WO2024064446A2 - Compositions and methods for the enhancement of immune cell activities against cancer - Google Patents

Abstract

Disclosed herein are compositions comprising a recombinant polypeptide targeting phosphatidylserine on the surface of cancer cells and methods of using the compositions to treat a cancer in a subject.

Description

COMPOSITIONS AND METHODS FOR THE ENHANCEMENT OF IMMUNE CELL ACTIVITIES AGAINST CANCER

I. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63/407,888 filed 19 September 2022, which is incorporated herein in its entirety.

II. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under CA251439 awarded by the National Institutes of Health. The government has certain rights in the invention.

III. REFERENCE TO THE SEQUENCE LISTING

[0003] The Sequence Listing submitted 25 July 2023 as an XML file named “23_2067_WO_Sequence_Listing”, created on 25 July 2023 and having a size of 1,490 kilobytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

IV. BACKGROUND

[0004] In cancer treatment, it is highly desirable to exploit cancer-specific markers for therapeutic development. Many such markers are cell surface markers. Phosphatidylserine (PS) is an abundant lipid molecule and an integral part of the cellular membrane. Unlike other abundant lipid molecules that evenly distribute in both the outer and inner cellular membranes, phosphatidylserine normally only resides in the inner cellular membrane. PS often ‘flips’ from the inner to the outer cellular membrane in dying cells, especially in apoptotic cells. As a result, cell surface levels of PS have become the most widely used molecular marker to quantify cellular apoptosis in research. Most of the cunent paradigms for cancer therapy assumes that PS- expressing tumor cells, either from exposure to internal or external stressors, are destined to die from apoptosis. However, PS-expressing tumor cells may survive the activation of the apoptosis cascade.

[0005] Thus, there remains an unmet medical need for ensuring the complete cell death of PS- expressing cancer cells including those from solid tumors cells as well as an unmet medical need for enhancing the efficacy of cancer therapies and/or treatments.

V. BRIEF DESCRIPTION OF THE FIGURES

[0006] FIG. 1A - FIG. 1C show the survival of phosphatidylserine-expressing AML cells. FIG. 1A shows FACS sorting of untreated cl498 cells (in DMEM with 8% FBS). The rectangles indicate the sorted cells. FIG. IB shows FACS sorting of c!498 cells treated with cytarabine (1 pM) for 24 hrs). FIG. 1C shows the estimated live cell numbers in the sorted cells. About 1000 each of the sorted cells were placed into multi-well plates. When the cells reached sufficient numbers, they were counted at different time points to measure their growth rates. The growth rates were then used to estimate the initial live cell numbers. The following formular was used to calculate the minimal initial live cell numbers: Minimal initial cell number = Total cell population/ (growth rate* t)(days to reach the current cell number).

[0007] FIG. 2A - FIG. 2E provide examples of different versions of phosphatidylserine (PS)- targeting annexin V-scFv (CD3(OKT3)/CD16A(3G8) bispecific or tri-specific fusion proteins. FIG. 2A shows a design for an AnnexinV-scFv(aCD3) and AnnexinV-scFv(aCD16A) recombinant proteins. FIG. 2B provides designs for PS-targeting trispecific protein. FIG. 2C shows a design for (AnnexinV-scFv(aCD3)-Fc)2. FIG. 2D shows a design for AnnexinV-aCD3- Fc with PC-targeting annexin V connected to the light chain of aCD3. FIG. 2E shows two tri- specific proteins targeting PS and CD16A with an IL15 cytokine or IL15 cytokine plus and IL15 receptor alpha sushi domain.

[0008] FIG. 3 shows a polyacrylamide gel analysis (PAGE) of bispecific recombinant AnnexinV- scFv(3G8) and AnnexinV-scFv(OKT3) produced in CHO cells and purified by Ni-NTA affinity purification.

[0009] FIG. 4 is a bar graph showing the activity of expanded NSCLC tumor-infiltrating lymphocytes (TILs) transfected with a secreted BITE against syngeneic lung cancer cells. The TILs were initially transduced with a lentivirus encoding a secreted BITE (Annexin V-scFv (OKT3) expanded in the presence of aAPC (artificial antigen presentation cells) and the cocultured w ith syngeneic NSCLC cells (transduced with luciferase) for 72 hrs. Survival of the tumor cells was measured by reading luciferase levels.

[0010] FIG. 5 is a bar graph showing the expansion rate ofNSCLC-derived and CRC (colorectal cancer)-derived tumor-infiltrating lymphocytes (TILs) in the presence of a PS-targeting BITE and aAPC. The TILs were expanded in the presence of aAPC (K562 cells expressing IL-15, CD86, and 4-BBL) and BITE protein (AnnexmV-scFv(OKT3), 100 ng/mL. T cells were co-cultured with irradiated aAPCs at different APC:TIL ratios for about 7 days and counted.

[0011] FIG. 6 is a bar graph showing the activity of expanded NSCLC tumor-infiltrating lymphocytes (TILs) expanded with a PS-targeting BITE or CD3+CD28+ beads. The TILs were expanded either in the presence of aAPC (K562 cells expressing IL- 15, CD86, and 4-BBL) and BITE (AnnexinV-scFv(OKT3), or in the presence of CD3+CD28+ beads (TransAct) for 8 days. The cells were then cocultured with syngeneic NSCLC cells (transduced with luciferase) for 72 hrs. Survival of the tumor cells was measured by reading luciferase levels.

[0012] FIG. 7 is a bar graph showing the activity of expanded CRC (colorectal cancer) tumorinfiltrating lymphocytes (TILs) expanded with a PS-targeting BITE. The TILs were expanded in the presence of APC (K562 cells expressing IL-15, CD86, and 4-BBL) and a PS-targeting BITE protein (AnnexinV-scFv(0KT3). The cells were then cocultured with syngeneic CRC cells (transduced with luciferase) for 72 hrs. Survival of the tumor cells was measured by reading luciferase levels.

[0013] FIG. 8A - FIG. 8D shows flow cytometry analysis (FACS) of NSCLC-derived TILs expanded in aAPC cells without or with PS-targeting A5-scFv(OKT3) BITE. FIG. 8A shows the FACS analysis for activation markers. FIG. 8B shows the FACS analysis for maturation markers. FIG. 8A shows the FACS analysis for proliferation markers. FIG. 8A shows the FACS analysis for markers for tumor cell reactive TILs.

VI. BRIEF SUMMARY

[0014] Disclosed herein is a recombinant polypeptide, comprising a phosphatidylserine (PS) binding domain, a CD3 or a CD16A binding domain, and an optional third domain that binds a tumor-specific antigen or an immune cell-specific antigen.

[0015] Disclosed herein is a recombinant polypeptide, comprising a phosphatidylserine (PS) binding domain, a CD3 or a CD16A binding domain, an optional third domain that binds a tumorspecific antigen or an immune cell-specific antigen, and further comprising an immune stimulatory cytokine domain.

[0016] Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a disclosed recombinant polypeptide.

[0017] Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide, comprising a phosphatidylserine (PS) binding domain, a CD3 or a CD16A binding domain, and an optional third domain that binds a tumor-specific antigen or an immune cell-specific antigen.

[0018] Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide, comprising a phosphatidylsenne (PS) binding domain, a CD3 or a CD16A binding domain, an optional third domain that binds a tumor-specific antigen or an immune cell-specific antigen, and further comprising an immune stimulatory cytokine domain. In an aspect, a disclosed polynucleotide can be an isolated DNA molecule, an isolated RNA molecule, a vector, plasmid vector, a transposon, or a viral vector.

[0019] Disclosed herein is an engineered cell, comprising a disclosed recombinant polypeptide a disclosed polynucleotide or both.

[0020] Disclosed herein is an engineered immune cell comprising a disclosed recombinant polypeptide, a disclosed polynucleotide, or both. Disclosed herein is an engineered tumor infiltration lymphocyte comprising a disclosed recombinant polypeptide a disclosed polynucleotide or both. [0021] Disclosed herein is a method of treating a subject in need thereof, the method comprising administering to the subject a disclosed polypeptide, a disclosed polynucleotide or a disclosed engineered cell or a combination thereof.

[0022] Disclosed herein is a method of treating a subject in need thereof, the method comprising contacting an immune cell with a recombinant polypeptide to activate the immune cell, thereby obtaining an immune cell population, and administering the immune cell population into a subject in need thereof

[0023] Disclosed herein is a method of treating a subject in need thereof, the method comprising obtaining or having obtained autologous TILs from a subject, transforming or transfecting the TILs with a disclosed polynucleotide to obtain a population of engineered autologous TILs and administering to the subject a pharmaceutical composition comprising the engineered autologous TILs.

[0024] In an aspect, the subject has cancer or a metastatic cancer. In an aspect, the subject has a solid tumor. Disclosed herein is a method of improving the efficacy of cancer therapy, the method comprising treating a subject in need thereof by admimstenng to the subject in need thereof a therapeutically effective amount of one or more a disclosed polypeptide, a disclosed polynucleotide, or a disclosed engineered cell.

VII. DETAILED DESCRIPTION

[0025] The present disclosure describes compositions, compounded compositions, kits, capsules, containers, and/or methods thereof. It is to be understood that the inventive aspects of which are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0026] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

A. Definitions

[0027] Before the present compounds, compositions, articles, sy stems, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0028] This disclosure describes inventive concepts with reference to specific examples. However, the intent is to cover all modifications, equivalents, and alternatives of the inventive concepts that are consistent with this disclosure.

[0029] As used in the specification and the appended claims, the singular fomis “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0030] The phrase ‘consisting essentially of limits the scope of a claim to the recited components in a composition or the recited steps in a method as well as those that do not materially affect the basic and novel characteristic or characteristics of the claimed composition or claimed method. The phrase ‘consisting of excludes any component, step, or element that is not recited in the claim. The phrase ‘compnsmg’ is synonymous with ‘including’, ‘containing’, or ‘characterized by’, and is inclusive or open-ended. ‘Comprising’ does not exclude additional, unrecited components or steps.

[0031] As used herein, when referring to any numerical value, the term ‘about’ means a value falling within a range that is ± 10% of the stated value.

[0032] Ranges can be expressed herein as from ‘about’ one particular value, and/or to ‘about’ another particular value. When such a range is expressed, a further aspect 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 a further aspect. 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 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.

[0033] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0034] As used herein, the terms ‘optional’ or ‘optionally’ means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. In an aspect, a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.

[0035] As used herein, the term ‘subject’ refers to the target of administration, e.g., a human being. The term ‘subject’ also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). Thus, the subj ect of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex, and thus, adult and child subjects, as well as fetuses, whether male or female, are intended to be covered. In an aspect, a subject can be a human patient. In an aspect, a subject can have cancer, be suspected of having cancer, or be at risk of developing cancer.

[0036] As used herein, the term ‘diagnosed’ means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by one or more disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, or a combination thereof, or by one or more disclosed methods. For example, ‘diagnosed with a disease or disorder’ means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as a hematological cancer) that can be treated by one or more disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, or a combination thereof, or by one or more disclosed methods. For example, “suspected of having a disease or disorder” can mean having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as a hematological cancer) that can likely be treated by one or more disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, or a combination thereof, or by one or more disclosed methods. In an aspect, an examination can be physical, can involve various tests (e.g., blood tests, genotyping, biopsies, etc.), scans (e.g., CT scans, PET scans, etc.), and assays (e.g., enzymatic assay), or a combination thereof. [0037] A “patient” refers to a subject afflicted with a disease or disorder (e.g., a cancer). In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder such as a cancer. In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder and is seeking treatment or receiving treatment for a disease or disorder (such as cancer).

[0038] As used herein, the phrase “identified to be in need of treatment for a disease or disorder,” or the like, refers to selection of a subject based upon need for treatment of the disease or disorder. For example, a subject can be identified as having a need for treatment of a disease or disorder (e.g., a cancer) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the cancer. In an aspect, the identification can be performed by a person different from the person making the diagnosis. In an aspect, the administration can be performed by one who performed the diagnosis.

[0039] As used herein, “activated” and “activation” can refer to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production and detectable effector functions. The term “activated T cells” can refer to T cells that are proliferating. Signals generated through the TCR alone can be insufficient for full activation of the T cell and one or more secondary or costimulatory signals may also be required. Thus, T cell activation comprises a primary stimulation signal through the TCR/CD3 complex and one or more secondary costimulatory signals. Co-stimulation can be evidenced by proliferation and/or cytokine production by T cells that have received a primary activation signal, such as stimulation through the TCR/CD3 complex.

[0040] As used herein, “inhibit,” “inhibiting”, and “inhibition” mean to diminish or decrease an activity, level, response, condition, severity, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, level, response, condition, severity, disease, or other biological parameter. This can also include, for example, a 10% inhibition or reduction in the activity, level, response, condition, severity, disease, or other biological parameter as compared to the native or control level (e.g., a subject not having received one or more of a disclosed recombinant polypeptide, a disclosed isolated nucleic acid molecules, disclosed vectors, a disclosed cells, a disclosed pharmaceutical compositions, or a combination thereol). Thus, in an aspect, the inhibition or 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. In an aspect, the inhibition or reduction can be 10-20%, 20-30%, 30-40%, 40-50%, 50- 60%, 60-70%, 70-80%, 80-90%, or 90-100% as compared to a native or control level (e.g., a subject not having received one or more of a disclosed recombinant polypeptide, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed cell, a disclosed pharmaceutical composition, or a combination thereof). In an aspect, the inhibition or reduction can be 0-25%, 25-50%, 50-75%, or 75-100% as compared to native or control levels. In an aspect, a native or control level can be a pre-disease or pre-disorder level (such as a pre-cancer state).

[0041] The words “treat” or “treating” or “treatment” include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder (e.g., a cancer); preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder (e.g., a cancer); and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder (e.g., a cancer). In an aspect, the terms cover any treatment of a subject, including a mammal (e.g. , a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, i.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease. For example, in an aspect, treating a disease or disorder can reduce the severity of an established a disease or disorder in a subject by l%-100% as compared to a control (such as, for example, an individual not having cancer). In an aspect, treating can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of a disease or disorder (e.g., a cancer). For example, treating a disease or disorder can reduce one or more symptoms of a disease or disorder in a subject by l%-100% as compared to a control (such as, for example, an individual not having cancer). In an aspect, treating can refer to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms of an established a disease or disorder (e.g., a cancer). It is understood that treatment does not necessarily refer to a cure or complete ablation or eradication of a disease or disorder. However, in an aspect, treatment can refer to a cure or complete ablation or eradication of a disease or disorder (such as a cancer).

[0042] As used herein, the term “prevent” or “preventing” or “prevention” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. In an aspect, preventing a disease or disorder having chromatin deregulation and/or chromatin dysregulation is intended. The words “prevent”, “preventing”, and “prevention” also refer to prophylactic or preventative measures for protecting or precluding a subject (e.g., an individual) not having a given a disease or disorder (such as a cancer) or related complication from progressing to that complication. In an aspect, preventing metastasis is intended.

[0043] As used herein, the terms “administering” and “administration” refer to any method of providing one or more of a disclosed recombinant polypeptide, a disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, or a combination thereof, or by one or more of disclosed methods to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, the following: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intratumoral administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, mtra-CSF administration, intra-artenal administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intraarterial administration or administration through the hepatic portal vein (HPV). Administration of one or more disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, or any combination thereof can comprise administration directly into the CNS or the PNS. Administration can be continuous or intermittent. Administration can comprise a combination of one or more routes.

[0044] In an aspect, the skilled person can determine an efficacious dose, an efficacious schedule, and an efficacious route of administration one or more disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, or any combination thereof to treat or prevent a disease or disorder (such as a cancer). In an aspect, the skilled person can also alter, change, or modify an aspect of an administering step to improve efficacy of one or more disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, or a combination thereof.

[0045] By “determining the amount” is meant both an absolute quantification of a particular analyte (e.g., biomarker for cancer, for example a solid tumor) or a determination of the relative abundance of a particular analyte (e.g., a cancer biomarker). The phrase includes both direct or indirect measurements of abundance or both. [0046] As used herein, “operably linked” means that expression of a gene or a transgene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5’ (upstream) or 3’ (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

[0047] As used herein, “promoter” or “promoters” are known to the art. Depending on the level and tissue-specific expression desired, a variety of promoter elements can be used. A promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native (endogenous) or foreign (exogenous) and can be a natural or a synthetic sequence. By foreign or exogenous, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0048] As used herein, an “enhancer” such as a transcription or transcriptional enhancer refers to regulatory DNA segment that is typically found in multicellular eukaryotes. An enhancer can strongly stimulate (“enhance”) the transcription of a linked transcription unit, re., it acts in cis. An enhancer can activate transcription over very long distances of many thousand base pairs, and from a position upstream or downstream of the site of transcription initiation. An enhancer can have a modular structure by being composed of multiple binding sites for transcriptional activator proteins. Many enhancers control gene expression in a cell type-specific fashion. Several remote enhancers can control the expression of a singular gene while a singular enhance can stimulate the transcription of one or more genes.

[0049] As used herein, “modifying the method” can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. In an aspect, a method can be altered by changing the amount of the one or more disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, or any combination thereof administered to a subject, or by changing the frequency of administration of the one or more disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, or a combination thereof to a subject, by changing the duration of time that one or more of disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, or any combination thereof is administered to a subj ect, or by substituting for one or more disclosed components and/or reagents with a similar or equivalent component and/or reagent. The same applies to all disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors disclosed cells, disclosed pharmaceutical compositions, and any combination thereof.

[0050] As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions j ust prior to use. Examples of suitable aqueous and nonaqueous earners, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate, hi an aspect, a pharmaceutical carrier employed can be a solid, liquid, or gas. In an aspect, examples of solid carriers can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. In an aspect, examples of liquid carriers can include sugar syrup, peanut oil, olive oil, and water. In an aspect, examples of gaseous carriers can include carbon dioxide and nitrogen. In preparing a disclosed composition for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable compositions are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The injectable compositions can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of stenle solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0051] As used herein, the term “excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, cap ry late, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See, also, for reference, Remington’s Pharmaceutical Sciences, (1990) Mack Publishing Co., Easton, Pa., which is hereby incorporated by reference in its entirety.

[0052] A “transposon” is a mobile genetic element that efficiently moves between vectors and chromosomes using the “cut and paste” or “copy and paste” mechanism. During transposase transposition (for example, PB transposase in the PiggyBac transposon system) recognizes transposon-specific sequences of inverted terminal repeats (ITRs) located at both ends of the transposon (there are 5’- and 3’-ITRs in any transposon system), it moves the contents from source sites and embeds them in chromosomal sites, such as TTAA chromosomal sites. In an aspect, the powerful activity of the Piggy Bac transposon system makes it easy to transfer genes of interest located between two ITRs to target genomes. The transposon can be divided into Class I transposon (retrotransposon) and Class II transposon (DNA transposon). In Class I transposon, after RNA is transcribed from nucleic acid in a cell or from transposon DNA on the animal genome, the DNA reverse-transcribed from the RNA is transferred to another location on the animal genome. It works by inserting it. Class II transposon cuts nucleic acid in cells or transposon DNA on the animal genome, and then inserts the cut transposon DNA into another location on the animal genome. The Class II transposon may include a first polynucleotide at a 5 ’ end, a second polynucleotide at a 3’ end, and a third polynucleotide. The first polynucleotide and the second polynucleotide may include an inverted terminal repeat (ITR) sequence. The third polynucleotide can be located between the first polynucleotide and the second polynucleotide. The third polynucleotide may include an exo-polynucleotide. The third polynucleotide may include a polynucleotide encoding a transposase. Unless otherwise specified below, the term “transposon” assumes the case of Class II transposon, but even if the term “transposon” is interpreted as Class I transposon, it is technically If there is no problem, it will not be necessary to limit the interpretation to Class II transposon.

[0053] As used herein, “concurrently” means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.

[0054] The term “contacting” as used herein refers to bringing one or more disclosed recombinant polypeptides, disclosed polynucleotides, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, disclosed anti-chemokines, disclosed anti-cancer agents, disclosed chemotherapeutics, or any combination thereof together with a target area or intended target area in such a manner that disclosed recombinant polypeptides, disclosed polynucleotides molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, disclosed anti- chemokines, disclosed anti-cancer agents, disclosed chemotherapeutics, or any combination thereof can exert an effect on the intended target or targeted area either directly or indirectly. A target area or intended target area can be one or more of a subject’s organs (e.g., lungs, heart, liver, kidney, brain, etc.) hosting cancerous cells. In an aspect, a target area or intended target area can be any cell or any organ infected by a disease or disorder (such as a cancer). In an aspect, a target area or intended target area can be any organ, tissue, or cells that are affected by a disease or disorder (such as a cancer).

[0055] As used herein, “determining” can refer to measuring or ascertaining the presence and severity of a disease or disorder, such as, for example, a cancer. Methods and techniques used to determine the presence and/or severity of a disease or disorder are typically known to the medical arts. For example, the art is familiar with the ways to identify and/or diagnose the presence, severity, or both of a disease or disorder (such as, for example, cancer).

[0056] As used herein, “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired result such as, for example, the treatment and/or prevention of a disease or disorder (e.g., a cancer) or a suspected disease or disorder (e.g., a cancer). As used herein, the terms “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired an effect on an undesired condition (e.g., a cancer). For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. In an aspect, “therapeutically effective amount” means an amount disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, disclosed anti-chemokines, disclosed anti-cancer agents, disclosed chemotherapeutics, or any combination thereof that (i) treats the particular disease, condition, or disorder (e.g., a cancer), (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder e.g., a cancer), or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein (e.g., a cancer). The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; one or more of disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, or any combination thereof employed; disclosed methods employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of one or more of disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, or any combination thereof employed; the duration of the treatment; drugs used in combination or coincidental with the one or more of disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, or any combination thereof employed, and other like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, disclosed anti-chemokines, disclosed anti-cancer agents, disclosed chemotherapeutics, or any combination thereof at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, then the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, a single dose of disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, disclosed anti-chemokines, disclosed anti-cancer agents, disclosed chemotherapeutics, or any combination thereof can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition, such as, for example, a cancer.

[0057] The term “antibody” (Ab) includes, without limitation, a glycoprotein immunoglobulin that binds specifically to an antigen. An antibody can comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof. Each H chain can comprise a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region can comprise three constant domains, CHI, CH2 and CH3. Each light chain can comprise a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region can comprise one constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL can comprise three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains can contain a binding domain that interacts with an antigen. The constant regions of the Abs can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Generally, human antibodies can be approximately 150 kD tetrameric agents composed of two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a “Y -shaped” structure. The heavy and light chains can be linked or connected to one another by a single disulfide bond and two other disulfide bonds can connect the heavy chain hinge regions to one another, so that the dimers can be connected to one another and the tetramer can be formed. Naturally produced antibodies are also glycosylated, e g., on the CH2 domain. The term “antibody” is used to mean an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing etc., through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multi-specific antibodies such as bispecific antibodies generated from at least two intact antibodies, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc. [0058] The term “variable region” or “variable domain” is used interchangeably. The variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen. In an aspect, the variable region can be a human variable region. In an aspect, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In an aspect, the variable region is a primate (e.g., non-human primate) variable region. In an aspect, the variable region comprises rodent or murine CDRs and primate framework regions (FRs). As used herein, “antigen binding region” can be used to define the region of an antibody or a single chain vanable region (scFv), a single chain antibody, a nanobody, a diabody, a Fab, or an F(ab')2 that interacts with the antigen. Typically, the antigen binding region can be minimally defined by one or more CDRs of the antibody.

[0059] The terms “VL” and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody or an antigen-binding molecule thereof. The terms “VH” and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody or an antigen-binding molecule thereof.

[0060] As used herein, “conjugate” or “conjugated” can be used to define the operative association of one disclosed component to another disclosed component. In an aspect, conjugated does not intend to refer solely to any type of operative association and is not particularly limited to chemical “conjugation”.

[0061] As known to the skilled person, IgG can bind to cell surface receptors on many types of cells to bring about an assortment of effects, for example, (i) the enabling of phagocytosis (e g., monocytes, macrophages, neutrophils), (ii) antibody-dependent cellular cytotoxicity (monocytes, macrophages and lymphocytes), or (iii) to effect feedback control on antibody synthesis (B and T lymphocytes). In an aspect, the properties of the IgG subclasses can very, and in most cases the Fc fragments can have the same property as the intact IgG; meaning that it does not appear to be modulated by the hinge or the Fab. In an aspect, cellular Fc receptors can be classified into three categories according to structure and affinity. All the sites on IgG that can interact with these separate receptors appear to be located in the Fc region, and, in the case of the FcyRI (which is the highest affinity receptor class), the site involves residues 233-237, at the N-terminal end of the Cy2 domain close to the hinge region but coded in the Cy2 exon.

[0062] “Endogenous” with reference to a gene, protein, and/or nucleic acid refers to the natural presence of that gene, protein, and/or nucleic acid in a cell, such as an immune cell.

[0063] “Exogenous” refers to an introduced agent, such as a nucleic acid, gene, or protein, into a cell, for example from an outside source. A nucleic acid introduced into a cell is exogenous even if it encodes a protein which is naturally found in the cell. Such exogenous introduction of a nucleic acid encoding a protein can be used to increase the expression of the protein over the level that would naturally be found in the cell under similar conditions, e.g., without introduction of the exogenous nucleic acid.

[0064] A “T cell receptor” or “TCR” refers to antigen-recognition molecules present on the surface of T cells. During normal T cell development, each of the four TCR genes, a, 0, y, and 6, can rearrange leading to highly diverse TCR proteins.

[0080] As used herein, “effector function” can refer to a biological result of interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions comprise, without limitation, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement mediated cytotoxicity (CMC). An effector function can be antigen binding dependent, antigen binding independent, or both. ADCC refers to lysis of antibody-bound target cells by immune effector cells. Without wishing to be bound by any theory, ADCC is generally understood to involve Fc receptor (FcR)-bearing effector cells recognizing and subsequently killing antibody-coated target cells (e.g., cells that express on their surface antigens to which an antibody is bound). Effector cells that mediate ADCC can comprise immune cells, comprising yet not limited to, one or more of natural killer (NK) cells, macrophages, neutrophils, eosinophils.

[0099] The term “immunotherapy” refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response. Examples of immunotherapy can include, but are not limited to, NK cells and T cell therapies. T cell therapy can include adoptive T cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACTTM), and allogeneic T cell transplantation. However, one of skill in the art would recognize that the conditioning methods disclosed herein would enhance the effectiveness of any transplanted T cell therapy. The T cells or NK cells of the immunotherapy can come from any source known in the art. For example, T cells and NK cells can be differentiated in vitro from a hematopoietic stem cell population (for example iPSCs) or can be obtained from a subject. T cells and NK cells can be obtained from, e.g., peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In addition, the T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood collected from a subject using techniques known to the skilled person.

[0065] As used herein, the term “humanized antibody” refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster, etc.) that have the desired specificity, affinity, and capability. In some instances, the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability. The humanized antibody can be further modified by the substitution of additional residue either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability. In general, the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.

[0066] That an antibody “selectively binds” or “specifically binds” to an epitope or receptor means that the antibody reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope or receptor than with alternative substances, including unrelated proteins. “Selectively binds” or “specifically binds” means, for instance, that an antibody binds to a protein with a KD of about 0. 1 mM or less, more usually about 1 pM or less. “Selectively binds” or “specifically binds” means at times that an antibody binds to a protein with a KD of about 0. 1 mM or less, at times about 1 pM or less, at times about 0. 1 pM or less, at times about 0.01 pM or less, and at times about 1 nM or less. It is understood that, in an aspect, an antibody or binding moiety that specifically binds to a first target may or may not specifically bind to a second target. As such, “specific binding” does not necessarily require (although it can include) exclusive binding, e.g., binding to a single target. [0067] A “target” or “target antigen” is any molecule bound by a binding motif (e.g., a PS- expressing cancer cell). A disclosed target can be cells and/or tissues in a subject.

[0068] “Antigen-specific targeting region” (ASTR) refers to the region of a disclosed recombinant polypeptide that targets specific antigens. In an aspect, the antigen-specific targeting regions comprise an antibody or a functional equivalent thereof or a fragment thereof or a derivative thereof and each of the targeting regions target a different antigen. The targeting regions can comprise full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, divalent single chain antibodies or diabodies, each of which are specific to the target antigen.

[0069] BITE as used herein is a recombinant polypeptide constructed from two different recognition domains connected by a protein linker. One of the domains recognizes a tumor- associated surface antigen, while the other domain recognizes a T cell surface marker, in order to activate and redirect cytotoxic T cells to tumor cells.

[0070] BIKE as used herein is a recombinant polypeptide constructed from two different recognition domains connected by a protein linker. One of the domains recognizes a tumor- associated surface antigen, while the other domain recognizes a NK cell surface marker, in order to activate and redirect cytotoxic NK-cells to tumor cells.

[0071] TRIKE as used herein is a recombinant polypeptide constructed from three different recognition domains connected by protein linkers. One of the domains recognizes a tumor- associated surface antigen, the second domain recognizes a NK cell surface marker, and a third domain is an immune stimulatory cytokine. The cytokine domain helps maintain a sustained NK cell activity against tumor cells.

[0072] An antigen-presenting cell (APC) or accessory' cell is a cell that displays antigen bound by major histocompatibility complex (MHC) proteins on its surface; this process is known as antigen presentation. T cells may recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T-cells. Antigen-presenting cells fall into two categories: those that express MHC class II molecules along with co-stimulatory molecules and those that express MHC class I molecules. Without being bound by theory, T cells must be activated before they can divide and perform their function. This is achieved by interacting with a APC which presents an antigen recognized by their T cell receptor. APCs can also present foreign and selflipids to T cells and NK cells by using the CD1 family of proteins, which are structurally similar to the MHC class I family. Artificial antigen presenting cells (aAPCs) are engineered platforms for T-cell activation.

[0073] “Transcriptional regulatory elements” refer to any nucleotide sequence that influences transcription initiation and rate, or stability and/or mobility of a transcript product. Regulatory sequences include, but are not limited to, promoters, promoter control elements, protein binding sequences, 5' and 3' UTRs, transcriptional start sites, termination sequences, polyadenylation sequences, introns, etc. Such transcriptional regulatory sequences can be located either 5'-, 3'-, or within the coding region of the gene and can be either promote (positive regulatory element) or repress (negative regulatory element) gene transcription.

[0074] A “translation regulatory sequence” refers to a specific region within an rnRNA (messenger RNA) molecule, usually located in the 5’ untranslated region (5’ UTR) or 3’ untranslated region (3’ UTR), that plays a crucial role in regulating the process of translation. Translation is the cellular process by which the ribosome translates the genetic information encoded in rnRNA into a sequence of amino acids to form a functional protein. Translation regulatory sequences contain various elements that can either enhance or inhibit the efficiency of translation initiation, elongation, or termination. These sequences function by interacting with specific proteins or RNA molecules, thereby influencing the rate of protein synthesis. They play a vital role in modulating gene expression, determining when and to what extent a protein is produced within the cell. There are several types of translation regulatory sequences, each with its specific functions. Some common examples include: 5’ Cap: A modified guanosine nucleotide added to the 5’ end of rnRNA, essential for translation initiation and ribosome binding. Kozak sequence: A sequence that surrounds the start codon (AUG) and helps in the proper recognition of the start codon by the ribosome. Internal Ribosome Entry Sites (IRES): Special sequences that enable ribosomes to initiate translation at internal sites within the rnRNA, bypassing the 5’ capdependent initiation. Poly(A) tail: A string of adenine nucleotides added to the 3’ end of rnRNA, involved in translation efficiency and rnRNA stability. MicroRNA Binding Sites: Specific sequences within the 3' UTR that can be targeted by microRNAs, leading to rnRNA degradation or translational repression. AU-nch elements (AREs): Sequence motifs in the 3’ UTR that influence rnRNA stability and translation efficiency. Alterations or mutations in translation regulatory sequences can lead to dysregulation of protein production, contributing to various diseases and disorders. Understanding these sequences and their regulatory mechanisms is crucial for deciphering the intricacies of gene expression and protein synthesis in living organisms.

[0075] The term “autologous” refers to any material derived from the same individual to which it is later to be re-introduced. For example, a subject’s own cells can be obtained, made to express one or more disclosed CARs, and then administered to the same subject.

[0076] As used herein, “RNA therapeutics” can refer to the use of oligonucleotides to target RNA. RNA therapeutics can offer the promise of uniquely targeting the precise nucleic acids involved in a particular disease with greater specificity, improved potency, and decreased toxicity. This could be particularly powerful for genetic diseases where it is most advantageous to aim for the RNA as opposed to the protein. In an aspect, a therapeutic RNA can comprise one or more expression sequences. As known to the art, expression sequences can comprise an RNAi, shRNA, mRNA, non-coding RNA (ncRNA), an antisense such as an antisense RNA, miRNA, morpholino oligonucleotide, peptide-nucleic acid (PNA) or ssDNA (with natural, and modified nucleotides, including but not limited to, LNA, BNA, 2’-0-Me-RNA, 2’-ME0-RNA, 2 -F-RNA), or analog or conjugate thereof. In an aspect, a disclosed therapeutic RNA can comprise one or more long non-coding RNA (IncRNA), such as, for example, a long intergenic non-coding RNA (lincRNA), pre-transcript, pre-miRNA, pre-mRNA, competing endogenous RNA (ceRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), pseudo-gene, rRNA, or tRNA. In an aspect, ncRNA can be pi wi -interacting RNA (piRNA), primary miRNA (pri-miRNA), or premature miRNA (pre-miRNA). In an aspect, a disclosed therapeutic RNA or an RNA therapeutic can comprise antisense oligonucleotides (ASOs) that inhibit mRNA translation, oligonucleotides that function via RNA interference (RNAi) pathway, RNA molecules that behave like enzymes (ribozymes), RNA oligonucleotides that bind to proteins and other cellular molecules, and ASOs that bind to mRNA and form a structure that is recognized by RNase H resulting in cleavage of the mRNA target. In an aspect, RNA therapeutics can comprise RNAi and ASOs that inhibit mRNA translation. Generally speaking, as known to the art, RNAi operates sequence specifically and post-transcriptionally by activating ribonucleases which, along with other enzymes and complexes, coordinately degrade the RNA after the original RNA target has been cut into smaller pieces while antisense oligonucleotides bind to their target nucleic acid via Watson-Crick base pairing, and inhibit or alter gene expression via steric hindrance, splicing alterations, initiation of target degradation, or other events.

[0077] As used herein, the terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer. [0078] The terms “proliferative disorder” and “proliferative disease” refer to disorders associated with abnormal cell proliferation such as cancer.

[0079] “Tumor” and “neoplasm” as used herein refer to any mass of tissue that result from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre-cancerous lesions.

[0080] “Metastasis” as used herein refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location. A “metastatic” or “metastasizing” cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures.

