Methods of treating lemptomeningeal disease

Abstract

Disclosed are compositions and methods for the delivery of anti-cancer therapies in a manner to cross the blood brain barrier or across the blood-CSF barrier across the choroid plexus in the treatment of a cancer, metastasis, and / or cancer complication including, but not limited to Leptomeningeal disease (LMD).

Description

METHODS OF TREATING LEMPTOMENINGEAL DISEASEThis application claims the benefit of U.S. Provisional Application No. 63 / 466,877, filed on May 16, 2023, which is incorporated herein by reference in its entirety.I. BACKGROUND

[0001] Leptomeningeal disease (LMD) is an aggressive late-stage metastasis of the CNS, in which tumor cells access the CSF and infiltrate the surface of the brain and spinal cord. The most common cancers that cause LMD include those of the breast and lung as well as melanoma. LMD results in a number of neurologic symptoms and signs such as headaches, cranial nerve palsies, stiff neck, and radiculopathies. The prognosis for patients with LMD is generally very poor (average survival is measured in weeks) and is universally fatal. Treatment with surgery, radiation, and systemic chemotherapy is palliative. Systemic therapy for LMD may fail because of inadequate drug penetration into the CSF across an intact blood brain barrier or blood-CSF barrier across the choroid plexus. What are needed are new treatments and methods that can facilitate drug penetration into the CSF across an intact blood brain barrier or blood-CSF barrier across the choroid plexus.II. SUMMARY

[0002] Disclosed are methods related to the treatment of cancers, metastasis, and cancer complications using a Ommaya reservoir for the intrathecal delivery of an anti-cancer therapy.

[0003] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer (such as, for example, breast cancer including, but not limited to triple negative breast cancer), metastasis, and / or cancer associated complication (such as, for example leptomeningeal disease (LMD)) in a subject comprising administering the anti-cancer therapy comprising at least one dendritic cell pulsed with at least one HER-3 CD4+ T cell epitope such as, for example, an epitope from the extracellular domain (ECD) of HER-3 (such as, for example, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO 8, and / or SEQ ID NO: 9) or intracellular domain (ICD) of HER-3 (such as, for example, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, and / or SEQ ID NO: 11) and / or at least one HER-2 CD4+ T cell epitope (such as, for example, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and / or SEQ ID NO: 17); wherein the anti-cancer therapy is administered intrathecally through an Ommaya reservoir. Thus, in one aspect, the dendritic cell can be pulsed with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7.SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and / or SEQ ID NO: 17. In some aspects, the HER-3 CD4+ T cell epitope pulsed dendritic cell and / or HER-2 CD4+ T cell epitope pulsed dendritic cell is activated with IL- 12 prior to administration.

[0004] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer (such as, for example, breast cancer including, but not limited to triple negative breast cancer), metastasis, and / or cancer associated complication (such as, for example leptomeningeal disease (LMD)) of any preceding aspect, wherein the method further comprises pulsing dendritic cells with one or two additional HER- 2 / neu class I binding peptides, including, but not limited to SEQ ID NO: 18 and / or SEQ ID NO: 19.III. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] 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.

[0006] Figures 1A and IB show injection of circulating tumor cells into the cisterna magna in a murine xenograft model to study leptomeningeal disease and central nervous system metastases. Figure 1A show an illustration showing the location of the cisterna magna and the CSF accessing site, in which CTCs are injected using a Hamilton syringe. Figure IB shows a representative IVIS image of mice that had developed leptomeningeal disease and central nervous system metastases (brain and along the spinal cord) after 2 weeks of injection with circulating tumor cells. Cells were labeled with a luciferase reporter gene. Control animals injected with saline did not develop tumors (n = 3), and the experiment was performed in triplicate. Abbreviations: CTCs = circulating tumor cells; IVIS = in vivo imaging system.

[0007] Figures 2A, 2B, 2C, and 2D show the implantation of the Murine Ommaya device. Figure 2A shows an illustration with an arrow pointing at the location of the bregma on the skull, and the approximate distance at which a burr hole is drilled in the skull (0.5 mm posterior / 1.1 mm lateral) from the bregma using a microdrill. Figure 2B shows a Murine Ommaya is assembled by combining a metal cannula and a 1 mm spacer as the base for glue attachment to the skull. Figure 2C shows representative images of mice that had Murine Ommayas implanted; these mice are monitored to ensure they are bright, alert, and reactive before receiving any injections. Figure 2D shows an example of the prototype magneticresonance imaging-compatible Murine Ommaya and representative brain magnetic resonance imaging images of Murine Ommaya implants.

[0008] Figures 3A and 3B show intraventricular (central nervous system) injection using the Murine Ommaya. Figure 3A shows an image of an injection, accessing the ventricle and central nervous system via the Murine Ommaya. Mice remain under anesthesia during the injection. Tn the example, the Murine Ommaya is connected to the miniature port that is attached to a prefilled Hamilton syringe. Injections are performed using an automatic injection set at an infusion rate of 1 pL / min and a volume of 5-7 pL. An image of a mouse brain injected with Evans Blue is shown. The circle shows where the Murine Ommaya was attached. No leakage of the dye was observed on the exterior of the brain. A cross section of the brain shows the lateral ventricles were filled with the dye; the dye did not penetrate brain parenchyma. Figure 3B shows images of mouse brains after 15 and 30 min following the injection of Evans Blue dye. The dye infiltrated the brain (15 min) and began to circulate on the spinal cord (30 min). Out of 5 mice, 4 received dye for visualization, and 1 served as control. The experiment was repeated in triplicate.

[0009] Figures 4A and 4B show direct-targeted immunotherapy using the Murine Ommaya increases the overall survival of breast cancer-associated leptomeningeal disease mice. Figure 4A shows BALB / c mice were injected with luciferase reporter-labeled human epidermal growth factor receptor 2-positive TUBO cells, a murine breast cancer cell line. Three days after cisterna magna injections, Murine Ommayas were implanted. Mice began to develop leptomeningeal disease (LMD) 1 week after injection. LMD mice were treated with either a human epidermal growth factor receptor antibody systemically via intraperitoneal injection or via intrathecal (Murine Ommaya) as a direct-targeted approach. Injections were given once a week for up to 4 weeks. Compared to untreated mice, mice receiving immunotherapy survived much longer. Murine Ommaya mice had complete disease regression by the fourth week, and these mice were eventually cured of disease. Figure 4B shows these mice also had significantly better median survival (Mantel-Cox test; P = 0.004; n = 5 mice per treatment arm) and better overall survival than systematically treated LMD mice. Abbreviations: LMD = leptomeningeal disease; IP = intraperitoneal

[0010] Figures 5 A, 5B, 5C, and 5D show intrathecal (IT) cDCls therapy optimization strategy, and comparison of IT to systemic cDCl therapy showing IT method was superior in treating mice with breast cancer LMD.

