Classifications

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Definitions

• DCIS Ductal carcinoma in situ
• Tib are tumors that are 5mm- lcm (see, e.g., American Joint Committee on Cancer (AJCC) Staging Manual-8 th Edition, Amin, M.B., et al. Eds., Springer Nature (2017)) and increased risk of ipsilateral breast recurrence.
• AJCC American Joint Committee on Cancer
• trastuzumab to decrease risk.
• trastuzumab can result in neurologic, cardiac, cognitive as well as other morbidities.
• anti-cancer combination therapies comprising at least one dendritic cell pulsed with an oncodriver (such as, for example, human epidermal growth factor receptor (HER) 2(HER2)) and at least one inhibitor of an immunoregulatory molecule (such as, for example, Semaphorin (SEMA) 4D (SEMA4D), or VEGF); wherein the immunoregulatory molecule being inhibited effects the vasculature of a tumor.
• an oncodriver such as, for example, human epidermal growth factor receptor (HER) 2(HER2)
• an immunoregulatory molecule such as, for example, Semaphorin (SEMA) 4D (SEMA4D), or VEGF
• a cancer such as, for example, breast cancer (including triple negative breast cancer, metastatic breast cancer (MBC), ductal carcinoma in situ (DCIS), and invasive breast cancer (IBC)), melanoma, colorectal cancer, pancreatic cancer, prostate cancer, bladder cancer, ovarian cancer, and stomach cancer, and including primary and distant tumors) in a subject comprising administering the anti-cancer combination therapy of any preceding aspect.
• a cancer such as, for example, breast cancer (including triple negative breast cancer, metastatic breast cancer (MBC), ductal carcinoma in situ (DCIS), and invasive breast cancer (IBC)
• MCC metastatic breast cancer
• DCIS ductal carcinoma in situ
• IBC invasive breast cancer
• a cancer in a subject comprising administering to the subject at least one dendritic cell pulsed with an oncodriver (such as, for example, human epidermal growth factor receptor (HER) 2 (HER2)) and at least one inhibitor of an immunoregulatory molecule (such as, for example, Semaphorin (SEMA) 4D (SEMA4D), or VEGF); wherein the immunoregulatory molecule being inhibited effects the vasculature of a tumor.
• an oncodriver such as, for example, human epidermal growth factor receptor (HER) 2 (HER2)
• an immunoregulatory molecule such as, for example, Semaphorin (SEMA) 4D (SEMA4D), or VEGF
• anti-cancer combination therapies methods treating, preventing, reducing, and/or inhibiting a cancer of any preceding aspect; wherein the at least one immunoregulatory molecule inhibitor is administered systemically and/or the oncodriver pulsed dendritic cell is administered intratumorally.
• anti-cancer combination therapies methods treating, preventing, reducing, and/or inhibiting a cancer of any preceding aspect; wherein the oncodriver pulsed dendritic cell is activated with IL-12 prior to administration.
• anti-cancer combination therapies methods treating, preventing, reducing, and/or inhibiting a cancer of any preceding aspect; wherein the at least one immunoregulator molecule inhibitor comprises an antibody or functional fragment thereof which binds to SEMA4D (also referred to herein as CD100) such as, for example, the anti- SEMA4D antibodies Mab 67 or VX15/2503 (Pepinemab). See, e.g., U.S. Patent No. 8,496,938, incorporated herein by reference.
• anti-cancer combination therapies methods treating, preventing, reducing, and/or inhibiting a cancer of any preceding aspect; wherein the dendritic cells are removed from the subject and pulsed with oncodriver ex vivo.
• anti-cancer combination therapies methods treating, preventing, reducing, and/or inhibiting a cancer of any preceding aspect; wherein the pulsed dendritic cells are administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36 hours, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 31, 45 days, 2, 3, 4, 5, or 6 months prior to administration of the at least one immunoregulatory molecule inhibitor; are administered concurrently with the at least one immunoregulatory molecule inhibitor; or wherein the at least one immunoregulatory molecule inhibitor is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36 hours, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 31, 45 days, 2, 3, 4, 5, or 6 months prior to administration of the pulsed dendritic cells.
• anti-cancer combination therapies methods treating, preventing, reducing, and/or inhibiting a cancer of any preceding aspect; wherein the at least one pulsed dendritic cell is administered at least 1, 2, 3, 4, 5,6 ,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 times per day or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 times per week for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks.
• anti-cancer combination therapies methods treating, preventing, reducing, and/or inhibiting a cancer of any preceding aspect; wherein the at least one immunoregulatory molecule inhibitor is administered at least 1, 2, 3, 4, 5,6 ,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 times per day or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 times per week for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks.
• FIG. 1A shows that loss of HER-2 Thl response with HER- 2 positive disease progression
• FIG. IB shows that vaccine, but not standard therapy helps restore anti-HER-2 Thl immunity
• FIG. 1C shows that Thl immunity to HER-2 predicts clinical response to standard therapy
• FIG. ID shows that HER-2 Thl responsivity predicts disease-free survival after standard therapy.
• DCIS ductal carcinoma in situ
• IBC invasive breast cancer
• Tx treatment
• pCR pathologic complete response
• ER estrogen receptor
• TNBC triple-negative breast cancer.
• FIG. 2A shows short-course anti-estrogen therapy concurrent with DC1 vaccination improves pCR rate (FIG. 2A) and anti-HER2 Thl immunity (FIG. 2B) for subjects with hormone-dependent (ER-positive) disease, and that pathologic complete response (pCR) predicts long-term freedom from subsequent breast events (SBE) for all vaccinated subjects (FIG. 2C).
• FIG. 3A-3G show Intratumoral DC1 in combination with anti-SEMA4D antibody induced tumor regression in HER2 positive TUBO model.
• FIG. 3A SEMA4D expression in TUBO cells as measured by immunohistochemistry, compared to an IgG isotype control antibody;
• FIG. 3B Intratumoral class II HER2 peptide pulsed DC1 intratumoral injection in combination with anti-SEMA4D antibody given intraperitoneally in single tumor model (FIG. 3B1) and survival curve (FIG. 3B2);
• FIG. 3C Efficacy of intratumoral DC1 in combination with anti- SEMA4D in bilateral model (FIG. 3C1 and FIG.