[0081] The terms “cancer stem cell” or “tumor stem cell” or “solid tumor stem cell” are used interchangeably herein and refer to a population of cells from a solid tumor that: (1 ) have extensive proliferative capacity; (2) are capable of asymmetric cell division to generate one or more kinds of differentiated progeny with reduced proliferative or developmental potential; and (3) are capable of symmetnc cell divisions for self-renewal or self-maintenance. These properties of “cancer stem cells” or “tumor stem cells” or “solid tumor stem cells” confer on those cancer stem cells the ability to form palpable tumors upon serial transplantation into an immunocompromised mouse compared to the majority of tumor cells that fail to form tumors. Cancer stem cells undergo self-renewal versus differentiation in a chaotic manner to form tumors with abnormal cell types that can change over time as mutations occur.

[0082] The terms “cancer cell” or “tumor cell” and grammatical equivalents refer to the total population of cells derived from a tumor including both non-tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells).

[0083] As used herein “tumorigenic” refers to the functional features of a solid tumor stem cell including the properties of self-renewal (giving rise to additional tumorigenic cancer stem cells) and proliferation to generate all other tumor cells (giving rise to differentiated and thus non- tumorigenic tumor cells) that allow solid tumor stem cells to form a tumor.

[0084] A “solid tumor” is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign (not cancer), or malignant (cancer). Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms’ tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).

[0085] As used herein, “tumor infiltrating lymphocytes” of “TILs” or “TIL” Tumor-infiltrating lymphocytes (TILs) are a type of immune cell that can be found within the tumor microenvironment of solid tumors. They are lymphocytes, a class of white blood cells responsible for the body’s immune response against foreign invaders and abnormal cells, including cancer cells. TILs play a critical role in the interaction between the immune system and cancer. When cancer develops, tumor cells can sometimes be recognized as abnormal by the immune system. Immune cells, including TILs, can then infiltrate the tumor mass, attempting to recognize and eliminate the cancer cells. The presence and activity of TILs in the tumor microenvironment can have significant implications for the progression and treatment of cancer. There are different types of lymphocytes that can infiltrate tumors: Cytotoxic T cells: These T cells have the ability to directly recognize and kill cancer cells by releasing cytotoxic molecules that induce cell death. Helper T cells: These T cells assist other immune cells in their functions and help orchestrate a broader immune response against the tumor. Regulatory' T cells (Tregs): These T cells have a suppressive function, dampening the immune response. In the context of cancer, Tregs can limit the immune system’s ability to attack the tumor, promoting immune evasion. Natural Killer (NK) cells: NK cells can also infiltrate tumors and directly kill cancer cells without the need for prior sensitization, making them important players in the innate immune response against cancer. A high density of activated TILs often correlates with better outcomes, as it indicates an active and effective anti-tumor immune response. On the other hand, some tumors can develop mechanisms to evade the immune system, leading to lower TIL infiltration and immune tolerance. Immunotherapies, such as immune checkpoint inhibitors, are designed to boost the activity of TILs and enhance the immune response against cancer.

[0086] “Autologous TILs” refers to tumor-infiltrating lymphocytes (TILs) that are collected from a patient’s own tumor tissue and then used in personalized cancer immunotherapy. Autologous TIL therapy is a form of adoptive cell therapy, where the patient’s immune cells are isolated, expanded, and reinfused back into the same patient to enhance the body’s ability to fight cancer. Without being bound by theory, the process of autologous TIL therapy typically involves the following steps: Tumor Biopsy: A small piece of the patient’s tumor is surgically removed through a biopsy or excision. Isolation of TILs: TILs are extracted from the tumor tissue. These are the lymphocytes that have infiltrated the tumor, indicating their potential to recognize and target cancer cells. TIL Activation and Expansion: The isolated TILs are cultured in the laboratory' and exposed to specific growth factors and cytokines to stimulate their activation and expansion. This process leads to a significant increase in the number of TILs, making them more effective in attacking cancer cells. Preparatory Treatment: Before reinfusing the expanded TILs back into the patient, the patient can receive lymphodepleting chemotherapy or radiation. This helps create space within the immune system for the infused TILs to proliferate and exert their anticancer effects. TIL Infusion: The expanded and activated TILs are then infused back into the patient through intravenous infusion. Once the TILs are back in the patient’s body, they can target and attack cancer cells, thus potentially leading to tumor regression or elimination.

[0087] “Allogeneic TILs” refers to tumor-infiltrating lymphocytes (TILs) that are collected from a genetically different individual (donor) and then used in the treatment of another individual (recipient) with cancer. These TILs are harvested from the tumor tissue of the donor, expanded and activated in the laboratory, and then infused into the recipient to boost their immune response against cancer.

[0088] As used herein, the “tumorigenicity” of a tumor refers to the ability of a random sample of cells from the tumor to form palpable tumors upon serial transplantation into immunocompromised mice.

[0089] As used herein, “lipid nanoparticles” or “LNPs” can deliver nucleic acid (e.g., DNA or RNA), protein (e.g., RNA-guided DNA binding agent), or nucleic acid together with protein. LNPs can comprise biodegradable, ionizable lipids. For example, LNPs can comprise (9Z,12Z)- 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-di enoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-

(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-di enoate) or another ionizable lipid. In an aspect, the term cationic and ionizable in the context of LNP lipids can be used interchangeably, e.g., wherein ionizable lipids are cationic depending on the pH.

[0090] An amino acid sequence that is “derived from” a wild-type sequence or other amino acid sequence disclosed herein can refer to an amino acid sequence that differs by one or more ammo acids compared to the reference amino acid sequence, for example, containing one or more amino acid insertions, deletions, or substitutions as disclosed herein. The terms “derivative,” “variant,” “variations” and “fragment,” when used herein with reference to a polypeptide, refers to a polypeptide related to a wild-type polypeptide, for example either by amino acid sequence, structure (e.g., secondary and/or tertiary), activity (e.g., enzymatic activity) and/or function. A part or fragment of a polypeptide can correspond to at least 1%, at least 2%, at least 3 %, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40% ofthe length of a polypeptide, such as a polypeptide having an amino acid sequence identified by a specific SEQ ID NO., or having at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the length (in amino acids) of the polypeptide.

[0091] Within the context of the present application, a protein is represented by an amino acid sequence, and correspondingly a nucleic acid molecule or a polynucleotide is represented by a nucleic acid sequence. Identity and similarity between sequences: throughout this application, it should be understood that for each reference to a specific amino acid sequence using a unique sequence identifier (SEQ ID NO.), the sequence can be replaced by: a polypeptide represented by an ammo acid sequence compnsmg a sequence that has at least 60% sequence identity or similanty with 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, or at least 1000 fold he reference amino acid sequence. Another preferred level of sequence identity or similarity is 65%. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 75%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 85%. Another preferred level of sequence identity or similarity' is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 98%. Another preferred level of sequence identity or similarity is 99%.

[0092] Each ammo acid sequence described herein by virtue of its identity or similanty percentage with a given amino acid sequence respectively has in a further preferred embodiment an identity or a similarity of at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% with the given nucleotide or amino acid sequence, respectively. The terms “homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is described herein as a relationship between two or more amino acid (polypeptide or protein) sequences, or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred embodiment, sequence identity is calculated based on the full length (in amino acids or nucleotides) of two given SEQ ID NOs or based on a portion thereof. A portion of a full-length sequence can be referred to as fragment, and preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of the length (in amino acids or nucleotides) of a reference sequence. “Identity” also refers to the degree of sequence relatedness between two amino acid or between two nucleic acid sequences, as the case can be, as determined by the match between strings of such sequences. The degree of sequence identity between two sequences can be determined, for example, by comparing the two sequences using computer programs commonly employed for this purpose, such as global or local alignment algorithms. Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, or another suitable method or algorithm. A Needleman and Wunsch global alignment algorithm can be used to align two sequences over their entire length or part thereof (part thereof may mean at least 50%, 60%, 70%, 80%, 90% of the length of the sequence), maximizing the number of matches and minimizes the number of gaps. Default settings can be used and preferred program is Needle for pairwise alignment (in an embodiment, EMBOSS Needle 6.6.0.0, gap open penalty 10, gap extent penalty: 0.5, end gap penalty: false, end gap open penalty: 10 , end gap extent penalty: 0.5 is used) and MAFFT for multiple sequence alignment ( in an embodiment, MAFFT v7Default value is: BLOSUM62 [bl62], Gap Open: 1.53, Gap extension: 0.123, Order: aligned , Tree rebuilding number: 2, Guide tree output: ON [true], Max iterate: 2 , Perform FFTS: none is used).

[0093] “Similarity” between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. Similar algorithms used for determination of sequence identity can be used for determination of sequence similarity. Optionally, in determining the degree of ammo acid similarity, the skilled person may also take into account so-called conservative amino acid substitutions. As used herein, “conservative” amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below:

[0094] Alternative conservative amino acid residue substitution classes:

[0095] Alternative physical and functional classifications of amino acid residues:

[0096] For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is pheny lalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; He to Leu or Vai; Leu to He or Vai; Lys to Arg; Gin or Glu; Met to Leu or He; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Vai to He or Leu. [0097] As used herein, the term “chimeric antigen receptor” or “CAR” refers to an artificial exogenous antigen recognition receptor that can induce signaling in an engineered cell that expresses the CAR upon binding of the CAR to an antigen, for example, an antigen associated with a cancer or infectious disease. A CAR generally induces signaling in the engineered cell that expresses the CAR but not in a cell that expresses or presents the antigen bound by the CAR. A CAR comprises at least one extracellular targeting domain, at least one transmembrane domain, and at least one intracellular signaling domain. In some cases, a CAR comprises a hinge domain. A CAR extracellular targeting domain can be, comprise, or be derived from, for example, a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or a functional derivative, variant or fragment thereof, including, but not limited to, a heavy chain variable domain (VH), alight chain variable domain (VL), a Fab, a Fab’, aF(ab’)2, an Fv, a singlechain Fv (scFv), a minibody, a diabody, a single-domain antibody such as a VHH, and any combination thereof. A CAR extracellular targeting domain can be, comprise, or be derived from, for example, a D ARPin, a non-antibody domain (e. g. , from or derived from a receptor or a receptor ligand, for example, APRIL). The intracellular signaling domain of a CAR can induce or reduce activity of an engineered cell comprising the CAR. An intracellular signaling domain of a CAR can be or can comprise a truncated portion of a signaling domain of another molecule. In some cases, intracellular domain of the CAR can be involved in regulating primary activation of a TCR complex in either a stimulatory manner or an inhibitory manner. In some embodiments, the intracellular signaling domain of the CAR is involved in inducing T cell activation and/or a cytotoxic response against cells that express the antigen that is bound by the CAR. In some cases, CARs are also referred to as artificial T cell receptors, chimeric immunoreceptors, or chimeric T cell receptors.

[0098] As used herein, “immune-modulating” refers to the ability' of one or more of disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, or a combination thereof to alter (modulate) one or more aspects of the immune system. The immune system functions to protect the organism from infection and from foreign antigens by cellular and humoral mechanisms involving lymphocytes, macrophages, and other antigen-presenting cells that regulate each other by means of multiple cell-cell interactions and by elaborating soluble factors, including lymphokines and antibodies, that have autocrine, paracrine, and endocrine effects on immune cells.

[0099] As used herein, “immune modulator” refers to an agent that is capable of adjusting a given immune response to a desired level (e.g., as in immunopotentiation, immunosuppression, or induction of immunologic tolerance). Examples of immune modulators include but are not limited to, a disclosed immune modulator can comprise aspirin, azathioprine, belimumab, betamethasone dipropionate, betamethasone valerate, bortezomib, bredinin, cyazathioprine, cyclophosphamide, cyclosporine, deoxy spergualin, didemnin B, fluocinolone acetonide, folinic acid, ibuprofen, IL6 inhibitors (such as sarilumab) indomethacin, inebilizumab, intravenous gamma globulin (IVIG), methotrexate, methylprednisolone, mycophenolate mofetil, naproxen, prednisolone, prednisone, prednisolone indomethacin, rapamycin, rituximab, sirolimus, sulindac, synthetic vaccine particles containing rapamycin (SVP-Rapamycin or ImmTOR), thalidomide, tocilizumab, tolmetin, triamcinolone acetonide, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD19 antibodies, anti- CD20 antibodies, anti-CD22 antibodies, anti-CD40 antibodies, anti-FcRN antibodies, anti-IL6 antibodies, anti-IGFIR antibodies, an IL2 mutein, a BTK inhibitor, or a combination thereof. In an aspect, a disclosed immune modulator can comprise one or more Treg (regulatory T cells) infusions (e.g., antigen specific Treg cells to AAV). In an aspect, a disclosed immune modulator can be bortezomib or SVP-Rapamycin. In an aspect, an immune modulator can be administered by any suitable route of administration including, but not limited to, in utero, intra-CSF, intrathecally, intravenously, subcutaneously, transdermally, intradermally, intramuscularly, orally, transcutaneously, intraperitoneally (IP), or intravaginally . In an aspect, a disclosed immune modulator can be administered using a combination of routes. Administration can also include hepatic intra-arterial administration or administration through the hepatic portal vein (HPV). Administration of an immune modulator can be continuous or intermittent, and administration can comprise a combination of one or more routes.

[0100] As used herein, the term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

[0101] As used herein, the term “in combination” in the context of the administration of other therapies (e.g., other agents) includes the use of more than one therapy (e.g., drug therapy). Administration “in combination with” one or more further therapeutic agents includes simultaneous (e.g., concurrent) and consecutive administration in any order. The use of the term “in combination” does not restrict the order in which therapies are administered to a subject. By way of non-limiting example, a first therapy (e.g., one or more of disclosed recombinant polypeptides, disclosed isolated nucleic acid molecules, disclosed vectors, disclosed cells, disclosed pharmaceutical compositions, or a combination thereof) can be administered prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks), concurrently, or after (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or longer) the administration of a second therapy to a subject having or diagnosed with cancer.

[0102] Disclosed are the components to be used to prepare disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical compositions as well as disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed phamraceutical compositions 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 cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific claimed compositions and/or claimed methods.

[0103] “Annexins” are Ca2+-regulated phospholipid-binding proteins that play an important role in the cell life cycle, exocytosis, and apoptosis. Annexin is a common name for a group of cellular proteins, mostly found in eukaryotic organisms, defined by their ability to bind negatively charged phospholipids in a calcium dependent manner and containing a 70 amino acid repeat sequence called an annexin repeat. Several proteins consist of annexin with other domains like gelsolin. ANXA1 (Annexin Al) is a protein-coding gene. This gene encodes a membrane-localized protein that binds phospholipids. This protein inhibits phospholipase A2 and has anti-inflammatory activity. Loss of function or expression of this gene has been detected in multiple tumors. Diseases associated with ANXA1 include Shoulder Impingement Syndrome and Brain Edema. Among its related pathways are GPCR downstream signaling and Class A/l (Rhodopsin-like receptors). Gene Ontology (GO) annotations related to this gene include calcium ion binding and signaling receptor binding. An important paralog of this gene is ANXA2. ANXA1 plays important roles in the innate immune response as effector of glucocorticoid-mediated responses and regulator of the inflammatory process. ANXA1 has anti-inflammatory activity. ANXA1 plays a role in glucocorticoid-mediated down -regulation of the early phase of the inflammatory' response (by similarity). ANXA1 contributes to the adaptive immune response by enhancing signaling cascades that are triggered by T-cell activation, regulates differentiation and proliferation of activated T- cells. ANXA1 promotes the differentiation of T-cells into Thl cells and negatively regulates differentiation into Th2 cells. ANXA1 has no effect on unstimulated T cells. ANXA1 negatively regulates hormone exocytosis via activation of the formyl peptide receptors and reorganization of the actin cytoskeleton. ANXA1 has high affinity for Ca(2+) and can bind up to eight Ca(2+) ions (by similarity). ANXA1 displays Ca(2+)-dependent binding to phospholipid membranes. ANXA1 plays a role in the formation of phagocytic cups and phagosomes and plays a role in phagocytosis by mediating the Ca(2+)-dependent interaction between phagosomes and the actin cytoskeleton (by similarity). ANXA1 functions at least in part by activating the formyl peptide receptors and downstream signaling cascades. ANXA1 promotes chemotaxis of granulocytes and monocytes via activation of the formyl peptide receptors and promotes rearrangement of the actin cytoskeleton, cell polarization and cell migration. ANXA1 promotes resolution of inflammation and wound healing. ANXA1 acts via neutrophil N-formyl peptide receptors to enhance the release of CXCL2.

[0104] ANXA2 (Annexin A2) is a protein coding gene. This gene encodes a member of the annexm family. Members of this calcium-dependent phospholipid-binding protein family play a role in the regulation of cellular growth and in signal transduction pathways. This protein functions as an autocrine factor which heightens osteoclast formation and bone resorption. This gene has three pseudogenes located on chromosomes 4, 9 and 10, respectively. Multiple alternatively spliced transcript variants encoding different isoforms have been found for this gene. Annexin A2 expression has been found to correlate with resistance to treatment against various cancer forms. Diseases associated with ANXA2 include Antiphospholipid Syndrome and Acute Promyelocytic Leukemia. Among its related pathways are Interleukin- 12 family signaling and Innate Immune System. Gene Ontology (GO) annotations related to this gene include RNA binding and small GTPase binding. An important paralog of this gene is ANXA1. Calcium- regulated membrane-binding protein whose affinity for calcium is greatly enhanced by anionic phospholipids. It binds two calcium ions with high affinity. May be involved in heat-stress response. Inhibits PCSK9-enhanced LDLR degradation, probably reduces PCSK9 protein levels via a translational mechanism but also competes with LDLR for binding with PCSK9. ANXA2 binds M. pneumoniae CARDS toxin, probably serves as one receptor for this pathogen. When ANXA2 is down-regulated by siRNA, less toxin binds to human cells and less vacuolization (a symptom of M. pneumoniae infection) is seen.

[0105] ANXA3 (Annexin A3) is a protein coding gene. This gene encodes a member of the annexin family. Members of this calcium-dependent phospholipid-binding protein family play a role in the regulation of cellular growth and in signal transduction pathways. This protein functions in the inhibition of phospholipase A2 and cleavage of inositol 1,2-cyclic phosphate to form inositol 1 -phosphate. This protein may also play a role in anti -coagulation. Diseases associated with ANXA3 include ovarian cancer and prostate cancer. Among its related pathways are prostaglandin synthesis and regulation. Gene Ontology (GO) annotations related to this gene include calcium ion binding and calcium-dependent phospholipid binding. An important paralog of this gene is ANXA11. Inhibitor of phospholipase A2, also possesses anti-coagulant properties. Also cleaves the cyclic bond of inositol 1,2-cyclic phosphate to form inositol 1 -phosphate.

[0106] ANXA4 (Annexin A4) is a protein coding gene. Annexin IV (ANXA4) belongs to the annexin family of calcium-dependent phospholipid binding proteins. Although their functions are still not clearly defined, several members of the annexin family have been implicated in membrane-related events along exocytotic and endocytotic pathways. ANXA4 has 45 to 59% identity with other members of its family and shares a similar size and exon-intron organization. Isolated from human placenta, ANXA4 encodes a protein that has possible interactions with ATP and has in vitro anticoagulant activity and also inhibits phospholipase A2 activity. ANX4 is almost exclusively expressed in epithelial cells. Several transcript vanants encoding different isoforms have been found for this gene. Among its related pathways are prostaglandin synthesis and regulation. Gene Ontology (GO) annotations related to this gene include calcium ion binding and calcium-dependent protein binding. An important paralog of this gene is ANXA11. Calcium/phospholipid-binding protein which promotes membrane fusion and is involved in exocytosis.

[0107] ANXA5 (Annexin A5) is a protein coding gene. The Annexin 5 gene spans 29 kb containing 13 exons, and encodes a single transcript of approximately 1.6 kb and a protein product with a molecular w eight of about 35 kDa. The protein encoded by this gene belongs to the annexin family of calcium-dependent phospholipid binding proteins some of which have been implicated in membrane-related events along exocytotic and endocytotic pathways. Annexin 5 is a phospholipase A2 and protein kinase C inhibitory protein with calcium channel activity and a potential role in cellular signal transduction, inflammation, growth and differentiation. Annexin 5 has also been described as placental anticoagulant protein I, vascular anticoagulant-alpha, endonexin II, lipocortin V, placental protein 4 and anchorin CII. Polymorphisms in this gene have been implicated in various obstetric complications. Diseases associated with ANXA5 include Pregnancy Loss, Recurrent 3 and Antiphospholipid Syndrome. Among its related pathways are Response to elevated platelet cytosolic Ca2+ and Regulation of CFTR activity (norm and CF). Gene Ontology (GO) annotations related to this gene include calcium ion binding and calciumdependent phospholipid binding. An important paralog of this gene is ANXA4. This protein is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade.

[0108] ANXA6 (Annexin A6) is a protein coding gene. Annexin VI belongs to a family of calcium-dependent membrane and phospholipid binding proteins. Several members of the annexin family have been implicated in membrane-related events along exocytotic and endocytotic pathways. The annexin VI gene is approximately 60 kbp long and contains 26 exons. It encodes a protein of about 68 kDa that consists of eight 68-amino acid repeats separated by linking sequences of variable lengths. It is highly similar to human annexins I and II sequences, each of which contain four such repeats. Annexin VI has been implicated in mediating the endosome aggregation and vesicle fusion in secreting epithelia during exocytosis. Alternatively spliced transcript variants have been described. Diseases associated with ANXA6 include Kwashiorkor and Malignant Hyperthermia. Among its related pathways are Cardiac conduction and Myometrial relaxation and contraction pathways. Gene Ontology (GO) annotations related to this gene include calcium ion binding and GTP binding. An important paralog of this gene is ANXA11.

[0109] ANXA7 (Annexm A7) is a protein coding gene. Annexin Vll is a member of the annexin family of calcium-dependent phospholipid binding proteins. The Annexin VII gene contains 14 exons and spans approximately 34 kb of DNA. An alternatively spliced cassette exon results in two mRNA transcripts of 2.0 and 2.4 kb which are predicted to generate two protein isoforms differing in their N-terminal domain. The alternative splicing event is tissue specific and the mRNA containing the cassette exon is prevalent in brain, heart and skeletal muscle. The transcripts also differ in their 3 ’-non-coding regions by the use of two alternative poly(A) signals. Annexin VII encodes a protein with a molecular weight of approximately 51 kDa with a unique, highly hydrophobic N-terminal domain of 167 amino acids and a conserved C-terminal region of 299 amino acids. The latter domain is composed of alternating hydrophobic and hydrophilic segments. Structural analysis of the protein suggests that Annexin VII is a membrane binding protein with diverse properties, including voltage-sensitive calcium channel activity, ion selectivity and membrane fusion. Among its related pathways are Cytoskeletal Signaling and Ca, cAMP and Lipid Signaling. Gene Ontology (GO) annotations related to this gene include RNA binding and integrin binding. An important paralog of this gene is ANXA11.

[0110] ANXA8 (Annexin A8) is a protein-coding gene. This gene encodes a member of the annexin family of evolutionarily conserved Ca2+ and phospholipid-binding proteins. The encoded protein may function as an anticoagulant that indirectly inhibits the thromboplastin-specific complex. Overexpression of this gene has been associated with acute myelocytic leukemia. A highly similar duplicated copy of this gene is found in close proximity on the long ami of chromosome 10. Diseases associated with ANXA8 include Breast Adenocarcinoma and Heterophy iasis. Gene Ontology (GO) annotations related to this gene include calcium ion binding and calcium-dependent phospholipid binding. An important paralog of this gene is ANXA8L1. This protein is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastinspecific complex, which is involved in the blood coagulation cascade.

[Oil 1] ANXA8L1 (Annexin A8 Like 1) is a protein-coding gene. Among its related pathways are Prostaglandin synthesis and regulation. Gene Ontology (GO) annotations related to this gene include calcium ion binding and calcium-dependent phospholipid binding. An important paralog of this gene is ANXA8. This gene encodes a member of the annexin family of evolutionarily conserved Ca2+ and phospholipid-binding proteins. The encoded protein may function as an anticoagulant that indirectly inhibits the thromboplastin-specific complex. Overexpression of this gene has been associated with acute myelocytic leukemia. A highly similar duplicated copy of this gene is found in close proximity on the long arm of chromosome 10.

[0112] ANXA9 (Annexin A9) is a protein coding gene. The annexins are a family of calciumdependent phospholipid- binding proteins. Members of the annexin family contain 4 internal repeat domains, each of which includes a type II calcium-binding site. The calcium-binding sites are required for annexins to aggregate and cooperatively bind anionic phospholipids and extracellular matrix proteins. This gene encodes a divergent member of the annexin protein family in which all four homologous type II calcium-binding sites in the conserved tetrad core contain amino acid substitutions that ablate their function. However, structural analysis suggests that the conserved putative ion channel formed by the tetrad core is intact. Diseases associated with ANXA9 include Pemphigus and Acantholytic Acanthoma. Gene Ontology (GO) annotations related to this gene include calcium ion binding and phospholipid binding. An important paralog of this gene is ANXA2. Low affinity receptor for acetylcholine known to be targeted by disease-causing pemphigus vulgaris antibodies in keratinocytes. [0113] ANXA10 (Annexin A10) is a protein coding gene. This gene encodes a member of the annexin family. Members of this calcium-dependent phospholipid-bmding protein family play a role in the regulation of cellular growth and in signal transduction pathways. The function of this gene has not yet been determined. Diseases associated with ANXA10 include Hepatocellular Carcinoma. Gene Ontology (GO) annotations related to this gene include calcium ion binding and calcium-dependent phospholipid binding. An important paralog of this gene is ANXA4.

[0114] ANXA1 1 (Annexin Al 1 ) is a protein coding gene This gene encodes a member of the annexin family, a group of calcium-dependent phospholipid-binding proteins. Annexins have unique N-terminal domains and conserved C -terminal domains, which contain calcium-dependent phospholipid-binding sites. The encoded protein is a 56-kD antigen recognized by sera from patients with various autoimmune diseases. Several transcript variants encoding two different isoforms have been identified. Diseases associated with ANXA11 include Amy otrophic Lateral Sclerosis 23 and Inclusion Body Myopathy and Brain White Matter Abnormalities. Gene Ontology (GO) annotations related to this gene include RNA binding and calcium-dependent protein binding. An important paralog of this gene is ANXA7. Binds specifically to calcychn in a calcium-dependent manner (By similarity). Required for midbody formation and completion of the terminal phase of cytokinesis.

[0115] ANXA13 (Annexin Al 3) is a protein coding gene. This gene encodes a member of the annexin family. Members of this calcium-dependent phospholipid-binding protein family play a role in the regulation of cellular growth and in signal transduction pathways. The specific function of this gene has not yet been determined; however, it is associated with the plasma membrane of undifferentiated, proliferating endothelial cells and differentiated villus enterocytes. Alternatively spliced transcript variants encoding different isoforms have been identified. Gene Ontology (GO) annotations related to this gene include calcium ion binding and phosphatidylsenne binding. An important paralog of this gene is ANXA8.

B. Compositions for Use in Disclosed Methods

1. Recombinant Polypeptides

[0116] Disclosed herein are recombinant polypeptides. Disclosed herein are recombinant polypeptides comprising a phosphatidylserine (PS) binding domain. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain and a CD3 binding domain. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain and a CD16A binding domain. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and a third domain that binds tumor specific antigens. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and a third domain that binds an immune cell-specific antigen. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and an immune stimulatory cytokine domain. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and an immune stimulatory cytokine domain. Disclosed herein is a recombinant polypeptide comprising a phosphatidylsenne (PS) binding domain, a CD1 A binding domain, and a third domain that binds tumor specific antigens. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD 16A binding domain, and a third domain that binds an immune cell-specific antigen. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, and an immune stimulatory cytokine domain. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and an immune stimulatory cytokine domain.

[0117] In an aspect, the recombinant polypeptides disclosed herein may further comprise one or more linker peptides. In an aspect, the one or more linker peptides may operably link the domains of the recombinant polypeptide as disclosed herein. Thus, in an aspect, disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain and a CD3 binding domain, and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain and a CD16A binding domain, and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, a third domain that binds tumor specific antigens, and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, a third domain that binds an immune cell-specific antigen, and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, an immune stimulatory cytokine domain, and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, an immune stimulatory cytokine domain, and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, a third domain that binds tumor specific antigens, and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, a third domain that binds an immune cell-specific antigen, and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, an immune stimulatory cytokine domain and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, an immune stimulatory cytokine domain, and a signal peptide.

[0118] In an aspect, the recombinant polypeptides disclosed herein may further comprise a signal peptide. In an aspect, the signal peptide is a secretory signal peptide. In an aspect, the secretory signal peptide is operable to facilitate extracellular secretion of the polypeptide. Thus, in an aspect, disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain and a CD16A binding domain, and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, a third domain that binds tumor specific antigens, and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, a third domain that binds an immune cell-specific antigen, and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, an immune stimulatory cytokine domain, and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, an immune stimulatory cytokine domain, and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, a third domain that binds tumor specific antigens, and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, a third domain that binds an immune cell-specific antigen, and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, an immune stimulatory cytokine domain and a signal peptide. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserme (PS) binding domain, a CD3 binding domain, an immune stimulatory cytokine domain, and a signal peptide.

[0119] In an aspect, the recombinant polypeptides disclosed herein can comprise a signal peptide and one or more linker peptides. In an aspect, the signal peptide is a secretory signal peptide. In an aspect, the secretory' signal peptide is operable to facilitate extracellular secretion of the polypeptide. In an aspect, the one or more linker peptides may operably link the domains of the recombinant polypeptide as disclosed herein. Thus, in an aspect, disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, a signal peptide, and one or more linker peptides. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain and a CD16A binding domain, a signal peptide, and one or more linker peptides. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, a third domain that binds tumor specific antigens, a signal peptide, and one or more linker peptides. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, a third domain that binds an immune cell-specific antigen, a signal peptide, and one or more linker peptides. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, an immune stimulatory cytokine domain, a signal peptide, and one or more linker peptides. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, an immune stimulatory cytokine domain, a signal peptide, and one or more linker peptides. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, a third domain that binds tumor specific antigens, a signal peptide, and one or more linker peptides. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, a third domain that binds an immune cell-specific antigen, a signal peptide, and one or more linker peptides. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserme (PS) binding domain, a CD16A binding domain, an immune stimulatory cytokine domain a signal peptide, and one or more linker peptides. Disclosed herein is a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, an immune stimulatory cytokine domain, a signal peptide, and one or more linker peptides.

[0120] In an aspect of a disclosed recombinant polypeptide, a disclosed phosphatidylserine (PS) binding domain can comprise Annexin Al (ANXA1) or the PS-binding core domain thereof, Annexin A2 (ANXA2), Annexin A3 (ANXA3), Annexin A4 (ANXA4), Annexin A5 (ANXA5), Annexin A6 (ANXA6), Annexin A7 (ANXA7), Annexin A8 (ANXA8), Annexin A8 Like 1 (ANXA8L1), Annexin A9 (ANXA9), Annexin A10 (ANXA10), Annexin Al l (ANXA11), Annexin A13 (ANXA13), Coagulation Factor II (F2), Coagulation Factor VII (F7), Coagulation Factor IX (F9), Coagulation Factor X (F10), Advanced Glycosylation End-Product Specific Receptor (AGER/RAGE) or the extracellular domain thereof, Growth Arrest Specific 6 (GAS6), Milk Fat Globule EGF And Factor V/VIII Domain Containing (MFGE8), Adhesion G Protein Coupled Receptor Bl (BAI1/ADGRB1) or the extracellular domain thereof, Protein S (PROS), Hepatitis A Virus Cellular Receptor 1 (HAVCR1) or the extracellular domain thereof, Hepatitis A Virus Cellular Receptor 2 (HAVCR2) or the extracellular domain thereof, T-Cell Immunoglobulin And Mucin Domain Containing 4 (TIMD4), Protein Kinase C Alpha (PRKCA) or the C2 domain thereof, Synaptotagmin (SYT1) or the C2A domain thereof, Jumonji Domain- Containing Protein 6 (JMJD6), Apolipoprotein H (APO-H), Stabilin-1 (STAB1) or the extracellular domain thereof, Stabilin-2 (STAB2) or the extracellular domain thereof, Killer Cell Lectin Like Receptor Kl(KLRKl), a variant thereof, a fragment thereof, or any combination thereof.

[0121] In an aspect, a disclosed ANXA1 can comprise the amino acid sequence set forth in SEQ ID NO: 1, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 1 , or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA1 can comprise the amino acid sequence set forth in SEQ ID NO:2, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:2, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA2 can comprise the amino acid sequence set forth in SEQ ID N0:3, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA2 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:3, or a vanant thereof, or a fragment thereof. In an aspect, a disclosed ANXA3 can comprise the amino acid sequence set forth in SEQ ID NO:4, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA3 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:4, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA4 can comprise the amino acid sequence set forth in SEQ ID N0:5, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA4 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:5, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA5 can comprise the ammo acid sequence set forth in SEQ ID N0:6, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA5 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:6, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA6 can comprise the amino acid sequence set forth in SEQ ID N0:7, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA6 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:7, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA7 can comprise the amino acid sequence set forth in SEQ ID N0:8, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA7 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 8, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA8 can comprise the amino acid sequence set forth in SEQ ID N0:9, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA8 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:9, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA8L1 can comprise the amino acid sequence set forth in SEQ ID NO: 10, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA8L1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 10, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA9 can comprise the amino acid sequence set forth in SEQ ID NO: 11, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA9 signal peptide can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 11, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA10 can comprise the ammo acid sequence set forth in SEQ ID NO: 12, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA10 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 12, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA11 can comprise the amino acid sequence set forth in SEQ ID NO: 13, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA11 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 13, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA13 can comprise the amino acid sequence set forth in SEQ ID NO: 14, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ANXA13 can compnse a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 14, or a variant thereof, or a fragment thereof.