[0011] Figures 6A, 6B, 6C, 6D, 6E, and 6F show intrathecal (IT) HER2- and HER3-pulsed cDCas therapy cured between 30% to 70% of HER2+ and triple negative breastcancer LMD mice, and IT cDCl-elicited proinflammatory response (i.e. INF-y, IL-6, IL-18) in mice cerebral spinal fluid.

[0012] Figure 7: Apheresis scheme to generate dendritic cell therapy for intrathecal treatment in LMD patients.

[0013] Figure 8 shows that breast cancer LMD patients receiving intrathecal (IT) cDCl therapy revealed high elevated proinflammatory cytokines (i.e. IFN-y, IL-6, IL- 12, TNF- a) in cerebral spinal fluid post-IT cDCl dosing.

[0014] Figures 9A and 9B show IT HER2- / HER3-DC immunotherapy eradicated HER2+ and TNBC-LMD and surviving mice became disease free. IT DC treatment prolongs survival and produces cures in murine HER2+ and TNBC-LMD as observed in TUBO-LMD (9A) and 4T1-LMD (9B) mice.

[0015] Figures 10A, 10B, and 10C show IT HER2- / HER3-DC immunotherapy eradicated HER2+ and TNBC-LMD and surviving mice became disease free. Figure 10A shows CD4+ and CD8+ T cells were increased in LMD mice treated with IT DC. Figure 10B shows that Immune activity is likely CD4+, but not CD8+, essential. Figure 10C shows that IT DCV is effective against LMD in BALB / c mice. BALB / c mice with Her2+ TUBO-LMD were given treatment: 1) HER2-DCV systemically, 2) HER2-DCV IT, or 2) HER2- and HER3-DCV IT (in alternation), once a week up to four weeks. All untreated mice succumbed to disease by week 4. Systemic therapy prolonged survival of LMD-mice but none survived by the end of study (Day 91). HER-DCV IT and HER2 / HER3-DCV IT treated mice performed significantly better with an overall survival of 44% and 78% respectively (N = 10 mice per group; Mantel- Cox test; P value < 0.0001). These mice remained bright, active, and alert.

[0016] Figure 11 shows Evidence of elevated IFN-g and IL18 in CSF in response to IT DC, an indication of compartmentalized antitumor effect in CSF tumor microenvironment (TME). High levels of IFN-y and IL- 18 were detected in CSF of treatment mice compared to normal and untreated controls, but not in serum.

[0017] Figures 12A and 12B show that IT HER2- / HER3-DC ’’cured” mice displayed protection (memory response) against TUBO-LMD recurrence.IV. DETAILED DESCRIPTION

[0018] 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 tobe understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.A. Definitions

[0019] 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.

[0020] 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 the 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.

[0021] 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:

[0022] “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.

[0023] 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 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.

[0024] 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.

[0025] '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.

[0026] 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.

[0027] 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.

[0028] 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 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.

[0029] 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.

[0030] 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.

[0031] "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.

[0032] "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 thecompositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0033] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0034] “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.

[0035] 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.

[0036] "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.

[0037] “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.

[0038] “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. 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.

[0039] “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.

[0040] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into thisapplication 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. Method of treating cancer

[0041] Oncodriver expression, such as HER3 overexpression in triple negative breast cancer (TNBC), has been shown to be involved in tumor progression. Previous attempts using peptide vaccines to boost CD8 immune responses have shown some efficacy but have not generated complete tumor regression. One possibility is that CD8s function sub-optimally without CD4s. Interestingly, there is a loss of the anti-HER3 CD4+ Thl immune response during breast tumorigenesis. Using the extracellular domain (ECD) of HER3 as a candidate "oncodriver" tumor antigen, immunogenic HER3 peptides that demonstrate class II promiscuity and generate anti-HER3 CD4 immunity were investigated for inclusion in dendritic cell (DC) vaccine development. However, systemic administration was unable to overcome to cross the blood brain barrier or blood-CSF barrier across the choroid plexus.

[0042] Administering cancer therapeutics (e.g., drugs and antibody -based treatments including checkpoint inhibitors and cellular therapies) directly into the CSF may overcome limitation for crossing the blood brain barrier or blood-CSF barrier across the choroid plexus. Accessing and sampling CSF from patients is possible through an Ommaya reservoir that is implanted beneath the scalp. This device allows for the administration of cancer agents (e.g., methotrexate and trastuzumab) as well as the sampling of CSF for diagnostic studies (e.g., the cytological diagnosis of LMD to monitor for responses to treatment) without performing a spinal tap.

[0043] As shown here the administration of an anti-cancer therapy (such as, for example, an anti-cancer therapy comprising at least one dendritic cell pulsed with at least one HER-3 CD4+ T cell epitope and / or at least one HER-2 CD4+ T cell epitope) can have significantly better effects on patient outcome than systemic delivery of the same agent. Peptide libraries of overlapping 15 amino- acid long peptide fragments were generated from the HER3 ECD and ICD (Table 1) and from HER2 (Table 2). Thus, it is understood and herein contemplated that the HER3 CD4+ T cell epitope can be generated from the ECD (such as, for example, SEQ ID NO: I, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 9) or the ICD (such as, for example, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 11) of HER3. In one aspect, the HER2+ T cell epitope canbe SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.Table 1: HER3 CD4+ T cell epitopesTTT^_ .T 1,r. _ . . . _ _ Homology withHER3 Ep ritopes Identified Amino Acid Sequencesw„ >7™^ Mouse HER3HER3 ECD P12 (aa 57-71) EVVMGNLEIVLTGHN (SEQ ID NO: 1) 100%HER3 ECD P81 (aa 401-415) SWPPHMHNFSVFSNL (SEQ ID NO: 2) 100%HER3 ECD P84 (aa 416-430) TTIGGRSLYNRGFSL (SEQ ID NO: 3) 100%HER3 ECD P91 (aa 450-464) SAGRIYISANRQLCY (SEQ ID NO: 4) 87%HER3 ICD P38 (aa 850-864) VADFGVADLLPPDDK (SEQ ID NO: 5) 100%HER3 ICD P41 (aa 865-879) QLLYSEAKTPIKWMA (SEQ ID NO: 6) 93%HER3 ICD P52 (aa 920-934) VPDLLEKGERLAQPQ (SEQ ID NO: 7) 93%HER ECD P12 (aa 56-70) CEVVMGNLEIVLTGH (SEQ ID NO: 8)HER3 ECD P91 (aa 451 -465) AGRIYIS ANRQLCYH (SEQ ID NO: 9)HER3 ICD P86 (aa 1090-1114) GCLASESSEGHVTGS (SEQ ID NO: 10)HER3 ICD P89 (aa 1115-1129) EAELQEKVSMCRSRS (SEQ ID NO: 11)Table 2: Her2 CD4+ T cell epitopesHER2 Epitopes Identified Amino Acid SequencesHER2 P42-56 HLDMLRHLYQGCQVV (SEQ ID NO: 12)HER2 P98-114 RLRIVRGTQLFEDNYAL (SEQ ID NO: 13)HER2 P328-345 TQRCEKCSKPCARVCYGL (SEQ ID NO: 14)HER2 P776-790 GVGSPYVSRLLGICL (SEQ ID NO: 15)HER2 P927-941 PAREIPDLLEKGERL (SEQ ID NO: 16)HER2 Pl 166-1180 TLERPKTLSPGKNGV (SEQ ID NO: 17)