• FIG. 3C2 TUBO bearing mice were treated with either with unpulsed or class II HER2 peptide pulsed activated DC1 alone or in combination with anti-SEMA4D antibody;
• FIG. 3D IHC staining of SEMA4D in DCIS and IBC patients, the numbers indicate the number of patients with positive SEMA4D staining;
• FIG. 3E Myeloid-derived suppressor cell (MDSC) infiltration per mg of tumor from treated with DC1 alone, anti-SEMA4D alone or in combination;
• FIG. 3F CD4 T cell infiltration per mg of tumor;
• FIG. 3G CD4 T cell infiltration (absolute number) in lymph node.
• FIG. 4A Fold expansion after three weeks of in vitro CD4 T cell expansion.
• CD4+ T cells were isolated from isolated splenocytes from vaccinated mice using EASYSEPTM (Stemcell Technologies) and co-cultured with HER2 peptide pulsed DC1 followed by expansion with IL-2 and IL-7 for three weeks;
• FIG. 4A Fold expansion after three weeks of in vitro CD4 T cell expansion.
• CD4+ T cells were isolated from isolated splenocytes from vaccinated mice using EASYSEPTM (Stemcell Technologies) and co-cultured with HER2 peptide pulsed DC1 followed by expansion with IL-2 and IL-7 for three weeks;
• FIG. 4A Fold expansion after three weeks of in vitro CD4 T cell expansion.
• FIG. 4B Expanded T cells from DC1+ anti- SEMA4D treated mice were more antigen specific for the HER2/neu peptide antigens p5, p435, and pl209 compared to T cells from DC1 vaccinated mice;
• FIG. 4C Cumulative IFN-g response of expanded T cells.
• FIG. 5A Flow staining of DCC in bone marrow (BM) by cytokeratin 8/18 and HER2 expression
• FIG. 5B Immunofluorescence staining of HER2, cytokeratin and ki67
• FIG. 5C Detection of DCC in different organs of neu T mice.
• FIG. 6A Immunofluorescence staining of bone marrow DCC from control and DC1 vaccinated neu T mice
• FIG. 6B Detectable tumor mass in mammary glands using ultrasound
• FIG. 6C Percent DCC in bone marrow measured using flow cytometry
• FIG. 6D Detection of senescent cells using b-gal assay. Representative images after the b-gal staining.
• FIG. 7A 8 weeks old NeuT mice were treated with HER2 peptide pulsed DC1 vaccine (two injections per week for three weeks) or anti-SEMA4D antibody.
• mice were sacrificed, and mammary glands were collected. The single cell suspension of mammary glands was prepared and stained for CD 19 positive B cells then analyzed by flow cytometry. Increased levels of CDal9+ B cells were observed in DC1 vaccinated mice and anti-SEMA4D treated mice compared to untreated control;
• FIG. 7A 8 weeks old NeuT mice were treated with HER2 peptide pulsed DC1 vaccine (two injections per week for three weeks) or anti-SEMA4D antibody.
• mice were sacrificed, and mammary glands were collected. The single cell suspension of mammary glands was prepared and stained for CD 19 positive B cells then analyzed by flow cytometry. Increased levels of CDal9+ B cells were observed in DC1 vaccinated mice and anti-SEMA4D treated mice compared to untreated control;
• the bone marrow cells derived from the control and DC1 vaccinated NeuT mice were stained for CD4 and CD8 T cell markers and analyzed by flow cytometry. Increased numbers of CD4T cells and CD8 T cells were observed in the bone marrow of DC1 vaccinated NeuT mice compared to untreated control mice.
• FIG. 8 A Dual blockade of HER2/HER3 in SK-BR-3 cells combined with Thl cytokines TNF-a and IFN-g enhance the number of senescent cells, higher SA ⁇ -gal staining was observed in cells;
• FIG. 8 A Dual blockade of HER2/HER3 in SK-BR-3 cells combined with Thl cytokines TNF-a and IFN-g enhance the number of senescent cells, higher SA ⁇ -gal staining was observed in cells;
• FIG. 8 A Dual blockade of HER2/HER3 in SK-BR-3 cells combined with Thl cytokines TNF-a and IFN-g enhance the number of senescent cells, higher SA ⁇ -gal staining was observed in cells;
• FIG. 8 A Dual blockade of HER2/HER3 in SK-BR-3 cells combined with Thl cytokines TNF-a and IFN-g enhance the number of senescent cells, higher SA ⁇ -gal staining was observed in cells;
• FIG. 8B SK-BR-3 cells untreated (1), treated with TNF-a and IFN-g (2), or treated with trastuzumab (Herceptin, H (TZm)) and pertuzumab (Per) (3), or treated with TNF-a, IFN-g and TZm and Per (4);
• FIG. 8C Western blot analysis of SK-BR-3 cells treated with Thl cytokines in combination with Tzm and Per induced Cyclin-dependent kinase 4 inhibitor B, also known as p!5 INK4b and cleaved caspase-3 expression (treatments numbered as in panel B);
• FIG. 8D and FIG. 8E Apoptosis by Annexin V/PI staining (treatments numbered as in panels D& E).
• FIG. 9A HCC-1419 and JIMT-1 cells; 1) untreated; 2) treated with TNF-a and IFN-g; 3) treated with TZm and Per , or 4) treated with TNF-a, IFN-g, Tzm, and Per.
• FIG. 9A % of SA- -gal-positive cells
• FIG. 9B Western blots analysis of pl5 INKb and cleaved caspase-3 expression of HCC-1419 post treatment
• FIG. 9C Western blot analysis of JIMT-1 cells. Vinculin was used as a control.
• FIG. 10A shows HER2-specific CD4 + Thl-mediated senescence and apoptosis of HER2-ovexpressing human breast cancer cells.
• FIG. 10A SK-BR-3 cells co-cultured with CD4 + T-cells alone (CD4 + only (1)), CD4 + T-cells + HER2 peptide-pulsed immature dendritic cells (CD4 + IDC H (2)), CD4 + T-cells + HER2 peptide-pulsed mature dendritic cells (CD4 + DC H (3)), or CD4 + DC H with trastuzumab (Tzm) and pertuzumab (Per) (4), or CD4 + T-cells + irrelevant peptide-pulsed mature dendritic cells (BRAF (CD4 + DC B) (5); or survivin (CD4 + DC S)(6)), with Tzm and Per.
• FIG. 10B Western blot analysis of tumor cells showed increase in pl5 INK4b and cleaved caspase-3 expression suggests induced senescence and apoptosis, respectively, when co-cultured with the DC H/CD4 + T-cells in presence of Tzm and Per, but not from DC B, DC S and iDC H groups. Vinculin was used as loading control
• FIG 11 shows IFN-g administered subcutaneously twice weekly with weekly Taxol and standard dose trastuzumab and pertuzumab in patients with first line metastatic breast cancer was safe and resulted in disease stabilization of partial responses.