[0122] In an aspect, a disclosed ADGRB1 can comprise the amino acid sequence set forth in SEQ ID NO: 15, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ADGRB1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 15, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ADGRB1 can comprise the amino acid sequence set forth in SEQ ID NO: 16, or a variant thereof, or a fragment thereof. In an aspect, a disclosed ADGRB1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 16, or a variant thereof, or a fragment thereof. In an aspect, a disclosed APO-H can comprise the amino acid sequence set forth in SEQ ID NO: 17, or a variant thereof, or a fragment thereof. In an aspect, a disclosed APO-H can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 17, or a variant thereof, or a fragment thereof. In an aspect, a disclosed F2 can comprise the amino acid sequence set forth in SEQ ID NO: 18, or a variant thereof, or a fragment thereof. In an aspect, a disclosed F2 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 18, or a variant thereof, or a fragment thereof. In an aspect, a disclosed F7 can comprise the amino acid sequence set forth in SEQ ID NO: 19, or a variant thereof, or a fragment thereof. In an aspect, a disclosed F7 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 19, or a variant thereof, or a fragment thereof. In an aspect, a disclosed F9 can comprise the amino acid sequence set forth in SEQ ID NO:20, or a variant thereof, or a fragment thereof. In an aspect, a disclosed F9 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 20, or a variant thereof, or a fragment thereof. In an aspect, a disclosed F10 can comprise the amino acid sequence set forth in SEQ ID NO: 21, or a variant thereof, or a fragment thereof. In an aspect, a disclosed F10 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 21, or a variant thereof, or a fragment thereof. In an aspect, a disclosed GAS6 can comprise the amino acid sequence set forth in SEQ ID NO:22, or a variant thereof, or a fragment thereof. In an aspect, a disclosed GAS6 an comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:22, or a variant thereof, or a fragment thereof. In an aspect, a disclosed MFGE8 can comprise the amino acid sequence set forth in SEQ ID NO:23, or a variant thereof, or a fragment thereof. In an aspect, a disclosed MFGE8 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:23, or a variant thereof, or a fragment thereof. In an aspect, a disclosed AGER can comprise the amino acid sequence set forth in SEQ ID NO:24, or a variant thereof, or a fragment thereof. In an aspect, a disclosed AGER can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:24, or a variant thereof, or a fragment thereof. In an aspect, a disclosed AGER can comprise the amino acid sequence set forth in SEQ ID NO:25, or a variant thereof, or a fragment thereof. In an aspect, a disclosed AGER can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:25, or a variant thereof, or a fragment thereof. In an aspect, a disclosed PROS1 can comprise the amino acid sequence set forth in SEQ ID NO:26, or a variant thereof, or a fragment thereof. In an aspect, a disclosed PROS 1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:26, or a variant thereof, or a fragment thereof. In an aspect, a disclosed STAB1 can comprise the amino acid sequence set forth in SEQ ID NO:27, or a variant thereof, or a fragment thereof. In an aspect, a disclosed STAB1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:27, or a variant thereof, or a fragment thereof. In an aspect, a disclosed STAB1 can comprise the amino acid sequence set forth in SEQ ID NO:28, or a variant thereof, or a fragment thereof. In an aspect, a disclosed STAB1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:28, or a variant thereof, or a fragment thereof. In an aspect, a disclosed STAB2 can comprise the amino acid sequence set forth in SEQ ID NO:29, or a variant thereof, or a fragment thereof. In an aspect, a disclosed STAB2 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:29, or a variant thereof, or a fragment thereof. In an aspect, a disclosed STAB2 can comprise the amino acid sequence set forth in SEQ ID NO:30, or a variant thereof, or a fragment thereof. In an aspect, a disclosed STAB2 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:30, or a variant thereof, or a fragment thereof.

[0123] In an aspect, a disclosed HAVCR1 can comprise the ammo acid sequence set forth in SEQ ID NO:31, or a variant thereof, or a fragment thereof. In an aspect, a disclosed HAVCR1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 31, or a variant thereof, or a fragment thereof. In an aspect, a disclosed HAVCR1 can comprise the amino acid sequence set forth in SEQ ID NO:32, or a variant thereof, or a fragment thereof. In an aspect, a disclosed HAVCR1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 32, or a variant thereof, or a fragment thereof. In an aspect, a disclosed HAVCR2 can comprise the amino acid sequence set forth in SEQ ID NO: 33, or a variant thereof, or a fragment thereof. In an aspect, a disclosed HAVCR2 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:33, or a variant thereof, or a fragment thereof. In an aspect, a disclosed HAVCR2 can comprise the ammo acid sequence set forth in SEQ ID NO: 34, or a variant thereof, or a fragment thereof. In an aspect, a disclosed HAVCR2 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:34, or a variant thereof, or a fragment thereof. In an aspect, a disclosed TIMD4 can comprise the ammo acid sequence set forth in SEQ ID NO:35, or a variant thereof, or a fragment thereof. In an aspect, a disclosed TIMD4 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:35, or a variant thereof, or a fragment thereof. In an aspect, a disclosed TIMD4 can comprise the amino acid sequence set forth in SEQ ID NO: 36, or a variant thereof, or a fragment thereof. In an aspect, a disclosed TIMD4 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:36, or a variant thereof, or a fragment thereof. In an aspect, a disclosed PRKCA can comprise the amino acid sequence set forth in SEQ ID NO:37, or a variant thereof, or a fragment thereof. In an aspect, a disclosed PRKCA can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:37, or a variant thereof, or a fragment thereof. In an aspect, a disclosed PRKCA can comprise the amino acid sequence set forth in SEQ ID NO: 38, or a variant thereof, or a fragment thereof. In an aspect, a disclosed PRKCA can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:38, or a variant thereof, or a fragment thereof. In an aspect, a disclosed SYT1 can comprise the amino acid sequence set forth in SEQ ID NO: 39, or a variant thereof, or a fragment thereof. In an aspect, a disclosed SYT1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 39, or a variant thereof, or a fragment thereof. In an aspect, a disclosed SYT1 can comprise the amino acid sequence set forth in SEQ ID NO:40, or a variant thereof, or a fragment thereof. In an aspect, a disclosed SYT1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:40, or a variant thereof, or a fragment thereof. In an aspect, a disclosed JMJD6 can comprise the amino acid sequence set forth in SEQ ID NO:41, or a variant thereof, or a fragment thereof. In an aspect, a disclosed JMJD6 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:41, or a variant thereof, or a fragment thereof. [0124] In an aspect, a disclosed KLRK1 can comprise the amino acid sequence set forth in SEQ ID NO:205, or a variant thereof, or a fragment thereof. In an aspect, a disclosed KLRK1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:205, or a variant thereof, or a fragment thereof. In an aspect, a disclosed KLRK1 can comprise the amino acid sequence set forth in SEQ ID NO:206, or a variant thereof, or a fragment thereof. In an aspect, a disclosed KLRK1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:206, or a variant thereof, or a fragment thereof.

[0125] In an aspect a disclosed PS binding domain can comprise the VH domain or the VL domain or both, or the single-chain variable domain of bavituximab. In an aspect, a disclosed PS binding domain can comprise the sequence set forth in SEQ ID NO:55, or a variant thereof, or a fragment thereof. In an aspect, a disclosed PS binding domain can comprise the sequence set forth in SEQ ID NO: 84, or a variant thereof, or a fragment thereof. In an aspect, a disclosed PS binding domain can comprise the sequence set forth in SEQ ID NO: 85, or a variant thereof, or a fragment thereof. In an aspect, a disclosed PS binding domain can comprise the sequence set forth in SEQ ID NO: 84, or a variant thereof, or a fragment thereof and the sequence set forth in SEQ ID NO: 85, or a variant thereof, or a fragment thereof. In an aspect, a disclosed PS binding chain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 55, or a variant thereof, or a fragment thereof. In an aspect, a disclosed PS binding chain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 84, or a variant thereof, or a fragment thereof. In an aspect, a disclosed PS binding chain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 85, or a variant thereof, or a fragment thereof. In an aspect, a disclosed PS binding domain can comprise the single-chain variable domain of PGN632. In an aspect, a single-chain variable domain of PGN632 can comprise a yl heavy chain and a light chain. In an aspect, a single-chain variable domain of PGN632 can bind to cardiolipin/PS. In an aspect, a disclosed PS binding domain can comprise the single-chain variable domain of Pl. In an aspect, a single-chain variable domain of Pl can comprise a yl heavy chain and a light chain. In an aspect, a single-chain variable domain of Pl can bind to cardiolipin/PS. In an aspect, a disclosed PS binding domain can comprise the singlechain variable domain of IS4. In an aspect, a single-chain variable domain of IS4 can comprise a y3 VH1 heavy chain and a X VX2 light chain. In an aspect, a single-chain variable domain of IS4 can bind to cardiohpin/PS. In an aspect, a disclosed PS binding domain can comprise the single- chain variable domain of CL1. In an aspect, a single-chain variable domain of CL1 can comprise a y3 VH1 heavy chain and a VX3 light chain. In an aspect, a single-chain variable domain of IS4 can bind to cardiohpin/PS. In an aspect, PGN632, Pl, IS4, and CL1 are described in Moody et al. (2010) J. Exp. Med. 207(4):763-776, which is incorporated herein by reference for its teachings of these antibodies and their characteristics.

[0126] In an aspect, the recombinant polypeptide can comprise a CD3 binding domain. In an aspect of the recombinant polypeptide, a disclosed CD3 binding domain can be attached, linked, and/or conjugated by any operative means to a disclosed PS binding domain. The CD3 antigen is a surface structure associated with the T-cell receptor (TCR) to form a complex involved in antigen recognition and signal transduction. CD3 comprises four distinct chains. In mammals, the CD3 complex can comprise a CD3y chain, a CD35 chain, and two CD3e chains. These chains associate with the T-cell receptor (TCR) and the CD3-zeta (^-chain) to generate an activation signal in T lymphocytes. The TCR, CD3-zeta, and the other CD3 molecules together constitute the TCR complex. In an aspect, disclosed CD3 binding domain can comprise one or more antigen binding regions of an anti-CD3 antibody, for example a CDR, a VH region, a VL region, a heavy chain, a light chain or any combinations thereof. In an aspect, a disclosed CD3 binding domain can comprise a single chain variable region (scFv), a single chain antibody, a nanobody, a diabody, a Fab, or an F(ab')2 that specifically targets CD3. In an aspect, a disclosed CD3 binding domain can comprise a single chain variable region (scFv), a single chain antibody, a nanobody, a diabody, a Fab, or an F(ab')2 that specifically targets human CD3.

[0127] In an aspect, a disclosed CD3 binding domain can comprise a single-chain antibody (scFv) to a human CD3 protein. In an aspect, a disclosed recombinant polypeptide can comprise a homozygous dimer of a disclosed PS-binding domain linked to an scFv against CD3, which can be fused with an Fc domain. In an aspect, a disclosed recombinant polypeptide can comprise a homozygous dimer of a disclosed PS-binding domain linked to an scFv against CD3 connected through a Fc hinge region (but not a Fc domain). In an aspect, a disclosed recombinant polypeptide can comprise a disclosed PS-bindmg domain linked to an scFv against CD3, which can be fused with an Fc domain only. In an aspect, a disclosed recombinant polypeptide can comprise a heterozygous dimer of a PS-binding domain and an anti-CD3 scFv but not a Fc domain. In an aspect, a disclosed recombinant polypeptide can comprise a heterozy gous dimer of a PS-binding domain and an anti-CD3 scFv fused with Fc domain.

[0128] In an aspect, an anti-CD3 antibody can comprise OKT3 (muromab), blincyto (blinatumomab), ChAglyCD3 (otelixizumab), hOKT3yl (teplizumab), nuvion (visilizumab), NI- 0401 (foralumab), a UCHTl or humanized UCHT1 antibody, tepilizumab, TRX4 (otlixizumab), or a variant thereof, or a fragment thereof, or any combination thereof. In an aspect, an anti-CD3 antibody can comprise a OKT3 or humanized OKT3 antibody or a vanant thereof, or a fragment thereof. In an aspect, a disclosed anti-CD3 binding domain can comprise one or more anti-CD3 antibodies.

[0129] In an aspect, a disclosed CD3 binding domain can comprise CDR, a VH region, a VL region, a heavy chain, a light chain, or any combinations thereof, of an OKT3 antibody. In an aspect, a disclosed CD3 binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of a humanized OKT3 antibody. In an aspect, a disclosed CD3 binding domain can comprise the sequence set forth in any one of SEQ ID NO: 86- 88 or a variant thereof, or a fragment thereof. In an aspect, a disclosed CD3 binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 86, 87, or 88, or a fragment thereof. In an aspect, a disclosed CD3 binding domain can comprise CDR, a VH region, a VL region, a heavy chain, a light chain, or any combinations thereof, of an UCTH1 antibody. In an aspect, a disclosed CD3 binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of a humanized UCTH1 antibody. In an aspect, a disclosed CD3 binding domain can compnse the sequence set forth in any one of SEQ ID NO: 89- 91 or a variant thereof, or a fragment thereof. In an aspect, a disclosed CD3 binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO:89, 90, or 91, or a fragment thereof. In an aspect, a disclosed CD3 binding domain can comprise CDR, a VH region, a VL region, a heavy chain, a light chain, or any combinations thereof of Blinatumomab. In an aspect, a disclosed CD3 binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of a humanized Blinatumomab antibody. In an aspect, a disclosed CD3 binding domain can comprise the sequence set forth in any one of SEQ ID NO: 92-94 or a fragment thereof. In an aspect, a disclosed CD3 binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO:92, 93, or 94, or a fragment thereof. In an aspect, a disclosed CD3 binding domain can comprise CDR, a VH region, a VL region, a heavy chain, a light chain, or any combinations thereof of Otelixizumab. In an aspect, a disclosed CD3 binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of a humanized Otelixizumab antibody. In an aspect, a disclosed CD3 binding domain can comprise the sequence set forth in any one of SEQ ID NO: 95- 97 or a fragment thereof. In an aspect, a disclosed CD3 binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO:95-97, or a fragment thereof. In an aspect, a disclosed CD3 binding domain can comprise CDR, a VH region, a VL region, a heavy chain, a light chain, or any combinations thereof of Foralumab. In an aspect, a disclosed CD3 binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of a humanized Foralumab antibody. In an aspect, a disclosed CD3 binding domain can comprise the sequence set forth in any one of SEQ ID N0:98-100 or a fragment thereof. In an aspect, a disclosed CD3 binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO:98, 99, or 100, or a fragment thereof. In an aspect, a disclosed CD3 binding domain can comprise CDR, a VH region, a VL region, a heavy chain, a light chain, or any combinations thereof of Tepilizumab. In an aspect, a disclosed CD3 binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of ahumanized Tepilizumab antibody. In an aspect, a disclosed CD3 binding domain can comprise the sequence set forth in any one of SEQ ID NO: 101-103 or a fragment thereof. In an aspect, a disclosed CD3 binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO:101, 102, or 103, or a fragment thereof.

[0130] In an aspect, the recombinant polypeptide can comprise a CD16 binding domain. In an aspect of the recombinant polypeptide, a disclosed CD16 binding domain can be attached, linked, and/or conjugated by any operative means to a disclosed PS binding domain. CD 16 is the type III Fey receptor and is a cluster of differentiation molecule found on the surface of natural kller cells, neutrophils, monocyes, macrophages and certain T cells. In humans, it exists in two different forms: FcyRIIIa (CD 16a) and FcyRIIIb (CD 16b), which have 96% sequence similarity in the extracellular immunoglobulin binding regions. These receptors bind the Fc portion of IgG antibodies, which then activates antibody-dependent cell-mediated cytotoxicity (ADCC) in NK cells. Natural killer (NK) cells are lymphocytes that can mediate lysis of certain tumor cells and virus-infected cells without previous activation. They can also regulate specific humoral and cell- mediated immunity. In an aspect, disclosed recombinant polypeptide comprising a CD16 binding domain can direct NK cells to tumor cells. In addition, CD 16 can mediate the direct killing of some virally infected and cancer cells without antibodies. In an aspect, the recombinant polypeptide can comprise a CD16A binding domain. In an aspect, the recombinant polypeptide can comprise a CD16B binding domain.

[0131] In an aspect, a disclosed CD16 binding domain can comprise one or more antigen binding regions of an anti-CD16 antibody, for example a CDR, a VH region, a VL region, a heavy chain, a light chain or any combinations thereof. In an aspect, a disclosed CD 16 binding domain can comprise a single chain variable region (scFv), a single chain antibody, a nanobody, a diabody, a Fab, or an F(ab')2 that specifically targets CD16. In an aspect, a disclosed CD16 binding domain can comprise a single chain variable region (scFv), a single chain antibody, a nanobody, a diabody, a Fab, or an F(ab')2 that specifically targets human CD16A. In an aspect, a disclosed CD 16 binding domain can comprise a single-chain antibody (scFv) to a human CD3 protein. In an aspect, a disclosed recombinant polypeptide can comprise a homozygous dimer of a disclosed PS-bindmg domain linked to an scFv against CD16A, which can be fused with an Fc domain. In an aspect, a disclosed recombinant polypeptide can comprise a homozygous dimer of a disclosed PS-binding domain linked to an scFv against CD16A connected through a Fc hinge region (but not a Fc domain). In an aspect, a disclosed recombinant polypeptide can comprise a disclosed PS-binding domain linked to an scFv against CD16A, which can be fused with an Fc domain only. In an aspect, a disclosed recombinant polypeptide can comprise a heterozy gous dimer of a PS-binding domain and an anti-CD16A scFv but not a Fc domain. In an aspect, a disclosed recombinant polypeptide can comprise a heterozygous dimer of a PS-binding domain and an anti-CD16A scFv fused with Fc domain.

[0132] In an aspect, a disclosed CD16 binding domain can comprise a single-chain antibody (scFv) to a human CD 16A protein. In an aspect, a disclosed recombinant polypeptide can comprise a homozygous dimer of a disclosed PS-binding domain linked to an scFv against CD16A, which can be fused with an Fc domain. In an aspect, a disclosed recombinant polypeptide can comprise a homozygous dimer of a disclosed PS-binding domain linked to an scFv against CD16A connected through a Fc hinge region (but not a Fc domain). In an aspect, a disclosed recombinant polypeptide can comprise a disclosed PS-binding domain linked to an scFv against CD16A, which can be fused with an Fc domain only. In an aspect, a disclosed recombinant polypeptide can comprise a heterozygous dimer of a PS-binding domain and an anti-CD!A6 scFv but not a Fc domain. In an aspect, a disclosed recombinant polypeptide can comprise a heterozygous dimer of a PS-binding domain and an anti-CD16A scFv fused with Fc domain.

[0133] In an aspect, a disclosed CD16 binding domain can comprise an anti-CD16A antibody. In an aspect, a disclosed CD 16 binding domain can comprise one or more antigen binding regions of an anti-CD16A antibody, for example a CDR, a VH region, a VL region, a heavy chain, a light chain or any combinations thereof. In an aspect, an anti-CD16A antibody can comprise a 3G8, humanized 3G8-5, humanized 3G8-22, humanized 3G8-43, CB16, B73.1, DJ130c, MEM-154, A9, GMA161, AFM13, D6, El l, C21 or C28 antibody, or a variant thereof, or fragment thereof. [0134] In an aspect, a disclosed CD16 binding domain can comprise CDR, a VH region, a VL region, a heavy chain, alight chain, or any combinations thereof, of an 3G8 antibody. In an aspect, a disclosed CD 16 binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of a humanized 3G8 antibody. Examples of humanized 3G8 antibodies include but are not restricted to 3G8-5, 3G8-22 or 3G8-43. In an aspect, a disclosed CD3 binding domain can comprise the sequence set forth in any one of SEQ ID NO:104-115 or a variant thereof, or a fragment thereof. In an aspect, a disclosed CD16A binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 104-1 15, or a variant thereof, or a fragment thereof. In an aspect, a disclosed CD16A binding domain can comprise CDR, a VH region, a VL region, a heavy chain, a light chain, or any combinations thereof, of an A9 antibody. In an aspect, a disclosed CD16A binding domain can comprise a CDR, VH region, a VL region, a heavy chain, alight chain or any combinations thereof of a humanized A9 antibody. In an aspect, a disclosed CD16A binding domain can comprise the sequence set forth in any one of SEQ ID NO: 116-118 or a variant thereof, or a fragment thereof. In an aspect, a disclosed CD16A binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 116-118, or a fragment thereof. In an aspect, a disclosed CD16A binding domain can comprise CDR, a VH region, a VL region, a heavy chain, a light chain, or any combinations thereof, of an AFM13 antibody. In an aspect, a disclosed CD16A binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of a humanized AFM13 antibody. In an aspect, a disclosed CD16A binding domain can comprise the sequence set forth in any one of SEQ ID NO : 119- 121 or a variant thereof, or a fragment thereof. In an aspect, a disclosed CD16A binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ !D NO:119-121, or a fragment thereof.

[0135] In an aspect, a disclosed CD16 binding domain can comprise a anti-CD16A nanobody. In an aspect, the CD16A binding domain can comprise a nanobody against the extracellular domain of the human CD16A protein. In an aspect, the nanobody can comprise a CDR, a VH region, a VL region of one or more of 3G8, humanized 3G8-5, humanized 3G8-22, humanized 3G8-43, CB16, B73.1, DJ130c, MEM-154, A9, GMA161, or AFM13. In such embodiments, the nanobody can comprise (i) the variable regions (VH and VL) or scFv version of the 3G8 mouse anti-human CD16A antibody, or other versions that are at least 80% similar to 3G8; (ii) the variable regions (VH and VL) or scFv version of humanized scFv version of 3G8 anti-human CD16A antibody, or other versions that are at least 80% similar to humanized 3G8 (for example, GMA161 (Macrogenics)); (iii) the variable regions (VH and VL) or scFv version of the A9 mouse anti- human CD16A antibody, or other versions that are at least 80% similar to A9; (iv) the variable regions (VH and VL) or scFv version of humanized scFv version of A9 anti-human CD16A antibody, or other versions that are at least 80% similar to A9 (for example, GMA161 (e g., Schlapschy et al, Protein Engineering, Design, and Selection, 22:175-188, 2009); (v) the variable regions (VH and VL) or scFv version of fully human anti-CD16 similar to those of AFM13; (vi) ahuman single domain (VH) with the amino acid sequences of D6 and El 1 (Li et al, Experimental and Molecular Pathology, 101 : 281 -289, 2016 ); or (vii) a camel-derived single domain nanobody with the amino acid sequences of C21 and C28 (see e.g., Behar et al, Protein Engineering, Design, and Selection, 21: 1-10, 2008); and combinations thereof. Non-limiting examples of anti-CD16A nanobodies include C21, C28, D6 and El 1. In an aspect, a disclosed CD16A binding domain can comprise the sequence set forth in any one of SEQ ID NO: 122-125 or a variant thereof, or a fragment thereof. In an aspect, a disclosed CD16A binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 122-125, or a fragment thereof.

[0136] In an aspect, the recombinant polypeptide can compnse an optional third domain that binds a tumor specific antigen (TSA). In an aspect, the TSA can be a solid or a metastatic tumor TSA. Non-limiting examples of TSA include Mesothelin, TSHR, Colorectal associated antigen (CRC)- C017-1A/GA733, T-cell receptor/CD3-zeta chain, CD19, CD123, CD22, CD30, CD138, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, prostate specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, interleukin- 11 receptor a (IL-1 IRa), PSCA, PRSS21, CD24, platelet-derived growth factor receptorbeta (PDGFR-beta), S SEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1, epidermal growth factor receptor (EGFR), NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, ty rosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl- GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE, MAGE-A1, legumain, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP- 2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, NKG2D, CD38, CD33, CD72, CD79a, CD79b, RORi, MUC-16, LICAM, CD20, CD23, CD37, CD47, CA125, CD56, c-Met, EGFR, GD-3, HPV E6, HPV, E7, MUC-1, HER2, folate receptor alpha, CD97, CD171, CD179a, CD44v6, Claudin 18.2, WTI, VEGF a, VEGFR1, VEGFR2, IL-3Ral, IL-3Ra2, IL-iRa, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, ephrin A2, ephrin B2, Lewis A antigen, Lewis Y antigen, GPC3, RAGE-1, folate receptor, TR0P2, EGFRviii, LGR5, SSX2,AKAP-4, FLT3, fucosyl GMi, GM3, o-acetyl-GD2, GD2, a fragment thereof, a variant thereof, or any combination thereof In an aspect, the recombinant polypeptide can comprise a third domain comprising one or more antigen binding regions of an anti-TSA antibody, wherein the TS As are as disclosed herein. In an aspect, the recombinant polypeptide can comprise a third domain comprising a VH region and/or a VL region of the anti-TSA antibody. In an aspect, the optional third domain can comprise a single chain variable region (scFv), single chain antibody, nanobody, diabody, Fab, dsFv diabody, or an F(ab')2 of an anti-TSA antibody. In an aspect, the optional third domain can comprise an scFv antibody that targets a TSA.

[0137] In an aspect, the recombinant polypeptide can comprise an optional third domain that binds an immune cell-specific antigen. In an aspect the optional third domain may bind a T-cell, a B- cell, a dendritic cell, a tumor infiltrating lymphocyte (TIL), a CD3⁺ T-cells, genetically modified T-cells, a y5T, a double negative (CD4-CD8-) T cells, natural killer T (NKT) cells, natural killer (NK) cells, macrophages, or variants thereof, and any combination thereof, optional third domain can comprise one or more antigen binding regions of an anti-immune cell specific antibody. In an aspect, the optional third domain can comprise aVH region and/or a VL region of the anti-immune cell specific antibody. In an aspect, the optional third domain can comprise a single chain variable region (scFv), single chain antibody, nanobody, diabody, Fab, dsFv diabody, or an F(ab')2 that targets an immune cell-specific antigen.

[0138] In an aspect, a disclosed recombinant polypeptide can further comprise an immune stimulatory cytokine domain, or a variant or a fragment thereof. In an aspect, the immune stimulatory cytokine domain can comprise an L2, IL4, IL5, IL7, IL10, IL12, IL15, IL17, or IL21 domain, or a variant thereof, or a fragment thereof. In an aspect, the immune stimulatory cytokine domain can comprise an L2, IL7, IL12, IL15, or IL21 domain, or a variant thereof, or a fragment thereof. In an aspect, the immune stimulatory cytokine domain can comprise an IL15, IL15-IL15R or a variant thereof, or a fragment thereof. In an aspect, the immune stimulatory cytokine domain can comprise the sequence set forth in any one of SEQ ID NO: 126-132 or a variant thereof, or a fragment thereof. In an aspect, a disclosed immune stimulatory cytokine domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 126-132, or a variant thereof, or a fragment thereof.

[0139] In an aspect of the recombinant polypeptide, disclosed domains can be attached, linked, and/or conjugated by any operative means. Thus, the various essential and optional elements or domains of the recombinant polypeptide, for example the PS binding domain, the CD3 binding domain or the CD 16 binding domain, a tumor-specific antigen binding domain, the immune cellspecific antigen or the immune stimulatory cytokine domain can be present in any order and attached, linked, and/or conjugated by any operative means. In an aspect, the various essential and optional elements or domains of the recombinant polypeptide can be linked by one or more linker peptides in any order. Thus, in an aspect, the recombinant polypeptide can comprise one or more linker peptides. In an aspect, a disclosed linker can comprise a flexible linker. In an aspect, a flexible linker can comprise (GGGGS)n, wherein n = 1 - 8 (SEQ ID NO: 168 - SEQ ID NO: 175). In an aspect, a flexible linker can comprise (G)n, wherein n = 1 - 8 (for example, G, GG, GGG, or SEQ ID NO: 176 - SEQ ID NO: 180). In an aspect, a disclosed linker can comprise a rigid linker. In an aspect, a rigid linker can comprise (EAAAK)n, wherein n = 1 - 8 (SEQ ID NO: 181 - SEQ ID NO: 188). In an aspect, a disclosed linker can comprise a Whitlow linker. In an aspect, a Whitlow tinker can comprise (GSTSGSGKSSEGKG)n, wherein n = 1 - 8 (SEQ ID NO: 189 - SEQ ID NO: 196). In an aspect, a Whitlow linker can comprise (GSTSGSGKPGSGEGSTKG)n, wherein n = 1 - 8 (SEQ ID NO: 197 - SEQ ID NO:204). Linker peptides are known to the skilled person in the art. In an aspect, a disclosed operative means of linking the one or more domains can comprise a direct covalent bond, for example a peptide bond, a disulfide bridge or a chemical cross-linker. In an aspect, a disclosed operative means can comprise direct attachment (e g., avidimbiotin). In an aspect, one or more of disclosed domains can comprise a Fc domain of an immunoglobulin. In an aspect, a disclosed immunoglobulin can be a human immunoglobulin. In an aspect, a disclosed human immunoglobulin can be IgGl, IgG2, IgG3, IgG4, or any combination thereof. In an aspect, one or more of disclosed domains can comprise the Fc domain of IgGl. In an aspect, one or more of disclosed domains can comprise the Fc domain of IgG2. In an aspect, one or more of disclosed domains can comprise the Fc domain of IgG3. In an aspect, one or more of disclosed domains can comprise the Fc domain of IgG4. In an aspect, a disclosed recombinant polypeptide can comprise a PS-binding domain operably linked to the Fc domain of IgGl. In an aspect, a disclosed recombinant polypeptide can comprise a PS-binding domain operably linked to the Fc domain of IgG2. In an aspect, a disclosed recombinant polypeptide can comprise a PS- binding domain operably linked to the Fc domain of IgG3. In an aspect, a disclosed recombinant polypeptide can comprise a PS-binding domain operably linked to the Fc domain of IgG4. [0140] In an aspect, the recombinant polypeptides disclosed herein may further comprise a signal peptide. In an aspect, the signal peptide is a secretory signal peptide. In an aspect, the secretory signal peptide is operable to facilitate extracellular secretion of the polypeptide. In an aspect, a disclosed secretory signal peptide can be any secretion signal known to the art. In an aspect, a disclosed secretory' signal peptide can comprise the sequence set forth in SEQ ID NO: 163 - SEQ ID NO: 167.

[0141] In an aspect, the domains of a disclosed recombinant protein can retain functionality. For example, a disclosed PS binding domain protein operatively linked to disclosed CD3 or CD 16 binding domain can retain one or more desired effector functions. In an aspect, a disclosed PS binding domain protein operatively linked to a disclosed CD3 or CD 16 binding domain can retain one or more desired effector functions including, for example, pro-apoptotic and/or anti- angiogenic functions. In an aspect, a disclosed PS binding domain protein operatively linked to disclosed domains can stimulate host effector functions, can target phosphatidylserine on tumor cells (including dividing, injured, and/or stressed), can localize to targeted cells and/or targeted sites (such as a cancer cell), can exert any therapeutic effect (e.g., anti-cancer and/or anti-viral effects), or any combination thereof. In an aspect, a disclosed recombinant polypeptide can be humanized or one or more humanized components.

[0142] In an aspect, a disclosed recombinant polypeptide can comprise a homozygous dimer of a disclosed PS-binding domain linked to an scFv against CD3, which can be fused with an Fc domain. In an aspect, a disclosed recombinant polypeptide can comprise a homozygous dimer of a disclosed PS-binding domain linked to an scFv against CD3 connected through a Fc hinge region (but not a Fc domain). In an aspect, a disclosed recombinant polypeptide can comprise a disclosed PS-binding domain linked to an scFv against CD3, which can be fused with an Fc domain only. In an aspect, a disclosed recombinant polypeptide can comprise a heterozygous dimer of a PS- binding domain and an anti-CD3 scFv but not a Fc domain. In an aspect, a disclosed recombinant polypeptide can comprise a heterozygous dimer of a PS-binding domain and an anti-CD3 scFv fused with Fc domain.

[0143] In an aspect, a disclosed recombinant polypeptide can comprise a bispecific T cell engager (BITE), wherein the BITE comprises: a phosphatidylserine (PS) binding domain as disclosed herein operably linked to an anti-CD3 antibody, or a variant thereof, or a fragment thereof. A disclosed BITE can be recombinant polypeptide constructed from two different recognition domains connected by a protein linker. One of the domains recognizes a tumor surface antigen (TS A), while the other domain recognizes a T cell surface marker, in order to activate and redirect cytotoxic T cells to tumor cells. In an aspect, a disclosed recombinant polypeptide can comprise a bispecific T cell engager (BITE), wherein the BITE comprises: a phosphatidylserine (PS) binding domain as disclosed herein operably linked to an anti-CD3 scFv or a variant thereof, or a fragment thereof. In an aspect, the BITE can comprise a PS binding domain as disclosed herein operably linked to an anti-CD3 antibody comprising 0KT3 (muromab), humanized 0KT3, blincytomab (blinatumomab), ChAglyCD3 (otelixizumab), hOKT3yl (teplizumab), nuvion (visilizumab), NI-0401 (foralumab), a UCHT1 or humanized UCHT1 antibody, tepilizumab, TRX4 (otlixizumab), or a variant thereof, or a fragment thereof In an aspect, a BITE can comprise a 0KT3 or humanized 0KT3 antibody or a variant thereof, or a fragment thereof operably linked to a PS binding domain. In an aspect, the can BITE comprises the sequence set forth in any one of SEQ ID NOS: 133-146. In an aspect, a disclosed BITE can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 133-146, or a variant thereof. In an aspect, the BITE can comprise a commercially available CD3-antibody or a variant thereof, or a fragment thereof, for example blincyto (blinatumomab), ChAglyCD3 (otelixizumab), hOKT3yl (teplizumab), nuvion (visilizumab), NI-0401 (foralumab), a UCHT1 or humanized UCHT1 antibody, tepilizumab, or TRX4 (otlixizumab). In an aspect, the BITE can comprise the sequence set forth in any one of SEQ ID NOS: 145-150. In an aspect, a disclosed BITE can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 145-150, or a variant thereof, or a fragment thereof. In an aspect, a disclosed BITE can comprise a signal sequence. In an aspect, a disclosed BITE can comprise a secretory signal peptide. In an aspect, the secretory signal peptide is operable to facilitate extracellular secretion of the BITE. In an aspect, a disclosed secretory signal peptide can be any secretion signal known to the art. In an aspect, a secretory signal peptide can comprise the sequence set forth in SEQ ID NO: 163- SEQ ID NO: 167. In an aspect, the BITE can comprise one or more linker sequences. In an aspect, any linker sequence known in the art can be used to link the domains in a BITE. In an aspect, a linker can comprise the sequence set forth in SEQ ID NO: 168 - SEQ ID NO:204.

[0144] In an aspect, a disclosed recombinant polypeptide comprises is a BIKE. In an aspect, a disclosed recombinant polypeptide can comprise a bispecific NK-cell engager (BIKE), wherein the BIKE can comprise: a phosphatidylserine (PS) binding domain as disclosed herein operably linked to binding domain specific to a NK cell specific antigen, or a variant thereof, or a fragment thereof. In an aspect, a disclosed recombinant polypeptide can comprise a bispecific NK-cell engager (BIKE), wherein the BIKE can comprise: a phosphatidylserine (PS) binding domain as disclosed herein operably linked to an anti-CD16A antibody, or a variant thereof, or a fragment thereof. BIKE as used herein is a recombinant polypeptide constructed from two different recognition domains connected by a protein linker. One of the domains recognizes a tumor- associated surface antigen, while the other domain recognizes a NK cell surface marker, in order to activate and redirect cytotoxic NK-cells to tumor cells. In an aspect, a disclosed recombinant polypeptide is a BIKE, wherein the BIKE can comprise: a phosphatidylserine (PS) binding domain as disclosed herein operably linked to an anti- CD16A scFv or a variant thereof, or a fragment thereof. In an aspect, the BIKE can comprise a PS binding domain as disclosed herein operably linked to an anti-CD16A antibody comprising 3G8, humanized 3G8-5, humanized 3G8-22, humanized 3G8-43, CB16, B73.1, DJ130c, MEM-154, A9, GMA161, AFM13, D6, El l, C21 or C28 antibody, or a variant thereof, or fragment thereof. In an aspect, a BIKE can comprise a 3G8 antibody or a variant thereof, or a fragment thereof operably linked to a PS binding domain. In an aspect, a BIKE can comprise a humanized 3 G8 antibody or a variant thereof, or a fragment thereof. In an aspect, the BIKE can comprise the sequence set forth in any one of SEQ ID NOS: 151-154, or SEQ ID NO: 209. In an aspect, a disclosed BIKE can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 151-154, or SEQ ID NO: 209. In an aspect, a BIKE can comprise a AFM13 or an A9 antibody or a variant thereof, operably linked to a PS binding domain. In an aspect, a BIKE can comprise the sequence set forth in any one of SEQ ID NOS: 155 or 156. In an aspect, a disclosed BIKE can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 155 or 156. In an aspect, a BIKE can comprise a C21, C28, D6 or El 1 nanobody or a variant thereof, operably linked to a PS binding domain. In an aspect, the BIKE can comprise the sequence set forth in any one of SEQ ID NOS: 157-160. In an aspect, a disclosed BIKE can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 157 or 160. In an aspect, the BIKE can comprise a signal sequence. In an aspect, the BIKE can comprise a secretory signal peptide. In an aspect, the BIKE can comprise a signal sequence. In an aspect, the BIKE can comprise a secretory signal peptide. In an aspect, the secretory signal peptide is operable to facilitate extracellular secretion of the BIKE. In an aspect, a disclosed secretory signal peptide can be any secretion signal known to the art. In an aspect, a secretory signal peptide can comprise the sequence set forth in SEQ ID NO: 163 - SEQ ID NO: 167. In an aspect, the BIKE can comprise one or more linker sequences. In an aspect, any linker sequence known in the art can be used to link the domains in a BIKE. In an aspect, a linker can comprise the sequence set forth in SEQ ID NO: 168 - SEQ ID NO:204. In an aspect, a linker can comprise the amino acid sequence G, GG or GGG.