[0044] Accordingly, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer (such as, for example, breast cancer including, but not limited to triple negative breast cancer), metastasis, and / or cancer associated complication (such as, for example leptomeningeal disease (LMD)) in a subject comprising administering the anti-cancer therapy comprising at least one dendritic cell pulsed with at least one HER-3 CD4+ T cell epitope such as, for example, an epitope from the extracellular domain (ECD) of HER-3 (such as, for example, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO 8, and / or SEQ ID NO: 9) or intracellular domain (ICD) of HER-3 (such as, for example, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, and / or SEQ ID NO: 11) and / or at least one HER-2 CD4+ T cell epitope (such as, for example, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and / or SEQ ID NO: 17); wherein the anti-cancer therapy is administered intrathecally through an Ommaya reservoir. Thus, in one aspect, the dendritic cell can be pulsed with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7. SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and / or SEQ ID NO: 17. In some aspects, the HER-3 CD4+ T cell epitope pulsed dendritic cell and / or HER-2 CD4+ T cell epitope pulsed dendritic cell is activated with IL- 12 prior to administration.

[0045] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer (such as, for example, breast cancer including, but not limited to triple negative breast cancer), metastasis, and / or cancer associated complication (such as, for example leptomeningeal disease (LMD)), wherein the method further comprises pulsing dendritic cells with one or two additional HER-2 / neu class I binding peptides, including, but not limited to SEQ ID NO: 18 and / or SEQ ID NO: 19.

[0046] 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 cellcarcinomas 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. In one aspect, the method can be used for the treatment of a cancer associated complication including, but not limited to leptomeningeal disease.

[0047] 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™ 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-AB V, 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 ETOPOPHOS® (Etoposide Phosphate), Etoposide, Etoposide Phosphate, EV ACET® (Doxorubicin 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, FOLFIRLBEVACIZUMAB, 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, GEMCITABINEOXALIPLATIN, 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, IB RANCE® (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 1 131 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 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, 0N1VYDE® (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® (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 SYNRIBO® (Omacetaxine Mepesuccinate), TABLOID® (Thioguanine), TAC, TAFINLAR® (Dabrafenib), TAGRISSO® (Osimertinib), Talc, Talimogene Laherparepvec,Tamoxifen Citrate, TARAB1NE 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, TORTSEL® (Temsirolimus), Tositumomab and Iodine T 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), VelP, VELBAN® (Vinblastine Sulfate), VELCADE® (Bortezomib), VELSAR® (Vinblastine Sulfate), Vemurafenib, VENCLEXTA® (Venetoclax), Venetoclax, VERZENIO® (Abemaciclib), VIADUR® (Leuprolide Acetate), VID AZA® (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, cemiplimab , 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, omburtamab), B7-H4, B7-H3, T cell immunoreceptor with Ig and ITEM 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).

[0048] 'Concurrent administration", "administration in combination", "simultaneous administration" or "administered simultaneously" as used herein, 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.

[0049] “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.

[0050] By contrast, “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. For example, 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.

[0051] It is understood and herein contemplated that while a single administration of the components of the disclosed anti-cancer combination therapies (i.e., the pulsed dendritic cells and / or the immunoregulator molecule inhibitor) would be ideal, not every patient will respond in the same manner. Thus, in one aspect, disclosed herein are 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. 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 anti-cancer agent 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. It is further understood and herein contemplated that the order and duration of the administered components can vary as appropriate for the subject being treated. In one aspect, disclosed herein are 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 anti-cancer agent; are administered concurrently with the at least one anti-cancer agent; or wherein the at least one anti-cancer agent 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.C. Compositions

[0052] 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 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 immunoregulator molecule inhibitor or oncodriver pulsed dendritic cell is disclosed and discussed and a number of modifications that can be made to a number of molecules including the immunoregulator molecule inhibitor or oncodriver pulsed dendritic cell are discussed, specifically contemplated is each and every combination and permutation of immunoregulator molecule inhibitor or oncodriver pulsed dendritic cell 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 subgroup 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 itis understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.1. Peptides a) Protein variants

[0053] As discussed herein there are numerous variants of the HER3 ECD and ICD peptide epitopes in Table 1 and HER2 peptide epitopes in Table 2 that are known and herein contemplated. Protein variants and derivatives are well understood to those of skill in the art and in can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and / or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Immunogenic fusion protein derivatives, such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 3 and 4 and are referred to as conservative substitutions.TABLE 3:Amino Acid Abbreviations_ Amino Acid _ Abbreviations Alanine Ala A allosoleucine AlleArginine Arg R asparagine Asn N aspartic acid Asp DCysteine Cys C glutamic acid Glu EGlutamine Gin QGlycine Gly GHistidine His HIsolelucine He ILeucine Leu LLysine Lys K phenylalanine Phe F proline Pro P pyroglutamic acid pGluSerine Ser SThreonine Thr TTyrosine Tyr YTryptophan Trp WValine Vai VTABLE 4:Amino Acid SubstitutionsOriginal Residue Exemplary Conservative Substitutions, others are known in the art.Ala SerArg Lys; GinAsn Gin; HisAsp GluCys SerGin Asn, LysGlu AspGly ProHis Asn;Gln lie Leu; VaiLeu He; VaiLys Arg; GinMet Leu; liePhe Met; Leu; TyrSer ThrThr SerTrp TyrTyr Trp; PheVai He; Leu

[0054] Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 4, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) acysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation and / or glycosylation.

[0055] For example, the replacement of one amino acid residue with another that is biologically and / or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as, for example, Gly, Ala; Vai, lie, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.

[0056] Substitutional or deletional mutagenesis can be employed to insert sites for N-glycosylation (Asn-X-Thr / Ser) or O-glycosylation (Ser or Thr). Deletions of cysteine or other labile residues also may be desirable. Deletions or substitutions of potential proteolysis sites, e.g. Arg, is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.

[0057] Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post- translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86

[1983] ), acetylation of the N- terminal amine and, in some instances, amidation of the C-terminal carboxyl.

