Attorney Docket Number 10110-437WO1 METHODS OF TREATING CANCER COMPRISING ADMINISTRATION OF INTRATUMORAL DCs IN COMBINATION WITH SYSTEMIC IgG MONOCLONAL ANTIBODY I. CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.63/441,298, filed on January 26, 2023, which is incorporated herein by reference in its entirety. II. STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under Grant Nos. W81XWH-16-1- 0385 and W81XWH-19-1-0675 awarded by the Department of Defense. The government has certain rights in the invention. III. BACKGROUND 1. Most breast cancer (BC) patients with high-risk, locally-advanced tumors require treatment with chemotherapy, which carries significant toxicities including alopecia, nerve damage, cognitive changes, cardiac toxicities, and potential for hematologic toxicities including leukemias. To date the only immunotherapies approved for BC (anti-PD1 for triple-negative, and trastuzumab/pertuzumab for HER2 BC) still require addition of chemotherapy for effectiveness. Chemotherapies are well known to change the tumor microenvironment (TME) and are dependent on the immune response, including lymphocytes, for efficacy. Immunotherapies alone have had minimal effects. Therefore, identifying alternative therapies that can alter the TME and favor current immunotherapies would be of tremendous benefit to BC patients. In addition, in triple negative breast cancer (TNBC) there are still about 40% of patients that are refractory to current chemotherapy and do not achieve pathologic complete response (pCR) to current neoadjuvant therapy. For HER2 there is also room for improving pCR, especially in the hormone receptor (HR) positive group (50% pCR) and about a third of the HR-negative group. What are needed are new cancer therapies. IV. SUMMARY 2. Disclosed are methods and compositions related to novel combination therapies comprising oncodriver pulsed dendritic cells and antibody-dependent cellular cytotoxicity (ADCC) competent antibodies and/or fragments thereof. 3. In one aspect, disclosed herein are anti-cancer combination therapies comprising at least one dendritic cell pulsed with an oncodriver (such as, for example, human epidermal growth factor receptor (HER) 1(HER1), HER2, HER3, EGFR, c-MET, B-Rapidly Accelerated
Attorney Docket Number 10110-437WO1 Fibrosarcoma (BRAF), KIT, Androgen Receptor (AR), Estrogren Receptor (ER), KRAS, TP53, or APC ) and at least one antibody-dependent cellular cytotoxicity (ADCC) competent antibody that binds to a target on a tumor or in a tumor microenvironment (including, but not limited to an antibody that binds an immunoregulatory molecule (such as, for example, Semaphorin (SEMA) 4D (SEMA4D) (such as, for example, pepinemab), SEMA4A, SEMA4B, SEMA4C, SEMA4F, SEMA4G, SEMA3A, SEMA3B, SEMA3C, SEMA3D, SEMA3E, SEMA3F, SEMA3G, or VEGF) or HER-2. In some embodiments the anti-cancer combination therapies of any preceding aspect can simultaneously reduce immune checkpoint inhibition and increase anti- tumor immune responses. In one aspect, the anti-cancer combination therapies of any preceding aspect provide systemic immunity for distal tumors of the same type as the primary tumor being treated. 4. In one aspect, disclosed herein are methods of treating, preventing, reducing, and/or inhibiting 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. In some aspects, the cancer is melanoma. Thus, in one aspect, disclosed herein are methods of treating 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) 1(HER1), HER2, HER3, EGFR, c-MET, B-Rapidly Accelerated Fibrosarcoma (BRAF), KIT, Androgen Receptor (AR), Estrogren Receptor (ER), KRAS, TP53, or APC ) and at least one antibody-dependent cellular cytotoxicity (ADCC) competent antibody that binds to a target on a tumor or in a tumor microenvironment (including, but not limited to an antibody that binds to an immunoregulatory molecule (such as, for example, Semaphorin (SEMA) 4D (SEMA4D) (such as, for example, pepinemab), SEMA4A, SEMA4B, SEMA4C, SEMA4F, SEMA4G, SEMA3A, SEMA3B, SEMA3C, SEMA3D, SEMA3E, SEMA3F, SEMA3G, or VEGF) or HER-2). 5. Also disclosed are anti-cancer combination therapies methods treating, preventing, reducing, and/or inhibiting a cancer of any preceding aspect; wherein the at least ADCC competent antibody is administered systemically or intratumorally and/or the oncodriver pulsed dendritic cell is administered intratumorally. In one aspect, disclosed herein are anti-cancer combination therapies of any preceding aspect wherein the anti-cancer combination therapy provides systemic immunity for distal tumors of the same type as the primary tumor being treated.