• FIG. 12A, 12B, 12C, and 12D show immunohistochemical staining of lymphocyte infiltration before and after DC1 vaccine in DCIS.
• FIG. 12A and FIG. 12B show areas of dense lymphocyte infiltrate;
• FIG. 12C and FIG. 12D show areas with little or no response.
• Figure 13 shows accumulation of lymphocytes pre and post DC1 vaccination in DCIS. CD4, CD8 and CD20 infiltration is shown.
• Figure 14 shows Lymphocytes infiltration in DCIS ducts after DC1 vaccination.
• FIG. 15A Slope of the DCE-MRI curves calculated after iv administration of Gadavist (a gadolinium-based MRI contrast agent, 0.2mmol/kg). Despite tumor volume, combo treatment shows a smaller slope which indicates less vessel leakage;
• FIG. 15B DCE-MRI curves of TUBO tumors showing that combination therapy has smaller DCE curve. Data is represented as relative value (respect to the first point of the curve) to show a reliable comparison among the tumors.
• Figure 16 shows CEST MRI (tumor pH) map of a TUBO tumor treated with anti-SEMA4D.
• T2-weighted image representing the ROI
• pH map the insertion shows the pH mean value and its Standard Deviation
• histogram representing pH values of all pixels calculated.
• FIG. 17A mean tumor size (mm 2 ) in a murine Her2 TUBO breast cancer model over time following treatment with Her2DCl, anti-SEMA4D, HER2 DC1 plus anti-SEMA4D, or Control IgG.
• FIG.17 B percent survival of Her2 TUBO tumor-bearing mice following treatment with Her2DCl, anti-SEMA4D, HER2 DC1 plus anti-SEMA4D, or Control IgG.
• FIG. 17A mean tumor size (mm 2 ) in a murine Her2 TUBO breast cancer model over time following treatment with Her2DCl, anti-SEMA4D, HER2 DC1 plus anti-SEMA4D, or Control IgG.
• FIG. 17B percent survival of Her2 TUBO tumor-bearing mice following treatment with Her2DCl, anti-SEMA4D, HER2 DC1 plus anti-SEMA4D, or Control IgG.
• FIG. 17C mean tumor size (mm 2 ) in CD4-depleted Her2 TUBO tumor-bearing mice over time following treatment with Her2DCl, anti-SEMA4D, HER2 DC1 plus anti-SEMA4D, or no treatment.
• FIG. 17D percent survival of CD4-depleted Her2 TUBO tumor-bearing mice following treatment with Her2DCl, anti-SEMA4D, HER2 DC1 plus anti-SEMA4D, or no treatment.
• FIG. 17E tumor size (mm 2 ) in individual Her2 TUBO tumor-bearing mice over time following treatment with Her2DCl, anti-SEMA4D, HER2 DC1 plus anti-SEMA4D, or Control IgG.
• FIG 17F tumor size (mm 2 ) in individual CD4-depleted Her2 TUBO tumor-bearing mice over time following treatment with Her2DCl, anti-SEMA4D, HER2 DC1 plus anti-SEMA4D, or Control IgG.
• FIG. 18 A mean tumor size (mm 2 ) in BALB/C. l29V2(B6)-Fcerlg tmlRav N12 tumor-bearing mice treated with HER2DC, anti- SEMA4D, HER2DC plus anti-SEMA4D; or no treatment.
• FIG. 18B percent survival of C.129P2(B6 )-Fcerlg tmlRav N12 tumor-bearing mice treated with HER2DC, anti-SEMA4D, HER2DC plus anti-SEMA4D; or no treatment.
• FIG. 18C Tumor growth curves for each mouse are shown. No complete tumor regressions are observed in FcRy-deficient mice. (* p ⁇ 0.05,
• FIG. 19 shows that Interferon gamma (IEN-g) is required for anti-tumor activity of DC1, anti- SEMA4D, and combination therapy.
• Mean tumor size (mm 2 ) in Balb/C IFN- g knock out (KO) (C.129S7 (B6)-IFNg tmlT 7J (IFN-y KO , Jackson Laboratories) mice that carried tumors generated from murine Her2 TUBO breast cancer cells is shown over time following treatment with Her2DCl, anti-SEMA4D, HER2 DC1 plus anti-SEMA4D, or Control IgG.
• Ranges can be expressed herein as from“about” one particular value, and/or to“about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. For example, if the value“10” is disclosed, then“about 10” is also disclosed.
• a particular data point“10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
• a “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
• a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance.
• a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
• a decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.
• the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.
• “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
• “reduce” or other forms of the word, such as“reducing” or“reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.
• “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.
• By“prevent” or other forms of the word, such as“preventing” or“prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.
• Biocompatible generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.
• compositions, methods, etc. include the recited elements, but do not exclude others.
• Consisting essentially of' when used to define compositions and methods shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
• Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and/or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.
• A“control” is an alternative subject or sample used in an experiment for comparison purposes.
• a control can be "positive” or "negative.”
• “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect.
• the amount of agent that is“effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified“effective amount.” However, an appropriate“effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an“effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts.
• an “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
• a "pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.
• “Pharmaceutically acceptable carrier” means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use.
• carrier or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.
• carrier encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.
• “Pharmacologically active” (or simply“active”), as in a“pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.
• Polymer refers to a relatively high molecular weight organic compound, natural or synthetic, whose structure can be represented by a repeated small unit, the monomer.
• Non-limiting examples of polymers include polyethylene, rubber, cellulose. Synthetic polymers are typically formed by addition or condensation polymerization of monomers.
• copolymer refers to a polymer formed from two or more different repeating units (monomer residues).
• a copolymer can be an alternating copolymer, a random copolymer, a block copolymer, or a graft copolymer. It is also contemplated that, in certain aspects, various block segments of a block copolymer can themselves comprise copolymers.
• a "binding molecule” or "antigen binding molecule” refers in its broadest sense to a molecule that specifically binds an antigenic determinant ⁇
• the binding molecule specifically binds to an immunoregulator molecule (such as for example, a transmembrane SEMA4D (CD 100) polypeptide of about 150 kDa or a soluble SEMA4D polypeptide of about 120 kDa).