[0058] It is understood that one way to define the variants and derivatives of the disclosed proteins herein is through defining the variants and derivatives in terms of homology / identity to specific known sequences. Specifically disclosed are variants of these and other proteins herein disclosed which have at least, 70% or 75% or 80% or 85% or 90% or 95% homology to the stated sequence. Those of skill in the art readily understand how to determine the homology of two proteins. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

[0059] Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

[0060] The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989.

[0061] It is understood that the description of conservative mutations and homology can be combined together in any combination, such as embodiments that have at least 70% homology to a particular sequence wherein the variants are conservative mutations.

[0062] As this specification discusses various proteins and protein sequences it is understood that the nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence.

[0063] It is understood that there are numerous amino acid and peptide analogs which can be incorporated into the disclosed compositions. For example, there are numerous D amino acids or amino acids which have a different functional substituent then the amino acids shown in Table 3 and Table 4. The opposite stereo isomers of naturally occurring peptides are disclosed, as well as the stereo isomers of peptide analogs. These amino acids can readily be incorporated into polypeptide chains by charging tRNA molecules with the amino acid of choice and engineering genetic constructs that utilize, for example, amber codons, to insert the analog amino acid into a peptide chain in a site specific way.

[0064] Molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage. For example, linkages for amino acids or amino acid analogs can include CH2NH-, — CH2S— , — CH2— CH2 — , — CH=CH— (cis and trans), — COCH2 - -, — CH(OH)CH2— , and — CHH2SO — (These and others can be found in Spatola, A. F. inChemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp. 463- 468; Hudson, D. et al., Int J Pept Prot Res 14:177-185 (1979) (— CH2NH— , CH2CH2-); Spatola et al. Life Sci 38: 1243-1249 (1986) (—CH H2— S); Hann J. Chem. Soc Perkin Trans. I 307-314 (1982) (— CH-CH— , cis and trans); Almquist et al. J. Med. Chem. 23:1392-1398 (1980) (— COCH2-); Jennings-White et al. Tetrahedron Lett 23:2533 (1982) (-COCH2-); Szelke et al. European Appln, EP 45665 CA (1982): 97:39405 (1982) (-CH(OH)CH2-); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (-C(OH)CH2-); and Hruby Life Sci 31:189-199 (1982) (— CH2-S-); each of which is incorporated herein by reference. A particularly preferred nonpeptide linkage is — CH2NH— . It is understood that peptide analogs can have more than one atom between the bond atoms, such as b-alanine, g- aminobutyric acid, and the like.

[0065] Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad- spectrum of biological activities), reduced antigenicity, and others.

[0066] D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used to generate more stable peptides. Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations.D. Examples

[0067] 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: A Murine Ommaya Xenograft Model to Study Direct- Targeted Therapy of Leptomeningeal Disease

[0068] A murine Ommaya reservoir was designed to mimic those used clinically. The reservoir requires the assembly of an accessing port and spacer parts and a modification ofthe mouse cannulation technique, which allows the device to remain permanently intact throughout the duration of the drug study. This device has been designated as the "Murine Ommaya".

[0069] In contrast to the osmotic infusion pump technique, which requires the preparation of excess liquid volumes to prefill the empty space in the tubing and continuous infusion over frequent injections , the Murine Ommaya minimizes the wastage of drug solutions. It permits effective administration of multiple single doses of treatments at any given time in small quantities (3-7 pL) into the CSF using a Hamilton syringe, a miniature access port, and an automatic injector. In real time, the efficacy of test drugs against LMD can be determined by imaging. Using this approach, a variety of chemotherapies, antibodies, and cell immunotherapies (as single or combined agents) can be tested against LMD to translate in vivo findings into rational treatment strategies for patients. To further improve the imaging capacity for a patient derived xenograft (PDX) model of LMD, a collaboration was undertaken with a manufacturer to develop a magnetic resonance imaging (MRI)-compatible version of the Murine Ommaya, which requires no assembly and is ready to use. MRI capability is beneficial, especially for PDX models in which the quantity of circulating tumor cells (CTCs) from CSF is sometimes the limiting factor, and often when prelabeling CTCs is infeasible.

[0070] In a detailed protocol that begins with the injection of CTCs to render mice with LMD. The Murine Ommaya is then surgically implanted, and multiple drug treatment steps via the Murine Ommaya are performed. As a proof of concept for demonstration, an in vivo side-by-side comparison was performed, in which the murine human epidermal growth factor receptor 2 (Her2) antibody called clone 7.16.4 (the human equivalent of trastuzumab) was delivered . The antibody targets Her2+ breast cancer cells either via the Murine Ommaya (direct-targeted or intrathecal therapy) or by intraperitoneal injection (systemic therapy).

[0071] The results showed that mice with LMD that received direct intrathecal immunotherapy lived significantly longer than those treated with the same therapy systemically. The CNS metastases in mice treated via the Murine Ommaya wereregressed by the third dose of the third week of treatment, resulting in improved overall survival.a) Protocol(1) Injection of CTCs into CSF to generate a mouse LMD model(a) Preparation of CTCs

[0072] Calculate the number of CTCs needed for injection, and prepare a singlecell suspension at 1.0 x 104 cells / pL in sterile phosphate-buffered saline (PBS). Place the cell suspension on ice or at 4 °C throughout the procedure.(b) Presurgical procedure1. Inject the mouse subcutaneously with 5 mg / kg buprenorphine sustained-release (Bup-SR).2. Anesthetize the mouse with 2-3% isoflurane until it shows no signs of the righting reflex. In addition, check for tail and / or paw pinch reflex to confirm the state of anesthesia.3. Prepare the mouse for surgery in a location remote from the operating area. Clip the surgical site (i.e., the dorsal surface of the skull) with enough border area to keep fur from contaminating the incision site; then, saturate the site with germicidal skin antiseptic, working from the center of the site to the periphery, and then allow to dry. Apply either sterile drape or a sterile adhesive-backed plastic drape material to protect the surgical site from contamination.4. Shave the fur of the entire ventral surface of the head, and prepare the skin using sterile technique.5. Situate the nose with a modified L-shaped nose cone of the stereotactic apparatus, ensuring the nares remain clear and open. Using tape across the ventral surfaces of both pinnae, gently pull the skin forward to secure it to the nose cone, and bend the neck at an approximately 90° angle once secured. Administer 1.5% isoflurane to maintain anesthesia.6. Position the body to ensure the spine is kept level with the cistema magna, and while applying slight traction to the tail, place tape at the tail base to secure.(c) Surgical cisterna magna injection1. With the neck in full extension, and beginning just between the pinnae, run the surgical scissor tips downward with slight pressure across the occipital bone.2. Make a small 3-5 mm midline incision just above the palpated concavity. Draw 5 pL of cell suspension at 1.0 x 104cells / pL (total 5.0 x 104cells) into a 30 G Hamilton syringe.3. Use blunt-tipped forceps with 1-2 mm tips to gently press down on the cisterna magna. Introduce tips in a closed position and open them while applying downward pressure on the dura.4. Repeat blunt dissection described in the previous step until the dural membrane is easily identified, and the associated blood vessels are visible in the exposed area.5. While holding the forceps open to retract surrounding musculature, introduce a 30 G non- coring needle under the dura to visualize the bevel. Ensure the needle is only introduced just beyond the bevel itself. Slowly deploy a syringe plunger, and deliver cells just below the dura.6. Properly place the bevel to observe the injection beneath the dura. Carefully administer the technique, and use the 30 G non-coring needle to prevent damage to the membrane and ensure minimal leakage. If leakage is noted, apply gentle pressure with a cotton-tipped applicator.7. Close the skin by applying a wound clip or micro-drop of skin adhesive. Once the injection is successfully performed, allow the mice to recover from anesthesia on a warm blanket, observing them continuously until they can maintain sternal position and demonstrate purposeful movement.8. Monitor the mice daily after the surgery for the first week. If a mouse appears to be in pain or distress, treat it with 10 mg / kg subcutaneous injection of carprofen once every 12 to 24 h for up to 5 days based on veterinary consultation and directive. Allow mice to recover and monitor them for at least 48 to 72 h before proceeding to the next step.