Attorney Docket Number 10110-437WO1 6. In one aspect, also disclosed herein are 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. 7. Also disclosed herein are 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 antibody-dependent cellular cytotoxicity (ADCC) competent antibody; are administered concurrently with the at least one antibody-dependent cellular cytotoxicity (ADCC) competent antibody; or wherein the at least one antibody-dependent cellular cytotoxicity (ADCC) competent antibody 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. 8. In one aspect, disclosed herein are 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. 9. Also disclosed herein are anti-cancer combination therapies methods treating, preventing, reducing, and/or inhibiting a cancer of any preceding aspect; wherein the at least one ADCC competent antibody 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. 10. In one aspect, disclosed herein are anti-cancer combination therapies methods treating, preventing, reducing, and/or inhibiting a cancer of any preceding aspect further comprising administering to the subject T cells (including, but not limited to the subject’s own T cells, TILs, or CAR T cells). In one aspect the T cells are CD4 T cells. 11. In one aspect, the anti-cancer therapy disclosed herein, help reduce immune check points while increasing anti-tumor activity.
Attorney Docket Number 10110-437WO1 V. BRIEF DESCRIPTION OF THE DRAWINGS 12. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods. 13. Figure 1 shows the overall strategy of sensing-conditioning, migration, activation and return-trafficking steps in a circular arrangement, referred to as a “smart circuit”. 14. Figures 2A, 2B, 2C, 2D and 2E show that activation of murine CD103+ cDC1, prepared from bone marrow stem cells, with interferon gamma (IFN- γ) and toll like receptor agonists (TLR) induce complete regression of HER2 tumors when the cDC1 are injected intratumorally in murine mammary carcinoma. Total elimination of tumor requires both specific antigen-pulsed DC be delivered intratumorally (i.t.) plus systemic co-administration of a monoclonal antibody such as those targeting HER2 (in the case of HER2/ErbB2pos mammary carcinoma) as shown in Figure 2A or the TME-like anti-semaphorin 4D, a ligand that binds Plexin B1 as shown in Figure 2B. Figure 2B shows that not only do treated tumors regress, but a remarkable and consistent abscopal response (i.e. regression of untreated tumor in a two-tumor model) is driven by CD103+ cDC1that actually extensively migrate from site of administration Figure 2C shows that cDC1 express XCR1, CCR7 and CD103 and are found to have migrated to distant tumor sites and lymph nodes. Figure 2D shows migration of CD4 Th1 into both treated and untreated tumor, while Figure 2E shows migration of CD8, NK, and B cells migrate into treated tumor. 15. Figure 3 shows the anti-tumor efficacy of CD40/CD40L signaling activated CD103+ HER2-DC in HER2+ TUBO model. BALB/c mice bearing TUBO tumors were treated with CD103+ HER2-DC or CD40/CD40L signaling activated (licensed) CD103+ HER2-DC. Enhanced anti-tumor response was observed in TUBO bearing mice that were treated with licensed CD103+ HER2-DC intratumoral delivery (i.t.). 16. Figures 4A and 4B show that using the breakthrough purification method and culture system, two populations of CD141+ DC with distinct, yet complementary properties can be identified from human peripheral monocytes. Figure 4A shows one subpopulation, which is larger in size and although apparently mature with DC and costimulatory markers, does not express significant levels of migration-associated marker CCR7 and also expresses little CD103 or XCR1. Figure 4A also shows a second population that is smaller but expresses CCR7, CD103 and XCR1. Figure 4B shows that patients achieving pCR demonstrated the highest expression of
Attorney Docket Number 10110-437WO1 CD141 by their DC, while those with residual cancer burden (RCB) 1-3 were almost uniformly lower. Figure 4C shows that a greater percentage of CD141+ cells can be seen in the post- treatment biopsy confirmed in the TME when specimens and biopsies were taken prior to and following cDC1 therapy. Figure 4C also shows clearly a resultant increase in CD4 Th, CD8 and NK cells as a result of the intratumoral CD141+ cDC1 treatment. Figure 4D shows that both populations of CD141+ cDC1 express CD40 at equivalent levels, and when cDC1 are licensed by CD4 Th1 signals, (like CD40 ligation) and survive for greater than four days. 17. Figures 5A and 5B show that CD141+ cDC1, when given intratumorally (but not subcutaneously), at dosage of either 50 million (5A) or 100 million (5B) CD141+ cDC1 cause tumor regression when combined with anti-HER2 antibody therapy even prior to the start of Taxol trastuzumab, pertuzumab therapy as shown in the post-immunotherapy panels. 18. Figure 6 is a schematic that shows the role for CD4 Th1 in tumor therapy: CD4 Th1 are responsible for licensing DC, providing help for CD8, direct effects on tumor through cytokine production, and class switching in B cells. 19. Figures 7A and 7B show the efficacy of CD103+ HER2-DC primed CD4Th1 response on primary tumors and metastatic seeding potential of DCCs. Figure 1A shows that BALB-HER2/neu transgenic mice were depleted with or without CD4 T cells or CD8 T cells, then treated with CD103+ HER2-DC intratumorally. Representative MRI images shows the capability of CD103+ HER2-DC in priming CD4Th1 cells to control spontaneous primary tumor growth in the mammary glands. Figure 1B shows representative IHC HER2 & cytokeratin 8/18 double staining images for experimental BALB-HER2/neuT mice showing no evidence for the development of micrometastases in lung, liver and brain at the age of week 24. 20. Figure 8 shows immunofluorescent staining of tissue sections of HER-2pos breast cancer after treatment with CD141pos DC therapy. VI. DETAILED DESCRIPTION 21. Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
Attorney Docket Number 10110-437WO1 A. Definitions 22. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like. 23. 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. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if 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. 24. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings: 25. “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. 26. An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant
Attorney Docket Number 10110-437WO1 amount. Thus, the increase 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% increase so long as the increase is statistically significant. 27. 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. Also, for example, 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. Thus, 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. 28. "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. 29. By “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. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control. 30. 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. 31. The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can
Attorney Docket Number 10110-437WO1 also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. 32. 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. 33. The term “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. In addition, 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. 34. "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. 35. "Comprising" is intended to mean that the 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. 36. A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative." 37. “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,
Attorney Docket Number 10110-437WO1 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. 38. 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. 39. "Pharmaceutically acceptable carrier" (sometimes referred to as a “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. The terms "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. As used herein, the term "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. 40. “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. 41. “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
Attorney Docket Number 10110-437WO1 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. When the terms “therapeutic agent” is 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. 42. “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of type I diabetes. In some embodiments, 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. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years. 43. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. B. Compositions 44. Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each
Attorney Docket Number 10110-437WO1 various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular combination therapy is disclosed and discussed and a number of modifications that can be made to a number of molecules including the combination therapy are discussed, specifically contemplated is each and every combination and permutation of combination therapy and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods. 45. Thus, this project will not just reduce the need for chemotherapy but rescue those patients’ refractory to it that do not achieve pCR. The Challenges undertaken in this application include: (1) Reduce the use of toxic therapies and replacing them with safer, more effective and less toxic regimens; (2) Reduce mortality from breast cancer. Dendritic cells (DC), which have not achieved real success to date, remain prime candidates to alter the TME and lead to favorable immunotherapies if used effectively. We propose a novel dual-population conventional type I dendritic cell (cDC1) using intratumoral delivery to effectively condition the TME, as well as distant sites of disease. 46. In one aspect, disclosed herein are anti-cancer combination therapies comprising at least one dendritic cell pulsed with an oncodriver (such as, for example, human epidermal growth factor receptor (HER) 1(HER1), HER2, HER3, EGFR, c-MET, B-Rapidly Accelerated Fibrosarcoma (BRAF), KIT, Androgen Receptor (AR), Estrogren Receptor (ER), KRAS, TP53, and APC) or epitope therefrom and at least one antibody-dependent cellular cytotoxicity (ADCC) competent antibody that binds to a target on a tumor or in a tumor microenvironment (such as for example, an ADCC competent antibody that binds to an immunoregulatory molecule inhibitor (including, but not limited to Semaphorin (SEMA) selected from the group consisting of SEMA4D (such as, for example, pepinemab), SEMA4A, SEMA4B, SEMA4C, SEMA4F, SEMA4G, SEMA3A, SEMA3B, SEMA3C, SEMA3D,
Attorney Docket Number 10110-437WO1 SEMA3E, SEMA3F, SEMA3G, or VEGF) or HER-2. In one aspect, the oncodriver pulsed dendritic cell is activated with IL-12 prior to administration. 47. It is understood and herein contemplated that the disclosed anti-cancer combination therapies can simultaneously reduce immune checkpoint inhibition and increase anti-tumor immune responses. Additionally, it is understood and herein contemplated that the disclosed anti-cancer therapies provide systemic immunity for distal tumors of the same type as the primary tumor being treated. 1. Antibodies (1) Antibodies Generally 48. The term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to engage in antibody-dependent cellular cytotoxicity (ADCC). The antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. 49. 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. 50. The disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies. For example, disclosed monoclonal antibodies can be
Attorney Docket Number 10110-437WO1 prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In 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. Alternatively, the lymphocytes may be immunized in vitro. 51. 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. 52. 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. 53. As used herein, 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, Fv, sFv, scFv, and the like, including hybrid fragments. Thus, fragments of the antibodies that retain the ability to bind their specific antigens are provided. For example, fragments of antibodies which maintain target binding activity and ADCC activity are included within the meaning of the term “antibody or fragment thereof.” 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). 54. Also included within the meaning of “antibody or fragments thereof” are conjugates of antibody fragments and antigen binding proteins (single chain antibodies).
