding fragments and mimetics thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23808587

Country of ref document: EP

Kind code of ref document: A2