[0145] In an aspect, a disclosed recombinant polypeptide comprises is a TRIKE. TRIKE as used herein is a recombinant polypeptide constructed from three different recognition domains connected by protein linkers. One of the domains recognizes a tumor-associated surface antigen, the second domain recognizes a NK cell surface marker, and a third domain is an immune stimulatory cytokine. The cytokine domain helps maintain a sustained NK cell activity against tumor cells. In an aspect, a disclosed recombinant polypeptide is a TRIKE that can comprise: a phosphatidylserine (PS) binding domain as disclosed herein operably linked to binding domain specific to a NK cell specific antigen, or a variant thereof, or a fragment thereof and am immune stimulatory cytokine domain. In an aspect, a disclosed recombinant polypeptide can comprise a tri-specific NK-cell engager (TRIKE), wherein the TRIKE can comprise: a phosphatidylserine (PS) binding domain; an scFv against CD16A or a variant thereof; and an immune stimulatory cytokine domain. In an aspect, a disclosed recombinant polypeptide can comprise a BIKE as provided herein operably linked to an immune stimulatory cytokine domain. In an aspect, the immune stimulatory cytokine domain can comprise an L2, IL4, IL5, IL7, IL10, IL12, IL15, IL17, or IL21 or a variant thereof, or a fragment thereof. In an aspect, the immune stimulatory cytokine domain can comprise an IL15, IL15-IL15R or a variant thereof, or a fragment thereof. In an aspect, the TRIKE can comprise the sequence set forth in SEQ ID NOS: 161 or 162. In an aspect, a disclosed TRIKE can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 161 or 162. In an aspect, the TRIKE can comprise a signal sequence. In an aspect, the TRIKE can comprise a secretory signal peptide. In an aspect, the secretory signal peptide is operable to facilitate extracellular secretion of the TRIKE. In an aspect, a disclosed secretory signal peptide can be any secretion signal known to the art. In an aspect, a disclosed secretory signal peptide can comprise the sequence set forth in SEQ ID NO: 163 - SEQ ID NO: 167. In an aspect, the TRIKE can comprise one or more linker sequences. In an aspect, any linker sequence known in the art can be used to link the domains in a TRIKE. In an aspect, a linker can comprise the sequence set forth in SEQ ID NO: 168- SEQ ID NO:204.

[0146] In an aspect, a disclosed recombinant polypeptide can further comprise a conjugated and/or fused small molecule. In an aspect, a disclosed small molecule can be a disclosed tubulin interactor, a disclosed DNA modifier, a disclosed RNA inhibitor, or any combination thereof. In an aspect, a disclosed small molecule can be a disclosed tubulin interactor, a disclosed DNA modifier, a disclosed RNA inhibitor, or any combination thereof. [0147] In an aspect, a disclosed therapeutically effective amount or effective dose of a disclosed recombinant polypeptide or pharmaceutical composition thereof can be about 0.01 pg/kg to about 10 mg/kg per subject.

[0148] In an aspect, a disclosed recombinant polypeptide can comprise the configuration set forth in FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, or FIG. 2E. In an aspect, a disclosed recombinant polypeptide is bi-specific (e g., PS binding domain and anti-CD3) with the ability to engage a T- cell (BITE) via a binding domain that targets a T-cell specific marker, for example CD3. In an aspect, a disclosed recombinant polypeptide is bi-specific (e.g., PS binding domain and antiCD 16 A) and has the ability to engage NK cells (BIKE) via a binding domain that targets a NK cell specific marker, for example CD 16. In an aspect, a disclosed recombinant polypeptide is tn- specific (e g., PS binding domain, anti-CD16A and IL15 or IL15-IL15R) and has the ability to engage NK cells (TRIKE) via a binding domain that targets a NK cell specific marker for example CD16.

[0149] Disclosed herein is a recombinant polypeptide, comprising a phosphatidylserine (PS) binding domain, a linker, and a CD3 or CD 16 binding domain. Disclosed herein is a recombinant polypeptide, comprising a phosphatidylserine (PS) binding domain, a CD3 or CD 16 binding domain, additional tumor antigen or immune cell specific binding domains and one or more linker peptides, operably linking the domains. The domains can be linked in any order. Disclosed herein is a recombinant polypeptide, comprising a phosphatidylserme (PS) binding domain, a linker, a CD3 or CD16 binding domain and additional tumor antigen or immune cell specific binding domains. Disclosed herein is a recombinant polypeptide, comprising a phosphatidylserine (PS) binding domain, a CD3 or CD 16 binding domain, an immune stimulatory cytokine domain and one or more linker peptides, operably linking the domains. The domains can be linked in any order. In an aspect, disclosed recombinant polypeptides may also comprise a signal peptide, for example a secretory signal peptide.

[0150] In an aspect, a disclosed recombinant polypeptide can be part of an antibody drug conjugate (ADCs). In an aspect, a disclosed ADC can enable the delivery of cytotoxic payloads to PS-expressing cancer cells. In an aspect, a disclosed ADCs can act by directly targeting and then killing selected cells, affecting both the growth and behavior of cells. In an aspect, a disclosed ADC can comprise three parts including (i) a disclosed antibody specific to the target associated antigen (i.e., phosphatidylserine on a tumor cell), (ii) a payload designed to kill target cancer cells, and (iii) a chemical linker to attach the pay load to the antibody.

[0151] In an aspect, a disclosed recombinant polypeptide can be used in a disclosed method 2. Polynucleotides

[0152] Disclosed herein are polynucleotides compnsing a nucleic acid sequence encoding one or more disclosed recombinant polypeptides. Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain. Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain and a CD3 binding domain. Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain recombinant polypeptide comprising a phosphatidylserine (PS) binding domain and a CD16A binding domain. Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and a third domain that binds a tumor specific antigen. Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and a third domain that binds an immune cell-specific antigen. Disclosed herein is a polynucleotide compnsing a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and an immune stimulatory cytokine domain. Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, and an immune stimulatory cytokine domain. Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, and a third domain that binds tumor specific antigens. Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, and a third domain that binds am immune cell-specific antigen. Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD 16 binding domain, and an immune stimulatory cytokine domain. Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and an immune stimulatory cytokine domain. In an aspect, the polynucleotides disclosed herein can comprise a nucleic acid sequence that further encodes one or more linker peptides that operably link the domains of the encoded recombinant polypeptides. In an aspect, the polynucleotides disclosed herein can comprise a nucleic acid sequence that further encodes one or more linker peptides that operably link the domains of the encoded recombinant polypeptides. In an aspect, the polynucleotides disclosed herein can comprise a nucleic acid sequence that further encodes a signal peptide, for example a secretory signal peptide.

[0153] Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a PS-binding domain operably linked to a single-chain antibody (scFv) to a human CD3 protein. Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising an Annexin-bindmg domain operably linked to a single-chain antibody (scFv) to a human CD3 protein. Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a single-chain antibody (scFv) to a human CD3 protein and one or more additional immunostimulatory domains. Disclosed herein is a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising an Annexin-binding domain operably linked to a single-chain antibody (scFv) to a human CD3 protein and one or more additional immunostimulatory domains.

[0154] In an aspect, a disclosed nucleic sequence can be a DNA sequence or an RNA sequence.

[0155] In an aspect of a disclosed polynucleotide encoding a recombinant polypeptide, a disclosed PS binding domain can comprises Annexin Al (ANXA1) or the PS-bindmg core domain thereof. Annexin A2 (ANXA2), Annexin A3 (ANXA3), Annexin A4 (ANXA4), Annexin A5 (ANXA5), Annexin A6 (ANXA6), Annexin A7 (ANXA7), Annexin A8 (ANXA8), Annexin A8 Like 1 (ANXA8L1), Annexin A9 (ANXA9), Annexin A10 (ANXA10), Annexin All (ANXA11), Annexin A13 (ANXA13), Coagulation Factor II (F2), Coagulation Factor VII (F7), Coagulation Factor IX (F9), Coagulation Factor X (F10), Advanced Glycosylation End-Product Specific Receptor (AGER/RAGE) or the extracellular domain thereof, Growth Arrest Specific 6 (GAS6), Milk Fat Globule EGF And Factor V/VIII Domain Containing (MFGE8), Adhesion G Protein Coupled Receptor Bl (BAI1/ADGRB1) or the extracellular domain thereof, Protein S (PROS), Hepatitis A Virus Cellular Receptor 1 (HAVCR1) or the extracellular domain thereof. Hepatitis A Virus Cellular Receptor 2 (HAVCR2) or the extracellular domain thereof, T Cell Immunoglobulin And Mucin Domain Containing 4 (TIMD4), Protein Kinase C Alpha (PRKCA) or the C2 domain thereof, Synaptotagmin (SYT1) or the C2A domain thereof, Jumonji Domain- Containing Protein 6 (JMJD6), Apolipoprotein H (APO-H), Stabilin-1 (STAB1) or the extracellular domain thereof, Stabilin-2 (STAB2) or the extracellular domain thereof, Killer Cell Lectin Like Receptor Kl(KLRKl), a variant thereof, a fragment thereof, or any combination thereof.

[0156] In an aspect, a disclosed ANXA1 can comprise the amino acid sequence set forth in SEQ ID NO: 01 or a fragment thereof. In an aspect, a disclosed ANXA1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:01 or a fragment thereof. In an aspect, a disclosed ANXA1 can comprise the amino acid sequence set forth in SEQ ID NO:02 or a fragment thereof. In an aspect, a disclosed ANXA1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:02 or a fragment thereof. In an aspect, a disclosed ANXA2 can comprise the amino acid sequence set forth in SEQ ID NO: 03 or a fragment thereof. In an aspect, a disclosed ANXA2 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:03 or a fragment thereof. In an aspect, a disclosed ANXA3 can comprise the amino acid sequence set forth in SEQ ID NO: 04 or a fragment thereof. In an aspect, a disclosed ANXA3 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:04 or a fragment thereof. In an aspect, a disclosed ANXA4 can comprise the amino acid sequence set forth in SEQ ID NO: 05 or a fragment thereof. In an aspect, a disclosed ANXA4 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 05 or a fragment thereof. In an aspect, a disclosed ANXA5 can comprise the amino acid sequence set forth in SEQ ID NO:06 or a fragment thereof. In an aspect, a disclosed ANXA5 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:06 or a fragment thereof. In an aspect, a disclosed ANXA6 can comprise the ammo acid sequence set forth in SEQ ID NO: 07 or a fragment thereof. In an aspect, a disclosed ANXA6 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:07 or a fragment thereof. In an aspect, a disclosed ANXA7 can comprise the amino acid sequence set forth in SEQ 1D NO:08 or a fragment thereof. In an aspect, a disclosed ANXA7 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity' to the sequence set forth in SEQ ID NO:08 or a fragment thereof. In an aspect, a disclosed ANXA8 can comprise the amino acid sequence set forth in SEQ ID NO:09 or a fragment thereof. In an aspect, a disclosed ANXA8 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:09 or a fragment thereof. In an aspect, a disclosed ANXA8L1 can comprise the amino acid sequence set forth in SEQ ID NO: 10 or a fragment thereof. In an aspect, a disclosed ANXA8L1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 10 or a fragment thereof. In an aspect, a disclosed ANXA9 can comprise the amino acid sequence set forth in SEQ ID NO: 11 or a fragment thereof. In an aspect, a disclosed ANXA9 signal peptide can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 11 or a fragment thereof. In an aspect, a disclosed ANXA10 can comprise the amino acid sequence set forth in SEQ ID NO: 12 or a fragment thereof. In an aspect, a disclosed ANXA10 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 12 or a fragment thereof. In an aspect, a disclosed ANXA1 1 can comprise the amino acid sequence set forth in SEQ ID NO: 13 or a fragment thereof. In an aspect, a disclosed ANXA11 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 13 or a fragment thereof. In an aspect, a disclosed ANXA13 can comprise the amino acid sequence set forth in SEQ ID NO: 14 or a fragment thereof. In an aspect, a disclosed ANXA13 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 14 or a fragment thereof.

[0157] In an aspect, a disclosed ADGRB1 can comprise the ammo acid sequence set forth in SEQ ID NO: 15 or a fragment thereof. In an aspect, a disclosed ADGRB1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 15 or a fragment thereof. In an aspect, a disclosed ADGRB1 can comprise the amino acid sequence set forth in SEQ ID NO: 16 or a fragment thereof. In an aspect, a disclosed ADGRB1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 16 or a fragment thereof. In an aspect, a disclosed APO-H can comprise the amino acid sequence set forth in SEQ ID NO: 17 or a fragment thereof. In an aspect, a disclosed APO-H can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 17 or a fragment thereof. In an aspect, a disclosed F2 can comprise the amino acid sequence set forth in SEQ ID NO: 18 or a fragment thereof. In an aspect, a disclosed F2 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 18 or a fragment thereof. In an aspect, a disclosed F7 can comprise the amino acid sequence set forth in SEQ ID NO: 19 or a fragment thereof. In an aspect, a disclosed F7 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 19 or a fragment thereof. In an aspect, a disclosed F9 can comprise the amino acid sequence set forth in SEQ ID NO: 20 or a fragment thereof. In an aspect, a disclosed F9 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:20 or a fragment thereof. In an aspect, a disclosed F10 can comprise the amino acid sequence set forth in SEQ ID NO:21 or a fragment thereof. In an aspect, a disclosed F10 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 21 or a fragment thereof. In an aspect, a disclosed GAS6 can comprise the amino acid sequence set forth in SEQ ID NO:22 or a fragment thereof. In an aspect, a disclosed GAS6 an comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:22 or a fragment thereof. In an aspect, a disclosed MFGE8 can comprise the amino acid sequence set forth in SEQ ID NO:23 or a fragment thereof. In an aspect, a disclosed MFGE8 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:23 or a fragment thereof. In an aspect, a disclosed AGER can comprise the amino acid sequence set forth in SEQ ID NO:24 or a fragment thereof. In an aspect, a disclosed AGER can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identify to the sequence set forth in SEQ ID NO:24 or a fragment thereof. In an aspect, a disclosed AGER can comprise the amino acid sequence set forth in SEQ ID NO:25 or a fragment thereof. In an aspect, a disclosed AGER can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identify to the sequence set forth in SEQ ID NO:25 or a fragment thereof. In an aspect, a disclosed PROS1 can comprise the amino acid sequence set forth in SEQ ID NO:26 or a fragment thereof. In an aspect, a disclosed PROS 1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identify to the sequence set forth in SEQ ID NO:26 or a fragment thereof. In an aspect, a disclosed STAB1 can compnse the ammo acid sequence set forth in SEQ ID NO:27 or a fragment thereof. In an aspect, a disclosed STAB1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identify to the sequence set forth in SEQ ID NO:27 or a fragment thereof. In an aspect, a disclosed STAB1 can comprise the amino acid sequence set forth in SEQ ID NO:28 or a fragment thereof. In an aspect, a disclosed STAB1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identify to the sequence set forth in SEQ ID NO:28 or a fragment thereof. In an aspect, a disclosed STAB2 can comprise the amino acid sequence set forth in SEQ ID NO:29 or a fragment thereof. In an aspect, a disclosed STAB2 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identify to the sequence set forth in SEQ ID NO:29 or a fragment thereof. In an aspect, a disclosed STAB2 can comprise the amino acid sequence set forth in SEQ ID NO:30 or a fragment thereof. In an aspect, a disclosed STAB2 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 30 or a fragment thereof. [0158] In an aspect, a disclosed HAVCR1 can comprise the amino acid sequence set forth in SEQ ID NO:31 or a fragment thereof. In an aspect, a disclosed HAVCR1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:31 or a fragment thereof. In an aspect, a disclosed HAV CR1 can comprise the amino acid sequence set forth in SEQ ID NO: 32 or a fragment thereof. In an aspect, a disclosed HAVCR1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 32 or a fragment thereof. In an aspect, a disclosed HAVCR2 can comprise the amino acid sequence set forth in SEQ ID NO:33 or a fragment thereof. In an aspect, a disclosed HAVCR2 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 33 or a fragment thereof. In an aspect, a disclosed HAVCR2 can comprise the ammo acid sequence set forth in SEQ ID NO:34 or a fragment thereof. In an aspect, a disclosed HAVCR2 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 34 or a fragment thereof. In an aspect, a disclosed TIMD4 can comprise the amino acid sequence set forth in SEQ ID NO: 35 or a fragment thereof. In an aspect, a disclosed TIMD4 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:35 or a fragment thereof. In an aspect, a disclosed TIMD4 can comprise the amino acid sequence set forth in SEQ ID NO: 36 or a fragment thereof. In an aspect, a disclosed TIMD4 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identify to the sequence set forth in SEQ ID NO: 36 or a fragment thereof. In an aspect, a disclosed PRKCA can comprise the amino acid sequence set forth in SEQ ID NO:37 or a fragment thereof. In an aspect, a disclosed PRKCA can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identify to the sequence set forth in SEQ ID NO:37 or a fragment thereof. In an aspect, a disclosed PRKCA can comprise the amino acid sequence set forth in SEQ ID NO:38 or a fragment thereof. In an aspect, a disclosed PRKCA can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identify to the sequence set forth in SEQ ID NO:38 or a fragment thereof. In an aspect, a disclosed SYT1 can comprise the amino acid sequence set forth in SEQ ID NO:39 or a fragment thereof. In an aspect, a disclosed SYT1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:39 or a fragment thereof. In an aspect, a disclosed SYT1 can comprise the amino acid sequence set forth in SEQ ID NO:40 or a fragment thereof. In an aspect, a disclosed SYT1 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:40 or a fragment thereof. In an aspect, a disclosed JMJD6 can comprise the amino acid sequence set forth in SEQ ID NO:41 or a fragment thereof. In an aspect, a disclosed JMJD6 can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:41 or a fragment thereof.

[0159] In an aspect of a disclosed polynucleotide, a disclosed encoded PS binding domain can comprise the antigen binding domain of an antibody. In an aspect, the encoded PS binding domain can comprise the VH domain or the VL domain or both, or the single-chain variable domain of bavituximab. In an aspect, the encoded PS binding domain can comprise the sequence set forth in SEQ ID NO:55, or a variant thereof, or a fragment thereof. In an aspect, the encoded PS binding domain can comprise the sequence set forth in SEQ ID NO:84, or a variant thereof, or a fragment thereof. In an aspect, an encoded PS binding domain can comprise the sequence set forth in SEQ ID NO: 85, or a variant thereof, or a fragment thereof. In an aspect, the encoded PS binding domain can comprise the sequence set forth in SEQ ID NO:84, or a variant thereof, or a fragment thereof and the sequence set forth in SEQ ID NO: 85, or a variant thereof, or a fragment thereof. In an aspect, the encoded PS binding chain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 55, or a variant thereof, or a fragment thereof. In an aspect, the encoded PS binding chain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO:84, or a variant thereof, or a fragment thereof. In an aspect, the encoded PS binding chain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in SEQ ID NO: 85, or a variant thereof, or a fragment thereof. In an aspect, an encoded PS binding domain can comprise the single-chain variable domain of PGN632. In an aspect, a single-chain variable domain of PGN632 can comprise a > 1 heavy chain and a > light chain. In an aspect, a single-chain variable domain of PGN632 can bind to cardiolipin/PS. In an aspect, an encoded PS binding domain can comprise the single-chain variable domain of Pl. In an aspect, a single-chain variable domain of Pl can comprise a D I heavy chain and a > light chain. In an aspect, a single-cham vanable domain of Pl can bind to cardiolipin/PS. In an aspect, an encoded PS binding domain can comprise the single-chain variable domain of IS4. In an aspect, a single-chain variable domain of IS4 can comprise a D3 VH1 heavy chain and a > VD2 light chain. In an aspect, a single-chain variable domain of IS4 can bind to cardiolipin/PS. In an aspect, an encoded PS binding domain can comprise the single-chain variable domain of CL1. In an aspect, a single-chain variable domain of CL1 can comprise a > 3 VH1 heavy chain and a > VD 3 light chain. In an aspect, a single-chain variable domain of IS4 can bind to cardiolipin/PS. In an aspect, PGN632, Pl, IS4, and CL1 are described in Moody et al. (2010) J. Exp. Med. 207(4):763- 776, which is incorporated herein by reference for its teachings of these antibodies and their characteristics.

[0160] In an aspect, a disclosed polynucleotide can comprise a nucleic acid sequence encoding a PS binding domain and a CD3 binding domain. In an aspect, a disclosed encoded CD3 binding domain can comprise CDR, a VH region, a VL region, a heavy chain, a light chain, or any combinations thereof, of an OKT3 antibody. In an aspect, the encoded CD3 binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of a humanized OKT3 antibody. In an aspect, the encoded CD3 binding domain can comprise the sequence set forth in any one of SEQ ID NO: 86-88 or a variant thereof, or a fragment thereof. In an aspect, the encoded CD3 binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 86, 87, or 88, or a fragment thereof. In an aspect, the encoded CD3 binding domain can comprise CDR, a VH region, a VL region, a heavy chain, a light chain, or any combinations thereof, of an UCTH1 antibody. In an aspect, the encoded CD3 binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of a humanized UCTH1 antibody. In an aspect, the encoded CD3 binding domain can comprise the sequence set forth in any one of SEQ ID NO: 89-91 or a variant thereof, or a fragment thereof. In an aspect, the encoded CD3 binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 89, 90, or 91, or a fragment thereof. In an aspect, the encoded CD3 binding domain can comprise CDR, a VH region, a VL region, a heavy chain, a light chain, or any combinations thereof of Blinatumomab. In an aspect, the encoded CD3 binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of a humanized Blinatumomab antibody. In an aspect, the encoded CD3 binding domain can comprise the sequence set forth in any one of SEQ ID NO:92-94 or a fragment thereof. In an aspect, the encoded CD3 binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO:92, 93, or 94, or a fragment thereof. In an aspect, the encoded CD3 binding domain can comprise CDR, a VH region, a VL region, a heavy chain, a light chain, or any combinations thereof of Otelixizumab. In an aspect, the encoded CD3 binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of a humanized Otelixizumab antibody. In an aspect, the encoded CD3 binding domain can comprise the sequence set forth in any one of SEQ ID NO:95-97 or a fragment thereof. In an aspect, the encoded CD3 binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 95-97, or a fragment thereof. In an aspect, the encoded CD3 binding domain can comprise CDR, a VH region, a VL region, a heavy chain, a light chain, or any combinations thereof of Foralumab. In an aspect, the encoded CD3 binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of a humanized Foralumab antibody. In an aspect, the encoded CD3 binding domain can comprise the sequence set forth in any one of SEQ ID NO: 98-100 or a fragment thereof. In an aspect, the encoded CD3 binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO:98, 99, or 100, or a fragment thereof. In an aspect, the encoded CD3 binding domain can comprise CDR, a VH region, a VL region, a heavy chain, a light chain, or any combinations thereof of Tepilizumab. In an aspect, the encoded CD3 binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of a humanized Tepilizumab antibody. In an aspect, the encoded CD3 binding domain can comprise the sequence set forth in any one of SEQ ID NO: 101-103 or a fragment thereof. In an aspect, the encoded CD3 binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 101, 102, or 103, or a fragment thereof.

[0161] In an aspect, a disclosed polynucleotide can comprise a nucleic acid sequence encoding a PS binding domain and CD16A binding domain and a PS binding domain. In an aspect, a disclosed polynucleotide can comprise a nucleic acid sequence encoding a CD16A binding domain as set forth in any one of the sequences in SEQ ID NOS: 104-125 or a variant thereof, or a fragment thereof. In an aspect, the encoded CD16 binding domain can comprise a anti-CD16A antibody. In an aspect, the encoded CD16 binding domain can comprise one or more antigen binding regions of an anti-CD16A antibody, for example a CDR, a VH region, a VL region, a heavy chain, a light chain or any combinations thereof. In an aspect, an anti-CD16A antibody can comprise a 3G8, humanized 3G8-5, humanized 3G8-22, humanized 3G8-43, CB16, B73.1, DJ130c, MEM-154, A9, GMA161, AFM13, D6, El 1, C21, or C28 antibody, or a variant thereof, or fragment thereof. [0162] In an aspect, the encoded CD 16 binding domain can comprise CDR, a VH region, a VL region, a heavy chain, alight chain, or any combinations thereof, of an 3G8 antibody. In an aspect, the encoded CD 16 binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of a humanized 3G8 antibody. Examples of humanized 3G8 anitbodies include but are not restricted to 3G8-5, 3G8-22 or 3G8-43. In an aspect, the encoded CD3 binding domain can comprise the sequence set forth in any one of SEQ ID NO: 104- 115 or a variant thereof, or a fragment thereof. In an aspect, the encoded CD1 A binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 104-115, or a variant thereof, or a fragment thereof. In an aspect, the encoded CD16A binding domain can comprise CDR, a VH region, a VL region, a heavy chain, a light chain, or any combinations thereof, of an A9 antibody. In an aspect, the encoded CD16A binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of a humanized A9 antibody. In an aspect, the encoded CD16A binding domain can comprise the sequence set forth in any one of SEQ ID N 0 : 116- 118 or a variant thereof, or a fragment thereof. In an aspect, the encoded CD16A binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 116-118, or a fragment thereof. In an aspect, the encoded CD16A binding domain can comprise CDR, a VH region, a VL region, a heavy chain, a light chain, or any combinations thereof, of an AFM13 antibody. In an aspect, the encoded CD16A binding domain can comprise a CDR, VH region, a VL region, a heavy chain, a light chain or any combinations thereof of a humanized AFM13 antibody. In an aspect, the encoded CD16A binding domain can comprise the sequence set forth in any one of SEQ ID NO: 119-121 or a variant thereof, or a fragment thereof. In an aspect, the encoded CD16A binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 119-121, or a fragment thereof. [0163] In an aspect, a disclosed CD16 bidning domain can comprise a anti-CD16A nanobody. In an aspect, the CD16A binding domain can comprise a nanobody against the extracellular domain of the human CD16A protein. In an aspect, the nanobody can comprise a CDR, a VH region, a VL region of one or more of 3G8, humanized 3G8-5, humanized 3G8-22, humanized 3G8-43, CB16, B73.1, DJ130c, MEM-154, A9, GMA161, or AFM13. In such embodiments, the nanobody can comprise (i) the variable regions (VH and VL) or scFv version of the 3G8 mouse anti-human CD16A antibody, or other versions that are at least 80% similar to 3G8; (ii) the variable regions (VH and VL) or scFv version of humanized scFv version of 3G8 anti-human CD16A antibody, or other versions that are at least 80% similar to humanized 3G8 (for example, GMA161(Macrogenics)); (iii) the variable regions (VH and VL) or scFv version of the A9 mouse anti -human CD16A antibody, or other versions that are at least 80% similar to A9; (iv) the variable regions (VH and VL) or scFv version of humanized scFv version of A9 anti-human CD16A antibody, or other versions that are at least 80% similar to A9 (for example, GMA161( se, e g., Schlapschy et al, Protein Engineering, Design, and Selection, 22:175-188, 2009); (v) the variable regions (VH and VL) or scFv version of fully human anti-CD16 similar to those of AFM13; (vi) ahuman single domain (VH) with the amino acid sequences of D6 and El 1 (Li et al, Experimental and Molecular Pathology, 101:281-289, 2016 ); or (vii) a camel-derived single domain nanobody with the amino acid sequences of C21 and C28 (see e.g., Behar et al, Protein Engineering, Design, and Selection, 21: 1-10, 2008); and combinations thereof. Non-limiting examples of anti-CD16A nanobodies include C21, C28, D6 and El 1. In an aspect, the encoded CD16A binding domain can comprise the sequence set forth in any one of SEQ ID NO: 122-125 or a variant thereof, or a fragment thereof. In an aspect, the encoded CD16A binding domain can comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 122-125, or a fragment thereof.

[0164] In an aspect, a disclosed polynucleotide can comprise a nucleic acid sequence encoding a tumor specific antigen binding domain in addition to the PS binding domain and the CD3 or CD16A binding domain. In an aspect the encoded tumor specific antigen binding domain may bind to Mesothelin, TSHR, Colorectal associated antigen (CRC)-C017-lA/GA733, T-cell receptor/CD3-zeta chain, CD19, CD123, CD22, CD30, CD138, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, prostate specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, interleukin- 11 receptor a (IL-llRa), PSCA, PRSS21, CD24, platelet-denved growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1, epidermal growth factor receptor (EGFR), NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF -I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE, MAGE-A1, legumain, HPV E6, E7, ETV6- AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, 0Y-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, NKG2D, CD38, CD33, CD72, CD79a, CD79b, RORi, MUC-16, LICAM, CD20, CD23, CD37, CD47, CA125, CD56, c- Met, EGFR, GD-3, HPV E6, HPV, E7, MUC-1, HER2, folate receptor alpha, CD97, CD171, CD179a, CD44v6, Claudin 18.2, WTI, VEGF a, VEGFR1, VEGFR2, IL-3Ral, IL-3Ra2, IL-iRa, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, ephrin A2, ephrin B2, Lewis A antigen, Lewis Y antigen, GPC3, RAGE-1, folate receptor, TROP2, EGFRviii, LGR5, SSX2, AKAP-4, FLT3, fucosyl GMi, GM3, o-acetyl-GD2, GD2, a fragment thereof, a variant thereof, or any combination thereof.

[0165] In an aspect, a disclosed polynucleotide can comprise a nucleic acid sequence encoding an immune cell specific antigen binding domain in addition to the PS binding domain and the CD3 or CD16A binding domain. In an aspect, the immune cell specific antigen binding domain may bind a T-cell, a B-cell, a dendritic cell, a tumor infiltrating lymphocyte (TIL), a CD3⁺ T-cells, genetically modified T-cells, a y5T, a double negative (CD4-CD8-) T cells, natural killer T ( KT) cells, natural killer (NK) cells, macrophages, or variants thereof, and any combination thereof.

[0166] In an aspect, a disclosed polynucleotide can comprise a nucleic acid sequence encoding an immune stimulatory cytokine domain. In an aspect, a disclosed polynucleotide can comprise a nucleic acid sequence encoding an immune stimulatory cytokine domain as set forth in any one of the sequences in SEQ ID NOS: 127-132 or a variant thereof, or a fragment thereof. In an aspect, a disclosed polynucleotide can comprise a nucleic acid sequence encoding an immune stimulatory cytokine domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any one of the sequences set forth in SEQ ID NOS: 127-132 or a fragment thereof.

[0167] In an aspect, the polynucleotides disclosed herein can comprise a nucleic acid sequence that further encodes one or more linker peptides that operably link the domains of the encoded recombinant polypeptides. In an aspect, the linker peptide can be any linker peptide known in the art. In an aspect, the encoded linker peptide can be a flexible linker. In an aspect, a flexible linker can comprise (GGGGS)n, wherein n = 1 - 8 (SEQ ID NO: 168 - SEQ ID NO: 175). In an aspect, a flexible linker can comprise (G)n, wherein n = 1 - 8 (for example, SEQ ID NO: 176 - SEQ ID NO: 180). In an aspect, a disclosed linker can comprise a rigid linker. In an aspect, a rigid linker can comprise (EAAAK)n, wherein n = 1 - 8 (SEQ ID NO: 181 - SEQ ID NO: 188). In an aspect, a disclosed linker can comprise a Whitlow linker. In an aspect, a Whitlow linker can comprise (GSTSGSGKSSEGKG)n, wherein n = 1 - 8 (SEQ ID NO: 189 - SEQ ID NO: 196). In an aspect, a Whitlow linker can comprise (GSTSGSGKPGSGEGSTKG)n, wherein n = 1 - 8 (SEQ ID NO: 197 - SEQ ID NO:204). In an aspect, the polynucleotides disclosed herein can comprise a nucleic acid sequence that further encodes a signal peptide, for example a secretory signal peptide. In an aspect, an encoded secretory signal peptide can be any secretion signal known to the art. In an aspect, a encoded secretory signal peptide can comprise the sequence set forth in SEQ ID NO:163 - SEQ ID NO: 167.

[0168] In an aspect, a disclosed polynucleotide may further comprise a nucleic acid sequence that encodes a chimeric antigen receptor (CAR), a recombinant TCR, aP-TCR, or a yd-TCR. In an aspect, the chimeric antigen receptor (CAR) may target CD138, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, RORi, MUC-16, LICAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CD47, CA125, CD56, c-Met, EGFR, GD-3, HPV E6, HPV E7, MUC-1, HER2, folate receptor alpha, CD97, CD171, CD179a, CD44v6, Claudin 18.2, WTI, VEGF a, VEGFR1, IL-3Ral, IL-3Ra2, IL-iRa, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG- 72, CEA, ephrin A2, ephrin B2, Lewis A antigen, Lewis Y antigen, MAGE, MAGE-Ai, RAGE- 1, folate receptor , EGFRvm, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GMi, GM3, o- acetyl- GD2, TROP2, GD2, or any combination thereof.

[0169] In an aspect, a disclosed polynucleotide may further comprise one or more nucleic acid sequences that are operable as a regulatory sequence. A regulatory sequence refers to any genetic element that is known to the skilled person to drive or otherwise regulate expression of nucleic acids in a cell. Such sequences include without limitation promoters, transcription terminators, enhancers, repressors, silencers, kozak sequences, polyA sequences, and the like. A regulatory sequence can, for example, be inducible, non-inducible, constitutive, cell-cycle regulated, metabolically regulated, and the like.