Attorney Docket Number 10110-437WO1 55. The fragments, whether attached to other sequences or not, 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. In any case, the antibody or antibody fragment must possess a bioactive property, such as a 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. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, M.J. Curr. Opin. Biotechnol.3:348-354, 1992). 56. As used herein, 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. (2) Human antibodies 57. The disclosed human antibodies can be prepared using any technique. The disclosed human antibodies can also be obtained from transgenic animals. For example, 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 al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)). Specifically, 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. (3) Humanized antibodies 58. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an
Attorney Docket Number 10110-437WO1 antibody molecule. Accordingly, a humanized form of a non-human antibody (or a fragment thereof) 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. 59. To generate 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). In some instances, 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. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. In practice, 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). 60. Methods for humanizing non-human antibodies are well known in the art. For example, 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.5,565,332 (Hoogenboom et al.), U.S. Patent No.5,721,367 (Kay et al.), U.S. Patent No.5,837,243 (Deo et al.), U.S. Patent No.5, 939,598 (Kucherlapati et al.), U.S. Patent No.6,130,364 (Jakobovits et al.), and U.S. Patent No.6,180,377 (Morgan et al.). 2. Pharmaceutical carriers/Delivery of pharmaceutical products 61. As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By "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
Attorney Docket Number 10110-437WO1 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. 62. The 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. As used herein, "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. The exact amount of the 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. 63. 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. 64. 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 al., 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. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). 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
Attorney Docket Number 10110-437WO1 of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. 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)). a) Pharmaceutically Acceptable Carriers 65. The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier. 66. 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. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of 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. 67. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
Attorney Docket Number 10110-437WO1 68. Pharmaceutical 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. 69. 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. 70. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of 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, inert gases and the like. 71. 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. 72. 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. 73. Some of the 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.
Attorney Docket Number 10110-437WO1 b) Therapeutic Uses 74. 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. Generally, 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. For example, 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 al., eds., Noges Publications, Park Ridge, N.J., (1985) ch.22 and pp.303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp.365-389. A typical daily dosage of the antibody used alone might range from about 1 µg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above. C. Method of treating cancer 75. The disclosed compositions can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphomas such as B cell lymphoma and T cell lymphoma; mycosis fungoides; Hodgkin’s Disease; myeloid leukemia (including, but not limited to acute myeloid leukemia (AML) and/or chronic myeloid leukemia (CML)); bladder cancer; brain cancer; nervous system cancer; head and neck cancer; squamous cell carcinoma of head and neck; renal cancer; lung cancers such as small cell lung cancer, non- small cell lung carcinoma (NSCLC), lung squamous cell carcinoma (LUSC), and Lung Adenocarcinomas (LUAD); neuroblastoma/glioblastoma; ovarian cancer; pancreatic cancer; prostate cancer; skin cancer; hepatic cancer; melanoma; squamous cell carcinomas of the mouth, throat, larynx, and lung; cervical cancer; cervical carcinoma; breast cancer including, but not limited to triple negative breast cancer; genitourinary cancer; pulmonary cancer; esophageal carcinoma; head and neck carcinoma; large bowel cancer; hematopoietic cancers; testicular cancer; and colon and rectal cancers.
Attorney Docket Number 10110-437WO1 76. Also disclosed herein are methods of treating, decreasing, inhibiting, ameliorating, reducing, and/or preventing a cancer and/or metastasis (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 any of the anti-cancer combination therapies disclosed herein. For example, disclosed herein are methods of treating, reducing, decreasing, inhibiting, and/or preventing a cancer and/or metastasis (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 to the subject an oncodriver (such as, for example, human epidermal growth factor receptor (HER) 1(HER1), HER2, HER3, EGFR, c-MET, B-Rapidly Accelerated Fibrosarcoma (BRAF), KIT, Androgen Receptor (AR), Estrogren Receptor (ER), KRAS, TP53, and APC) pulsed dendritic cell and at least Antibody-dependent cellular cytotoxicity (ADCC) competent antibody that binds to a target on a tumor or in a tumor microenvironment (such as for example, an ADCC competent antibody that binds to an immunoregulatory molecule inhibitor (including, but not limited to Semaphorin (SEMA) selected from the group consisting of SEMA4D (such as, for example, pepinemab), SEMA4A, SEMA4B, SEMA4C, SEMA4F, SEMA4G, SEMA3A, SEMA3B, SEMA3C, SEMA3D, SEMA3E, SEMA3F, SEMA3G, or VEGF) or HER-2. It is understood and herein contemplated that the disclosed anti-cancer combination therapies can simultaneously reduce immune checkpoint inhibition and increase anti-tumor immune responses. 77. Also disclosed are methods of treating, decreasing, inhibiting, ameliorating, reducing, and/or preventing a cancer and/or metastasis; wherein the at least ADCC competent antibody is administered systemically or intratumorally and/or the oncodriver pulsed dendritic cell is administered intratumorally. Additionally, it is understood and herein contemplated that the disclosed anti-cancer therapies provide systemic immunity for distal tumors of the same type as the primary tumor being treated. Accordingly, in one aspect, disclosed herein are methods of treating, decreasing, inhibiting, ameliorating, reducing, and/or preventing a distal tumor comprising comprising administering any of the anti-cancer combination therapies disclosed herein. 78. In one aspect, also disclosed herein are methods of treating, decreasing, inhibiting, ameliorating, reducing, and/or preventing a cancer; wherein the dendritic cells are removed from the subject and pulsed with oncodriver ex vivo.