[0073] Bup-SR, given preemptively, will last up to 72 h, so additional analgesics are not normally necessary. However, animals will be provided additional analgesic as necessary (if lethargic, not eating, ruffled). If a surgical site develops signs of postoperative infection (i.e., redness, swelling, tenderness, allodynia, hyperalgesia, hyperpathia, or suppuration), or if the mouse is guarding the affected area, euthanize the mouse. If cancer cells are successfully injected into the CSF, LMD and tumor progression will develop in the CNS within 1 or 2 weeks (depending on the types of CTCs or cell line used) (Figure 1).(2) Murine Ommaya assembly and implantation(a) Preparation of stationDisinfect the station surface. Place a blue covering over the surface, and keep ready all sterilized tools and supplies.(b) Presurgical rocedureApply analgesia (Bup-SR), and maintain sterile technique as described herein.(c) Surgical implantation of Murine Ommaya1. Assemble the Murine Ommaya injection device using a 25 G (0.51 mm outer diameter) miniature injection port and a 1 mm spacer disc. Use a cyanoacrylate sterile adhesive to ensure the penetration of approximately 2.5 mm of the metal cannula into the right cerebral hemisphere (Figure 2A-C).An MRI-compatible Mouse Ommaya prototype was developed in this laboratory, which already has both parts (miniature injection port and spacer) 3D-printed together as a single unit (Figure 2D). The single unit version was tested by MRI and can save time by eliminating the assembly step.2. Anesthetize the mouse with 2-3% isoflurane until there are no signs of the righting reflex. Further, check for tail and / or paw pinch reflex to confirm the state of anesthesia.3. Shave the entire ventral surface of the head of fur, and prepare the skin according to the sterile technique, as previously described in step 1.2.3. Place the mouse in the stereotactic apparatus with a nose cone to continue isoflurane administration during the procedure; decrease the isoflurane to 1.5%. Gently tighten the ear bars to secure the head, and apply eye lubricant to cover the eyes of the mouse.4. Make a small skin incision (3 mm), followed by blunt dissection of the underlying subcutaneous tissues to expose the skull. Dry the skull using hydrogen peroxide-soaked cotton-tipped applicator sticks.5. Drill a burr hole in the skull 0.5 mm posterior and 5.1 mm lateral of the bregma- the anatomical point on the skull where the coronal suture is intersected perpendicularly by the sagittal suture (Figure 2A)- as well as a 0.9 mm burr hole to expose the dura mater. Move the microdrill aside, and gently score the bone immediately surrounding the burr hole prior to inserting an injection port (depth of approximately 2.5 mm), affixed to the skull using a cyanoacrylate sterile adhesive. State suture around the injection spotusing 4-0 no-absorbance nylon sutures in an interrupted stitch pattern or purse string suture.6. House post-surgery mice in individual cages for surgery recovery.(3) Murine Ommaya treatment(a) Dosing mice using the Murine Ommaya1. Anesthetize the mouse with 2-3% isoflurane, until there are no signs of righting reflex. Further, check for tail and / or paw pinch reflex to confirm maintenance of anesthesia.2. Access the Murine Ommaya using a port injection adapter and a Hamilton syringe. Using forceps, hold the top of the miniature injection port and gently insert the port injector adaptor fully into the port's septum.

[0074] The port injector pierces the septum of the port in a manner that reduces dead space to a minimum and allows for controlled injection via a motorized syringe pump (Figure 3A). Targeted / novel treatments are injected at a flow rate of 1 pL / min while under anesthesia. The treatment is to be given at specified intervals (daily / weekly) for a set duration (weeks / months), according to the researcher's discretion.

[0075] A volume between 3 and 7 p L is optimal, as a volume < 3 pL is unreliable, and a volume > 7 pL may cause too much pressure. The size of the Hamilton syringe can range from 10 to 100 pL, depending on the number of mice in each treatment arm. Preloading the appropriate volume into the Hamilton syringe will prevent repeated replacement of the syringe, thereby minimizing errors. It is best to dedicate 1 Hamilton syringe per treatment arm.3. Once the injection is made, detach the Murine Ommaya from the port injection adaptor using forceps, and return the mouse to the cage to recover from anesthesia, as described herein.(b) Euthanasia

[0076] Euthanize the mice by inhalation of carbon dioxide (CO2) from a compressed tank source. Expose the mice to increasing concentrations of CO2 (i.e., a displacement rate from 10% to 30% of the chamber volume / min is to be used) to avoid or minimize discomfort or distress. Monitor the mice until assurance of cessation of cardiovascular and respiratory movements.b) Results:

[0077] In mice, the total volume of CSF is approximately 35-40 L and is produced at a rate of about 350 nL / min; it turns over 12-13 times a day. For the purpose of visualizing the route of the injection, 2% Evans Blue was injected via the Murine Ommaya model, following which 15 min and 30 min were allowed to elapse before harvesting the brains for analysis. The dye successfully infiltrated the ventricles and the brain in 15 min. Within 30 min, the dye became visible on the spinal cord (Figure 3).

[0078] As a proof of concept, BALB / c mice were injected with a luciferase- labeled Her2+ TUBO breast cancer cell line intracistemally, and the Murine Ommayas were implanted. Approximately 1 week after the injection of cancer cells, the mice began to develop LMD. These mice were treated once a week for up to 4 weeks with Her2-antibody immunotherapy, either through systemic therapy via intraperitoneal injection or intrathecally via the Murine Ommaya (Figure 4A).