[001] A regulatory sequence can be a promoter. A disclosed promoter can comprise a ubiquitous promoter, a constitutive promoter, or a tissue specific promoter. In an aspect, a disclosed promoter can be operably linked to a disclosed nucleic acid sequence encoding a disclosed recombinant polypeptide. Promoters are known to the art. In an aspect, a disclosed promoter can be a promoter/enhancer. An enhancer element is a nucleic acid sequence that functions to enhance transcription. As used herein, the terms “enhance” and “enhancement” with respect to nucleic acid expression or polypeptide production, refers to an increase and/or prolongation of steady-state levels of the indicated nucleic acid or polypeptide, e g., by at least about 2%, 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 2-fold, 2.5-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50- fold, 100-fold or more. Promoter/enhancers are known to the art. In an aspect, a disclosed promoter can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed promoter or a disclosed promoter/enhancer can be used for constitutive and efficient expression of a disclosed recombinant polypeptide. In an aspect, a disclosed promoter or a disclosed promoter/enhancer can be used for constitutive and efficient expression of a disclosed recombinant polypeptide. A promoter can be an inducible promoter (e.g, a heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc ). The promoter can be a constitutive promoter (e.g, CMV promoter, UBC promoter, CAG promoter). In some cases, the promoter can be a spatially restricted and/or temporally restricted promoter (e.g., a tissue specific promoter, a cell type specific promoter, etc.). [002] The promoter can be chosen so that it will function in the target cell(s) of interest. Tissuespecific promoters refer to promoters that have activity in only certain cell types. The use of a tissue-specific promoter in a nucleic acid expression cassette can restrict unwanted transgene expression in the unaffected tissues as well as facilitate persistent transgene expression by escaping from transgene induced host immune responses. Tissue specific promoters include, but are not limited to, immune cell specific, neuron-specific promoters, muscle-specific promoters, liver-specific promoters, skeletal muscle-specific promoters, and heart-specific promoters. Examples of liver-specific promoters include, but are not limited to, the alphal- microglobulin/bikunin enhancer/thyroid hormone-binding globulin promoter, the human albumin (hALB) promoter, the thyroid hormone-binding globulin promoter, the a- 1 -antitrypsin promoter, the bovine albumin (bAlb) promoter, the murine albumin (mAlb) promoter, the human al- antitrypsin (hAAT) promoter, the ApoEhAAT promoter composed of the ApoE enhancer and the hAAT promoter, the transthyretin (TTR) promoter, the liver fatty acid binding protein promoter, the hepatitis B virus (HBV) promoter, the DC 172 promoter consisting of the hAAT promoter and the al -microglobulin enhancer, the DC 190 promoter containing the human albumin promoter and the prothrombin enhancer, and other natural and synthetic liver-specific promoters..

[003] In other aspects, the promoter can be a constitutive promoter. Constitutive promoters refer to promoters that allow for continual transcription of its associated gene. Constitutive promoters are always active and can be used to express genes in a wide range of cells and tissues. Examples of constitutive promoters include, but are not limited to, a CMV major immediate-early enhancer/chicken beta-actin promoter, a cytomegalovirus (CMV) major immediate-early promoter, an Elongation Factor 1-a (EFl -a) promoter, a simian vacuolating virus 40 (SV40) promoter, an AmpR promoter, a PgK promoter, a human ubiquitin C gene (Ubc) promoter, a MFG promoter, a human beta actin promoter, a CAG promoter, a EGR1 promoter, a FerH promoter, a FerL promoter, a GRP78 promoter, a GRP94 promoter, a HSP70 promoter, a b-kin promoter, a murine phosphoglycerate kinase (mPGK) or human PGK (hPGK) promoter, a ROSA promoter, human Ubiquitin B promoter, a Rous sarcoma virus promoter, or any other natural or synthetic ubiquitous promoters. In an aspect, the constitutively active promoter can comprise human b- actin, human elongation factor- la, chicken b-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, or herpes simplex virus thymidine kinase. [004] Inducible promoters refer to promoters that can be regulated by positive or negative control. Factors that can regulate an inducible promoter include, but are not limited to, chemical agents (e.g., the metallothionein promoter or a hormone inducible promoter), temperature, and light.

[005] The promoters can be operably linked to one or more (e g., 2, 3, 4, 5, 6, 7, or 8) enhancer elements (e.g., a neuron-specific promoter fused to a cytomegalovirus enhancer) or combined to form a tandem promoter (e.g., neuron-specific/constitutive tandem promoter). When two or more tissue-specific promoters are present, the isolated nucleic acid can be targeted to two or more different tissues at the same time.

[006] As discussed above, a disclosed promoter can be an endogenous promoter. Endogenous refers to a disclosed promoter or disclosed promoter/enhancer that is naturally linked with its gene. In an aspect, a disclosed endogenous promoter can generally be obtained from anon-coding region upstream of a transcription initiation site of a gene (such as, for example, a disclosed phosphorylase kinase, phosphorylase, or some other enzy me involved in the glycogen metabolic pathway). In an aspect, a disclosed endogenous promoter can be used for constitutive and efficient expression of a disclosed transgene (e g., a nucleic acid sequence encoding a polypeptide capable of preventing glycogen accumulation and/or degrading accumulated glycogen). In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer.

[007] As discussed above, a disclosed promoter can be an exogenous promoter. Exogenous (or heterologous) refers to a disclosed promoter or a disclosed promoter/enhancer that can be placed in juxtaposition to a gene by means of molecular biology techniques such that the transcription of that gene can be directed by the linked promoter or linked promoter/enhancer.

[0170] In an aspect, a disclosed polynucleotide can comprise one or more cis-acting regulatory elements. Cis-regulatory elements as described herein are facilitators of co-expression and include sequences that ensure that the component nucleic acid sequences (for example, the nucleic acid encoding a disclosed recombinant polypeptide and the nucleic acid encoding a CAR or a TCR as described herein) are translated from the single mRNA transcribed from the polynucleotide. A cis-acting regulatory element can be, for example, selected from (but is not limited to) an internal ribosome entry site (IRES) sequence or a sequence encoding a 2A-self cleaving peptide. In an aspect, the nucleotide sequence inserted between each of the nucleic acid sequence is a sequence encoding a 2A self-cleaving peptide or is an IRES sequence. An IRES sequence functions by allowing the assembly of a new translation initiation complex after the ribosome dissociates from the mRNA following the synthesis of the first polypeptide. Suitable IRES sequences will be known to the skilled person and examples are further available in public databases such as IRESite: The database of experimentally verified IRES structures, described in Mokrejs et al, Nucleic Acids Res. 2006; 34(Database issue): D125-DI30, which is incorporated herein by reference in its entirety. Non-limiting examples of IRES ’s known in the art include Picomavirus IRES, Apthovirus IRES, Hepatitis A IRES, Pestivirus IRES, and Hepesvirus IRES.

[0171] In an aspect, the nucleotide sequence inserted between each of the nucleic acids is a sequence encoding a 2A self-cleaving peptide. 2A self-cleaving peptides (abbreviated herein as ”2 A peptides”) can be advantageous for expression of multi cistronic polynucleotides described herein due to their small size and self-cleavage ability, which allows for facilitation of polypeptide co-expression. 2A peptides are typically composed of 16-22 amino acids and originate from viral RNA. 2A peptide-mediated polypeptide cleavage is typically triggered by ribosomal skipping of the peptide bond between the proline (P) and glycine (G) in the C-termmal of a 2A peptide, resulting in the polypeptide located upstream of the 2A peptide to have extra amino acids on its C-terminal end while the peptide located downstream the 2A peptide has an extra proline on its N-terminal end. Examples of nucleic acid sequences encoding 2A peptides can be found in Xu Y , et al (2019), and Pincha M., et al, (2011) (supra). Non-limiting examples of suitable 2A peptides are F2A (2A peptide derived from the foot-and-mouth disease virus), E2A (2A peptide derived from the equine rhinitis virus), P2A (2A peptide derived from the porcine teschovirus-1), or T2A (2A peptide derived from the Thosea asigna virus). In an aspect, the 2A self-cleaving peptide is a F2A peptide. In an aspect, the 2A self-cleaving peptide is an E2A peptide. In an aspect, the 2A self-cleaving peptide is a P2A peptide. In an aspect, the 2A self-cleaving peptide is a T2A peptide. The skilled person understands that a polynucleotide described herein may also comprise nucleotide sequences encoding different 2A self-cleaving peptides. As anon-limiting example, in a tri cistronic construct, a P2A peptide-encoding sequence can be inserted between the nucleic acid encoding the first and the second polypeptide, and a T2A peptide-encoding sequence can be inserted between the nucleic acid encoding the second and third polypeptide. Accordingly, polynucleotides comprising nucleotide sequences encoding multiple different 2A self-cleaving peptides are also provided. A disclosed polynucleotide can comprise a P2A peptide-encoding sequence and a T2A peptide-encoding sequence.

[0172] In an aspect, a disclosed polynucleotide provided herein can comprise at least one or at least two cis-acting regulatory' elements. In an aspect, at least one cis-acting regulatory element can comprise F2A, E2A, P2A. or T2A. In an aspect at least one cis-acting regulatory element can comprise a Picomavirus IRES, Apthovirus IRES, Hepatitis A IRES, Pestivirus IRES, Hepesvirus IRES. In an aspect the polynucleotide can comprise at least a first cis-acting regulatory element and a second cis-acting regulatory element such that the first and the second cis-acting regulatory elements are each independently selected F2A, E2A, P2A, T2A or any combination thereof. In an aspect the polynucleotide can comprise at least a first cis-acting regulatory element and a second cis-acting regulatory elements such that the first and second cis-acting regulatory elements are each independently selected from Picomavirus IRES, Apthovirus IRES, Hepatitis A IRES, Pestivirus IRES, Hepesvirus IRES, and any combination thereof.

[0173] In an aspect, a disclosed polynucleotide can further comprise a transcriptional termination signal. A transcnptional termination signal can be a nucleic acid sequence that marks the end of a gene during transcription. Examples of a transcriptional termination signal include, but are not limited to, bovine growth hormone polyadenylation signal (BGHpA), Simian virus 40 polyadenylation signal (Sv40 Poly A), and a synthetic polyadenylation signal. A polyadenylation sequence can compnse the nucleic acid sequence AATAAA.

[0174] A disclosed polynucleotide can be “codon optimized” to ensure expression in a target cell or organism. As used herein, “codon optimization” can refer to a process of modifying a nucleic acid sequence for enhanced expression in disclosed engineered cells of interest by replacing one or more codons or more of the native sequence with codons that are more frequently or most frequently used in the genes of that engineered cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. As contemplated herein, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database.” Many methods and software tools for codon optimization have been reported previously. (See, for example, genomes.urv.es/OPTIMIZER/).

[0175] In an aspect, a disclosed polynucleotide can be an RNA sequence or a mRNA sequence. In an aspect the mRNA sequence can encode any one of a disclosed recombinant polypeptide.

[0176] In an aspect, a disclosed polynucleotide can be introduced to T cells and/or NK cells. In an aspect, a disclosed polynucleotide can be used to activated one or more types of immune cells (e.g., naive T cells, central memory' T cells, effector memory T cells, NK cells or combination thereol) upon antigen binding.

[0177] In an aspect, a disclosed polynucleotide can encode a recombinant polypeptide that can stimulate an effector cell mediated immune modulator response to PS-expressing tumor cells. In an aspect, a disclosed polynucleotide can encode a recombinant polypeptide that can induce a tumor reducing immune response. In an aspect, a disclosed polynucleotide can encode a recombinant polypeptide that can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed polynucleotide can encode a recombinant polypeptide that can cross-prime an antitumor T cell response. In an aspect, a disclosed polynucleotide can encode a recombinant polypeptide that can induce a tumor eliminating immune response. In an aspect, a disclosed polynucleotide can encode a recombinant polypeptide that can effect cell death of PS-expression cells. In an aspect, a disclosed polynucleotide can treat cancer.

[0178] In an aspect, a disclosed polynucleotide can encode a recombinant polypeptide that can induce cell death in the targeted cell. In an aspect, a disclosed polynucleotide can encode a recombinant polypeptide that can stimulate an effector cell mediated immune modulator response to PS-expressing tumor cells. In an aspect, a disclosed polynucleotide can encode a recombinant polypeptide that can induce a tumor reducing immune response. In an aspect, a disclosed polynucleotide can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed polynucleotide can encode a recombinant polypeptide that can cross-prime an anti-tumor T cell response. In an aspect, a disclosed polynucleotide can encode a recombinant polypeptide that can induce a tumor eliminating immune response.

[0179] In an aspect, disclosed polynucleotide can be used to treat cancer. In an aspect, a disclosed polynucleotide can improve and/or extend the survivability of the subject, can improve a subject’s quality of life, can increase and/or prolong a subject’s life span, or any combination thereof. In an aspect, a disclosed polynucleotide can be used as a DNA or an RNA therapeutic molecule in the treatment of cancer. In an aspect, a disclosed polynucleotide can be used as a DNA or an RNA therapeutic molecule in the treatment of solid and metastatic tumors. In an aspect, a disclosed polynucleotides can be transduced into an engineered cell for producing a recombinant polypeptide therapeutic. In an aspect, a disclosed polynucleotides can be transduced into a cell ex vivo and a disclosed engineered cell can be used as a cell therapeutic.

[0180] In an aspect, a disclosed polynucleotide can be used in a disclosed method.

3. Vectors

[008] Disclosed herein is a vector comprising a disclosed polynucleotide. Disclosed herein is a vector comprising a polynucleotide encoding a disclosed polypeptide. Disclosed herein is a vector comprising a polynucleotide encoding a disclosed recombinant polypeptide. Disclosed herein is a recombinant vector comprising a disclosed polynucleotide. Disclosed herein is a recombinant vector comprising a polynucleotide encoding a disclosed polypeptide. Disclosed herein is a recombinant vector comprising a polynucleotide encoding a disclosed recombinant polypeptide. [009] In any of the aspects of the present disclosure, the polynucleotide comprising the nucleic acid sequence encoding a disclosed recombinant polypeptide as provided herein can be packaged or provided in a vector (e.g., a recombinant expression vector). The term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. It will be apparent to those skilled in the art that any suitable vector can be used to deliver the isolated nucleic acids of the disclosure to the target cell(s) or subject of interest. The choice of delivery vector can be made based on a number of factors known in the art, including age and species of the target host, in vitro vs. in vivo delivery, level and persistence of expression desired, intended purpose (e.g., for therapy or enzyme production), the target cell or organ, route of delivery, size of the isolated nucleic acid, safety concerns, and the like.

[0010] The vectors can be viral or non-viral as described further below. Suitable vectors that are known in the art and that can be used to deliver, and optionally, express the nucleic acid sequence of the disclosure (e.g., viral and non-viral vectors), including, virus vectors (e.g., retrovirus, adenovirus, AAV, lentiviruses, or herpes simplex virus), non-virus vectors (e.g. plasmids, or transposons), lipid vectors, poly -lysine vectors, synthetic polyamino polymer vectors that are used with nucleic acid molecules, such as a plasmid, and the like. In some embodiments, the non-viral vector can be a polymer-based vector (e.g., polyethyleimine (PEI), chitosan, poly (DL-Lactide) (PLA), or poly (DL-lactide-co-glycoside) (PLGA), dendrimers, polymethacrylate) a peptide- based vector, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid based vector.

[0011] Other types of vectors include “plasmids”, which are circular double-stranded DNA loops into which additional nucleic acid segments can be ligated and viral vectors wherein additional nucleic acid segments can be ligated into the viral genome and which comprises the vector genome (e.g., viral DNA) packaged within a virion. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bactenal vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In some examples, the vectors, like the nucleic acid expression cassettes above, can be capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors”, or more simply “expression vectors”, which serve equivalent functions.

[0012] In an aspect, a disclosed polynucleotide comprising the nucleic acid sequence encoding a disclosed recombinant polypeptide can be incorporated into a recombinant viral vector. As used herein, the term “viral vector” refers to a virus (e.g., AAV) particle that functions as a nucleic acid delivery vehicle, and which comprises the vector genome (e.g., viral DNA) packaged within a virion. Alternatively, in some contexts, the term “vector” is used to refer to the vector genome/viral DNA alone.

[0181] In an aspect, a disclosed vector can be a transposon-based vector such as Sleeping Beauty and PiggyBac, both of which are known in the art. In an aspect, a first plasmid can be loaded with a disclosed polynucleotide encoding a disclosed recombinant polypeptide, named transposon, surrounded by inverted repeats (IRs) that contain short direct repeats (DRs), while a second plasmid encodes the enzyme (transposase) that can recognize the sequences from the first plasmid and cut the transposon out of the first plasmid. Then a disclosed sequence encoded a disclosed recombinant polypeptide can be successfully delivered into the targeted cell (e.g., a T cell or a NK cell or a macrophage) cytoplasm and inserted randomly into TA dinucleotide base pairs of the recipient DNA sequence.

[0182] In an aspect, a disclosed vector can comprise lipid and/or polymer-based nanoparticles loaded with mRNA encoding a disclosed recombinant polypeptide. In an aspect, a disclosed vector can comprise lipid and/or polymer-based nanoparticles loaded with transposon-based plasmids such as, for example, transposon-based plasmids compnsing a sequence encoding a disclosed recombinant polypeptide. In an aspect, a disclosed vector can comprise mRNA encoding a disclosed recombinant polypeptide. In an aspect, a disclosed vector can comprise lipid and/or polymer-based nanoparticles loaded with mRNA encoding a disclosed recombinant polypeptide. [0183] In an aspect, a disclosed viral vector can be an adenovirus vector, an AAV vector, aherpes simplex virus vector, a retrovirus vector, a lentivirus vector, and alphavirus vector, a flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease viral vector, a poxvirus vector, or a picomavirus vector. In an aspect, a disclosed viral vector can be an adeno-associated virus (AAV) vector. In an aspect, a disclosed AAV vector can include naturally isolated serotypes including, but not limited to, AAV1, AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAVrh39, AAVrh43, AAVcy.7 as well as bovine AAV, caprine AAV, canine AAV, equine AAV, ovine AAV, avian AAV, primate AAV, non-primate AAV, and any other virus classified by the International Committee on Taxonomy of Viruses (ICTV) as an AAV. In an aspect, an AAV capsid can be a chimera either created by capsid evolution or by rational capsid engineering from a naturally isolated AAV variants to capture desirable serotype features such as enhanced or specific tissue tropism and/or a host immune response escape. Naturally isolated AAV variants include, but not limited to, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV- 1829, AAV2 Y/F, AAV2 T/V, AAV218, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A- String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS, AAV- PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, and AAVcc.81. In an aspect, a disclosed AAV vector can be AAV-Rh74 or a related variant (e.g., capsid variants like RHM4-1). In an aspect, a disclosed AAV vector can be a self-complementary AAV as disclosed herein.

[0184] In an aspect, a disclosed vector can be a recombinant vector comprising a disclosed nucleic acid sequence. Recombinant vectors (such as recombinant viral vectors) are known to the art.

[0185] Disclosed herein is a vector comprising a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain. Disclosed herein is a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain and a CD3 binding domain. Disclosed herein is a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain recombinant polypeptide comprising a phosphatidylserine (PS) binding domain and a CD16A binding domain. Disclosed herein is a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylsenne (PS) binding domain, a CD3 binding domain, and a third domain that binds a tumor specific antigen. Disclosed herein is a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and a third domain that binds am immune cell-specific antigen. Disclosed herein is a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and an immune stimulatory cytokine domain. Disclosed herein is a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, and an immune stimulatory cytokine domain. Disclosed herein is a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, and a third domain that binds tumor specific antigens. Disclosed herein is a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD 16A binding domain, and a third domain that binds an immune cell-specific antigen. Disclosed herein is a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD 16 binding domain, and an immune stimulatory cytokine domain. Disclosed herein is a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and an immune stimulatory cytokine domain. In an aspect, the polynucleotides disclosed herein can comprise a nucleic acid sequence that further encodes one or more linker peptides that operably link the domains of the encoded recombinant polypeptides. In an aspect, the polynucleotides disclosed herein can comprise a nucleic acid sequence that further encodes one or more linker peptides that operably link the domains of the encoded recombinant polypeptides. In an aspect, the polynucleotides disclosed herein can comprise a nucleic acid sequence that further encodes a signal peptide, for example a secretory signal peptide.

[0186] In an aspect, disclosed herein is a vector comprising an polynucleotide sequence comprising a nucleic acid sequence encoding a recombinant polypeptide comprising the sequence set forth in any one of SEQ ID NOS: 133-162, or SEQ ID NO: 209. Disclosed herein is a vector comprising an polynucleotide sequence comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO: 131- 162, or SEQ ID NO: 209.

[0187] In an aspect, a disclosed vector can comprise a disclosed polynucleotide molecule encoding a disclosed recombinant polypeptide having a structure set forth in FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, or FIG. 2E

[0188] In an aspect, a disclosed vector can be used to introduce a disclosed polynucleotide to one or more cells to obtain engineered cells. In an aspect, engineered cells are discussed infra. In an aspect, a disclosed cell can comprise T cells, NK cells, PBMC, tumor infiltrating lymphocytes (TILs), macrophages, or iPSCs. In an aspect, a disclosed cell can comprise any cell capable of generating a disclosed recombinant polypeptide.

[0189] In an aspect, a disclosed vector can be used to introduce a disclosed polynucleotide comprising a nucleic acid sequence encoding a disclosed recombinant polypeptide to one or more cells to obtain engineered cells. In an aspect, a disclosed vector can be used to introduce a disclosed polynucleotide comprising a nucleic acid sequence encoding a disclosed recombinant polypeptide to one or more T cells, or NK cells or TILs.

[0190] In an aspect, a disclosed vector can be an integrating vector or a non-integrating vector. In an aspect, integration can mean that the nucleotides of nucleic acid sequence can be stably inserted into the cellular genome (e.g., covalently linked to the nucleic acid sequence within the cell’s chromosomal DNA).

[0191] In an aspect, a disclosed vector can stimulate an effector cell mediated immune modulator response to PS-expressing tumor cells. In an aspect, a disclosed vector can induce a tumor reducing immune response. In an aspect, a disclosed vector can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed vector can cross-prime an anti -tumor T cell response. In an aspect, a disclosed vector can induce a tumor eliminating immune response. In an aspect, a disclosed vector can treat cancer.

[0192] In an aspect, a disclosed vector can induce cell death in the targeted cell. In an aspect, a disclosed vector can stimulate an effector cell mediated immune modulator response to PS- expressing tumor cells. In an aspect, a disclosed vector can induce a tumor reducing immune response. In an aspect, a disclosed vector can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed vector can cross-prime an anti-tumor T cell response. In an aspect, a disclosed vector can induce a tumor eliminating immune response. In an aspect, a disclosed vector can improve and/or extend the survivability of the subject, can improve a subject’s quality of life, can increase and/or prolong a subject’s life span, or any combination thereof.

[0193] In an aspect, a disclosed vector can be used in a disclosed method.

4. Engineered Cells

[0194] Disclosed herein are engineered cells transformed or transfected with one or more of a disclosed polynucleotide or a vector comprising a disclosed polynucleotide. Thus, in an aspect, a disclosed engineered cell can comprise a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain. Disclosed herein is an engineered cell comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain and a CD3 binding domain. Disclosed herein is an engineered cell comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain recombinant polypeptide comprising a phosphatidylserme (PS) binding domain and a CD16A binding domain. Disclosed herein is an engineered cell comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and a third domain that binds a tumor specific antigen. Disclosed herein is an engineered cell comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and a third domain that binds an immune cell-specific antigen. Disclosed herein is a cell comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and an immune stimulatory cytokine domain. Disclosed herein is an engineered cell comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, and an immune stimulatory cytokine domain. Disclosed herein is an engineered cell comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, and a third domain that binds tumor specific antigens. Disclosed herein is an engineered cell comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD16A binding domain, and athird domain that binds an immune cell-specific antigen. Disclosed herein is an engineered cell comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD 16 binding domain, and an immune stimulatory cytokine domain. Disclosed herein is an engineered cell comprising a polynucleotide comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a phosphatidylserine (PS) binding domain, a CD3 binding domain, and an immune stimulatory cytokine domain. In an aspect, the polynucleotides disclosed herein can comprise a nucleic acid sequence that further encodes one or more linker peptides that operably link the domains of the encoded recombinant polypeptides. In an aspect, the polynucleotides disclosed herein can comprise a nucleic acid sequence that further encodes one or more linker peptides that operably link the domains of the encoded recombinant polypeptides. In an aspect, the polynucleotides disclosed herein can comprise a nucleic acid sequence that further encodes a signal peptide, for example a secretory signal peptide.

[0195] In an aspect, disclosed herein is an engineered cell comprising a polynucleotide sequence comprising a nucleic acid sequence encoding a recombinant polypeptide comprising the sequence set forth in any one of SEQ ID NOS: 133-162, or SEQ ID NO: 209. Disclosed herein is an engineered cell comprising a polynucleotide sequence comprising a nucleic acid sequence encoding a recombinant polypeptide comprising a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the sequence set forth in any one of SEQ ID NO:133-162, or SEQ ID NO: 209.

[0196] In an aspect, a disclosed engineered cell can comprise a disclosed polynucleotide molecule encoding a disclosed recombinant polypeptide having a structure set forth in FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, or FIG. 2E

[0197] In an aspect, a disclosed engineered cell is a prokaryotic cell comprising the polynucleotide or vector disclosed herein. In an aspect, a disclosed engineered cell in a eukaryotic cell comprising the polynucleotide or vector disclosed herein. In an aspect, a disclosed engineered cell is a mammalian cell, comprising a polynucleotide or vector described herein. In an aspect, a disclosed engineered cell is a human cell comprising a polynucleotide or vector described herein. In an aspect, a disclosed engineered cell is an immune cell comprising a polynucleotide described herein. As used herein, an “immune cell” means any cell of the immune system that originate from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells). Exemplary immune cells include CD4+ T cells, CD8+ T cells, CD4- CD8- double negative T cells, y5 T cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells can be referred to as “antigen presenting cells” or “APCs,” which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC interacts with a TCR on the surface of a T cell. In an aspect, an engineered immune cell provided herein can comprise additional edits and or modifications in comparison to naturally occurring counterparts of the same cell. In an aspect, disclosed cells can comprise T cells, B cells, natural killer (NK) cells, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, monocytes, macrophages, platelets, thymocytes, myeloid cells, or any combination thereof. In an aspect, disclosed cells can comprise any cell capable of generating a disclosed chimeric fusion protein. In an aspect, disclosed T cells and NK cells can be differentiated in vitro from a hematopoietic stem cell population (for example iPSCs) or can be obtained from a subject. In an aspect, T cells and NK cells can be obtained from, for example, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, tumors, or any combination thereof. In an aspect, disclosed T cells can be derived from one or more T cell lines available in the art. In an aspect, disclosed T cells can also be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person.

[0198] In an aspect, a disclosed engineered cell can be atumor infiltrating lymphocyte comprising the polynucleotide or vector disclosed herein. By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Thl and Thl7 CD4+ T cells, natural killer cells, dendritic cells and Ml macrophages. TILs include both primary and secondary TILs. “Primary TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs and expanded TILs (“REP TILs” or “post-REP TILs”). In certain aspect, the term “Primary TILs” can include rTILs and mixtures of eTILs and rTILs. A TIL can be isolated from an organ afflicted with a cancer. “Allogeneic TILs " refers to tumor-infiltrating lymphocytes (TILs) that are collected from a genetically different individual (donor) and then used in the treatment of another individual (recipient) with cancer. These TILs are harvested from the tumor tissue of the donor, expanded and activated in the laboratory, and then infused into the recipient to boost their immune response against cancer. “Autologous TILs” refers to tumor-infiltrating lymphocytes (TILs) that are collected from a patient’s own tumor tissue and then used in personalized cancer immunotherapy. Autologous TIL therapy is a form of adoptive cell therapy, where the patient’s immune cells are isolated, expanded, and reinfused back into the same patient to enhance the body’s ability to fight cancer. One or more cells can be isolated from an organ with a cancer that can be a brain, heart, lungs, eye, stomach, pancreas, kidneys, liver, intestines, uterus, bladder, skin, hair, nails, ears, glands, nose, mouth, lips, spleen, gums, teeth, tongue, salivary' glands, tonsils, pharynx, esophagus, large intestine, small intestine, rectum, anus, thyroid gland, thymus gland, bones, cartilage, tendons, ligaments, suprarenal capsule, skeletal muscles, smooth muscles, blood vessels, blood, spinal cord, trachea, ureters, urethra, hypothalamus, pituitary, pylorus, adrenal glands, ovaries, oviducts, uterus, vagina, mammary glands, testes, seminal vesicles, penis, lymph, lymph nodes or lymph vessels. One or more TILs can be from a brain, heart, liver, skin, intestine, lung, kidney, eye, small bowel, or pancreas. TILs can be from a pancreas, kidney, eye, liver, small bowel, lung, or heart. TILs can be from a pancreas. The one or more cells can be pancreatic islet cells, for example, pancreatic cells. In some cases, a TIL can be from a gastrointestinal cancer. A TIL culture can be prepared a number of ways. For example, a tumor can be trimmed from non- cancerous tissue or necrotic areas. A tumor can then be fragmented to about 2-3 mm in length. In some cases, a tumor can be fragmented from about 0.5 mm to about 5 mm in size, from about 1 mm to about 2 mm, from about 2 mm to about 3 mm, from about 3 mm to about 4 mm, or from about 4 mm to about 5 mm. Tumor fragments can then be cultured in vitro utilizing media and a cellular stimulating agent such as a cytokine. In some cases, IL-2 can be utilized to expand TILs from a tumor fragment. A concentration of IL-2 can be about 6000 lU/mL. A concentration of IL- 2 can also be about 2000 lU/mL, 3000 lU/mL, 4000 lU/mL, 5000 lU/mL, 6000 lU/mL, 7000 lU/mL, 8000 lU/mL, 9000 lU/mL, or up to about 10000 lU/mL. Once TILs are expanded they can be subject to in vitro assays to determine tumor reactivity. For example, TILs can be evaluated by FACs for CD3, CD4, CD8, and CD58 expression. TILs can also be subjected to cocultured, cytotoxicity, ELISA, or ELISPOT assays. In an aspect, TIL cultures can be cryopreserved or undergo a rapid expansion. A cell, such as a TIL, can be isolated from a donor of a stage of development including, but not limited to, fetal, neonatal, young and adult. In an aspect, the TIL can be transduced or transfected with a polynucleotide comprising a nucleic acid sequence expressing a disclosed polypeptide. Thus, in an aspect, a disclosed engineered cell can be a TIL.

[0199] In an aspect, any of a disclosed engineered cells can further comprise a nucleic acid sequence expressing a chimeric antigen receptor (CAR), a recombinant TCR, a^-TCR, or y3- TCR. In such an aspect, a disclosed engineered cell can express any CAR known in the art, nonlimiting examples of which include CARs targeting CD138, CD38, CD33, CDI23, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, RORi, MUC-16, LICAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CD47, CA125, CD56, c-Met, EGFR, GD-3, HPV E6, HPV E7, MUC-1, HER2, folate receptor alpha, CD97, CD 171, CD 179a, CD44v6, Claudin 18.2, WTI, VEGF a, VEGFR1, IL-3Ral, IL-3Ra2, IL-iRa, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, ephrin A2, ephrin B2, Lewis A antigen, Lewis Y antigen, MAGE, MAGE-Ai, RAGE-1, folate receptor , EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GMi, GM3, o-acetyl- GD2, TROP2, GD2, or any combination thereof.

[0200] In an aspect, a disclosed engineered cell can be transfected with a disclosed polynucleotide or vector in vitro, ex vivo or in vivo depending on the projected use of a disclosed engineered cell. For example, cells can be transduced or transfected in vitro for experimental purposes to study expression and properties of the recombinant polypeptide disclosed herein. In an aspect, engineered cells can be transduced or transfected in vitro to produce the recombinant polypeptide for laboratory ortherapeutic use. Thus, in an aspect, following transformation, transfection, and/or transduction, disclosed engineered cells can be used to generate a disclosed recombinant polypeptide. In an aspect, engineered cells can be transduced ex vivo for therapeutic purposes and used in allogenic or autogenic therapies. For example, in a aspect, a population of immune cells, for example TILs obtained from a subject can be transformed or transduced ex vivo with a disclosed polynucleotide or disclosed vector and used for adoptive cell therapy. In an aspect a cell can be contacted in vivo with a disclosed polynucleotide or vector for therapeutic purposes.

[0201] In an aspect, the current disclosure also encompasses a population of engineered cells at least one engineered cell of which comprises a polynucleotide disclosed here. In an aspect the population can only comprise one engineered cell or plurality of engineered cells as provided herein. In an aspect, cell populations can further comprise additional cells not comprising the polynucleotide provided herein. For example, in certain aspects, the cell population comprises y8 T cells, a[3 T cells, NK cells or TILs comprise one or more polynucleotides provided herein. In an aspect, a cell population as described herein can comprise engineered, TILs, T cells and other engineered or non-engineered immune system cells. In an aspect, a disclosed cell population can comprise at least 5% to 10%, or 10% to 20%, or 20% to 30%, or 30% to 40%, or 40% to 50%, or 50% to 60%, or 60% to 70%, or 70% to 80%, or 80% to 90%, or 90% to 100% of disclosed engineered cells provided herein.

[0202] In an aspects, a disclosed population of cells that co-express the CAR and y5 TCR monomers or fragments thereof exhibit enhancement of at least one or more of: proliferation, cellular survival, cytotoxicity, antitumor activity, persistence and/or tumor cell killing ability, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20- fold, at least 50-fold, at least 100-fold, or at least 1000-fold as compared to a corresponding population of cells that do not express the recombinant protein. Methods of making and using a disclosed cells or cell populations are provided infra.

[0203] In an aspect, a disclosed engineered cell can be used in a disclosed method.

5. Pharmaceutical Compositions

[0204] Disclosed herein is a pharmaceutical composition comprising, consisting, or consisting essentially of a disclosed recombinant polypeptide, and a suitable carrier. Disclosed herein is a pharmaceutical and/or pharmaceutical composition compnsmg, consisting, or consisting essentially of a disclosed polynucleotide or a disclosed vector provided herein and a suitable carrier. Disclosed herein is a pharmaceutical and/or pharmaceutical composition comprising, consisting, or consisting essentially of a disclosed engineered cell and a suitable carrier. Disclosed herein is a pharmaceutical and/or pharmaceutical composition comprising, consisting, or consisting essentially of a disclosed population of cells and a suitable carrier.

[0205] In an aspect, a disclosed pharmaceutical composition can comprise, consist of, or consist essentially of a disclosed recombinant polypeptide (for example, as provided in Section VII(B)(1)) and a pharmaceutically acceptable carrier optionally, other excipients, medicinal agents, pharmaceutical agents, stabilizing agents, buffers, earners, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. For other methods of administration, the carrier can be either solid or liquid. For inhalation administration, the carrier will be respirable, and optionally can be in solid or liquid particulate form.