Attorney Docket Number 10110-437WO1 79. Also disclosed herein are methods of treating, decreasing, inhibiting, ameliorating, reducing, and/or preventing 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 antibody- dependent cellular cytotoxicity (ADCC) competent antibody; are administered concurrently with the at least one antibody-dependent cellular cytotoxicity (ADCC) competent antibody; or wherein the at least one antibody-dependent cellular cytotoxicity (ADCC) competent antibody 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. 80. In one aspect, disclosed herein are methods of treating, decreasing, inhibiting, ameliorating, reducing, and/or preventing 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. 81. Also disclosed herein are methods of treating, decreasing, inhibiting, ameliorating, reducing, and/or preventing a cancer; wherein the at least one ADCC competent antibody 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. 82. In one aspect, disclosed herein are anti-cancer combination therapies and methods of treating, decreasing, inhibiting, ameliorating, reducing, and/or preventing a cancer further comprising administering to the subject T cells (including, but not limited to the subject’s own T cells, TILs, or CAR T cells). In one aspect the T cells are CD4 T cells. 83. In some aspects, it is understood and herein contemplated that the combination therapy can have an abscopal effect on cancers other than the cancer being initially targeted. 84. It is understood and herein contemplated that the disclosed treatment regimens can used alone or in combination with any anti-cancer therapy 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 (Copanlisib Hydrochloride), Alkeran
Attorney Docket Number 10110-437WO1 for Injection (Melphalan Hydrochloride), Alkeran Tablets (Melphalan), Aloxi (Palonosetron Hydrochloride), Alunbrig (Brigatinib), Ambochlorin (Chlorambucil), Amboclorin Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane),Arranon (Nelarabine), Arsenic Trioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, Avastin (Bevacizumab), Avelumab, Axitinib, Azacitidine, Bavencio (Avelumab), BEACOPP, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Besponsa (Inotuzumab Ozogamicin) , Bevacizumab, Bexarotene, Bexxar (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometyx (Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, Campath (Alemtuzumab), Camptosar , (Irinotecan Hydrochloride), Capecitabine, CAPOX, Carac (Fluorouracil--Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine Implant, Casodex (Bicalutamide), CEM, Ceritinib, Cerubidine (Daunorubicin Hydrochloride), Cervarix (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), CMF, Cobimetinib, Cometriq (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine Liposome, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Daratumumab, Darzalex (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), DTIC-Dome (Dacarbazine), Durvalumab, Efudex (Fluorouracil--Topical), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Elotuzumab, Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Empliciti (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride , EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi) , Ethyol (Amifostine), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin
Attorney Docket Number 10110-437WO1 Hydrochloride Liposome), Everolimus, Evista , (Raloxifene Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU (Fluorouracil Injection), 5-FU (Fluorouracil-- Topical), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil--Topical), Fluorouracil Injection, Fluorouracil--Topical, Flutamide, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI- CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE- CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (Carmustine Implant), Gliadel wafer (Carmustine Implant), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Hemangeol (Propranolol Hydrochloride), Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin (Topotecan Hydrochloride), Hydrea (Hydroxyurea), Hydroxyurea, Hyper-CVAD, Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Idamycin (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, Idhifa (Enasidenib Mesylate), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), Intron A (Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, Istodax (Romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), JEB, Jevtana (Cabazitaxel), Kadcyla (Ado- Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lartruvo (Olaratumab), Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Leustatin (Cladribine), Levulan (Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), Lomustine, Lonsurf (Trifluridine and Tipiracil Hydrochloride), Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine
Attorney Docket Number 10110-437WO1 Hydrochloride, Megestrol Acetate, Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide, Mexate (Methotrexate), Mexate-AQ (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride) , Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate), Necitumumab, Nelarabine, Neosar (Cyclophosphamide), Neratinib Maleate, Nerlynx (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide, Ninlaro (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride, Onivyde (Irinotecan Hydrochloride Liposome), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin- stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Portrazza (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride , Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, Relistor (Methylnaltrexone Bromide), R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, R-ICE, Rituxan (Rituximab), Rituxan Hycela (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and , Hyaluronidase Human, ,Rolapitant Hydrochloride, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, Rydapt