• an immunoregulator molecule such as for example, a transmembrane SEMA4D (CD 100) polypeptide of about 150 kDa or a soluble SEMA4D polypeptide of about 120 kDa.
• a binding molecule is an antibody or an antigen binding fragment thereof, e.g., MAb 67 or pepinemab.
• “Therapeutic agent” refers to any composition that has a beneficial biological effect.
• Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer).
• the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
• therapeutic agent when used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
• “Therapeutically effective amount” or“therapeutically effective dose” of a composition refers to an amount that is effective to achieve a desired therapeutic result.
• a desired therapeutic result is the control of type I diabetes.
• a desired therapeutic result is the control of obesity.
• Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief.
• DCIS patients presenting with DCIS in general have excellent prognosis however those presenting at age ⁇ 40, African American females, and ER neg DCIS have modestly increased risk of dying of subsequent BC that neither surgery nor radiation appears to prevent.
• a second problem is that many young patients also present with larger regions of HER2 expressing DCIS that contains areas of Tla/Tlb invasion. These patients are typically either treated with mastectomy because of the size of area of calcifications or treated with strong neoadjuvant chemotherapy regimens of carboplatin, taxotere with trastuzumab and pertuzumab (PTCH) or taxol and trastuzumab (TH) with good survival but to suffer the long term consequences of extensive surgery, radiation and chemotherapy.
• PTCH trastuzumab
• TH taxol and trastuzumab
• HER- 2/ neu over-expression plays a critical role in breast cancer (BC) development and its expression in ductal carcinoma in situ (DCIS) is associated with development of invasive BC (IBC).
• IBC ductal carcinoma in situ
• HER2-DC1 class II HER2 peptide-pulsed Type I polarized dendritic cell vaccine
• pCR pathologic complete response rate
• Semaphorin 4D is a family of soluble and transmembrane proteins that are essential for tissue and organ development and are involved in immune regulation. Overexpression of SEMA4D correlates with poor prognosis and tumor progression in various cancers.
• a murine anti-SEMA4D monoclonal antibody (provided by Vaccinex) in combination with DC1 vaccine was investigated to enhance anti-tumor immune response in a preclinical model of HER2 positive TUBO breast cancer.
• anti-cancer combination therapies comprising at least one dendritic cell pulsed with an oncodriver (such as, for example, human epidermal growth factor receptor HER2, and at least one inhibitor of an immunoregulatory molecule (such as, for example, Semaphorin (SEMA) 4D (SEMA4D), or VEGF).
• an oncodriver such as, for example, human epidermal growth factor receptor HER2
• an immunoregulatory molecule such as, for example, Semaphorin (SEMA) 4D (SEMA4D), or VEGF.
• SEMA Semaphorin
• VEGF vascular endothelial growth factor receptor
• the disclosed anti-cancer combination therapies can be used to treat, prevent, reduce, and/or inhibit any disease where uncontrolled cellular proliferation occurs such as cancers including primary and distant tumors.
• cancers including primary and distant tumors.
• lymphomas Hodgkins and non- Hodgkins
• leukemias carcinomas, carcinomas of solid tissues
• squamous cell carcinomas adenocarcinomas
• sarcomas gliomas
• high grade gliomas blastomas
• neuroblastomas plasmacytomas
• histiocytomas melanomas
• adenomas hypoxic tumors
• myelomas myelomas
• AIDS- related lymphomas or sarcomas metastatic cancers, or cancers in general.
• a representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer (including triple negative breast cancer, metastatic breast cancer (MBC), ductal carcinoma in situ (DCIS), and invasive breast cancer (IBC)), and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoi
• a cancer such as, for example, breast cancer (including triple negative breast cancer, metastatic breast cancer (MBC), ductal carcinoma in situ (DCIS), and invasive breast cancer (IBC)), melanoma, colorectal cancer, pancreatic cancer, and prostate cancer and including primary and distant tumors) in a subject comprising administering the anti-cancer combination therapy of any preceding aspect.
• a cancer such as, for example, breast cancer (including triple negative breast cancer, metastatic breast cancer (MBC), ductal carcinoma in situ (DCIS), and invasive breast cancer (IBC)
• MCC metastatic breast cancer
• DCIS ductal carcinoma in situ
• IBC invasive breast cancer
• a cancer in a subject comprising administering to the subject at least one dendritic cell pulsed with an oncodriver (such as, for example, human epidermal growth factor receptor (HER) HER2, and at least one inhibitor of an immunoregulatory molecule (such as, for example, Semaphorin (SEMA) 4D (SEMA4D), or VEGF).
• an oncodriver such as, for example, human epidermal growth factor receptor (HER) HER2
• an immunoregulatory molecule such as, for example, Semaphorin (SEMA) 4D (SEMA4D), or VEGF.
• SEMA Semaphorin
• VEGF vascular endothelial growth factor receptor
• the disclosed methods and anti-cancer combination therapies comprise inhibitor of an immunoregulatory molecules that have both an immunoregulatory effect, and in certain nonlimiting aspects can also affect the vasculature of a tumor.
• said inhibitors can comprise any small molecule, peptide, protein, antibody (including any functional fragments of an antibody or other binding molecule), and/or functional nucleic acid (siRNA, RNA, aptamer) that inhibits the immunoregulatory and/or vascular activity of the immunoregulatory molecule.
• the inhibitor of an immunoregulatory molecule comprises the SEMA4D inhibitor pepinemab (an anti-SEMA4D antibody)
• immunoregulatory molecule such as, for example, Semaphorin (SEMA) 4D (SEMA4D), or VEGF
• SEMA Semaphorin
• VEGF vascular endothelial growth factor
• the antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which in vivo therapeutic and/or prophylactic activities can be tested according to known clinical testing methods.
• Native antibodies are usually heterotetrameric glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V(H)) followed by a number of constant domains.
• V(H) variable domain
• Each light chain has a variable domain at one end (V(L)) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
• Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.
• the light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (k) and lambda (1), based on the amino acid sequences of their constant domains.
• immunoglobulins can be assigned to different classes.
• IgA human immunoglobulins
• IgD immunoglobulins
• IgE immunoglobulins
• IgG immunoglobulins
• variable is used herein to describe certain portions of the variable domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.
• variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains.
• CDRs complementarity determining regions
• FR framework
• the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a b-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the b-sheet structure.
• the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Rabat E. A. et al.,“Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1987)).
• the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
• the heavy chain portions of one polypeptide chain of a multimer are identical to those on a second polypeptide chain of the multimer.