[0206] In an aspect, a disclosed pharmaceutical composition can comprise, consist of, or consist essentially of a polynucleotide (for example, as provided in Section VII(B)(2)) encoding a disclosed recombinant polypeptide and a pharmaceutically acceptable carrier optionally, other excipients, medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. In an aspect, the polynucleotide can be a DNA molecule or a RNA molecule. In an aspect the polynucleotide can comprise one or more chemical modifications. Nonlimiting examples of chemical modifications can include terminal cap moieties, phosphate backbone modifications, and the like. Examples of classes of terminal cap moieties include, without limitation, inverted deoxy abasic residues, glyceryl modifications, 4', 5 '-methylene nucleotides, l-(P-D-erythrofuranosyl) nucleotides, 4'-thio nucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, a-nucleotides, modified base nucleotides, threo pentofuranosyl nucleotides, acyclic 3',4'-seco nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3 '-3 '-inverted nucleotide moieties, 3'-3'- inverted abasic moieties, 3 '-2 '-inverted nucleotide moieties, 3'-2'-inverted abasic moieties, 5'-5'- inverted nucleotide moieties, 5 '-5 '-inverted abasic moieties, 3'-5'-inverted deoxy abasic moieties, 5'-amino-alkyl phosphate, l,3-diamino-2-propyl phosphate, 3 aminopropyl phosphate, 6- aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxy propyl phosphate, 1,4-butanediol phosphate, 3'-phosphoramidate, 5' phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'- phosphate, 5'-amino, 3' -phosphor othioate, 5 '-phosphor othioate, phosphorodithioate, and bridging or non-bridging methylphosphonate or 5'-mercapto moieties. Non-limiting examples of phosphate backbone modifications (i.e., resulting in modified intemucleotide linkages) include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and alkylsilyl substitutions. For injection, the carrier will typically be a liquid. For other methods of administration, the carrier can be either solid or liquid. For inhalation administration, the carrier will be respirable, and optionally can be in solid or liquid particulate form.

[0207] In an aspect, a disclosed pharmaceutical composition can comprise, consist of, or consist essentially of a recombinant viral vector (for example, as provided in Section VII(B)(3)) encoding a disclosed recombinant polypeptide and a pharmaceutically acceptable earner optionally, other excipients, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. For other methods of administration, the carrier can be either solid or liquid. For inhalation administration, the carrier will be respirable, and optionally can be in solid or liquid particulate form.

[0208] In an aspect, a disclosed pharmaceutical composition can comprise, consist of, or consist essentially of a virus (for example, as provided in Section VII(B)(3)) encoding a disclosed recombinant polypeptide and a pharmaceutically acceptable carrier optionally, other excipients, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. For other methods of administration, the carrier can be either solid or liquid. For inhalation administration, the carrier will be respirable, and optionally can be in solid or liquid particulate form. [0209] In an aspect, a disclosed pharmaceutical composition can comprise, consist of, or consist essentially of a disclosed engineered cell (for example, as provided in Section VII(B)(4)) encoding a disclosed recombinant polypeptide and a pharmaceutically acceptable carrier optionally, other excipients, medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. For other methods of administration, the carrier can be either solid or liquid. For inhalation administration, the carrier will be respirable, and optionally can be in solid or liquid particulate form. Tn an aspect, a disclosed engineered cell can be a autologous or an allogenic immune cell. In an aspect, a disclosed engineered cell can be an autologous or an allogenic TIL.

[0210] In an aspect, a disclosed pharmaceutical composition can comprise, consist of, or consist essentially of a disclosed population of cells (for example, as provided in Section VII(B)(4)) encoding a disclosed recombinant polypeptide and a pharmaceutically acceptable carrier optionally, other excipients, medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. For other methods of administration, the earner can be either solid or liquid. For inhalation administration, the carrier will be respirable, and optionally can be in solid or liquid particulate form.

[0211] By “pharmaceutically acceptable” it is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject along with the isolated nucleic acid or vector without causing any undesirable biological effects such as toxicity. Thus, such a pharmaceutical composition can be used, for example, in transfection of a cell ex vivo or in administering an isolated nucleic acid or vector directly to a subject.

[0212] In certain aspects, compositions disclosed herein can further compromise one or more pharmaceutically acceptable diluent(s), excipient(s), and/or carrier(s). As used herein, a pharmaceutically acceptable diluent, excipient, or earner, refers to a matenal suitable for administration to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Pharmaceutically acceptable diluents, carriers, and excipients can include, but are not limited to, physiological saline, Ringer’s solution, phosphate solution or buffer, buffered saline, and other carriers known in the art.

[0213] In an aspect, pharmaceutical compositions herein can also include stabilizers, antioxidants, colorants, other medicinal or pharmaceutical agents, carriers, adjuvants, preserving agents, stabilizing agents, wetting agents, emulsifying agents, solution promoters, salts, solubilizers, antifoaming agents, antioxidants, dispersing agents, surfactants, or any combination thereof. Herein, the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for composition and administration of drugs can be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0214] In certain aspects, pharmaceutical compositions descnbed herein can be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries to facilitate processing of genetically modified endothelial progenitor cells into preparations which can be used phamiaceutically. In an aspect, any of the well-known techniques, carriers, and excipients can be used as suitable and/or as understood in the art.

[0215] In certain aspects, pharmaceutical compositions described herein can be an aqueous suspension comprising one or more polymers as suspending agents. In an aspect, polymers that can comprise pharmaceutical compositions described herein include: water-soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose; water-insoluble polymers such as cross-linked carboxyl-contaming polymers; mucoadhesive polymers, selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid/butyl acrylate copolymer, sodium alginate, and dextran; or a combination thereof. In an aspect, pharmaceutical compositions disclosed herein can comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of polymers as suspending agent(s) by total weight of the composition. In an aspect, pharmaceutical compositions disclosed herein can comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of polymers as suspending agent(s) by total weight of the composition.

[0216] In certain aspects, pharmaceutical compositions disclosed herein can comprise a viscous composition. In an aspect, viscosity of composition herein can be increased by the addition of one or more gelling or thickening agents. In an aspect, compositions disclosed herein can comprise one or more gelling or thickening agents in an amount to provide a sufficiently viscous composition to remain on treated tissue. In an aspect, pharmaceutical compositions disclosed herein can comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of gelling or thickening agent(s) by total weight of the composition. In an aspect, pharmaceutical compositions disclosed herein can comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of gelling or thickening agent(s) by total weight of the composition. In an aspect, suitable thickening agents for use herein can be hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate. In other aspects, viscosity enhancing agents can be acacia (gum arabic), agar, aluminum magnesium silicate, sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, Carbopol, xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia, chitin, carboxymethylated chitosan, chondrus, dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthum gum, gum tragacanth, ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether/maleic anhydride copolymer (PVM/MA), poly(methoxyethyl methacrylate). poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethylcellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® (dextrose, maltodextrin and sucralose), or any combination thereof.

[0217] In an aspect, a disclosed pharmaceutical compositions can comprise additional agents or additives selected from a group including surface-active agents, detergents, solvents, acidifying agents, alkalizing agents, buffering agents, tonicity modifying agents, ionic additives effective to increase the ionic strength of the solution, antimicrobial agents, antibiotic agents, antifungal agents, antioxidants, preservatives, electrolytes, antifoaming agents, oils, stabilizers, enhancing agents, and the like. In an aspect, pharmaceutical compositions disclosed herein can comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of one or more agents by total weight of the composition. In an aspect, pharmaceutical compositions disclosed herein can comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more agents by total weight of the composition. In an aspect, one or more of these agents can be added to improve the performance, efficacy, safety, shelf-life and/or other property of a disclosed muscarinic antagonist composition. In an aspect, additives can be biocompatible, without being harsh, abrasive, and/or allergenic.

[0218] In certain aspects, pharmaceutical compositions disclosed herein can comprise one or more acidifying agents. As used herein, “acidifying agents” refers to compounds used to provide an acidic medium. Such compounds include, by way of example and without limitation, acetic acid, amino acid, citric acid, fumaric acid and other alpha hydroxy acids, such as hydrochloric acid, ascorbic acid, and nitric acid and others known to those of ordinary skill in the art. In an aspect, any pharmaceutically acceptable organic or inorganic acid can be used. In an aspect, pharmaceutical compositions disclosed herein can comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more acidifying agents by total weight of the composition. In an aspect, pharmaceutical compositions disclosed herein can comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more acidifying agents by total weight of the composition.

[0219] In certain aspects, pharmaceutical compositions disclosed herein can comprise one or more alkalizing agents. As used herein, “alkalizing agents” are compounds used to provide alkaline medium. Such compounds include, by way of example and without limitation, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, and trolamine and others known to those of ordinary skill in the art. In an aspect, any pharmaceutically acceptable organic or inorganic base can be used. In an aspect, pharmaceutical compositions disclosed herein can comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more alkalizing agents by total weight of the composition. In an aspect, pharmaceutical compositions disclosed herein can comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more alkalizing agents by total weight of the composition.

[0220] In certain aspects, pharmaceutical compositions disclosed herein can comprise one or more antioxidants. As used herein, “antioxidants” are agents that inhibit oxidation and thus can be used to prevent the detenoration of preparations by the oxidative process. Such compounds include, by way of example and without limitation, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophophorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite and other materials known to one of ordinary skill in the art. In an aspect, pharmaceutical compositions disclosed herein can comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more antioxidants by total weight of the composition. In an aspect, pharmaceutical compositions disclosed herein can comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more antioxidants by total weight of the composition. [0221] In certain aspects, pharmaceutical compositions disclosed herein can comprise a buffer system. As used herein, a “buffer system” is a composition comprised of one or more buffering agents wherein “buffering agents” are compounds used to resist change in pH upon dilution or addition of acid or alkali. Buffering agents include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate and other materials known to one of ordinary' skill in the art. In an aspect, any pharmaceutically acceptable organic or inorganic buffer can be used. In an aspect, pharmaceutical compositions disclosed herein can comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more buffering agents by total weight of the composition. In an aspect, pharmaceutical compositions disclosed herein can comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more buffering agents by total weight of the composition.

[0222] In an aspect, the amount of one or more buffering agents can depend on the desired pH level of a composition. In an aspect, pharmaceutical compositions disclosed herein can have a pH of about 6 to about 9. In an aspect, pharmaceutical compositions disclosed herein can have a pH greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6.

[0223] In certain aspects, pharmaceutical compositions disclosed herein can comprise one or more preservatives. As used herein, “preservatives” refers to agents or combination of agents that inhibits, reduces or eliminates bacterial growth in a pharmaceutical dosage form. Non-limiting examples of preservatives include Nipagin, Nipasol, isopropyl alcohol and a combination thereof. In an aspect, any pharmaceutically acceptable preservative can be used. In an aspect, pharmaceutical compositions disclosed herein can comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more preservatives by total weight of the composition. In an aspect, pharmaceutical compositions disclosed herein can comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more preservatives by total weight of the composition.

[0224] In certain aspects, pharmaceutical compositions disclosed herein can comprise one or more surface-acting reagents or detergents. In an aspect, surface-acting reagents or detergents can be synthetic, natural, or semi-synthetic. In an aspect, compositions disclosed herein can comprise anionic detergents, cationic detergents, zwitterionic detergents, ampholytic detergents, amphoteric detergents, nonionic detergents having a steroid skeleton, or a combination thereof. In an aspect, pharmaceutical compositions disclosed herein can comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more surface-acting reagents or detergents by total weight of the composition. In an aspect, pharmaceutical compositions disclosed herein can comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more surface-acting reagents or detergents by total weight of the composition.

[0225] In certain aspects, pharmaceutical compositions disclosed herein can comprise one or more stabilizers. As used herein, a “stabilizer” refers to a compound used to stabilize an active agent against physical, chemical, or biochemical process that would otherwise reduce the therapeutic activity of the agent. Suitable stabilizers include, by way of example and without limitation, succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycols, sodium caprylate and sodium saccharin and others known to those of ordinary skill in the art. In an aspect, pharmaceutical compositions disclosed herein can comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more stabilizers by total weight of the composition. In an aspect, pharmaceutical compositions disclosed herein can comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more stabilizers by total weight of the composition.

[0226] In an aspect, pharmaceutical compositions disclosed herein can comprise one or more tonicity agents. As used herein, a “tonicity agents” refers to a compound that can be used to adjust the tonicity of the liquid composition. Suitable tonicity agents include, but are not limited to, glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose and others known to those or ordinary skill in the art. Osmolarity in a composition can be expressed in milliosmoles per liter (mOsm/L). Osmolarity can be measured using methods commonly known in the art. In an aspect, a vapor pressure depression method is used to calculate the osmolarity of the compositions disclosed herein. In an aspect, the amount of one or more tonicity agents comprising a pharmaceutical composition disclosed herein can result in a composition osmolarity of about 150 mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 350 mOsm/L, about 280 mOsm/L to about 370 mOsm/L or about 250 mOsm/L to about 320 mOsm/L. In an aspect, a composition herein can have an osmolality ranging from about 100 mOsm/kg to about 1000 mOsm/kg, from about 200 mOsm/kg to about 800 mOsm/kg, from about 250 mOsm/kg to about 500 mOsm/kg, or from about 250 mOsm/kg to about 320 mOsm/kg, or from about 250 mOsm/kg to about 350 mOsm/kg or from about 280 mOsm/kg to about 320 mOsm/kg. In an aspect, a pharmaceutical composition described herein can have an osmolarity of about 100 mOsm/L to about 1000 mOsm/L, about 200 mOsm/L to about 800 mOsm/L, about 250 mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 350 mOsm/L, about 250 mOsm/L to about 320 mOsm/L, or about 280 mOsm/L to about 320 mOsm/L. In an aspect, pharmaceutical compositions disclosed herein can comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more tonicity modifiers by total weight of the composition. In an aspect, pharmaceutical compositions disclosed herein can comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more tonicity modifiers by total weight of the composition.

[0227] The pharmaceutical carriers, diluents or excipients suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy synngabihty exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0228] For purposes of intramuscular injection, solutions in an adjuvant such as sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose. Solutions of recombinant viral vector (e.g., rAAV) as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxpropylcellulose. A dispersion of recombinant viral vector (e.g., rAAV) can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.

[0229] Pharmaceutical compositions can be prepared as injectable compositions or as topical compositions to be delivered to the subject by transdermal transport. Numerous compositions for both intramuscular injection and transdermal transport have been previously developed and can be used in the practice of the invention. The recombinant viral vector can be used with any pharmaceutically acceptable carrier and/or excipient for ease of administration and handling.

[0230] In an aspect, a disclosed pharmaceutical composition can comprise (i) one or more active agents, (ii) biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof.

[0231] In an aspect, a disclosed pharmaceutical composition can further comprise one or more anti-inflammatory agents. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and my cophenolate.

[0232] In an aspect, NSAIDs can comprise ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as rofecoxib and celecoxib, sialylates, or any combination thereof. In an aspect, analgesics can comprise acetaminophen, oxycodone, tramadol, proporxyphene hydrochloride, or any combination thereof. In an aspect, glucocorticoids can comprise cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, or any combination thereof. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists (e.g., etanercept, adalimumab, and infliximab, chemokine inhibitors and adhesion molecule inhibitors. In an aspect, biological response modifiers can comprise monoclonal antibodies as well as recombinant forms of molecules. In an aspect, exemplary disease-modifying anti-rheumatic drugs (DMARDs) can comprise include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), minocycline, or any combination thereof

[0233] In an aspect, a disclosed chemotherapeutic agent in a disclosed pharmaceutical composition can comprise an anthracy cline, a vmca alkaloid, an alkylating agent, an immune cell antibody, an antimetabolite, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor, an immunomodulator, or any combination thereof. In an aspect, a disclosed chemotherapeutic agent can comprise 5 -fluorouracil (Adrucil, Efudex), 6- mercaptopurine (Purinethol), 6-thioguanine, aclarubicin or aclacinomycin A, alemtuzamab (Lemtrada), anastrozole (Arimidex), bicalutamide (Casodex), bleomycin sulfate (Blenoxane), bortezomib (Velcade), busulfan (Myleran), busulfan injection (Busulfex), capecitabine (Xeloda), carboplatin (Paraplatin), carmustine (BiCNU), chlorambucil (Leukeran), cisplatin (Platinol), cladribine (Leustatin), Cosmegan, cyclophosphamide (Cytoxan or Neosar), cyclophosphamide, cytarabine liposome injection (DepoCyt), cytarabine, cytosine arabinoside (Cytosar-U), dacarbazine (DTIC-Dome), dactinomycin (Cosmegen), daunorubicin citrate liposome injection (DaunoXome), daunorubicin hydrochloride (Cerubidine), dexamethasone, docetaxel (Taxotere), doxorubicin hydrochloride (Adriamycin, Rubex), etoposide (Vepesid), fludarabine phosphate (Fludara), flutamide (Eulexin), folic acid antagonists, gemcitabine (difluorodeoxycitidine), gemtuzumab, gliotoxin, hydroxyurea (Hydrea), Idarubicin (Idamycin), ifosfamide (IFEX), ifosfamide, irinotecan (Camptosar), L-asparaginase (ELSPAR), lenalidomide), leucovorin calcium, melphalan (Alkeran), melphalan, methotrexate (Folex), mitoxantrone (Novantrone), mylotarg, N4-pentoxy carbonyl-5 deoxy-5 -fluorocytidine, nab-paclitaxel (Abraxane), paclitaxel (Taxol), pentostatin, phoenix (Y ttrium90/MX-DTPA), polifeprosan 20 with carmustine implant (Gliadel), purine analogs and adenosine deaminase inhibitors (fludarabine), pyrimidine analogs, rituximab, tamoxifen citrate (Nolvadex), temozolomide), teniposide (Vumon), tezacitibine, thalidomide or a thalidomide derivative, thiotepa, tirapazamine (Tirazone), topotecan hydrochloride for injection (Hy camptin), tositumomab), vinblastine (Velban), vinblastine, vincristine (Oncovin), vindesine, vinorelbine (Navelbine), or any combination thereof.

[0234] In an aspect, a disclosed pharmaceutical composition can comprise an anti-chemokine therapy that enhances the resident memory T cell formations in tumor-free tissues. In an aspect, a disclosed anti-chemokine therapy can comprise one or more antibodies against CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CCL25, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CCR2, CCR5, CCR7, CCR8, CCR9, CXCR3, CXCR4, CXCR5, CX3CL1, CX3CR1, or any combination thereof.

[0235] In an aspect, a disclosed pharmaceutical composition can further comprise abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomabm bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farietuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxelumomab, namatumab, naplumomab, necilumumab, nimotuzumab, nofetumomab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab, pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab, radretumab, rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab, siltuximab, simtuzumab, solitomab, tacatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49, 3F8, or any combination thereof.

[0236] In an aspect, a disclosed pharmaceutical composition can stimulate an effector cell mediated immune response to PS-expressing tumor cells. In an aspect, a disclosed pharmaceutical composition can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation in a subject having cancer. In an aspect, metabolic dysregulation can be associated with cancer or cancerous cells. In an aspect, following administration of a disclosed pharmaceutical composition, cell death of PS-expressing cancer cells can be effected.

[0237] In an aspect, a disclosed pharmaceutical composition can be prepared for systemic or direct administration. In an aspect, a disclosed pharmaceutical composition can be prepared for oral administration, intravenous administration, intratumoral administration, intraperitoneal administration, or any combination thereof. In an aspect, a disclosed pharmaceutical composition can be prepared for any method of administration disclosed herein. In an aspect, a disclosed pharmaceutical composition can be prepared for administration via multiple routes either concurrently or sequentially. For example, in an aspect, a disclosed pharmaceutical composition can be first administered intratumorally and then be administered intravenously. In an aspect, a disclosed pharmaceutical composition can be first administered intratumorally and then be administered orally. A skilled clinical can determine the best route of administration for a subject at a given time.

[0238] In an aspect, a disclosed pharmaceutical composition can comprise one or more immune modulators. In an aspect, a disclosed pharmaceutical composition can comprise one or more proteasome inhibitors. In an aspect, a disclosed pharmaceutical composition can comprise one or more immunosuppressives or immunosuppressive agents. In an aspect, an immunosuppressive agent can be anti-thymocyte globulin (ATG), cyclosporine (CSP), my cophenolate mofetil (MMF), or a combination thereof. In an aspect, a disclosed pharmaceutical composition can comprise an anaplerotic agent (such as, for example, C7 compounds like triheptanom or MCT).

[0239] In an aspect, a disclosed pharmaceutical composition can comprise an RNA therapeutic. An RNA therapeutic can comprise RNA-mediated interference (RNAi) and/or antisense oligonucleotides (ASO). In an aspect, a disclosed RNA therapeutic can be directed at any protein or enzy me that is overexpressed or is overactive due to a missing, deficient, and/or mutant protein or enzyme (such as, for example, a missing, deficient, and/or mutant protein or enzyme related to cancer and/or associated with cancerous cells). In an aspect, a disclosed RNA therapeutic can be directed at any protein or enzyme that is overexpressed or is overactive and related to cancer and/or associated with cancerous cells.

[0240] In an aspect, a disclosed therapeutically effective amount or effective dose of a disclosed recombinant polypeptide or pharmaceutical composition thereof can be about 0.01 pg/kg to about 10 mg/kg per subject.

[0241] In an aspect, a disclosed pharmaceutically acceptable carrier can comprise any disclosed carrier. In an aspect, a disclosed pharmaceutically acceptable carrier can comprise any disclosed excipient.

[0242] In an aspect, a disclosed pharmaceutical composition can stimulate an effector cell mediated immune modulator response to PS-expressing tumor cells. In an aspect, a disclosed pharmaceutical composition can induce a tumor reducing immune response. In an aspect, a disclosed pharmaceutical composition can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed pharmaceutical composition can cross-prime an anti-tumor T cell response. In an aspect a disclosed pharmaceutical composition can induce a tumor eliminating immune response. In an aspect, a disclosed pharmaceutical composition can treat cancer.

[0243] In an aspect, a disclosed pharmaceutical composition can induce cell death in the targeted cell. In an aspect, a disclosed pharmaceutical composition can stimulate an effector cell mediated immune modulator response to PS-expressing tumor cells. In an aspect, a disclosed pharmaceutical composition can induce a tumor reducing immune response. In an aspect, a disclosed pharmaceutical composition can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed pharmaceutical composition can cross-prime an anti-tumor T cell response. In an aspect, a disclosed pharmaceutical composition can induce a tumor eliminating immune response. In an aspect, a disclosed pharmaceutical composition can treat cancer. In an aspect, a disclosed pharmaceutical composition can improve and/or extend the survivability of the subject, can improve a subject’s quality of life, can increase and/or prolong a subject’s life span, or any combination thereof. [0244] In an aspect, a disclosed pharmaceutical composition can be used in a disclosed method.

6. Animals

[0245] Disclosed herein are animals used to validate the efficacy and/or safety of one or more disclosed recombinant polypeptides, one or more disclosed polynucleotide comprising a nucleic acid sequence, one or more disclosed vectors, one or more disclosed pharmaceutical compositions, or any combination thereof. In an aspect, a disclosed animal can be treated with one or more disclosed recombinant polypeptides, one or more disclosed polynucleotide comprising a nucleic acid sequence, one or more disclosed vectors, one or more disclosed pharmaceutical compositions, or any combination thereof. In an aspect, animals can be assessed and/or monitored for one or more biological and/or chemical functions prior to treatment, during treatment, after treatment, or any combination thereof, wherein treatment can comprise administering one or more disclosed recombinant polypeptides, one or more disclosed antibody drug conjugates, one or more disclosed polynucleotide comprising a nucleic acid sequences, one or more disclosed vectors, one or more disclosed pharmaceutical compositions, or any combination thereof. In an aspect, a disclosed treated subject can be a mouse or a rat. In an aspect, a disclosed treated subject can be a transgenic mouse or a transgenic rat. In an aspect, a disclosed treated subject can have one or more types of cancers and/or tumors (such as, for example, a solid cancer).

[0246] In an aspect, a disclosed animal can be used in a disclosed method.

C. Methods of Treating a Cancer

[0247] Disclosed herein is a method of treating a cancer, the method comprising administering to the subject in need thereof, a therapeutically effective amount of a disclosed pharmaceutical composition. Disclosed herein is a method of treating a cancer, the method comprising administering to the subject in need thereof, a therapeutically effective amount of a disclosed pharmaceutical composition as provided in Section VII(B)(5). Disclosed herein is a method of treating a cancer, the method comprising administering to the subject in need thereof, a therapeutically effective amount of a disclosed recombinant polypeptide that targets a phosphatidylserine-expressing tumor cell. Disclosed herein is a method of treating a cancer, the method comprising administering to the sub] ect in need thereof, a therapeutically effective amount of a disclosed polynucleotide that encodes a disclosed recombinant polypeptide that targets a phosphatidylserine-expressing tumor cell. Disclosed herein is a method of treating a cancer, the method comprising administering to the subj ect in need thereof, a therapeutically effective amount of a disclosed engineered cell wherein a disclosed engineered cell can comprise a disclosed polynucleotide or a disclosed recombinant polypeptide or both. In an aspect, a disclosed engineered cell can comprise a surface expressed recombinant polypeptide as provided herein. In an aspect, a disclosed engineered cell may secrete a disclosed recombinant polypeptide. In an aspect, a disclosed engineered cell can be obtained by transforming or transducing a cell with a polynucleotide or a vector comprising a nucleic acid sequence encoding a recombinant polypeptide that targets a phosphatidylserine-expressing tumor cell. Disclosed herein is a method of treating a cancer, the method comprising administering to the subject in need thereof, a therapeutically effective amount of a disclosed pharmaceutical composition as provided in Section VIT (B)(5) that targets or facilitates the targeting of a phosphatidylserine-expressing tumor cells.

[0248] Disclosed herein is a method of treating a subject in need thereof, the method comprising contacting an immune cell ex vivo with a disclosed recombinant polypeptide to activate the immune cell thus obtaining an immune cell population and administering the immune cell population into a subject in need thereof. As used herein, “activating” an immune cell comprises clonal expansion of the cell followed by differentiation into immune effector cells and induction of cell-mediated cytotoxicity and/or cytokine release. In an aspect, the immune cell can be an allogenic cell. In an aspect, the immune cell can be an autologous cell. In an aspect, the immune cell can be an immune effector cell. In an aspect, the immune effector cell can be a tumorinfiltrating lymphocyte (TIL), CD3+T cell from peripheral blood, genetically modified T cell expressing one or more chimeric antigen receptors targeting any tumor cell-specific antigens, y5T cells, double negative (CD4-CD8-) T cells, natural killer T (NKT) cell, natural killer (NK) cell, a B cell, a macrophage, or any combination thereof. In an aspect, the immune effector cell can be pre-treated with the recombinant fusion proteins ex vivo, or transduced with a disclosed polynucleotide encoding the recombinant protein ex vivo or both, and adoptively transferred into patients. In an aspect, the immune effector cell can be a T-cell. In an aspect, the immune effector cell can be an NK-cell. In an aspect, the immune cell can be a T-cell and the recombinant polypeptide can be a BITE (bi-specific T-cell engager). In an aspect, the immune cell can be a NK-cell and the recombinant polypeptide is a BIKE or a TRIKE. In an aspect, the method further can comprise expanding the immune cell in the presence of an antigen presenting cell (APC). In an aspect, the antigen presenting cell can be an artificial antigen presenting cell (aAPC). In an aspect, a disclosed APC can display a tumor antigen, wherein the tumor antigen can be an antigen commonly expressed by a tumor cell in a subject in need of treatment.

[0249] Tumor-infiltrating lymphocytes (TILs) are a type of immune cell that can be found within the tumor microenvironment of solid tumors. They are lymphocytes, a class of white blood cells responsible for the body’s immune response against foreign invaders and abnormal cells, including cancer cells. TILs play a critical role in the interaction between the immune system and cancer. When cancer develops, tumor cells can sometimes be recognized as abnormal by the immune system. Immune cells, including TILs, can then infiltrate the tumor mass, attempting to recognize and eliminate the cancer cells. The presence and activity of TILs in the tumor microenvironment can have significant implications for the progression and treatment of cancer. There are different types of lymphocytes that can infiltrate tumors: Cytotoxic T cells: These T cells have the ability to directly recognize and kill cancer cells by releasing cytotoxic molecules that induce cell death. Helper T cells: These T cells assist other immune cells in their functions and help orchestrate a broader immune response against the tumor. Regulatory T cells (Tregs): These T cells have a suppressive function, dampening the immune response, hi the context of cancer, Tregs can limit the immune system’s ability to attack the tumor, promoting immune evasion. Natural Killer (NK) cells: NK cells can also infiltrate tumors and directly kill cancer cells without the need for prior sensitization, making them important players in the innate immune response against cancer.

[0250] “Allogeneic TILs” refers to tumor-infiltrating lymphocytes (TILs) that are collected from a genetically different individual (donor) and then used in the treatment of another individual (recipient) with cancer. These TILs are harvested from the tumor tissue of the donor, expanded and activated in the laboratory, and then infused into the recipient to boost their immune response against cancer.

[0251] “Autologous TILs” refers to tumor-infiltrating lymphocytes (TILs) that are collected from a patient’s own tumor tissue and then used in personalized cancer immunotherapy. Autologous TIL therapy is a form of adoptive cell therapy, where the patient’s immune cells are isolated, expanded, and reinfused back into the same patient to enhance the body’s ability to fight cancer. Without being bound by theory, the process of autologous TIL therapy typically involves the following steps: Tumor Biopsy: A small piece of the patient’s tumor is surgically removed through a biopsy or excision. Isolation of TILs: TILs are extracted from the tumor tissue. These are the lymphocytes that have infiltrated the tumor, indicating their potential to recognize and target cancer cells. TIL Activation and Expansion: The isolated TILs are cultured in the laboratory and exposed to specific growth factors and cytokines to stimulate their activation and expansion. This process leads to a significant increase in the number of TILs, making them more effective in attacking cancer cells. Preparatory Treatment: Before reinfusing the expanded TILs back into the patient, the patient can receive lymphodepleting chemotherapy or radiation. This helps create space within the immune system for the infused TILs to proliferate and exert their anticancer effects. TIL Infusion: The expanded and activated TILs are then infused back into the patient through intravenous infusion. Once the TILs are back in the patient’s body, they can target and attack cancer cells, thus potentially leading to tumor regression or elimination.

[0252] A TIL can be isolated from an organ afflicted with a cancer. One or more cells can be isolated from an organ with a cancer that can be a brain, heart, lungs, eye, stomach, pancreas, kidneys, liver, intestines, uterus, bladder, skin, hair, nails, ears, glands, nose, mouth, lips, spleen, gums, teeth, tongue, salivary glands, tonsils, pharynx, esophagus, large intestine, small intestine, rectum, anus, thyroid gland, thymus gland, bones, cartilage, tendons, ligaments, suprarenal capsule, skeletal muscles, smooth muscles, blood vessels, blood, spinal cord, trachea, ureters, urethra, hypothalamus, pituitary, pylorus, adrenal glands, ovaries, oviducts, uterus, vagina, mammary glands, testes, seminal vesicles, penis, lymph, lymph nodes or lymph vessels. One or more TILs can be from a brain, heart, liver, skin, intestine, lung, kidney, eye, small bowel, or pancreas. TILs can be from a pancreas, kidney, eye, liver, small bowel, lung, or heart. TILs can be from a pancreas. The one or more cells can be pancreatic islet cells, for example, pancreatic P cells. In some cases, a TIL can be from a gastrointestinal cancer. A TIL culture can be prepared a number of ways. For example, a tumor can be trimmed from non-cancerous tissue or necrotic areas. A tumor can then be fragmented to about 2-3 mm in length. In some cases, a tumor can be fragmented from about 0.5 mm to about 5 mm in size, from about 1 mm to about 2 mm, from about 2 mm to about 3 mm, from about 3 mm to about 4 mm, or from about 4 mm to about 5 mm. Tumor fragments can then be cultured in vitro utilizing media and a cellular stimulating agent such as a cytokine. In some cases, IL-2 can be utilized to expand TILs from a tumor fragment. A concentration of IL-2 can be about 6000 lU/mL. A concentration of IL-2 can also be about 2000 lU/mL, 3000 lU/mL, 4000 lU/mL, 5000 lU/mL, 6000 lU/mL, 7000 lU/mL, 8000 lU/mL, 9000 lU/mL, or up to about 10000 lU/mL. Once TILs are expanded they can be subject to in vitro assays to determine tumor reactivity. For example, TILs can be evaluated by FACs for CD3, CD4, CD8, and CD58 expression. TILs can also be subjected to cocultured, cytotoxicity , ELISA, or ELISPOT assays. In some cases, TIL cultures can be cryopreserved or undergo a rapid expansion. A cell, such as a TIL, can be isolated from a donor of a stage of development including, but not limited to, fetal, neonatal, young and adult. TILs can be isolated from an adult human A human from whom cells can be isolated can be under the age of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year(s). For example, cells can be isolated from a human under the age of 6 years. Cells, such as TILs, can also be isolated from a human under the age of 3 years. In some cases, a human donor can be an adult from at least about 18 years of age. In some cases, a blood product can be stored. For example, a cryostore freezing bag can be utilized to store and freeze a blood product. In an aspect, disclosed herein is a method of treating cancer using allogenic or autogenic engineered tumor infiltrating lymphocytes (TIL). In an aspect, disclosed herein is a method of treating cancer using an allogenic or autogenic engineered tumor infiltrating lymphocytes (TIL) expressing a disclosed recombinant polypeptide. In an aspect, the method of treating a subject in need thereof comprises (i) transforming or transfecting allogenic TILs with a polynucleotide comprising a nucleic acid sequence encoding a disclosed recombinant polypeptide to obtain engineered TILs; and (ii) administering to the subject a pharmaceutical composition comprising the engineered TILs. In an aspect, the method of treating a subject in need thereof comprises (i) obtaining or having obtained autologous TILs from the subject; (ii) transforming or transfecting the TILs with a polynucleotide comprising a nucleic acid sequence encoding a disclosed recombinant polypeptide to obtain engineered TILs; (hi) administering to the subject a pharmaceutical composition comprising the engineered TILs. In an aspect, the method may further comprise, activating the allogenic or autogenic TILs ex vivo by contacting them with a disclosed recombinant polypeptide. In an aspect, the method of treating a subject in need thereof further comprises exposing the TILs to a disclosed recombinant protein and co-culturing with irradiated APCs (antigen presentation cells) to activate the T cells with the recombinant polypeptide to promote TIL expansion. In an aspect the APCs are artificial antigen presenting cells (aAPC).

[0253] In an aspect, the cancer can comprise a solid tumor. Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy , pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, W’lms’ tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases. In as aspect, the cancer can comprise colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, non-Hodgkin’s lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi’s sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers, or a combination of cancers.

[0123] In an aspect, a therapeutically effective amount or effective dose or effective amount or therapeutically effective dosage of a disclosed pharmaceutical composition can be any amount that, when used alone or in combination with another therapeutic agent, can attack and destroy PS-expressing tumor cells. In an aspect, a therapeutically effective amount or effective dose or effective amount or therapeutically effective of a disclosed pharmaceutical composition can be any amount that, when used alone or in combination with another therapeutic agent, can attack and destroy PS-expressing tumor cells. In an aspect, a therapeutically effective amount or effective dose or effective amount or therapeutically effective dosage of of a disclosed pharmaceutical composition can be any amount that can enhance the success rate of one or more conventional cytotoxic cancer therapies against PS-expressing tumor cells.