Attorney Docket Number 10110-437WO1 (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa-2b), Sylvant (Siltuximab), Synribo (Omacetaxine Mepesuccinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq , (Atezolizumab), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, Tolak (Fluorouracil--Topical), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and Iodine I 131 Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Unituxin (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride), Vectibix (Panitumumab), VeIP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, Venclexta (Venetoclax), Venetoclax, Verzenio (Abemaciclib), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, Vistogard (Uridine Triacetate), Voraxaze (Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride), Vyxeos (Daunorubicin Hydrochloride and Cytarabine Liposome), Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda (Capecitabine), XELIRI, XELOX, Xgeva (Denosumab), Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab), Yondelis (Trabectedin), Zaltrap (Ziv-Aflibercept), Zarxio (Filgrastim), Zejula (Niraparib Tosylate Monohydrate), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zofran (Ondansetron Hydrochloride), Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), Zydelig (Idelalisib), Zykadia (Ceritinib), and/or Zytiga (Abiraterone Acetate). The treatment methods can include or further include checkpoint inhibitors including, but are not limited to antibodies that block PD-1 (such as, for example, Nivolumab (BMS-936558 or MDX1106), pembrolizumab, CT-011, MK-3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX-1105 (BMS-936559), MPDL3280A, or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA-4 (such as, for example, Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009,
Attorney Docket Number 10110-437WO1 omburtamab), B7-H4, B7-H3, T cell immunoreceptor with Ig and ITIM domains (TIGIT)(such as, for example BMS-986207, OMP-313M32, MK-7684, AB-154, ASP-8374, MTIG7192A, or PVSRIPO), CD96, B- and T-lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA)(such as, for example, JNJ-61610588, CA-170), TIM3 (such as, for example, TSR-022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661), LAG-3 (such as, for example, BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013, and Immutep) D. Examples 85. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. 1. Example 1 E. References 86. The immune system evolved primarily to protect us from infection. DCs are the principal sentinel cells that are the first to sense biochemical signals of microbial pathogens in their immediate surroundings. They then secrete soluble factors called cytokines and chemokines that condition this local environment, preparing it to evict the invaders, and attract additional cells to achieve this. Many of the DC then migrate to regional lymph nodes where lymphocytes of the immune system are sensitized and activated against the specific microbial threat. After activation and expansion, lymphocytes return traffic, drawn back to the original site of infection by the DC-produced chemokines, finding a conditioned environment favorable for destroying the pathogen. Because this evolved process for successfully eliminating invaders contains these sensing-conditioning, migration, activation and return-trafficking steps in a circular arrangement, we refer to it as a “smart circuit”. Our overall strategy is to re-create and optimize this natural circuit to entice the immune system to attack cancer with the same ferocity it usually reserves for microbes (Fig 1). We do this by injecting directly into the tumor uniquely cultured antigen-pulsed DCs activated with biochemical signals of microbial infection (TLR ligands) which causes strong cytokine/chemokine secretion at site of administration (sensing- conditioning). DCs migrate to local lymph nodes and present the pulsed tumor peptides to T lymphocytes which once activated, are drawn back (return traffic) to the site of DC
Attorney Docket Number 10110-437WO1 administration (i.e. the tumor) where the implanted DC have altered the tumor microenvironment in ways that facilitates rejection. a) Effectiveness of CD103+ conventional DC1 (cDC1) in murine models: 87. Human dendritic cell (DC) as vaccines have thus far not typically demonstrated efficacy in the treatment of invasive breast cancer (BC). In contrast, we have shown, under the right conditions, murine CD103+ cDC1 prepared from bone marrow stem cells and activated with interferon gamma (IFN- γ) and toll like receptor agonists (TLR) induce complete regression of HER2 tumors when the cDC1 are injected intratumorally in murine mammary carcinoma. Total elimination of tumor requires both specific antigen-pulsed DC be delivered intratumorally (i.t.) plus systemic co-administration of a monoclonal antibody