• heavy chain portion-containing monomers are not identical.
• each monomer may comprise a different target binding site, forming, for example, a bispecific antibody.
• the heavy chain portions of a binding molecule for use in the diagnostic and treatment methods disclosed herein may be derived from different immunoglobulin molecules.
• a heavy chain portion of a polypeptide may comprise a Cm domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule.
• a heavy chain portion can comprise a hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule.
• a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an IgG4 molecule.
• the term“light chain portion” includes amino acid sequences derived from an immunoglobulin light chain, e.g., a kappa or lambda light chain.
• the light chain portion comprises at least one of a VL or CL domain.
• Anti- SEMA4D antibodies, or antigen-binding fragments, variants, or derivatives thereof disclosed herein can be described or specified in terms of the epitope(s) or portion(s) of an antigen, e.g. , a target polypeptide disclosed herein (e.g., SEMA4D) that they recognize or specifically bind.
• a target polypeptide may comprise a single epitope, but typically comprises at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen.
• an "epitope" on a target polypeptide may be or may include non-polypeptide elements, e.g. , an epitope may include a carbohydrate side chain.
• the minimum size of a peptide or polypeptide epitope for an antibody is thought to be about four to five amino acids.
• Peptide or polypeptide epitopes preferably contain at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids. Since a CDR can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids comprising an epitope need not be contiguous, and in some cases, may not even be on the same peptide chain.
• a peptide or polypeptide epitope recognized by anti-SEMA4D antibodies may contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or between about 15 to about 30 contiguous or non-contiguous amino acids of SEMA4D.
• an antibody binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind” to an epitope when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope.
• the term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope.
• antibody “A” may be deemed to have a higher specificity for a given epitope than antibody "B,” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.”
• preferentially binds it is meant that the antibody specifically binds to an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope.
• an antibody that "preferentially binds" to a given epitope would more likely bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.
• the term“monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.
• the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.
• the disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies.
• disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
• a hybridoma method a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
• the lymphocytes may be immunized in vitro.
• the monoclonal antibodies may also be made by recombinant DNA methods.
• DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
• Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al.
• In vitro methods are also suitable for preparing monovalent antibodies.
• Digestion of antibodies to produce fragments thereof, particularly, Fab fragments can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566.
• Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.
• the term“antibody or fragments thereof’ encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab’)2, Fab’, Fab, Fd, Fv, scFv, disulfide-linked Fvs (sdFv), fragments comprising either or both VH or VL domain, and the like, including hybrid fragments.
• fragments of the antibodies that retain the ability to bind their specific antigens are provided.
• fragments of antibodies which maintain immunoregulatory molecule such as, for example, Semaphorin (SEMA) 4D (SEMA4D), or VEGF) binding activity are included within the meaning of the term“antibody or fragment thereof.”
• immunoregulatory molecule such as, for example, Semaphorin (SEMA) 4D (SEMA4D), or VEGF
• SEMA Semaphorin
• SEMA4D Semaphorin 4D
• VEGF vascular endothelial growth factor 4D
• Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).
• antibody or fragments thereof conjugates of antibody fragments and antigen binding proteins (single chain antibodies).
• the fragments can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc.
• the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen.
• Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide.
• heavy chain portion includes amino acid sequences derived from an immunoglobulin heavy chain.
• a polypeptide comprising a heavy chain portion comprises at least one of: a CHI domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.
• the term“antibody” or“antibodies” can also refer to a human antibody and/or a humanized antibody.
• Many non-human antibodies e.g., those derived from mice, rats, or rabbits
• are naturally antigenic in humans and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.
• the disclosed human antibodies can be prepared using any technique.
• the disclosed human antibodies can also be obtained from transgenic animals.
• transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et ak, Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et ak, Nature, 362:255-258 (1993); Bruggermann et ak, Year in Immunol., 7:33 (1993)).
• the homozygous deletion of the antibody heavy chain joining region (J (H)) gene in these chimeric and germ- line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge.
• Antibodies having the desired activity are selected using Env-CD4-co-receptor complexes as described herein.
• Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule.
• a humanized form of a non-human antibody is a chimeric antibody or antibody chain (or a fragment thereof, such as an sFv, Fv, Fab, Fab’, F(ab’)2, or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody.
• a humanized antibody residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen).
• CDRs complementarity determining regions
• donor non-human antibody molecule
• Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues.
• Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
• a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human.
• humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
• Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).
• Fc antibody constant region
• humanized antibodies can be generated according to the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
• Methods that can be used to produce humanized antibodies are also described in U.S. Patent No. 4,816,567 (Cabilly et al.), U.S. Patent No.
• the inhibitor of an immunoregulatory molecule can be the SEMA4D inhibitor pepinemab (an anti-SEMA4D antibody) such as those described in US8496938, US8816058, US9605055, and US9676840, patents which are incorporated herein by reference for their teachings of anti-SEMA4D (anti-DClOO) antibodies.
• Anti- SEMA4D monoclonal antibodies have been developed to neutralize SEMA4D, including MAb 67, MAb 2503, and MAb 76.
• Anti-SEMA4D antibodies or antigen-binding fragments, variants, or derivatives thereof can include, e.g., MAb 2503, MAb 67, or MAb 76.
• the anti- SEMA4D antibodies bind human, murine, or both human and murine SEMA4D.
• the anti- SEMA4D antibodies block SEMA4D binding to its receptor, e.g., Plexin-Bl or Plexin-B2.
• the disclosed anti-cancer combination therapies and methods of treating, inhibiting, reducing, and/or preventing a cancer using said anti-cancer combination therapies can comprise more than one immunoregulator molecule inhibitor and more than one population of pulsed dendritic cells with each population of pulsed dendritic cells being pulsed with the same or different oncodrivers.
• subject refers to any individual who is the target of administration or treatment.
• the subject can be a vertebrate, for example, a mammal.
• the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline.
• the subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole.
• the subject can be a human or veterinary patient.
• patient refers to a subject under the treatment of a clinician, e.g., physician.
• the term“therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
• treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
• This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
• this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
• the pulsed dendritic cells can be activated prior to administration as well as prior to being pulsed with the oncodriver.
• Activation of the dendritic cells (DC1) can be achieved by contacting the cells with IFN-g, TNFoc, CD40, IL21, and/or IL-12.