[0254] In an aspect, a disclosed therapeutically effective amount or effective dose of a disclosed of a disclosed pharmaceutical composition can be about 0.01 pg/kg to about 10 mg/kg per subject. [0255] In an aspect, a therapeutically effective amount or effective dose or effective amount or therapeutically effective dosage of a disclosed pharmaceutical composition can protect a subject against the onset of a disease and/or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays. [0256] In an aspect, administering a disclosed of a disclosed pharmaceutical composition can comprise systemic or direct administration. In an aspect, administering can comprise oral administration, intravenous administration, intratumoral administration, intraperitoneal administration, intracranial administration, subcutaneous administration, intradermal administration, intrathecal administration, or any combination thereof. In an aspect, administering of a disclosed pharmaceutical composition can be by a method of administration disclosed herein. In an aspect, of a disclosed pharmaceutical composition can be administered via multiple routes either concurrently or sequentially.

[0257] For example, in an aspect, a disclosed pharmaceutical composition can be first administered intratumorally and then be administered intravenously. In an aspect, a disclosed pharmaceutical composition can be first administered intratumorally and then be administered orally. A skilled clinician can determine the best route of administration for a subject at a given time.

[0258] In an aspect, a disclosed method can comprise repeating the administering of a disclosed pharmaceutical composition. In an aspect, a disclosed method can comprise repeating the administering of a disclosed recombinant polypeptide, a disclosed cell, or any disclosed composition.

[0259] In an aspect, a disclosed method of treating a cancer or metastatic cancer can comprise protecting the subject from metastasis. In an aspect, a disclosed method of treating a cancer or metastatic cancer can comprise reducing the risk of developing metastasis. In an aspect, a disclosed method of treating a cancer or metastatic cancer can comprise preventing or inhibiting metastasis.

[0260] In an aspect, a disclosed method can comprise monitoring the subject for adverse effects. In an aspect, in the absence of adverse effects, a disclosed method can comprise continuing to treat the subj ect. In an aspect, continuing to treat the subj ect can comprise continuing to administer to the subject a disclosed pharmaceutical composition. In an aspect, in the presence of adverse effects, a disclosed method can comprise modifying one or more steps of the method. In an aspect, modifying one or more steps of a disclosed method can comprise modifying the administering step. In an aspect, modifying the administering step can comprise changing the amount of a disclosed pharmaceutical composition administered to the subject, changing the frequency of administration of a disclosed pharmaceutical composition, changing the duration of administration of a disclosed pharmaceutical composition, changing the route of administration of a disclosed pharmaceutical composition, or any combination thereof.

[0261] In an aspect, a disclosed method of treating a cancer or metastatic cancer can further comprise administering to the subject an immune checkpoint inhibitor (e.g., an anti-PDl molecule). In an aspect, a disclosed anti-PDl molecule can comprise an anti-PDl antibody, an anti-PDLl antibody, or any combination thereof. In an aspect, a disclosed anti-PDl antibody can comprise a monoclonal antibody, a humanized monoclonal antibody, or a fragment thereof. In an aspect, a disclosed anti-PDl antibody can comprise a polyclonal antibody, a humanized polyclonal antibody, or a fragment thereof. In an aspect, a disclosed anti-PDl antibody can comprise any antibody or antibody fragment that specifically recognizes PD1. In an aspect, a disclosed anti- PDLl antibody can comprise a monoclonal antibody, a humanized monoclonal antibody, or a fragment thereof. In an aspect, a disclosed anti-PDLl antibody can comprise a polyclonal antibody, a humanized polyclonal antibody, or a fragment thereof. In an aspect, a disclosed anti- PDLl antibody can comprise any antibody or antibody fragment that specifically recognizes PDL1. Antibodies and methods of preparing antibodies are known in the art. Similarly, recombinant antibodies and methods of preparing recombinant antibodies are known in the art. [0262] In an aspect, a disclosed method of treating a cancer or metastatic cancer can further comprise repeating the administering of the anti-PDl molecule. In an aspect, a disclosed anti-PDl molecule can be administered prior to, concurrent with, or after the administration of the interfering molecule. In an aspect of a disclosed method of treating a cancer or metastatic cancer administering a disclosed anti-PDl molecule can comprise systemic or direct administration. In an aspect, administering a disclosed anti-PDl molecule can comprise intravenous administration, intratumoral administration, intraperitoneal administration, or any combination thereof. In an aspect, administering a disclosed can be administered by any method of administration disclosed herein. In an aspect, a disclosed anti-PDl molecule can be administered via multiple routes either concurrently or sequentially. For example, in an aspect, a disclosed anti-PDl molecule can be first administered intratumorally and then be administered intravenously. In an aspect, administering a disclosed anti-PDl molecule can be first administered intratumorally and then be administered orally. A skilled clinician can determine the best route of administration for a subject at a given time.

[0263] For example, in an aspect, a disclosed anti-PDl molecule can be administered about 3 months, about 2 months, or about 1 month prior to the administering of a disclosed pharmaceutical composition. In an aspect, a disclosed anti-PDl molecule can be administered about 8 weeks, about 7 weeks, about 6 weeks, about 5 weeks, about 4 weeks, about 3 weeks, about 2 weeks, or about 1 week prior to the administering of a disclosed pharmaceutical composition. In an aspect, a disclosed anti-PDl molecule can be administered about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day prior to the administering of a disclosed pharmaceutical composition. In an aspect, a disclosed anti-PDl molecule can be administered about 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hours prior to the administering of a disclosed pharmaceutical composition.

[0264] In an aspect, a disclosed method of treating a cancer or metastatic cancer can further comprise repeating the administering of a disclosed anti-PDl molecule. In an aspect, a disclosed anti-PDl molecule can be administered prior to, concurrent with, or after the administration of a disclosed recombinant polypeptide.

[0265] In an aspect, a disclosed method of treating a cancer or metastatic cancer can comprise administering to the subject one or more additional anti-cancer therapies. Anti-cancer therapies are known to the art. In an aspect, a disclosed anti-cancer therapy can comprise endocrine therapy, radiotherapy, hormone therapy, gene therapy, thermal therapy, ultrasound therapy, or any combination thereof. In an aspect, a disclosed anti-cancer therapy can comprise one or more chemotherapeutic agents. In an aspect, a disclosed chemotherapeutic agent can comprise an anthracy cline, a vinca alkaloid, an alkylating agent, an immune cell antibody, an antimetabolite, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor, an immunomodulator, or any combination thereof. In an aspect, a disclosed chemotherapeutic agent can comprise 5 -fluorouracil (Adrucil, Efudex), 6-mercaptopurine (Purinethol), 6-thioguanine, aclarubicin or aclacinomycin A, alemtuzamab (Lemtrada), anastrozole (Arimidex), axitinib (Inlyta), bevacizumab (Avastin), bicalutamide (Casodex), bleomycin sulfate (Blenoxane), bortezomib (Velcade), busulfan (Myleran), busulfan injection (Busulfex), capecitabine (Xeloda), carboplatin (Paraplatin), carmustine (BiCNU), chlorambucil (Leukeran), cisplatin (Platinol), cladribine (Leustatin), Cosmegan, cyclophosphamide (Cytoxan or Neosar), cyclophosphamide, cytarabine liposome injection (DepoCyt), cytarabine, cytosine arabinoside (Cytosar-U), dacarbazine (DTIC-Dome), dactinomycin (Cosmegen), daunorubicin citrate liposome injection (DaunoXome), daunorubicin hydrochloride (Cerubidine), dexamethasone, docetaxel (Taxotere), doxorubicin hydrochloride (Adriamycin, Rubex), etoposide (Vepesid), fludarabine phosphate (Fludara), flutamide (Eulexin), folic acid antagonists, gemcitabine (difluorodeoxycitidine), gemtuzumab, gliotoxin, hydroxyurea (Hydrea), Idarubicin (Idamycin), ifosfamide (IFEX), ifosfamide, irinotecan (Camptosar), L-asparaginase (ELSPAR), lenalidomide), leucovorin calcium, melphalan (Alkeran), melphalan, methotrexate (Folex), mitoxantrone (Novantrone), mylotarg, N4-pentoxy carbonyl-5 deoxy-5 -fluorocytidine, nab-paclitaxel (Abraxane), paclitaxel (Taxol), pentostatin, phoenix (Y ttrium90/MX-DTPA), polifeprosan 20 with carmustine implant (Gliadel), purine analogs and adenosine deaminase inhibitors (fludarabine), pyrimidine analogs, rituximab, tamoxifen citrate (Nolvadex), temozolomide), teniposide (Vumon), tezacitibine, thalidomide or a thalidomide derivative, thiotepa, tirapazamine (Tirazone), topotecan hydrochloride for injection (Hy camptin), tositumomab), vinblastine (Velban), vinblastine, vincristine (Oncovin), vindesine, vinorelbine (Navelbine), or any combination thereof.

[0266] In an aspect, a disclosed method of treating a cancer or metastatic cancer can further comprise repeating the administering of a disclosed chemotherapeutic agent. In an aspect, a disclosed chemotherapeutic agent can be administered prior to, concurrent with, or after the administration of a disclosed recombinant polypeptide. The table below provides a non- comprehensive listing of treatments of cancers as known to the art.

[0267] In an aspect, a disclosed method of treating a cancer or metastatic cancer can comprise administering to the subject an anti-chemokine therapy. In an aspect, a disclosed anti-chemokine therapy can comprise one or more antibodies against CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CCL25, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CCR2, CCR5, CCR7, CCR8, CCR9, CXCR3, CXCR4, CXCR5, CX3CL1, CX3CR1, or any combination thereof. In an aspect, a disclosed method of treating a cancer or metastatic cancer can further comprise repeating the administering of a disclosed anti-PDl molecule. In an aspect, a disclosed anti-chemokine molecule can be administered prior to, concurrent with, or after the administration of a disclosed recombinant polypeptide.

[0268] In an aspect, a disclosed method of treating a cancer or metastatic cancer can comprise administering an oligonucleotide therapeutic agent. A disclosed oligonucleotide therapeutic agent can comprise a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, noncoding RNA (ncRNA), an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA), or an analog or conjugate thereof. In an aspect, a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise a CRISPR-based endonuclease. In an aspect, a disclosed endonuclease can be Cas9. CRISPR/Cas9 systems and methods are known to the art. In an aspect, a disclosed method of treating a cancer or metastatic cancer can further comprise repeating the administering of a disclosed anti-oligonucleotide therapeutic agent. In an aspect, a disclosed anti-oligonucleotide therapeutic agent can be administered prior to, concurrent with, or after the administration of a disclosed recombinant polypeptide.

[0269] In an aspect, a disclosed method of treating a cancer or metastatic cancer can further comprise preventing or inhibiting metastasis of cancer cells. In an aspect, a disclosed method can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of decrease and/or reduction in the risk of and/or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g., a disclosed recombinant polypeptide or a pharmaceutical composition thereof)). In an aspect, preventing or inhibiting metastasis of cancer cells can comprise a 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% or any amount of decrease and/or reduction in the risk of and/or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g., a disclosed recombinant polypeptide or a pharmaceutical composition thereof).

I l l [0270] In an aspect, a disclosed method of treating a cancer or metastatic cancer can comprise surgically resecting the tumor and/or cancer cells from the subject. In an aspect, following resecting the tumor and/or cancer cells from the subject, a disclosed method of treating a cancer or metastatic cancer can comprise continuing to administer to the subject a therapeutically effective amount of a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof and continuing to administer to the subject a therapeutically effective amount of an anti-PDl molecule, a disclosed anti-chemokine therapy, a disclosed chemotherapeutic agent, any disclosed therapeutic agent, or any combination thereof.

[0271] In an aspect, a disclosed method of treating a cancer or metastatic cancer can further comprise subjecting the subject to one or more invasive or non-invasive diagnostic assessments. Diagnostic assessments are known to the art. In an aspect, a disclosed non-invasive diagnostic assessment can comprise x-rays, computerized tomography (CT) scans, magnetic resonance imaging (MRI) scans, ultrasounds, positron emission tomography (PET) scans, or any combination thereof. In an aspect, a disclosed invasive diagnostic assessment can comprise a tissue biopsy or exploratory surgery.

[0272] In an aspect, a disclosed method of treating a cancer or metastatic cancer can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation in a subject, such as, for example, a subject having a cancer or metastatic cancer or cancerous cells. In an aspect, a disclosed pharmaceutical composition can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation in a subject having a cancer or metastatic cancer. In an aspect, metabolic dysregulation can be associated with a cancer or metastatic cancer or cancerous cells. In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (h) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi-systemic manifestations of a cancer; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a cancer, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity. In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level. In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise preventing or inhibiting metastasis of cancer cells in the subject.

[0273] In an aspect of a disclosed method of treating a cancer or metastatic cancer, techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are provided below.

[0274] In an aspect of a disclosed method of treating a cancer or metastatic cancer, techniques to monitor, measure, and/or assess the status of a subject’s disease and/or disorder and/or cancer can comprise qualitative (or subjective) means as well as quantitative (or objective) means. In an aspect, microscopy can be used for the visualization of cells to determine morphology and staining characteristics. In an aspect, immunohistochemistry can be used to identify specific molecules in different kinds of cells and/or tissues. The tissue is treated with antibodies that bind to the specific molecule. These are made visible under a microscope by using a color reaction, a radioisotope, or a fluorescent dye. In an aspect, flow cytometry can be used to sort and collect cells for additional analyses as flow cytometer is capable of rapid, quantitative, multiparameter analysis of heterozygous cell populations on a cell-by-cell basis. In an aspect, immunophenotyping can be used to detect specific antigens and/or markers that are expressed on various cells. Immunophenotyping uses fluorochrome-tagged monoclonal antibodies to analyze heterogenous populations of cells. Fluorescence in situ hybridization (FISH) can be used to characterize structural chromosome abnormalities and identify chromosomes of uncertain origin. Cytogenetics can be used to analyze the chromosomes during metaphase as it describes the number of chromosomes and their appearance, thereby identifying chromosomal abnormalities. PCR can be used to amplify DNA and/or RNA for analyses. Gene expression profiles can be used to measure the activity of genes.

[0275] In an aspect, a disclosed method can further comprise decreasing and/or reducing one or more symptoms related to and/or associated with the subject’s cancer or metastatic cancer. In an aspect, decreasing and/or reducing one or more symptoms related to and/or associated with the subject’s cancer or metastatic cancer can a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of decrease and/or reduction in the risk of and/or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g., a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof). In an aspect, preventing or inhibiting metastasis of cancer cells can comprise a 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% or any amount of decrease and/or reduction in the risk of and/or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g., a disclosed recombinant polypeptide or a pharmaceutical composition thereof).

[0276] In an aspect, a disclosed method of treating a cancer or metastatic cancer can comprise stimulating an effector cell mediated immune modulator response to PS-expressing tumor cells. In an aspect, a disclosed method of treating a cancer or metastatic cancer can induce a tumor reducing immune response. In an aspect, a disclosed method of treating a cancer or metastatic cancer can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed method of treating a cancer or metastatic cancer can cross-prime an anti-tumor T cell response. In an aspect, a disclosed method of treating a cancer or metastatic cancer can induce a tumor eliminating immune response. In an aspect, a disclosed method of treating a cancer or metastatic cancer can effect cell death of PS-expression cells. In an aspect, a disclosed method of treating a cancer or metastatic cancer can comprise improving the efficacy of one or more anti-cancer therapies and/or treatments.

[0277] In an aspect, a disclosed method of treating a cancer or metastatic cancer can comprise generating a disclosed pharmaceutical composition.

[0278] In an aspect, a disclosed method of treating a cancer or metastatic cancer can comprise generating a disclosed recombinant polypeptide using one or more disclosed vectors and/or one or more disclosed plasmids and/or one or most disclosed cells and/or any combination thereof.

[0279] In an aspect, a disclosed method of treating a cancer or metastatic cancer can comprise validating the efficacy and/or toxicity of disclosed recombinant polypeptide or pharmaceutical composition thereof. In an aspect, validating the efficacy and/or toxicity of a disclosed pharmaceutical composition can comprise using an in vivo model and/or in vitro model.

[0280] In an aspect, a disclosed method of treating a cancer or metastatic cancer can further comprise administering genetically modified cells expressing a chimeric antigen receptor targeting phosphatidylserine (PS) or a pharmaceutical composition thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting phosphatidylserine (PS) or a pharmaceutical composition thereof one or more times, administering one or more anticancer therapies one or more times, or administering any combination thereof one or more time. [0281] In an aspect, a disclosed method of treating a cancer or metastatic cancer can improve and/or extend the survivability of the subject, can improve a subject’s quality of life, can increase and/or prolong a subject’s life span, or any combination thereof. In an aspect, a disclosed method of treating a cancer or metastatic cancer can stimulate an effector cell mediated immune modulator response to PS-expressing tumor cells. In an aspect, a disclosed method of treating a cancer or metastatic cancer can induce a tumor reducing immune response. In an aspect, a disclosed method of treating a cancer or metastatic cancer can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed method of treating a cancer or metastatic cancer can cross-prime an antitumor T cell response. In an aspect, a disclosed method of treating a cancer or metastatic cancer can induce a tumor eliminating immune response. In an aspect, a disclosed pharmaceutical composition can treat cancer.

[0282] In an aspect of a disclosed method of treating a cancer or metastatic cancer, a disclosed recombinant polypeptide can be replaced by a disclosed vector. In an aspect of a disclosed method of treating a cancer or metastatic cancer, a disclosed recombinant polypeptide can be replaced by a disclosed nucleic acid molecule. In an aspect of a disclosed method of treating a cancer or metastatic cancer, a disclosed recombinant polypeptide can be replaced by a disclosed plasmid. In an aspect of a disclosed method of treating a cancer or metastatic cancer, a disclosed recombinant polypeptide can be replaced by a disclosed antibody drug conjugate. In an aspect of a disclosed method of treating a cancer or metastatic cancer, a disclosed recombinant polypeptide can be replaced by a disclosed pharmaceutical composition.

D. Methods of Improving the Efficacy of Cancer Therapy

[0283] Disclosed herein is a method of improving the efficacy of cancer therapy, the method comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of one or more cells transformed or transduced with a disclosed recombinant polypeptide or a pharmaceutical composition thereof. Disclosed herein is a method of improving the efficacy of cancer therapy, the method comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of a disclosed recombinant polypeptide or a pharmaceutical composition thereof. Disclosed herein is a method of improving the efficacy of cancer therapy, the method comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of a disclosed polynucleotide comprising a nucleic acid sequence encoding a disclosed recombinant polypeptide or a pharmaceutical composition thereof.

[0284] Disclosed herein is a method of improving the efficacy of cancer therapy, the method comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of a disclosed recombinant polypeptide targeting tumor cells expressing phosphatidylserine (PS) or a pharmaceutical composition thereof. Disclosed herein is a method of improving the efficacy of cancer therapy, the method comprising treating a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of a disclosed recombinant polypeptide targeting phosphatidylserine-expressing tumor cells or a pharmaceutical composition thereof.

[0285] In an aspect, a disclosed recombinant polypeptide can target phosphatidylserme (PS)- expressing cancer cells. In an aspect, a disclosed recombinant polypeptide can target phosphatidylserine (PS)-expressing solid tumors.

[0286] Disclosed herein is a method of improving the efficacy of cancer therapy, the method comprising contacting an immune cell ex vivo with a disclosed recombinant polypeptide to activate the immune cell thus obtaining an immune cell population and administering the immune cell population into a subject in need thereof. In an aspect, a disclosed immune cell can be an allogenic cell. In an aspect, a disclosed immune cell can be an autologous cell. In an aspect, a disclosed immune cell can be an immune effector cell. In an aspect, a disclosed immune effector cell can be a tumor-infiltrating lymphocyte (TIL), CD3+T cell from peripheral blood, genetically modified T cell expressing one or more chimeric antigen receptors targeting any tumor cell-specific antigens, y8T cells, double negative (CD4-CD8-) T cells, natural killer T (NKT) cell, natural killer (NK) cell, a B cell, a macrophage, or any combination thereof. In an aspect, a disclosed immune effector cell can be pre-treated with the recombinant fusion proteins ex vivo, or transduced with a disclosed polynucleotide encoding the recombinant protein ex vivo or both, and adoptively transferred into patients. In an aspect, the immune effector cell can be a T-cell. In an aspect, a disclosed immune effector cell can be an NK-cell. In an aspect, the immune cell can be a T-cell and the recombinant polypeptide is a BITE (bi-specific T-cell engager). In an aspect, the immune cell can be a NK-cell and the recombinant polypeptide is a BIKE or a TRIKE. In an aspect, the method can further comprise expanding the immune cell in the presence of an antigen presenting cell (APC). In an aspect, the antigen presenting cell can be an artificial antigen presenting cell (aAPC). In an aspect, a disclosed APC can display a tumor antigen, wherein the tumor antigen can be an antigen commonly expressed by a tumor cell in a subject in need of treatment.

[0287] In an aspect, a subject can be treatment-naive. In an aspect, a subject has received one or more treatments prior to the administering of a disclosed recombinant polypeptide or pharmaceutical composition thereof. In an aspect, one or more treatments can comprise one or more anti-cancer therapies and/or treatments.

[0288] In an aspect, a subject can have, be diagnosed with, or be suspected of having one or more cancers. In an aspect, a disclosed a method of improving the efficacy of cancer therapy can further comprise diagnosing the subject as have cancer or cancerous cells. In an aspect, a disclosed method of improving the efficacy of cancer therapy can further comprise diagnosing the subject as having a cancer or cancerous cells.

[0289] In an aspect, the cancer can comprise a solid tumor. Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy , pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, chonocarcmoma, Wilms’ tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases. In as aspect, the cancer can comprise colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, non-Hodgkin’s lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi’s sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers, or a combination of cancers.

[0123] In an aspect, a therapeutically effective amount or effective dose or effective amount or therapeutically effective dosage of a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof can be any amount that, when used alone or in combination with another therapeutic agent, can attack and destroy PS-expressing tumor cells. In an aspect, a therapeutically effective amount or effective dose or effective amount or therapeutically effective dosage of a disclosed recombinant polypeptide or pharmaceutical composition thereof can be any amount that, when used alone or in combination with another therapeutic agent, can attack and destroy PS-expressing tumor cells. In an aspect, a therapeutically effective amount or effective dose or effective amount or therapeutically effective dosage of a disclosed recombinant polypeptide or pharmaceutical composition thereof can be any amount that can enhance the success rate of one or more conventional cytotoxic cancer therapies against PS-expressing tumor cells.

[0290] In an aspect, a disclosed therapeutically effective amount or effective dose of a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof can be about 0.01 pg/kg to about 10 mg/kg per subject.

[0291] In an aspect, a therapeutically effective amount or effective dose or effective amount or therapeutically effective dosage of a disclosed recombinant polypeptide or pharmaceutical composition thereof can protect a subject against the onset of a disease and/or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety' of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0292] In an aspect, administering a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof can comprise systemic or direct administration. In an aspect, administering can comprise oral administration, intravenous administration, intratumoral administration, intraperitoneal administration, intracranial administration, subcutaneous administration, intradermal administration, intrathecal administration, or any combination thereof. In an aspect, administering a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof can be administered by any method of administration disclosed herein. In an aspect, a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof can be administered via multiple routes either concurrently or sequentially.

[0293] For example, in an aspect, a disclosed recombinant polypeptide or pharmaceutical composition thereof can be first administered intratumorally and then be administered intravenously. In an aspect, a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof can be first administered intratumorally and then be administered orally. A skilled clinician can determine the best route of administration for a subject at a given time.

[0294] In an aspect, a disclosed method can comprise repeating the administering of a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof.

[0295] In an aspect, a disclosed method of improving the efficacy of cancer therapy can comprise protecting the subject from metastasis. In an aspect, a disclosed method of improving the efficacy of cancer therapy can comprise reducing the risk of developing metastasis. In an aspect, a disclosed method of improving the efficacy of cancer therapy can comprise preventing or inhibiting metastasis.

[0296] In an aspect, a disclosed method of improving the efficacy of cancer therapy can comprise monitoring the subject for adverse effects. In an aspect, in the absence of adverse effects, a disclosed method can comprise continuing to treat the subject. In an aspect, continuing to treat the subject can comprise continuing to administer to the subject a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof. In an aspect, in the presence of adverse effects, a disclosed method can comprise modifying one or more steps of the method. In an aspect, modifying one or more steps of a disclosed method can comprise modifying the administering step. In an aspect, modifying the administering step can comprise changing the amount of a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof administered to the subject, changing the frequency of administration of a disclosed recombinant polypeptide or pharmaceutical composition thereof, changing the duration of administration of a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof, changing the route of administration of a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof, or any combination thereof. [0297] In an aspect, a disclosed method of improving the efficacy of cancer therapy can further comprise administering to the subject an immune checkpoint inhibitor (e.g., an anti-PDl molecule). In an aspect, a disclosed anti-PDl molecule can comprise an anti-PDl antibody, an anti-PDLl antibody, or any combination thereof. In an aspect, a disclosed anti-PDl antibody can comprise a monoclonal antibody, a humanized monoclonal antibody, or a fragment thereof. In an aspect, a disclosed anti-PD l antibody can comprise a polyclonal antibody, a humanized polyclonal antibody, or a fragment thereof. In an aspect, a disclosed anti-PDl antibody can comprise any antibody or antibody fragment that specifically recognizes PD1. In an aspect, a disclosed anti- PDLl antibody can comprise a monoclonal antibody, a humanized monoclonal antibody, or a fragment thereof. In an aspect, a disclosed anti-PDLl antibody can comprise a polyclonal antibody, a humanized polyclonal antibody, or a fragment thereof. In an aspect, a disclosed anti- PDLl antibody can comprise any antibody or antibody fragment that specifically recognizes PDL1. Antibodies and methods of preparing antibodies are known in the art. Similarly, recombinant antibodies and methods of preparing recombinant antibodies are known in the art.

[0298] In an aspect, a disclosed method of improving the efficacy of cancer therapy can further comprise repeating the administering of the anti-PDl molecule. In an aspect, a disclosed anti-PDl molecule can be administered prior to, concurrent with, or after the administration of the interfering molecule.

[0299] In an aspect of a disclosed method of improving the efficacy of cancer therapy, administering a disclosed anti-PDl molecule can comprise systemic or direct administration. In an aspect, administering a disclosed anti-PDl molecule can comprise intravenous administration, intratumoral administration, intraperitoneal administration, or any combination thereof. In an aspect, administering a disclosed can be administered by any method of administration disclosed herein. In an aspect, a disclosed anti-PDl molecule can be administered via multiple routes either concurrently or sequentially. For example, in an aspect, a disclosed anti-PDl molecule can be first administered intratumorally and then be administered intravenously. In an aspect, administering a disclosed anti-PDl molecule can be first administered intratumorally and then be administered orally. A skilled clinician can determine the best route of administration for a subject at a given time.

[0300] For example, in an aspect, a disclosed anti-PDl molecule can be administered about 3 months, about 2 months, or about 1 month prior to the administering of a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof. In an aspect, a disclosed anti-PDl molecule can be administered about 8 weeks, about 7 weeks, about 6 weeks, about 5 weeks, about 4 weeks, about 3 weeks, about 2 weeks, or about 1 week prior to the administering of a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof. In an aspect, a disclosed anti-PDl molecule can be administered about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day prior to the administering of a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof. In an aspect, a disclosed anti-PDl molecule can be administered about 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hours prior to the administering of a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof. In an aspect, a disclosed method of improving the efficacy of cancer therapy can further comprise repeating the administering of a disclosed anti-PDl molecule. In an aspect, a disclosed anti-PDl molecule can be administered prior to, concurrent with, or after the administration of a disclosed recombinant polypeptide.

[0301] In an aspect, a disclosed method of improving the efficacy of cancer therapy can comprise administering to the subject one or more additional anti-cancer therapies. Anti-cancer therapies are known to the art. In an aspect, a disclosed anti-cancer therapy can comprise endocrine therapy, radiotherapy, hormone therapy, gene therapy, thermal therapy, ultrasound therapy, or any combination thereof. In an aspect, a disclosed anti-cancer therapy can comprise one or more chemotherapeutic agents. In an aspect, a disclosed chemotherapeutic agent can comprise an anthracy cline, a vinca alkaloid, an alkylating agent, an immune cell antibody, an antimetabolite, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor, an immunomodulator, or any combination thereof. In an aspect, a disclosed chemotherapeutic agent can comprise 5 -fluorouracil (Adrucil, Efudex), 6-mercaptopurine (Purinethol), 6-thioguanine, aclarubicin or aclacinomycin A, alemtuzamab (Lemtrada), anastrozole (Arimidex), axitinib (Inlyta), bevacizumab (Avastin), bicalutamide (Casodex), bleomycin sulfate (Blenoxane), bortezomib (Velcade), busulfan (Myleran), busulfan injection (Busulfex), capecitabine (Xeloda), carboplatin (Paraplatin), carmustine (BiCNU), chlorambucil (Leukeran), cisplatin (Platinol), cladribine (Leustatin), Cosmegan, cyclophosphamide (Cytoxan or Neosar), cyclophosphamide, cytarabine liposome injection (DepoCyt), cytarabine, cytosine arabinoside (Cytosar-U), dacarbazine (DTIC-Dome), dactinomycin (Cosmegen), daunorubicin citrate liposome injection (DaunoXome), daunorubicin hydrochloride (Cerubidine), dexamethasone, docetaxel (Taxotere), doxorubicin hydrochloride (Adriamycin, Rubex), etoposide (Vepesid), fludarabine phosphate (Fludara), flutamide (Eulexin), folic acid antagonists, gemcitabine (difluorodeoxycitidine), gemtuzumab, gliotoxin, hydroxyurea (Hydrea), Idarubicin (Idamycin), ifosfamide (IFEX), ifosfamide, irinotecan (Camptosar), L-asparaginase (ELSPAR), lenalidomide), leucovorin calcium, melphalan (Alkeran), melphalan, methotrexate (Folex), mitoxantrone (Novantrone), mylotarg, N4-pentoxy carbonyl-5 deoxy-5 -fluorocytidine, nab-paclitaxel (Abraxane), paclitaxel (Taxol), pentostatin, phoenix (Y trium90/MX-DTPA), polifeprosan 20 with carmustine implant (Gliadel), purine analogs and adenosine deaminase inhibitors (fludarabine), pyrimidine analogs, rituximab, tamoxifen citrate (Nolvadex), temozolomide), teniposide (Vumon), tezacitibine, thalidomide or a thalidomide derivative, thiotepa, tirapazamine (Tirazone), topotecan hydrochloride for injection (Hy camptin), tositumomab), vinblastine (Velban), vinblastine, vincristine (Oncovin), vindesine, vinorelbine (Navelbine), or any combination thereof In an aspect, a disclosed method of improving the efficacy of cancer therapy can further comprise repeating the administering of a disclosed chemotherapeutic agent. In an aspect, a disclosed chemotherapeutic agent can be administered prior to, concurrent with, or after the administration of a disclosed recombinant polypeptide.

[0302] In an aspect, a disclosed method of improving the efficacy of cancer therapy can comprise administering to the subject an anti-chemokine therapy. In an aspect, a disclosed anti-chemokine therapy can comprise one or more antibodies against CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CCL25, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CCR2, CCR5, CCR7, CCR8, CCR9, CXCR3, CXCR4, CXCR5, CX3CL1, CX3CR1, or any combination thereof. In an aspect, a disclosed method of improving the efficacy of cancer therapy can further comprise repeating the administering of a disclosed anti-chemokine therapy. In an aspect, a disclosed anti- chemokine therapy can be administered prior to, concurrent with, or after the administration of a disclosed recombinant polypeptide.

[0303] In an aspect, a disclosed method of improving the efficacy of cancer therapy can comprise administering an oligonucleotide therapeutic agent. A disclosed oligonucleotide therapeutic agent can comprise a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, noncoding RNA (ncRNA), an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA), or an analog or conjugate thereof. In an aspect, a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise a CRISPR-based endonuclease. In an aspect, a disclosed endonuclease can be Cas9. CRISPR/Cas9 systems and methods are known to the art. In an aspect, a disclosed method of improving the efficacy of cancer therapy can further comprise repeating the administering of a disclosed oligonucleotide therapeutic agent. In an aspect, a disclosed oligonucleotide therapeutic agent can be administered prior to, concurrent with, or after the administration of a disclosed recombinant polypeptide. [0304] In an aspect, a disclosed method of improving the efficacy of cancer therapy can further comprise preventing or inhibiting metastasis of cancer cells. In an aspect, a disclosed method can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of decrease and/or reduction in the risk of and/or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g., a disclosed recombinant polypeptide or a pharmaceutical composition thereof)). In an aspect, preventing or inhibiting metastasis of cancer cells can comprise a 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% or any amount of decrease and/or reduction in the risk of and/or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g., a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereol).

[0305] In an aspect, a disclosed method of improving the efficacy of cancer therapy can comprise surgically resecting the tumor and/or cancer cells from the subject. In an aspect, following resecting the tumor and/or cancer cells from the subject, a disclosed method of improving the efficacy of cancer therapy can comprise continuing to administer to the subject a therapeutically effective amount of a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof and continuing to administer to the subject a therapeutically effective amount of an anti-PDl molecule, a disclosed anti-chemokine therapy, a disclosed chemotherapeutic agent, any disclosed therapeutic agent, or any combination thereof.

[0306] In an aspect, a disclosed method of improving the efficacy of cancer therapy can further comprise subjecting the subject to one or more invasive or non-invasive diagnostic assessments. Diagnostic assessments are known to the art. In an aspect, a disclosed non-invasive diagnostic assessment can comprise x-rays, computerized tomography (CT) scans, magnetic resonance imaging (MR1) scans, ultrasounds, positron emission tomography (PET) scans, or any combination thereof. In an aspect, a disclosed invasive diagnostic assessment can comprise a tissue biopsy or exploratory surgery.

[0307] In an aspect, a disclosed method of improving the efficacy of cancer therapy can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation in a subject, such as, for example, a subject having cancer or cancerous cells. In an aspect, a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation in a subject having cancer. In an aspect, metabolic dysregulation can be associated with cancer or cancerous cells. In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi-systemic manifestations of a cancer; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a cancer, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity. In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level. In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise preventing or inhibiting metastasis of cancer cells in the subject.

[0308] In an aspect of a disclosed a method of improving the efficacy of cancer therapy, techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are discussed supra.