such as those targeting HER2 (in the case of HER2/ErbB2pos mammary carcinoma) (Fig 2A) or the TME-like anti-semaphorin 4D, a ligand that binds Plexin B1 (Fig 2B). Not only do treated tumors regress, but a remarkable and consistent abscopal response (i.e. regression of untreated tumor in a two-tumor model) is driven by CD103+ cDC1that actually extensively migrate from site of administration (Fig 2B). These cDC1 express XCR1, CCR7 and CD103 and are found to have migrated to distant tumor sites and lymph nodes (Fig 2C). In addition, CD4 Th1 critically migrate into both treated and untreated tumor (Fig 2D), while CD8, NK, and B cells migrate into treated tumor (Fig2E) and complete regression is dependent on CD4 Th1 CD8, B cells (antibody) and NK cells. The systemically administered antibodies are essential for eliciting the CD4 Th1 migration when concomitantly administered (Fig 2D). Early CD4Th1 response is essential but the requirement for tumor antibody can be at least partially replaced using CD40L activation of the cDC1 indicating the need for CD4 Th1 licensing of the CD103+ cDC1 (Fig 3). The tumor response is also dependent on IFN-γ and FcR mediated antibody dependent cytotoxicity. Mice that reject these tumors are completely resistant to tumor challenge, indicating a strong protective anti- tumor immune response. Overall, this is an extremely effective immune response that in murine models can effectively replace the need for chemotherapy. 88. The problem for translation of such findings has been CD103+ or equivalent CD141+ cDC1 have been difficult to obtain from humans in large numbers. In addition, anti- HER2 CD4 Th1 may not be abundant enough in the tumor microenvironment to induce regression. Using a breakthrough purification method and culture system, we can now identify from human peripheral monocytes two populations of CD141+ DC with distinct, yet complementary properties (Fig 4A). One subpopulation is larger in size (Red), and although apparently mature with DC and costimulatory markers (Fig 4A), does not express significant
Attorney Docket Number 10110-437WO1 levels of migration-associated marker CCR7 and also expresses little CD103 or XCR1 (Fig 4A). The second population (Blue) is smaller but expresses CCR7, CD103 and XCR1 (Fig 4A). Interestingly, when HER2 MHC Class II peptide-pulsed DC were administered intratumorally prior to standard TCHP, a link was established between the level of CD141 expressed by the DC and attainment of pCR (Fig 4B). Specifically, patients achieving pCR demonstrated the highest expression of CD141 by their DC, while those with residual cancer burden (RCB) 1-3 were almost uniformly lower (Fig 4B). In fact, the single patient in the RCB group that had high expression of CD141 by their DC began with a 7.7 cm tumor bed and showed only a single microscopic focus remaining after therapy, indicating a response just short of achieving pCR. In any event, the CD141 expression on the larger cDC1 population was the only DC marker that was statistically-associated with pCR (Fig 4B), and this CD141 expression was confirmed in the TME when specimens and biopsies were taken prior to and following cDC1 therapy as can be seen in Fig 4C as a greater percentage of CD141+ cells can be seen in the post-treatment biopsy. There is also clearly a resultant increase in CD4 Th, CD8 and NK cells as a result of the intratumoral CD141+ cDC1 treatment (Fig 4C). Both populations of CD141+ cDC1 express CD40 at equivalent levels, and when cDC1 are licensed by CD4 Th1 signals, (like CD40 ligation), survive for greater than four days (Fig 4D) and even as long as a week. The smaller cDC1 population, by virtue of its high CCR7 expression with migratory potential, may in fact migrate to regional sentinel nodes and distant tumor sites as nodal metastasis also resolve in response to therapy (Fig xx top panel). We show herein that HER2 DCIS complete pathologic responses to neoadjuvant HER2 pulsed DC1 was associated with high levels of anti-HER2 CD4Th1 in sentinel nodes. It is possible these cDC1 are encountering CD40L on CD4Th1 in nodes and then survive and migrate to other sites of disease. These 141+ cDC1 are the equivalent of CD103+ murine DC that cause eradication of tumors with antitumor HER2 antibodies described above. We have documented these CD141+ cDC1, when given intratumorally (but not subcutaneously), at dosage of either 50 million or 100 million CD141+ cDC1 (Fig 5A&5B), cause tumor regression when combined with anti-HER2 antibody therapy even prior to the start of Taxol trastuzumab, pertuzumab therapy (Fig 5A&5B post - immunotherapy panels). Collectively these studies indicate these two populations of CD141+cDC1s are key to unlocking effective anti-BC immunity, dramatically changing the TME and thus raise the possibility of eliminating the need for chemotherapy. 89. Further defining and understanding the role of the different populations of cDC1 will be critical to understand the mechanism of this therapy, and how to improve upon outcomes. For instance, it is important to understand how to increase CD141+ expression on the larger DC