• the subject’s own dendritic cells can be removed and pulsed ex vivo and transferred back to the subject for use in the disclosed anti-cancer combination therapies for treating, preventing, reducing, and/or inhibiting a cancer.
• the disclosed anti-cancer combination therapies can be administered via any route determined to be appropriate by the attending physician.
• Administration to a subject includes any route of introducing or delivering to a subject an agent either locally and/or systemically.
• Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intratumoral, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like.
• parenteral e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intratumoral, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques, and the like.
• Constant administration means that the compounds are administered at the same point in time or essentially immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time.
• Systemic administration refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g. greater than 50% of the body), for example through entrance into the circulatory or lymph systems.
• local administration refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount.
• locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject’s body.
• Administration includes self-administration and the administration by another.
• anti-cancer combination therapies methods treating, preventing, reducing, and/or inhibiting a cancer of any preceding aspect; wherein the at least one immunoregulatory molecule inhibitor is administered systemically and/or the oncodriver pulsed dendritic cell is administered intratumor ally.
• anti-cancer combination therapies methods treating, preventing, reducing, and/or inhibiting a cancer; wherein the at least one pulsed dendritic cell is administered at least 1, 2, 3, 4, 5,6 ,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 times per day or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 times per week for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks.
• anti-cancer combination therapies methods treating, preventing, reducing, and/or inhibiting a cancer of any preceding aspect; wherein the at least one immunoregulatory molecule inhibitor is administered at least 1, 2, 3, 4, 5,6 ,7, 8, 9, 10, 11,
• anti-cancer combination therapies methods treating, preventing, reducing, and/or inhibiting a cancer; wherein the pulsed dendritic cells are administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36 hours, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 31, 45 days, 2, 3, 4, 5, or 6 months prior to administration of the at least one immunoregulatory molecule inhibitor; are administered concurrently with the at least one immunoregulatory molecule inhibitor; or wherein the at least one immunoregulatory molecule inhibitor is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36 hours, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 31, 45 days, 2, 3, 4, 5, or 6 months prior to administration of the pulsed dendritic cells.
• cancer can augmented with any therapeutic treatment of a cancer including, but not limited surgical, radiological, and/or pharmaceutical treatments of a cancer.
• “surgical treatment” refers to tumor resection of the tumor by any means known in the art.
• “pharmaceutical treatment” refers to the administration of any anti-cancer agent known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin- stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aliqopa (Copanlis
• chemotherapeutics that are PD1/PDL1 blockade inhibitors (such as, for example, lambrolizumab, nivolumab, pembrolizumab, pidilizumab, BMS-936559, Atezolizumab, Durvalumab, or Avelumab).
• PD1/PDL1 blockade inhibitors such as, for example, lambrolizumab, nivolumab, pembrolizumab, pidilizumab, BMS-936559, Atezolizumab, Durvalumab, or Avelumab.
• compositions can also be administered in vivo in a pharmaceutically acceptable carrier.
• pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
• the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
• compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant.
• topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector.
• Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
• compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
• Parenteral administration of the composition, if used, is generally characterized by injection.
• Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
• a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
• the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
• the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et ak, Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700- 703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol.
• Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
• the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).
• compositions including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.
• Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.
• an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
• the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
• the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
• Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
• compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
• compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
• Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
• the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
• the disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
• Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
• non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
• Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
• Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
• Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
• Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
• Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
• compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable..
• compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
• inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
• organic acids such as formic acid, acetic acid, propionic acid, glyco
• Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.
• the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are affected.
• the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
• the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
• the dosage can be adjusted by the individual physician in the event of any counterindications.
• Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
• Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
• guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et ak, eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et ak, Antibodies in Human Diagnosis and Therapy, Haber et ak, eds., Raven Press, New York (1977) pp. 365- 389.
• a typical daily dosage of the antibody used alone might range from about 1 pg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.
• oncodrivers including HER2, EGFR (ER+ and TNBC), C-MET (TNBC) and HER3 (ER+, HER2, and TNBC).
• MBC metastatic breast cancer
• targeted agents such as anti-estrogens, CDK4/6 inhibitors, HER2 directed therapies and AKT inhibitors.
• Most of these patients however, become resistant to therapies or stop responding and progress. Thus, these patients are in need of additional therapies.
• Checkpoint therapies have shown promising but limited effectiveness in MBC so identifying effective new immunotherapies that may be combined with targeted agents to make them more effective in MBC would be highly desirable.
• Oncodrivers may be critical appropriate targets of the immune response as indicated by the data below.
• HER2 pos IBC patients achieving a pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) demonstrate improved survival while those with residual disease at the time of surgery demonstrate increased risk of recurrence.
• pCR pathological complete response
• NAC neoadjuvant chemotherapy
• Those patients that regain or partially retain anti-HER2 Thl demonstrate higher pCR rate to neoadjuvant chemo/trastuzumab therapy, while those with residual disease display the lowest anti-HER2 CD4 Thl responses (Fig ID). Shown is the loss of repertoire but there is also significant difference in overall and cumulative responses.
• the loss of anti-oncodriver CD4 immune response represents a decrease in interferon gamma (IFN-g) production in the Tbet pos Thl population and not GATA 3 pos Th2 population, indicating the Thl immune response may be critical to mediating anti-oncodriver BC responses. Indeed, elevated immune gene expression in the tumor is associated with improved outcomes.
• Example 2 Boosting anti-HER-2 CD4 Thl using DC1 therapy in HER2 pos early breast cancer:
• Example 3 DC1 vaccines in combination with SEMA4D blockade:
• Semaphorin 4D is a member of family of cell surface molecules that are essential for tissue and organ development and are involved in immune regulation. Antibodies against SEMA4D have been shown to regulate lymphocyte infiltration into tumors (see, e.g., U.S. Patent No. 9,243,068).
• SEMA4D Semaphorin 4D
• Mab 67 see, e.g., U.S. Patent No. 8,496,938, with DC1 vaccinations in a HER2 murine TUBO tumor model as follows.
• the TUBO mouse mammary tumor cell line accesion No. CVCL_2A33, Rovero, S., et ak, J.
• Immunol 165: 5133-5142 (2000) was first shown to express SEMA4D by immunohistochemistry (FIG. 3A).
• FIG. 3B1 For single tumor model (FIG. 3B1), Balb/C mice received 2.5e5 TUBO cells subcutaneously on right flank on day 0. When tumors were palpable on day 7, mice were randomized into four groups. Mice received monotherapy with either control antibody or anti-sema4D antibody intraperitoneally until end point or intratumoral HER2-DC1 weekly for six weeks. For combination therapy, mice received anti- sema4D antibody prior to receiving first intratumoral HER2-DC1 injection once a week for six weeks.