[0079] Although untreated mice died by day 19, all mice that received intrathecal therapy through the Murine Ommaya survived (P = 0.004). By week 4, a complete regression of tumors was observed. In comparison to mice treated with systemic therapy, which had moderate success in treating LMD, mice that received intrathecal therapy had a much longer overall survival (Figure 4B, Figure 6) and revealed high elevated proinflammatory cytokines (i.e. IFN-g, IL-6, IL-12, TNF-a) in cerebral spinal fluid post-IT cDCl dosing (Figure 8).

[0080] We also included a cohort given a HER2 peptide-pulsed DCV and HER3 peptide-pulsed DCV IT regimen in our study. We found that the median survival of untreated LMD-mice was 10 days, and mice given systemic HER2-DCV therapy was 19 days. Neither of these cohorts survived. LMD-mice that received IT therapy performed significantly better than those that received systemic therapy (P value < 0.0001). The median survival of the Her2-DCV IT cohort was 63 days, with an overall survival of 44%. We observed that IT HER2- / HER3-DC immunotherapy eradicated HER2+ and TNBC-LMD and surviving mice became disease free. IT DC treatment prolongs survival and produces cures in murine HER2+ and TNBC-LMD. We also observed a complete tumor regression in some mice following 4 doses of vaccine treatments. Interestingly, the HER2-DCV / HER3-DCV IT cohort had the best overall survival of 78%. (Figure 10). In addition, we observed that increasing the number of DCls (from 7.0 x 104to 1.0 x 10scells) per injection IT or increasing the number of doses did not further impacted the survival of LMD mice. Upon sampling mice CSF that received IT DCV therapies, we found an elevation of IFN-y and IL18 through Thl cytokine screening, indicating IT DCV could elicit an immune response in CSF (Figure 11). All of the surviving mice at the end of the trial (91days) remained disease free and healthy. Therefore, in this setting, using DC Is pulsed with both HER2 and HER3 peptides and delivered IT prolongs survival in this immunocompetent animal model of LMD. Furthermore, results from functional analysis (Figure 12) showed peripheral lymphoid organs from disease- free mice responded to HER2-peptide by producing an elevated level of IFN-y. This suggests an adaptive immune response in our cured animals. c) Discussion:

[0081] The Murine Ommaya is a reliable model, which allows for repeated administration of anti- cancer agents into the CSF space in preclinical models of LMD and other CNS-related diseases. CSF was sampled from mice while the device was still attached without interruption. This direct-targeted therapy xenograft model is an important step in developing and testing rational treatment strategies for LMD. The time from cistema magna injections to the initial sign of LMD development varies depending on the cancer cell type. LMD and CNS metastases begin to take form around 1 or 2 weeks after CTC inoculation. If a highly proliferative cancer cell line is used, it is possible that metastasis can occur in <7 days. In this case, vascularization due to tumor growth can sometimes makes implantation of the Murine Ommaya challenging. One solution to this challenge is to reduce the number of cancer cells for injection into the CSF space, allowing for more time before tumor development. Furthermore, this protocol has been optimized to implant the Murine Ommaya no later than 72 h after the cistema magna injection to ensure enough time for mice to recover from surgery before the first treatment. Researchers should calculate the growth rate of the CTCs in xenograft models before planning the treatment regimen.

[0082] Although there are other direct intraventricular delivery methods, such as using an osmotic pump system or an intracerebroventricular (ICV) bolus injection, as described previously9, there are several advantages to using the Murine Ommaya model. For example, an ICV bolus injection is done in a single delivery, whereas the Murine Ommaya allows for multiple doses of treatment at any time, whether given as a single agent or as combined therapies. The osmotic pump is designed to sustain for up to 14, 28, or 42 days before the pump needs replacement, and sometimes more frequently if a smaller mouse with a smaller pump is used. Changing the osmotic pump requires a surgical procedure, which adds stress to tumorbearing mice. A Murine Ommaya replacement is not necessary for long-duration experiments as long as the device remains intact. It also minimizes potential variability that results from changing the pump9. Implanted Murine Ommayas in the experimental mice remained intact for longer than 42 days, and this duration allowed for long-lived treatment regimens.

[0083] Previous findings suggest that a pulsatile intermittent dosing into the CSF has better efficacy against LMD than a prolonged drug delivery process by infusion13. It would be impossible to perform repeated single-dose injections using the osmotic pump system. There is no easy way to flush out the remaining trapped liquid after each injection. The osmotic pump is also limited to delivering mixtures of compatible or single drugs and typically requires higher volumes of drug preparation for continuous infusion. Tn contrast, the Murine Ommaya is designed for accurate micro-injections as little as 3 to 7 pL, without having to account for dead space, and there is no limitation on the type of drugs researchers can use, including immune cell therapy. The Murine Ommaya also minimizes reagent waste if a particular sample is precious and maximizes the use of that resource. For any treatment regimen that requires multiple doses of anti-cancer therapies, the Murine Ommaya is easy to use, and there is minimal risk of infection or surgical misadventure, with the alternative approaches of repeatedly accessing the CSF surgically or via repeated delivery with a needle. The Murine Ommaya provides researchers with the flexibility to adjust drug concentrations and dosing frequencies and to assess target modulation and duration of the study according to the research of interest.

[0084] As the Murine Ommaya can be difficult to implant in smaller mice, it is better to use mice that are at least 8 to 10 weeks of age. It is possible for the Murine Ommaya to come off during the treatment trial if the device is not secured to the skull during the implantation steps and the glue wears off, or if the mice have abrasively tampered with it. The latter scenario occurs more frequently when multiple mice were housed in the same cage. Hence, it is recommended to house no more than two Murine Ommaya-implanted mice per cage for the duration of the treatment schedule. This protocol was modified to apply cyanoacrylate sterile adhesive on the spacer, which was found to be the most effective glue for adhering the spacer to the skull surface and preventing the Murine Ommaya from coming off. The results showed that LMD mice benefited from direct intrathecal therapy via the Murine Ommaya, with increased overall survival. Single microliter volumes could be safely administered, bypassing the BBB, thereby reducing the amount of drug preparation. Most importantly, the mice that were cured of CNS metastases from the Her2-antibody immunotherapy study have remained healthy.