Attorney Docket Number 10110-437WO1 population to potentially increase the odds all patients can achieve pCR (Fig 5B). Mouse models indicate the CD103+ cDC1 are CCR7+ and do migrate to distant tumor sites, therefore defining the DC response in the murine models will be critical to gain insights into function and mechanism as well as interactions with CD4 Th1, CD8, NK and B cells, all of which are important in mediating response applicable to all breast cancers including TNBC and HR positive breast cancer. b) The role for CD4 Th1 in tumor therapy: 90. CD4 Th1 are responsible for licensing DC, providing help for CD8, direct effects on tumor through cytokine production, and class switching in B cells (Fig 6). A second problem is that many BC do not have adequate functionally active CD4Th1 cells in the tumor, and these cells, besides licensing CD141+ DC through CD40 ligand, are central to antibody responses, driving other innate effectors such as NK and NKT cells, and interferon gamma (IFN- γ) production, which we have shown in HER2 BC to cause ubiquination and loss of HER2 in tumors. IFN-γ also leads to apoptosis or senescence of disseminated cancer cells (DCC) and micrometastasis blocking active metastatic disease. This is mediated specifically by CD4 Th1 cells and not CD8 T cells (Fig.7A). Depletion of CD4 but not CD8 reverses the inhibition of micrometastasis formation in multiple organs (Fig 7B) Thus, the role of CD4 Th1 is to license CD141+DC early in the primary tumor, contribute to directing the effectors leading to control of primary tumors and importantly contributes to prevention and elimination of early metastatic disease (DCC and micrometastasis) something chemotherapy is often given to control. Systemic delivery of CD4 Th1 may be inferior to delivery of the cells with cDC1 directly into the TME. c) B cells, NK and Anti-tumor IgG and its role in tumor eradication: 91. Trastuzumab is an anti-HER IgG that both interferes with HER2 intracellular signaling but importantly drives antibody-dependent cytotoxicity (ADCC) through Fc receptors on NK cells. We have demonstrated that when anti-HER2 antibodies are administered systemically along with intratumorally-administered HER2 pulsed CD103+ DC, complete eradication of tumor ensues (Fig 2A). This requires a fully activated mature DC be pulsed with anti-tumor antigens. This therapy of combining anti-HER2 antibodies with intratumoral CD103+DC can drive abscopal effect and eradicate distant non-treated sites of disease. The intratumoral CD103+ DC were found not only to migrate to distant tumor sites, but these cells did not demonstrate significant numbers of apoptotic DC indicating these cells not only migrated but were functionally active. Given HER2 targeted antibodies can inhibit HER2 signaling we asked whether other tumor non-signaling IgG can cause the same effect. We selected anti- semaphorin 4D (Sema4D) which is expressed on HER2 expressing breast cancer and HER2
Attorney Docket Number 10110-437WO1 expressing TUBO cells and in the TME used in these models but not in TNBC. Systemic delivery of anti-Sema4D with intratumoral CD103+ HER2 pulsed DC results in complete eradication of tumors even larger advanced tumors (Fig 2 B). This combination resulted in an abscopal effect, and the response is mediated by IFN-γ, CD4, CD8 (Fig 2 D, E) and FcR through ADCC. The same mechanisms is observed in the HER2 antibody treatment. The response of anti-tumor antibodies is dependent on early involvement of CD4 Th1 (Fig 2D). In breast cancer patients’ administration of cDC1 results in upregulation of Semaphorin 4D in the TME on both tumor cells and infiltrating cells. We can validate whether any tumor antibody expressed in the tumor or TME can help to mediate these complete responses. In addition, we can explore whether this phenomenon can be mediated by tumor associated antibodies such as cetuximab or anti-PD1 in TNBC models. d) Human breast cancer and neoadjuvant therapy: 92. The ability to achieve a pCR to neoadjuvant therapy in HER2 and TNBC is associated with increased odds of survival. However, most of these patients require chemotherapy in addition to the immunotherapy (HER2; trastuzumab antibodies, TNBC; anti- PD1). We have data that intratumoral delivery of CD141+DC pulsed with tumor antigen can cause significant reduction of tumors. We have a Phase I trial ongoing that administers 6 weekly doses of 50 million or 100 million HER2-pulsed CD141+ DC with two doses of trastuzumab and pertuzumab followed by weekly Taxol trastuzumab and pertuzumab (reduce chemo from Taxotere, carboplatin and Herceptin and Perejeta (TCHP)) to patients with Stage II and III HER2 breast cancer. The first dose level led to 4/6 complete responses with anticipated rate of THP of 40% pCR indicating already a robust improvement in pCR rates. Importantly, one can demonstrate responses to immunotherapy alone (prior to THP) as seen in Fig 3 demonstrating response in both the large primary tumor and axillary nodes that were documented to be positive (Fig 3A). Of note, following THP this patient achieved pCR. Monoclonal antibodies such as trastuzumab (HER2) or potentially cetuximab (TNBC), when administered systemically could lead to influx of CD4 Th1 which are needed in the tumor microenvironment to license CD141+ DC and further prime B cells for antibody production. In this application, we will study and implement CD141+DC in combination with exogenously-delivered anti-tumor CD4Th1, in addition to anti-tumor antibodies, to reduce and subsequently eliminate the need for chemotherapy.