• B ALB/c mice received DC vaccines intratumorally once a week for six weeks. In those animals with two tumor locations, that vaccine was administered in only one of the two tumors.
• Mab 67 was given intraperitoneally at the concentration of lOmg/kg / body weight at weekly interval. Control mice received isotype control antibodies, DC treatment or Mab 67 alone.
• Tumors were measured every 2-3 days with a caliper until the endpoint. For comparison of in vitro measurements, a one-way ANOVA (followed by Tukey post hoc test) was performed. For comparison of in vivo measurements, the same test was performed using tumor measurement taken at each time point. A Mann- Whitney test was used to compare between two treatment groups. All statistical evaluations of data were performed using GraphPad Prism software. Statistical significance was achieved at p ⁇ 0.05.
• FIG. 3F The potential relevance to human tumors is highlighted by the fact that SEMA4D was expressed in about 60% of human HER2 DCIS or HER2 IBC as measured by immunohistochemistry (Fig 3D).
• Example 4 Administration of Intratumoral HER2 pulsed DC1 and systemic anti-SEMA4D antibody Resulted in Strong Anti-Tumor Thl responses.
• Spleens were harvested from mice that were rendered cured of TUBO tumors by the combination systemic administration of anti-SEMA4D antibody with intratumoral HER2 pulsed DC1 therapy as described in Example 3, as well from control mice vaccinated intratumorally with the DC1 vaccine alone or in combination with anti-SEMA4D antibody, and CD4+T cells were isolated from splenocytes were co-cultured with with HER2 pulsed DC1 for three-four days followed by expansion in the presence of IL-2 (lOU/ml) and IL-7 (20ng/ml for three weeks.
• IL-2 lOU/ml
• IL-7 20ng/ml for three weeks.
• FIG. 4 Cells expanded from spleens harvested from mice vaccinated with HER2 pulsed DC1 either alone or in combination with systemic anti-SEMA4D antibody treatment were compared (Fig 4).
• the data indicate the CD4 T cells from mice given combination therapy displayed an in vitro expansion of about 7-fold compared to that of the control mice of about 3 -fold (FIG. 4A).
• the CD4 T cells expanded from the spleens of mice given the combination therapy also displayed strong antigen specificity for HER2/neu-derived peptides p5, p435, and pl209 (see, e.g., Jalali et al., Nanomedicine ;8 692-701 (2012)) as compared to the CD4 T cells expanded from mice that received the DC1 vaccine alone (FIG.
• CD4 T cells expanded from the spleens of mice given the combination therapy displayed a strong production of IFN-g indicating a strong TH1 response (FIG. 4C).
• Example 5 DC1 treatment of B ALB/c Neu T transgenic mice Impacts Disseminated Cancer Cells in HER2 Expressing Mammary Carcinoma.
• the DC1 vaccines were tested in HER2 patients with residual disease following neoadjuvant chemotherapy /trastuzumab, the DC1 administration was safe and resulted in increased anti-
• NeuT is a transgenic mouse model of breast cancer in which the mouse mammary tumor virus (MMTV) promoter drives neuT expression and mice develop spontaneous tumors in the mammary fat pads of female mice.
• MMTV mouse mammary tumor virus
• Neoplastic change occurs, albeit asynchronously, in all mammary glands so that by about day
• one or more tumors are palpable and by about day 230 all 10 mammary glands contain palpable tumors.
• We and others have demonstrated there are disseminated cancer cells that can be identified in several organs prior to the appearance of mammary carcinomas (FIG. 5A and 5B). These HER2+ cytokeratin positive cells can be detected in the bone marrow, lungs and liver from week 9 on and they compose 10-15% of the cells and are in a reasonably low proliferative state as measured by Ki67 (FIG. 5C). Vaccination of mice with HER2 pulsed
• DC1 after week 9 when DCC are present but prior to the development of mammary carcinoma resulted in a decrease in DCC as well as a decreased expression of Ki67 (FIG. 6A).
• Vaccines reduced the development of mammary carcinoma at week 16 (FIG. 6B). There was also a diminished number of DCC (FIG. 6C) and increase in markers of tumor senescence in DCC from Neu T mice treated with DC1. Shown in Fig 6D is b-galactosidase expression a common marker of senescence. This latter data indicates that, besides the impact of therapy on primary or metastatic disease, with intratumoral therapy, DC1 can through induction of anti- HER2 CD4 T cells drive senescence and protect against tumor development and may have an impact on BC mortality.
• mice were treated at 8 weeks of age with subcutaneous injections of 1X10 6 rat neu peptide-pulsed DC1 twice a week or with anti-SEMA4D antibody administered weekly at a concentration of 10mg/kg/ body weight.
• the mice were sacrificed, and mammary glands were collected.
• the single cell suspension of mammary glands was prepared and stained for CD 19 positive B cells then analyzed by flow cytometry. An increased level of CD 19+ B cells was observed in DC1 vaccinated mice and in anti-SEMA4D antibody-treated mice compared to untreated control, indicating B cell infiltration into the mammary glands (FIG. 7A).
• the accumulation of B cells in the tumor region indicates that antitumor antibodies play a role in causing the effects seen in DC1 therapy with SEMA4D.
• the bone marrow cells derived from the control and DC1 vaccinated NeuT mice were stained for CD4 and CD 8 T cell markers and analyzed by flow cytometry. Increased numbers of CD4T cells and CD8 T cells were observed in the bone marrow of DC1 vaccinated NeuT mice compared to untreated control mice (FIG. 7B).
• Example 7 IFN-g and TNF-a from CD4 Thl cause Induction of Tumor Senescence in HER2 Expressing Breast Cancer:
• trastuzumab a humanized recombinant monoclonal antibody directed against the extracellular subdomain IV of HER2, inhibits ligand-independent dimerization, blocks downstream proliferation signaling pathways, and induces antibody-dependent cellular cytotoxicity (ADCC) and Pertuzumab, another humanized recombinant monoclonal antibody targeting the extracellular subdomain II of HER2, prevents ligand-dependent heterodimerization with other members of the HER family, which also inhibits proliferation signaling pathways and induces ADCC. Together both antibodies act in a complementary fashion.