[0085] A collaboration with a manufacturer aimed to develop an MRI-compatible version of the Murine Ommaya for the PDX model of LMD. Because this prototype version has a spacer incorporated, no assembly is required, allowing for better adherence to the skull. A limitation of this prototype is that although the device is MRI-compatible, it generates a shadow where the device is inserted, which decreases the visibility of the image for quantificationanalyses. The MRI- compatible version is a good alternative tool when ex vivo CTC sampling is a limiting factor, and prelabeling cells is not feasible. The combination of an LMD xenograft model and the Murine Ommaya technique is beneficial for studying direct- targeted drug efficacy bypassing the BBB. The results from these in vivo studies are clinically relevant to design rational therapeutic strategies for patients with LMD.2. Example 2:

[0086] A First in Human dose escalation of Dendritic Cell Vaccine (DCV) administered Intrathecally (IT) primed against HER2 / HER3 in Patients with Leptomeningeal Disease (LMD) from Triple-Negative Breast Cancer (TNBC) or HER2+ Breast Cancer (HER2+BC) a) Preparation of the Vaccine

[0087] Preparation of the vaccines are a multi-step process starting with leukapheresis and countercurrent centrifugal elutriation to obtain the relevant monocyte pool. This monocyte pool is treated with GM-CSF and IL-4. The culture is then divided into two separate cultures: one filtered and pulsed with HER-2 / neu peptides and the second with HER-3 peptides. Both cultures are matured with IFN-y, and LPS for conversion and harvested separately (Figure 7). b) Subject Leukapheresis

[0088] Subjectsfirst undergo leukapheresis to obtain lymphocyte and monocyte fractions. The leukapheresis is performed in the Apheresis facility on the Spectra Optia (Terumo BCT, Colorado), using the CMNC platform. Subjects undergo leukapheresis with a target processing volume of 20-24 L to optimize the TNC yield. The target TNC yield is 1-5 x 1010.. At the completion of leukapheresis, all IV access is removed. For subjects in whom leukapheresis did not yield adequate cells to make the required number of vaccines, additional leukaphereses is undertaken.

[0089] To separate the lymphocyte and monocyte fractions, the pheresis pool obtained is then be subject to countercurrent centrifugal elutriation on a Model J-6M centrifuge (Beckman Instruments) equipped with a JE-5.0 elutriation rotor operating at 1725g and 20°C or equivalent apparatus. The cells are loaded onto the elutriator at 120cc / minute flow rate, then fractions are collected using stepwise increases in flow rates to obtain lymphocyte-rich (120 and 140 cc / minute), transitional (150 cc / minute) and monocyte-rich (180 and 190 cc / minute) fractions. A final fraction is collected at zero rpm (rotor off).

[0090] The transitional (150 cc / minute) and monocyte-rich (180 and 190 cc / minute) fractions are pooled and cultured to produce sufficient number of DC1 cells for 10-12 vaccinations. Any remaining monocyte population as well as the lymphocyte fraction is reserved for research when possible. c) Cell Culture and Preparation of Dendritic Cell Vaccine

[0091] All work in the production of the monocytes is performed following strict guidelines for sterility anddone with Good Laboratory Practices (GLP). The vaccine activationtakes place in the Cell Therapy Facility (CTF) at the Moffitt Cancer Center under Good Manufacturing Practices (GMP). The CTF is accredited by the Foundation for the Accreditation of Cellular Therapy (FACT). The Moffitt Cancer Center Cell Therapies Coreoversees the vaccine manufacturing.

[0092] Vaccines are manufactured from both fresh (preferred) or cryopreserved monocytes. Monocytes are washed 1-2 times in Hanks Buffer (HBSS) post elutriation. Monocytes are cultured with GM-CSF, IL-4, IFN-y, and LPS for conversion. Monocytes at concentrations of 3x106cells / ml are cultured in serum free medium (CellGenix Macrophage SFM medium or equivalent) with 50 ng / ml (250 lU / ml) rh GM-CSF (preferred vendor: Sanofi), and 1000 lU / ml IL-4 (preferred vendor: Cellgenix). The cells aredivided into two groups: HER- 2 / neu vaccine and HER-3 vaccine. Ten - sixteen hours after setup the cultures are filtered and pulsed with either HER-2 / neu peptide sequences or HER-3 peptide sequences at a final concentration of 20 pg / ml. Each peptide sequence is filtered and then pulsed in separate wells or flask to avoid competitive inhibition. The recombinant HER- 2 / neu peptides sequences are as follows: 3 extracellular domain (ECD) p42-56 (HLDMLRHLYQGCQVV) (SEQ NO: 12), p98- 114 (RLRIVRGTQLFEDNYAL) (SEQ ID NO: 13), p328- 345(TQRCEKCSKPCARVCYGL) (SEQ ID NO: 14) and 3 intracellular domain (ICD) p776-790 (GVGSPYVSRLLGICL)(SEQ ID NO: 15), p927-941 (PAREIPDLLEKGERL)(SEQ ID NO: 16), and pll66-1180 (TLERPKTLSPGKNGV)(SEQ ID NO: 17). The recombinant HER-3 peptide sequences are as follows: 4 ECD P12 (aa 56-70, CEVVMGNLEIVLTGH)(SEQ ID NO: 8), P81 (aa 401-415, SWPPHMHNFSVFSNL)(SEQ ID NO: 2), P84 (aa 416-430, TTIGGRSLYNRGFSL)(SEQ ID NO: 3), P91 (aa 451-465, AGRIYISANRQLCYH)(SEQ ID NO: 9) and 5 ICD P38 (aa 850- 864, VADFGVADLLPPDDK)(SEQ ID NO: 5), P41 (aa 865-879, QLLYSEAKTPIKWMA) (SEQ ID NO: 6), P52 (aa 920-934, VPDLLEKGERLAQPQ) (SEQ ID NO: 7), P86 (aa 1090- 1114, GCLASESSEGHVTGS) (SEQ ID NO: 10), P89 (aa 1115-1129, EAELQEKVSMCRSRS) (SEQ ID NO: 11). Six - eight hours post peptide pulse IFN-y (preferred vendor: R&D Scientific) at a final concentration of 1000 u / ml is added. Six - eighteen hours later LPS (preferred vendor: US Pharmacopeia) at a final concentration of lOOu / mL is added and the cells are cultured an additional two to eight hours. For the HER2 / neuvaccine who are HLA-A2+ subjects, 50ug / ml of two additional HER-2 / neu class 1 binding peptides p369-377 (KIFGSEAFL)(SEQ ID NO: 18) and p689-697 (SEQ NO: X) (RLLQETELV) )(SEQ ID NO: 19) are filtered and pulsed into separate, previously un-pulsed wells one to two hours prior to harvest. For the HER2 / neu vaccine HLA-A3+ subjects, only peptides p369-377 (KIFGSEAFL) )(SEQ ID NO: 18) are filtered and pulsed into separate, previously un-pulsed wells one to two hour prior to harvest. In the event that HLA-A tissue typing results are not completed in time for the HLA-A2 peptide addition the HER2 / neu vaccine, the peptides will be added to the culture under the principle that if said patient is not HLA-A2+ or HLA-A3+ the peptides will not be able to bind properly and be washed away during the harvest of the vaccine.