• ADCC antibody-dependent cellular cytotoxicity
• Example 8 Clinical Activity of Thl cytokines (IFN-g) in HER2 Breast Cancer:
• this regimen is successful in at least maintaining the established pCR rate and thus, the regimen can replace the more toxic regimens with simpler potentially more effective regimen using a combination of immunotherapy with Thl cytokines and standard therapy.
• DC1 vaccines can drive a similar response in neoadjuvant HER2 patients to drive pCR in combination with PTCH.
• Example 9 Accumulation of B and T cells occurs in patients responding to HER2 -pulsed DC1 Vaccines:
• SEMA4D has been described as a promoter of angiogenesis of tumors and, therefore, has been associated with tumor progression (see, e.g., Zhou, et al. Methods Mol. Biol 1493:429-441 (2017)).
• DCE Dynamic Contrast Enhancement
• the murine monoclonal antibody Mab 67 described above, is disclosed, e.g., in U.S. Patent No. 8496938.
• MAb 67 VH Q V QLQQS GPEL VKPG AS VKISCKAS GYSFSDYYMHWVKQSPEN S LEWIGQINPTTGGASYN QKFKGKATLTVDKSSSTAYMQLKSLTSEESAVYYCTRYYYGRHFDVWGQGTTVTVSS
• Mab VX15/2503 (pepinemab) is a humanized version of MAb 67, and is also disclosed in U.S. Patent No. 8496938. The amino acid sequences of VX15/2503 are reproduced below.
• Example 11 Depletion of CD4+ T cells abrogates anti-SEMA4D activity, both alone and in combination with DC-1 treatment.
• CD4+ T cells play a major role in instigating and shaping adaptive immune responses.
• CD4 depleted tumor-bearing mice were generated and the immune response to treatment was compared to that of non-CD4 depleted (“normal”) tumor bearing mice.
• mice received 2.5e5 TUBO cells subcutaneously on the right flank on day 0. When tumors were palpable on day 7, the mice were randomized into four groups. Mice received monotherapy with either an IgG isotype control antibody or anti-SEMA4D antibody/MAb67 (10mg/kg/ body weight) intraperitoneally until end point or le6/ IOOmI intratumoral HER2- DC1 weekly for six weeks. For combination therapy, mice received anti-sema4D antibody prior to receiving a first intratumoral HER2-DC1 injection once a week for six weeks.
• CD4 depleted Balb/C mice were generated by administering 300pg of anti-CD4 depleting antibody (Clone GK1.4) intraperitoneally, starting three days before TUBO tumor implantation, and treatment with the anti-CD4 antibody continued with two injections per week until end point. (See Evans, E. E., et al. (2015). Cancer Immunol Res 3(6): 689-70) The CD4- depleted mice were treated with monotherapy, anti-SEMA4D antibody /MAb67, or combination therapy as described above for tumor-bearing non-CD4 depleted mice.
• anti-CD4 depleting antibody Clone GK1.4
• FIG. 17A -D - 17A and B Control, no CD4 depletion; tumor volume and survival, respectively
• 17C and D CD4 depletion
• Figures 17 E (control, no CD4 depletion) and 17 F (CD4 depletion) show tumor growth curves for each mouse. Complete tumor regression was observed following treatment with anti-SEMA4D and activity was further enhanced with the combination therapy. All of the combination-treated mice in the non-depleted group survived for the sixty-day testing period (FIG. 17B), and 4/5 of these mice showed complete tumor regression. (FIG. 17E).
• CD4+ T cells are required for a clinically effective response to anti-SEMA4D therapies.
• the FcyR mediated functions most commonly associated with therapeutic antibodies are those that mediate target cell elimination - antibody-dependent cellular cytotoxicity (ADCC), which is a mechanism of cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell, whose membrane- surface antigens have been bound by specific antibodies. These functions are triggered when antibody binding to antigen on the surface of a target cell generates sufficient avidity to trigger signaling through FcyRs on effector cells such as NK cells and macrophages, which then eliminate target cells through direct killing or phagocytosis. Preclinical models show that these forms of FcyR-mediated cytotoxicity are a significant component of the mechanism of action for certain tumor targeted antibodies. (Clynes RA et al., Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med. 2000, 6: 443-446).
• ADCC antibody-dependent cellular cytotoxicity
• FcyR knock-out mice were generated. Briefly, the model was created by targeted disruption of the Fcerlg gene via introduction of a new stop codon in E14 ES cells and injecting the targeted cells into C57BL/6 blastocysts. Heterozygotes on a C57BL/6 background were intercrossed to generate homozygous targeted mutation mice. The mice were then backcrossed twelve generations (N12) to a BALB/cByJ inbred background. (Takai T, Li M, Sylvestre D, Clynes R, Ravetch J. (1994) FcRy Chain Deletion results in Pleiotrophic Effector Cell Defects. ).
• BALB/C- Fcerlg KO mice (CA29F2(B6)-Fcerl g tmlRav N12) were injected at both flanks subcutaneously with 2.5X10 5 tumor cells/ site on day 0. DC were generated, matured to DC1 and pulsed with MHC class II neu peptides. Mice received le6/100pl DC1 vaccine intratumorally once a week for six weeks. In those animals with two tumor locations, the vaccine was administered in only one of the two tumors.
• Anti-SEMA4D Mab 67 was given intraperitoneally at a concentration of 10 mg/kg/ body weight at weekly intervals. Control mice received isotype control antibodies, DC treatment or Mab 67 alone.
• Interferon-gamma is a pleiotropie molecule with associated antiproliferative, pro- apoptotic and antitumor mechanisms. This effector cytokine is considered to be a major effector of immunity and is a key Thl cytokine relevant for anti-tumor immune response.
• mice were generated in Balb/C IFN-gamma knock out (KO) (C.129S7 (B6)-IFNg tmlTs /J (IFN-y K °, Jackson Laboratories) mice.
• the mice were administered 2.5e5 TUBO cells subcutaneously on the right flank on day 0.
• Dendritic cells were generated, matured to DC1 and pulsed with MHC class II neu peptides.
• mice On day 7 when tumors were palpable, mice were randomized into four groups.
• mice received monotherapy with either control antibody or anti-sema4D antibody (lOmg/kg / body weight) intraperitoneally until end point or le6/100pl intratumoral HER2- DC1 weekly for six weeks.
• mice received anti-sema4D antibody prior to receiving a first intratumoral HER2-DC1 injection once a week for six weeks. Tumors were measured every 2-3 days with a caliper until the endpoint. Mean tumor volume for each group is shown in Figure 19.

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