[0093] The cells are then harvested from the plates or flask, maintaining HER2 / neu culture separate from the HER- 3 culture. Each culture is washed one-two times with Hanks Buffer (HBSS). Samples are tested for cell count and viability. A sample for FACS analysis is removed from the DS but not tested prior to release.

[0094] Cells are centrifuged and the pellet resuspended at 400,000 to 50 million cells per mL of cryopreservation media (45% Plasmalyte, 5% HSA, 50% CS10) for cryopreservation per Moffitt SOP with a target of at least 6 final vaccines (HER-2 DCV DP and HER-3 DCV DP) for administration. Cell concentration is based on the total cells needed per vaccine dose. Vials are frozen in a controlled rate freezer and stored in monitored LN2 tanks until shipment or administration in compliance with Moffitt SOPs. For QC testing, which includes endotoxin, gram stain, and sterility testing, samples from HER-2 DCV DP and HER-3 DCV DP are used.

[0095] Samples from the clinical product are submitted to the Cell Therapy Facility Quality Control lab for quality control testing. Cell viability of the pre-cryopreserved vaccine is determined using trypan blue staining or equivalent. Cell viability of greater than 70% is required for release of vaccine. Additionally, a negative gram stain of the vaccine sample is required as well as an endotoxin level less than 0.2EU / kg in order to clear the vaccine for patient administration. The endotoxin test is performed according to Good Laboratory Practices. BacT Alert blood culture bottles are inoculated with a vaccine sample and sent to the Department of Microbiology for sterility testing. The results of endotoxin testing and microbiology cultures are checked when available and if not meeting specifications the vaccine is discarded. Sterility testing is performed in accordance with CFR 610.12 and a specification of no growth is required. A final quality analysis certificate is issued for each dose of vaccine administered. In addition, a sample of each vaccine lot manufactured is also removed for FACSanalysis of cell surface markers to confirm conversion of monocytes to DC. Since the activity of the vaccine does not depend on phenotype the vaccine will not be held for phenotype but will serve as an internal evaluation of the process of vaccine preparation.The requirements for vaccine release are:

[0096] If the culture test is not available until after vaccine administration the following contingency plan is in place for a positive culture:• Notification of both the primary attending physician and the patient.• Identification of the responsible microorganism and sensitivity testing.• A description of additional patient monitoring as required by the circumstances of the contamination incident.• Initiate an investigation of the event and take corrective actions to prevent future recurrences. The scope of the investigation and proposed corrections should include: o Evaluation of current procedures for collecting and processing the autologous peripheral blood product to determine the step at which contamination could be introduced. o Development of changes in the procedures that will assist in preventing sterility lapses in the future. o Development of appropriate testing to ensure that revised changes to the procedures produce a sterile product. This should include sterility testing at various points in the preparation of the product.• Report the incident as an adverse event within 15 working days.

[0097] The vaccines are made from freshly collected, elutriated cells and cryopreserved until administration preparation. Vaccinations are made from cryopreserved aliquots of the elutriated cells, if required.E. ReferencesChamberlain, M. C. Leptomeningeal metastasis. Current Opinion in Neurology. 22 (6), 665- 674 (2009).Davies, M. A. et al. Prognostic factors for survival in melanoma patients with brain metastases. Cancer. 117 (8), 1687-1696 (2011).DeVos, S. L., Miller, T. M. Direct intraventricular delivery of drugs to the rodent central nervous system. Journal of Visualized Experiments. 75, e50326 (2013).Dunn, L. et al. Murine model of wound healing. Journal of Visualized Experiments. 75, e50265 (2013).Glitza, I. C. et al. Leptomeningeal disease in melanoma patients: An update to treatment, challenges, and future directions. Pigment Cell & Melanoma Research. 33 (4), 527-541 (2020).Kodumudi, K. N. et al. Sequential anti-PDl therapy following dendritic cell vaccination improves survival in a HER2 mammary carcinoma model and identifies a critical role for CD4 T cells in mediating the response. Frontiers in Immunology. 10, 1939 (2019).Nayar, G. et al. Leptomeningeal disease: current diagnostic and therapeutic strategies. Oncotarget. 8 (42), 73312-73328 (2017).Raizer, J. J. et al. Brain and leptomeningeal metastases from cutaneous melanoma: survival outcomes based on clinical features. Neuro-oncology. 10 (2), 199-207 (2008).Shackleford, G. M. et al. Continuous and bolus intraventricular topotecan prolong survival in a mouse model of leptomeningeal medulloblastoma. PLoS One. 14 (1), e0206394 (2019).Shapiro, W. R., Johanson, C. E., Boogerd, W. Treatment modalities for leptomeningeal metastases. Seminars in Oncology. 36 (4 Suppl 2), S46-54 (2009).Simon, M. J., Iliff, J. J. Regulation of cerebrospinal fluid (CSF) flow in neurodegenerative, neurovascular and neuroinflammatory disease. Biochimica et Biophysica Acta. 1862 (3), 442- 451 (2016).Taillibert, S. et al. Leptomeningeal metastases from solid malignancy: a review. Journal of Neurooncology. 75 (1), 85-99 (2005).Znidaric, T. et al. Breast cancer patients with brain metastases or leptomeningeal disease: 10- year results of a national cohort with validation of prognostic indexes. Breast Journal. 25 (6), 1117-1125 (2019).

Claims

V. CLAIMSWhat is claimed is:

1. A method of treating a cancer, metastasis, and / or cancer associated complication in a subject comprising administering the anti-cancer therapy comprising at least one dendritic cell pulsed with at least one HER-3 CD4+ T cell epitope and / or at least one HER-2 CD4+ T cell epitope; wherein the anti-cancer therapy is administered intrathecally through an Ommaya reservoir.

2. The method of claim 1, wherein the HER-3 CD4+ T cell epitope is located in the extracellular domain of HER3.

3. The method of claim 2, wherein the HER-3 CD4+ T cell epitope comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 8, and / or SEQ IDNO: 9.

4. The method of claim 1, wherein the HER-3 CD4+ T cell epitope is located in the intracellular domain of HER3.

5. The method of claim 4, wherein the HER-3 CD4+ T cell epitope comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, and / or SEQ ID NO: 11.

6. The method of claim 1 , wherein the HER-2 CD4+ T cell epitope comprises SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and / or SEQ ID NO: 17.

7. The method of any of claims 1-6, further comprising at least one anti-cancer agent.

8. The method of claim 6, wherein the at least one anti-cancer agent comprises paclitaxel.

9. The method of any of claims 1-8, wherein the HER-3 CD4+ T cell epitope pulsed dendritic cell and / or HER-2 CD4+ T cell epitope pulsed dendritic cell is activated with IL- 12 prior to administration.