[0309] In an aspect of a disclosed method of improving the efficacy of cancer therapy, techniques to monitor, measure, and/or assess the status of a subject’s cancer can comprise qualitative (or subjective) means as well as quantitative (or objective) means. In an aspect, microscopy can be used for the visualization of cells to determine morphology and staining charactenstics. In an aspect, immunohistochemistry can be used to identify specific molecules in different kinds of cells and/or tissues. The tissue is treated with antibodies that bind to the specific molecule. These are made visible under a microscope by using a color reaction, a radioisotope, or a fluorescent dye. In an aspect, flow cytometry can be used to sort and collect cells for additional analyses as flow cytometer is capable of rapid, quantitative, multiparameter analysis of heterozygous cell populations on a cell-by-cell basis. In an aspect, immunophenotyping can be used to detect specific antigens and/or markers that are expressed on various cells. Immunophenotyping uses fluorochrome-tagged monoclonal antibodies to analyze heterogenous populations of cells. Fluorescence in situ hybridization (FISH) can be used to characterize structural chromosome abnormalities and identify chromosomes of uncertain origin. Cytogenetics can be used to analyze the chromosomes during metaphase as it describes the number of chromosomes and their appearance, thereby identifying chromosomal abnormalities. PCR can be used to amplify DNA and/or RNA for analyses. Gene expression profiles can be used to measure the activity of genes. [0310] In an aspect, a disclosed a method of improving the efficacy of cancer therapy can further comprise decreasing and/or reducing one or more symptoms related to and/or associated with the subject’s cancer. In an aspect, decreasing and/or reducing one or more symptoms related to and/or associated with the subject’s cancer can a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of decrease and/or reduction in the risk of and/or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g., a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof). In an aspect, preventing or inhibiting metastasis of cancer cells can comprise a 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% or any amount of decrease and/or reduction in the risk of and/or actual metastasis of cancer cells when compared to a control subject (such as, for example, a subject that has not received a disclosed treatment (e.g., a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereol).

[0311] In an aspect, a disclosed method of improving the efficacy of cancer therapy can comprise stimulating an effector cell mediated immune modulator response to PS-expressing tumor cells. In an aspect, a disclosed method of improving the efficacy of cancer therapy can induce a tumor reducing immune response. In an aspect, a disclosed method of improving the efficacy of cancer therapy can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed method of improving the efficacy of cancer therapy cross-prime an anti-tumor T cell response. In an aspect, a disclosed method of improving the efficacy of cancer therapy can induce a tumor eliminating immune response. In an aspect, a disclosed method of improving the efficacy of cancer therapy can effect cell death of PS-expression cells. In an aspect, a disclosed method of improving the efficacy of cancer therapy can comprise improving the efficacy of one or more anti-cancer therapies and/or treatments.

[0312] In an aspect, a disclosed method of improving the efficacy of cancer therapy can comprise generating a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof.

[0313] In an aspect, a disclosed method of improving the efficacy of cancer therapy can comprise generating a disclosed recombinant polypeptide using one or more disclosed vectors and/or one or more disclosed plasmids and/or one or most disclosed cells and/or any combination thereof. [0314] In an aspect, a disclosed method of improving the efficacy of cancer therapy can comprise validating the efficacy and/or toxicity of disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof. In an aspect, validating the efficacy and/or toxicity of a disclosed recombinant polypeptide or a disclosed pharmaceutical composition thereof can comprise using an in vivo model and/or in vitro model.

[0315] In an aspect, a disclosed method of improving the efficacy of cancer therapy can further comprise administering genetically modified cells expressing a chimeric antigen receptor targeting phosphatidylserine (PS) or a pharmaceutical composition thereof or engineered T cells or NK cells or macrophages expressing a chimeric antigen receptor targeting phosphatidylserine (PS) or a pharmaceutical composition thereof one or more times, administering an anti-PDl molecule one or more times, administering one or more anti-cancer therapies one or more times, or administering any combination thereof one or more time.

[0316] In an aspect, a disclosed method of treating a cancer can improve and/or extend the survivability of the subject, can improve a subject’s quality of life, can increase and/or prolong a subject’s life span, or any combination thereof. In an aspect, a disclosed method of improving the efficacy of cancer therapy can stimulate an effector cell mediated immune modulator response to PS-expressing tumor cells. In an aspect, a disclosed method of improving the efficacy of cancer therapy can induce a tumor reducing immune response. In an aspect, a disclosed method of improving the efficacy of cancer therapy can induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed method of improving the efficacy of cancer therapy can cross-prime an anti-tumor T cell response. In an aspect, a disclosed method of improving the efficacy of cancer therapy can induce a tumor eliminating immune response. In an aspect, a disclosed method of improving the efficacy of cancer therapy composition can treat cancer.

[0317] In an aspect of a disclosed method of improving the efficacy of cancer therapy, a disclosed recombinant polypeptide can be replaced by a disclosed vector. In an aspect of a disclosed method of improving the efficacy of cancer therapy, a disclosed recombinant polypeptide can be replaced by a disclosed nucleic acid molecule. In an aspect of a disclosed method of improving the efficacy of cancer therapy, a disclosed recombinant polypeptide can be replaced by a disclosed plasmid. In an aspect of a disclosed method of improving the efficacy of cancer therapy, a disclosed recombinant polypeptide can be replaced by a disclosed antibody drug conjugate. In an aspect of a disclosed method of improving the efficacy of cancer therapy, a disclosed recombinant polypeptide can be replaced by a disclosed pharmaceutical composition. E. Kits

[0318] Disclosed herein is a kit comprising one or more of a disclosed recombinant polypeptide, a disclosed pharmaceutical composition, a disclosed polynucleotide comprising a nucleic acid sequence, a disclosed vector, a disclosed engineered cell, or any combination thereof. Disclosed herein is a kit comprising one or more disclosed recombinant polypeptides, one or more disclosed pharmaceutical compositions, one or more disclosed polynucleotide comprising a nucleic acid sequence, one or more disclosed vectors, one or more disclosed engineered cells, or any combination thereof.

[0319] In an aspect, a disclosed kit can comprise one or more additional active agents and/or therapeutic agents. In an aspect, the one or more agents can treat, prevent, inhibit, and/or ameliorate one or more comorbidities in a subject. In an aspect, one or more active agents can treat, inhibit, prevent, and/or ameliorate cellular and/or metabolic complications related to cancer or cancer cells or cancerous cells.

[0320] In an aspect, a disclosed kit can comprise a disclosed recombinant polypeptide (e.g., SEQ ID NOS: 133-162, or 209, or a fragment thereof).

[0321] In an aspect, a disclosed kit can comprise a disclosed bispecific recombinant polypeptide as set forth FIG. 2A - FIG. 2E.

[0322] In an aspect, a disclosed kit can comprise at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose (such as, for example, treating a subject diagnosed with or suspected of having a disease or disorder such as cancer). Individual member components can be physically packaged together or separately. For example, a kit comprising an instruction for using the kit can or cannot physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which can be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. In an aspect, a disclosed kit for use in a disclosed method can comprise one or more containers holding a disclosed recombinant polypeptide, a disclosed pharmaceutical composition, a disclosed polynucleotide comprising a nucleic acid sequence, a disclosed vector, a disclosed engineered cell, or any combination thereof, and a label or package insert with instructions for use. In an aspect, suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers can be formed from a variety of materials such as glass or plastic. The container can hold a disclosed recombinant polypeptide, a disclosed pharmaceutical composition, a disclosed polynucleotide comprising a nucleic acid sequence, a disclosed vector, a disclosed engineered cell, or any combination thereof, and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert can indicate a disclosed recombinant polypeptide, a disclosed pharmaceutical composition, a disclosed polynucleotide comprising a nucleic acid sequence, a disclosed vector, a disclosed engineered cell, or any combination thereof can be used for treating, preventing, inhibiting, and/or ameliorating a disease or disorder or complications and/or symptoms associated with a disease or disorder such as cancer or metastatic cancer. A kit can comprise additional components necessary for administration such as, for example, other buffers, diluents, fdters, needles, and syringes.

[0323] In an aspect, a disclosed kit can be used to preventing or inhibiting metastasis of cancer cells. In an aspect, a disclosed kit can be used to risk of developing metastases. In an aspect, a disclosed kit can be used to validate the efficacy and/or toxicity of a disclosed recombinant polypeptide, a disclosed pharmaceutical composition, a disclosed polynucleotide comprising a nucleic acid sequence, a disclosed vector, a disclosed engineered cell, or any combination thereof. [0324] In an aspect, a disclosed kit can be used to improve and/or extend the survivability of the subject, can improve a subject’s quality of life, can increase and/or prolong a subject’s life span, or any combination thereof. In an aspect, a disclosed kit can be used to can stimulate an effector cell mediated immune modulator response to PS-expressing tumor cells. In an aspect, a disclosed kit can be used to induce a tumor reducing immune response. In an aspect, a disclosed kit can be used to induce phagocytosis of cancer cells in the subject. In an aspect, a disclosed kit can be used to cross-prime an anti-tumor T cell response. In an aspect, a disclosed kit can be used to induce a tumor eliminating immune response. In an aspect, a disclosed kit can be used to treat cancer.

F. Miscellaneous

[0325] Disclosed herein is a composition comprising recombinant polypeptides for use to enhance the ftinction of immune effector cells, the composition comprising: 1) a phosphatidylserme- binding domain, 2) a domain that binds either CD3 or CD16A, and 3) an optional third domain that binds another tumor-specific or immune cell-specific antigen. In an aspect, a disclosed composition is packaged into a viral vector. In an aspect, a disclosed composition is packaged into a liposome. In an aspect, a disclosed composition is packaged into a nanoparticle. Disclosed herein is a composition comprising recombinant polypeptides, comprising a phosphatidylserine-binding domain, a CD3 -binding domain or a CD16A binding domain, and a third optional antigen binding domain are linked through two linker regions and combinations thereof. In an aspect, the recombinant polypeptide can comprise no linker region, a flexile linker, a rigid linker, or a whitlow linker and combinations thereof. In an aspect, a disclosed PS binding domain can comprises Annexin Al (ANXA1) or the PS-binding core domain thereof, Annexin A2 (ANXA2), Annexin A3 (ANXA3), Annexin A4 (ANXA4), Annexin A5 (ANXA5), Annexin A6 (ANXA6), Annexin A7 (ANXA7), Annexin A8 (ANXA8), Annexin A8 Like 1 (ANXA8L1), Annexin A9 (ANXA9), Annexin A10 (ANXA10), Annexin Al l (ANXA11), Annexin A13 (ANXA13), Coagulation Factor II (F2), Coagulation Factor VII (F7), Coagulation Factor IX (F9), Coagulation Factor X (F10), Advanced Glycosylation End-Product Specific Receptor (AGER/RAGE) or the extracellular domain thereof, Growth Arrest Specific 6 (GAS 6), Milk Fat Globule EGF And Factor V/VIII Domain Containing (MFGE8), Adhesion G Protein Coupled Receptor Bl (BAI1/ADGRB1) or the extracellular domain thereof, Protein S (PROS), Hepatitis A Virus Cellular Receptor 1 (HAVCR1) or the extracellular domain thereof, Hepatitis A Virus Cellular Receptor 2 (HAVCR2) or the extracellular domain thereof, T Cell Immunoglobulin And Mucin Domain Containing 4 (TIMD4), Protein Kinase C Alpha (PRKCA) or the C2 domain thereof, Synaptotagmin (SYT1) or the C2A domain thereof, Jumonji Domain-Containing Protein 6 (JMJD6), Apolipoprotein H (APO-H), Stabilin-1 (STAB1) or the extracellular domain thereof, Stabilin-2 (STAB2) or the extracellular domain thereof, Killer Cell Lectin Like Receptor Kl(KLRKl), a variant thereof, a fragment thereof, or any combination thereof.

[0326] In an aspect, the PS binding domain comprises a heavy chain variable domain (VH), a light chain variable domain (VL), a Fab, a Fab’, a F(ab’)2, a Fv, a single-chain Fv (scFv), a minibody, a diabody, a single-domain antibody, and or any combination thereof, of the anti-PS antibody. In an aspect the PS binding domain comprises the single chain variable region (scFv) of the anti-PS antibody. In an aspect, the anti-PS antibody comprises Bavituximab, PGN632, Pl, IS4, or CL 1, a fragment thereof, a variant thereof, or any combination thereof.

[0327] In an aspect, the CD3 binding domain comprises one or more antigen binding regions of an anti-CD3 antibody. In an aspect, the CD3 binding domain comprises a heavy chain variable domain (VH), a light chain variable domain (VL), a Fab, a Fab’, a F(ab’)2, a Fv, a single-chain Fv (scFv), a minibody, a diabody, a single-domain antibody, and or any combination thereof, of the anti-CD3 antibody. In an aspect, the CD3 binding domain comprises the single chain variable region (scFv) of the anti-CD3 antibody. In an aspect, the anti-CD3 antibody comprises OKT3, humanized OKT3, UCHT1, humanized UCHT1, TRX4 (Otlixizumab), foralumab, visiluzumab, tepilizumab (hOKT3y), a fragment thereof, a variant thereof, or any combination thereof.

[0328] In an aspect, the CD16A binding domain comprises one or more antigen binding regions of an anti-CD16A antibody. In an aspect, the CD16A binding domain comprises a a heavy chain variable domain (VH), a light chain variable domain (VL), a Fab, a Fab’, a F(ab’)2, a Fv, a singlechain Fv (scFv), a minibody, a diabody, a single-domain antibody, and or any combination thereof, of the anti-CD16 antibody. In an aspect, the anti-CD16A domain comprises the single chain variable region (scFv) of the anti-CD16 antibody. In an aspect, the anti-CD16 antibody comprises 3G8, humanized 3G8-5, humanized 3G8-22, humanized 3G8-43, CB16, B73.1, DJ130c, MEM-154, A9, GMA161, AFM13, D6, El l, C21, or C28. In an aspect, a recombinant polypeptide comprises an optional third domain, comprising (i) Null, with no additional domains; (ii) the extracellular domain of the NKG2D protein, which can bind it ligands expressed on the surface of various tumor cells, (iii) the scFv versions of antibodies that can target various tumor antigens, and combinations thereof, iv) the immune stimulatory cytokines such as IL2, IL7, IL12, IL15, or IL21. In an aspect, he optional third domain comprises a tumor specific antigen (TSA) binding protein. In an aspect, the TSA binding protein is an antibody comprising the scFv version of an antibody against CD138, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, RORi, MUC-16, LICAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CD47, CA125, CD56, c-Met, EGFR, GD-3, HPV E6, HPVE7, MUC-1, HER2, folate receptor alpha, CD97, CD171, CD179a, CD44v6, Claudin 18.2, WTI, VEGF a, VEGFR1, IL-3Ral, IL-3Ra2, IL- iRa, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, ephrin A2, ephrin B2, Lewis A antigen, Lewis Y antigen, MAGE, MAGE-Ai, GPC3, RAGE-1, folate receptor, TROP2, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GMi, GM3, o-acetyl-GD2, or GD2; and combinations thereof. In an aspect, the composition is further linked with the human albumin protein or the Fc versions of human IgGl, IgG2, IgG3, and IgG4 that have no effector functions to boost the half-life of the fusion protein in vivo, or combination thereof. In an aspect, the recombinant polypeptide can comprise a PS-binding domain and a human Fc (Fragment crystallizable) domain with intact effector functions, with a linker in between. In an aspect the recombinant polypeptide can comprise a PS-binding domain a CD3-binding domain or a CD16A binding domain, and a third optional antigen binding domain linked through two linker regions while the Fc domain comprises a molecule selected from the group consisting of human IgGl, IgG2, IgG3, and IgG4.

[0329] Disclosed herein is a pharmaceutical composition comprising a disclosed recombinant polypeptide. Disclosed herein is a pharmaceutical composition comprising a disclosed recombinant polypeptide and a diluent, excipient and/or carrier.

[0330] Disclosed herein is a method of enhancing the function of immune effector cells by use of a disclosed recombinant polypeptide, the method comprising, consisting of, or consisting essentially of administering to a subject in need thereof a therapeutically effective amount of a disclosed composition such that the immune effector cell function is enhanced. In an aspect, the immune effector cells are (i) pre-treated with a disclosed recombinant fusion proteins ex vivo; (ii) and/or transduced with a polynucleotide comprising a nucleic acid sequence encoding a disclosed recombinant fusion protein ex vivo,' (iii) and adoptively transferred into patients. In an aspect, the immune effector cells comprise tumor-infiltrating lymphocytes (TILs), CD3+T cells from peripheral blood, genetically modified T cells expressing chimeric antigen receptors targeting any tumor cell-specific antigens, y6T cells, double negative (CD4-CD8-) T cells, Natural killer T (NKT) cells, natural killer (NK) cells, B cells, macrophages, or combinations thereof. In an aspect the polynucleotide comprising a nucleic acid sequence encoding the recombinant protein is packaged into viral vectors, liposomes, or nanoparticles. In an aspect, the polynucleotide is used ex vivo to transduce immune effector cells ex vivo. In an aspect the genetic construct or the recombinant polypeptide is administered to the subject in vivo.

[0331] In an aspect, the immune effector cells are T cells derived from TILs (tumor infiltrating lymphocytes) in solid tumors, and the polynucleotide encodes a bispecific T cell engager (BITE) comprises annexin A5 (annexin V) and an scFv against CD3. In an aspect, the T cells derived from TILs (tumor infiltrating lymphocytes) are exposed to the PS-binding recombinant fusion bispecific T cell engager (BITE) protein and co-cultured with irradiated aAPCs (artificial antigen presentation cells) to ‘"arm” the T cells with the BITE and to promote T cell grow th during TIL expansion. In an aspect the T cells are grown and “armed” together with artificial antigen presenting cells (aAPCs) expressing a secreted version of the BITE (bi-specific T cell engager) that binds PS (through Annexin V) and CD3 (through an scFv against CD3). In an aspect, the immune effector cells further include a T, y5T, double negative T (DNT), and NKT cells derived from autologous or allogenic PBMC. In an aspect, the immune effector cells can comprise CAR- T, CAR- NK, or CAR-macrophage cells. In an aspect, the immune effector cells are transduced with a chimeric antigen receptor targeting an antigen. In an aspect, the chimeric antigen receptor (CAR) comprises CD138, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothehn, PSMA, BCMA, RORi, MUC-16, L1CAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CD47, CA125, CD56, c-Met, EGFR, GD-3, HPV E6, HPV E7, MUC-1, HER2, folate receptor alpha, CD97, CD171, CD179a, CD44v6, Claudin 18.2, WTI, VEGF a, VEGFR1, IL-3Ral, IL-3Ra2, IL- iRa, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, ephrin A2, ephrin B2, Lewis A antigen, Lewis Y antigen, MAGE, MAGE-Ai, RAGE-1, folate receptor , EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GMi, GM3, o-acetyl- GD2, TROP2, GD2, and combinations thereof. In an aspect, the genetic construct encodes a BITE. In an aspect, the BITE and the CAR are expressed from the same or different gene-transducing vectors. In an aspect, the BITE and the CAR are expressed from a single or multiple promoters. In an aspect the BITE and the CAR are expressed from the same promoter, and the BITE and CAR encoding genes are optionally linked by an IRES nucleotide sequence, a P2A sequence, or T2A sequence. In an aspect, the immune effector cells comprise TCR-T cells expressing recombinant TCR targeting an antigen selected from the group consisting of: WT-1, mesothelin, MART- 1, NY-ESO-1, MAGE A3, HPV E7, mutant RAS (G12C, G12D, and G12V), surviving, alpha- fetoprotein, other tumor- specific antigens, and combinations thereof.

[0332] In an aspect the BITE and the TCR-T are expressed from the same or different gene- transducmg vectors and/or expressed from a single or multiple promoters. In an aspect the BITE and the TCR-T are expressed from the same promoter, the BITE and TCR-T encoding genes are optionally linked by an IRES nucleotide sequence, P2A sequence, or a T2A sequence.

[0333] In an aspect, the immune effector cells comprise NK cells and the recombinant fusion protein comprises a bi-specific NK cell engager (BIKE), the BIKE comprising an Annexin A5 (Annexin V) and an scFc against CD16A. Alternatively, the recombinant fusion protein comprises a tri-specific NK cells engager (TRIKE), the TRIKE comprising a Annexin A5, an scFv against CD16A, and IL15, or IL15 with the sushi domain. In an aspect, the NK cells are exposed to the PS- binding recombinant BIKE (bi-specific nature killer engager), or TRIKE protein and cocultured aAPCs (artificial antigen presenting cells) to “arm” the NK cells with the BIKE or TRIKE to promote NK cell growth.

[0334] In an aspect, the NK cells are derived from cells selected from the group consisting of: (i) autologous PBMC; (ii) allogenic cord blood (CB) derived NK cells; (iii) cells differentiated from induced pluripotent stem cells (iPSCs) derived from autologous or allogenic cells, and combinations thereof. In an aspect, the NK cells are grown and armed in aAPC cells transduced with a genetic construct expressing a secreted version of the PS- binding BIKE (bispecific natural killer engager) or TRIKE (trispecific natural killer engager).

[0335] In an aspect the method comprises, (i) transducing T cells with a polynucleotide expressing a secreted BITE (bi- specific T cell engager) that targets PS (through annexm A5 or scFv or other PS- binding protein domains) and CD3 (though an scFv); and/or (ii) transducing NK cells with a polynucleotide expressing a secreted BIKE (bi-specific natural killer engager) or TRIKE that targets PS (through annexin A5 or scFv or other PS-binding protein domains) and CD16A (through an scFv). In an aspect, the method further comprises: (i) expressing PS and CD3 targeting BITE on the T cells, or ex vivo “arming” with the BITE by further loading with another BITE targeting CD3 and a tumor specific antigen; and/or (ii) expressing the PS and CD16A-targeting BIKE or TRIKE on NK cells, or ex vivo “arming” the cells with the BIKE or TRIKE by further loading with another BIKE or TRIKE targeting CD16A and a tumor specific antigen. In an aspect, the tumor specific antigen are selected from the group consisting of: CD138, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, RORi, MUC-16, LICAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CD47, CA125, CD56, c-Met, EGFR, GD-3, HPV E6, HPV E7, MUC-1, HERZ, folate receptor alpha, CD97, CD171, CD179a, CD44v6, Claudin 18.2, WTI, VEGF a, VEGFR1, IL- 3Ral, IL-3Ra2, IL-iRa, PSA, FcRH5, NKG2D ligand, NY- ESO-1, TAG-72, CEA, ephrin A2, ephrin B2, Lewis A antigen, Lewis Y antigen, MAGE, MAGE- Ai, RAGE-1, folate receptor , EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GMi, GM3, o- acetyl-GD2, TR0P2, GD2 or combinations thereof.

G. List of Sequences

VIII. EXAMPLES

[0336] Most of the current paradigms for cancer therapy assume that PS-expressing tumor cells, either from exposure to internal or external stressors, are destined to die from apoptosis. But data provided herein showed that some of these PS-expressing tumor cells survive. As detailed herein, the compositions and methods described herein can significantly enhance the success rate of conventional cytotoxic cancer therapy.

[0337] The Examples that follow are illustrative of specific aspects of the invention, and various uses thereof. They set forth for explanatory purposes only and are not to be taken as limiting the invention.

Example 1 Examination of Survival of Phosphatidylserine-Positive Tumor Cells

[0338] To determine whether cells expression high-level of PS expression on the cell surface are destined to die, the viability of cl 498 murine AML (Acute Myeloid Leukemia) cells treated with cytarabine (part of a classical chemotherapy regimen for human AML) was examined. Specifically, whether cell surface PS-expressing acute myelocytic leukemia (AML) cells survived was examined. FACS was used to identify and sort cells with high cell surface PS-expression and the ability to survive was measured. FIG. 1A - FIG. IB). In FIG. 1A, FACS sorting of untreated C1498 cells (in DMEM with 8% FBS). The rectangles indicate the sorted cells. In FIG. IB, FACS sorting of c!498 cells treated with cytarabine (1 M) for 24 hrs). The data indicated the following: (i) about 22-25% of untreated cl498 cells are either PS+7AAD-(7AAD is a fluorescent dye used to detect membrane integrity), early apoptotic cells, or PS+7AAD+, late apoptotic cells, remained alive and could proliferate in a robust manner (FIG. 1C), and (ii) about 25% or 8% of cytarabine- treated PS+7AAD-C1498 cells or PS+7AAD+ c!498 cells remained viable and could proliferate (FIG. 1C). These results indicated that a substantial proportion of PS+ cells (which the art presumed should be dead and/or destined to die), survived the initiation of the apoptotic process. The fact that these cells could survive indicated that new agents and/or treatment methods are needed to prevent tumor relapse. In FIG. 1C, about 1000 each of the sorted cells were placed into multi-well plates. When the cells reached a sufficient number, the cells were counted at different time points to measure growth rates. The growth rates were then used to estimate the initial live cell numbers. The following formular was used to calculate the minimal initial live cell numbers: Minimal initial cell number = Total cell population I (growth rate* t (days to reach the current cell number).

Example 2 PS-targeting multi-specific proteins

[0339] FIG. 2 provides schematics of some versions of the phosphatidylserine-targeting multispecific recombinant proteins contemplated and used in these studies. FIG.2A shows abi-specific fusion proteins targeting PS, and CD3, or CD16A, which can activate CD3+ T cells or CD16A NK cells against PS-expressing cancer cells. FIG. 2B shows the design of trispecific proteins targeting PS, and CD3 (top) or CD16A (bottom), and a third tumor specific antigen. FIG. 2C provides examples of bispecific PS-(annexm V) and CD3-(scFv) targeting protein with an Fc domain. FIG. 2D shows bispecific PS-targeting recombinant fusion protein with a full anti-CD3 antibody with annexin V linked to the light chain of anti-CD3 antibody. FIG. 2E shows two trispecific proteins targeting PS and CD16A with an IL15 cytokine or IL15 cytokine plus and IL15 receptor alpha sushi domain.

Example 3 Production of PS-targeting bispecific T cell engager (BITE) and bispecific nature killer cell engager (BIKE)

[0340] DNA constructs encoding secreted A5-scFv(OKT3) (BITE) or A5-scFv(3G8) (BIKE) were assembled (SEQ IDNOS:133 and SEQ IDNO:209) and transduced into CHO-cells. A5-scFv(OKT3) and A5-scFv(3G8) were purified from the respective clones using a Ni-His column. FIG. 3 shows the production of expected protein as determined by PAGE gels stained by Coomassie blue.

Example 4

Efficacy of TIL cells transduced with a secreted BITE targeting phosphatidylserine in killing syngeneic non-small cell lung cancer (NCSLC) cells

[0341] In order to determine the efficacy of tumor-infiltrating lymphocyte (TILs) encoding a PS binding bi-specific T-cell engaging recombinant polypeptide (BITE) in killing syngeneic cancer cells, lentiviral vectors were used to transduce into NSCLC-derived TIL cells a secreted BITE gene encoding A5-scFv(OKT3). The cells were expanded in the presence of aAPCs (K562 cells expressing IL15, CD86, and 4-1BBL). The cells were then cocultured with synergistic NSCLC cells that were transduced with luciferase, for 72 hrs. Survival of the tumor cells was measured by reading luciferase levels. The survival data is provided in FIG. 4 and indicates a dose- dependent killing of NSCLC cells. Example 5 BITE-facilitated expansion of NSCLC- and CRC- derived TIL cells

[0342] To further characterize if the PS-targeting BITE can facilitate TIL expansion, TILs derived from a NSCLC patient and a colorectal cancer (CRC) patient were co-cultured with irradiated aAPCs (lOOGy) at different T celkaAPC ratios and cultured in the presence of the lOOng/mL recombinant A5-scFv(OKT3) BITE protein for 7 days. The NSCLC and CRC cells were then counted. The data indicated that at the T:APC ratio of 100, both NSCLC and CRC cells could be expanded over 130 - 160 fold (FIG. 5).

Example 6 Ex vivo expanded TILs are active against syngeneic cancer cells

[0343] Ex vivo expanded TILs were tested for their activity against syngeneic cancer cells. The TILs were expanded either in the presence of aAPC (K562 cells expressing IL-15, CD86, and 4- 1BBL) and BITE (Annexin V-scFv (OKT3), or in the presence of CD3+CD28+ beads (TrasAct) for 8 days. The cells were then co-cultured with syngeneic NSCLC cells (transduced with luciferase) for 72 hrs. Survival of the tumor cells was measured by reading luciferase levels. For NSCLC-derived TILs, the date showed that the BITE-exposed TILs killed syngeneic NSCLC cancer cells significantly more efficiently (-60% killing vs -40% killing) than those activated by CD3+CD28+ beads (FIG. 6). For CRC-derived TILs, almost all cancer cells were killed by BITE- expanded CRC TILs (FIG. 7), indicating robust cell killing activities of the TILs.

Example 7 Flow cytometry analysis of NSCLC-derived TILs

[0344] Flow cytometry was used to characterize the T cell status of TILs expanded in the presence of aAPCs in the absence or presence of PS- targeting BiTE (A5-scFv(OKT3). The analysis showed that TILs grown in the presence of PS- targeting BITE demonstrate significantly higher levels of activation, maturation, and proliferation markers (FIG. 8A - FIG. 8D). Importantly, they also showed higher levels of CD279(PDl)/CD39/CD103, which previous studies demonstrated mark tumor cell-reactive TILs.

Summary of Specific Examples

[0345] As demonstrated herein, recombinant polypeptides comprising a PS-binding domain and a CD3 or CD16 binding domain increase the efficacy of chemotherapy. The Examples showed clearly that Acute Myeloid Leukemia (AML) cells expressing PS were alive and proliferated in a robust manner (FIG. 1) These cells, previously thought to be destined to die and therefore irrelevant, were thus “survivors” that could propel the relapse of AML. The data provided herein shows that TILs cells expressing bispecific T-cells engaging (BITE) polypeptides are active against lung carcinoma (NSCLC) and colorectal carcinoma (CRC) suggesting that the bi-specific T-cell or NK-cell engagers targeting PS could significantly enhance the therapeutic efficacy of T immune effector cells, with or without concurrent chemotherapy against solid tumors. These data provided strong support for the notion that direct targeting of PS on cancer cells could be an effective therapeutic strategy, which is against the prevailing view in the field currently. In addition, the data shows that T cell engagers and NK cell engagers, based on PS targeting, could be effective novel therapeutics targeting multiple types of cancer cells. These can also be used either in vivo or in an ex vivo setting to boost T cell or NK cell activities.

Claims

IX. CLAIMS What is claimed is

1. A recombinant polypeptide, comprising: a phosphatidylserine (PS) binding domain; a CD3 or a CD16A binding domain; and an optional third domain that binds a tumor-specific antigen or an immune cell-specific antigen.

2. The recombinant polypeptide of Claim 1, wherein the PS binding domain comprises Annexin Al (ANXA1) or the PS-binding core domain thereof, Annexin A2 (ANXA2), Annexin A3 (ANXA3), Annexin A4 (ANXA4), Annexin A5 (ANXA5), Annexin A6 (ANXA6), Annexin A7 (ANXA7), Annexin A8 (ANXA8), Annexin A8 Like 1 (ANXA8L1), Annexin A9 (ANXA9), Annexin A10 (ANXA10), Annexin All (ANXA11), Annexin A13 (ANXA13), Coagulation Factor II (F2), Coagulation Factor VII (F7), Coagulation Factor IX (F9), Coagulation Factor X (F10), Advanced Glycosylation End-Product Specific Receptor (AGER/RAGE) or the extracellular domain thereof. Growth Arrest Specific 6 (GAS6), Milk Fat Globule EGF And Factor V/VIII Domain Containing (MFGE8), Adhesion G Protein Coupled Receptor Bl (BAI1/ADGRB1) or the extracellular domain thereof, Protein S (PROS), Hepatitis A Virus Cellular Receptor 1 (HAVCR1) or the extracellular domain thereof, Hepatitis A Virus Cellular Receptor 2 (HAVCR2) or the extracellular domain thereof, T Cell Immunoglobulin And Mucin Domain Containing 4 (TIMD4), Protein Kinase C Alpha (PRKCA) or the C2 domain thereof, Synaptotagmin (SYT1) or the C2A domain thereof, Jumonji Domain-Containing Protein 6 (JMJD6), Apolipoprotein H (APO-H), Stabilin-1 (STAB1) or the extracellular domain thereof, Stabilin-2 (STAB2) or the extracellular domain thereof. Killer Cell Lectin Like Receptor Kl(KLRKl), a variant thereof, a fragment thereof, a variant thereof, a fragment thereof, or any combination thereof.

3. The recombinant polypeptide of Claim 1, wherein the PS binding domain comprises one or more antigen binding regions of an anti-PS antibody.

4. The recombinant polypeptide of Claim 3, wherein the PS binding domain comprises a heavy chain variable domain (VH), a light chain variable domain (VL), a Fab, a Fab’, a F(ab’)2, a Fv, a single-chain Fv (scFv), a minibody, a diabody, a single-domain antibody, and or any combination thereof, of the anti-PS antibody.

5. The recombinant polypeptide of Claim 4, wherein the PS binding domain comprises the single chain variable region (scFv) of the anti-PS antibody. The recombinant polypeptide of any preceding claim, wherein the anti-PS antibody comprises Bavituximab, PGN632, Pl, IS4, or CL1, a fragment thereof, a variant thereof, or any combination thereof. The recombinant polypeptide of Claim 1, wherein the CD3 binding domain comprises one or more antigen binding regions of an anti-CD3 antibody. The recombinant polypeptide of Claim 7, wherein the CD3 binding domain comprises a heavy chain variable domain (VH), a light chain variable domain (VL), a Fab, a Fab’, a F(ab’)2, a Fv, a single-chain Fv (scFv), a minibody, a diabody, a single-domain antibody, and or any combination thereof, of the anti-CD3 antibody. The recombinant polypeptide of Claim 8, wherein the CD3 binding domain comprises the single chain variable region (scFv) of the anti-CD3 antibody. The recombinant polypeptide of any one of Claims 7-9, wherein the anti-CD3 antibody comprises OKT3, humanized OKT3, UCHT1, humanized UCHT1, TRX4 (Otlixizumab), foralumab, visiluzumab, tepilizumab (hOKT3y), a fragment thereof, a variant thereof, or any combination thereof. The recombinant polypeptide of Claim 1, wherein the CD16A binding domain comprises one or more antigen binding regions of an anti-CD16A antibody. The recombinant polypeptide of Claim 11, wherein the CD16A binding domain comprises a heavy chain variable domain (VH), a light chain variable domain (VL), a Fab, a Fab’, a F(ab’)2, a Fv, a single-chain Fv (scFv), a minibody, a diabody, a single-domain antibody, and or any combination thereof, of the anti-CD16 antibody. The recombinant polypeptide of Claim 12, wherein anti-CD16A domain comprises the single chain variable region (scFv) of the anti-CD16 antibody. The recombinant polypeptide of any one of Claims 11-13, wherein the anti-CD16 antibody comprises 3G8, humanized 3G8-5, humanized 3G8-22, humanized 3G8-43, CB16, B73.1, DJ130c, MEM-154, A9, GMA161, AFM13, D6, El l, C21, or C28. The recombinant polypeptide of any one of Claims 1-14, wherein the recombinant polypeptide does not comprise the optional third domain that binds a tumor-specific antigen or an immune cell-specific antigen. The recombinant polypeptide of any one of Claims 1-14, wherein the recombinant polypeptide comprises the optional third domain that binds a tumor-specific antigen or an immune cellspecific antigen. The recombinant polypeptide of Claim 16, wherein the optional third domain binds a tumorspecific antigen (TS A), and wherein the TS A is a part of a tumor cell surface protein. The recombinant polypeptide of Claim 16, wherein the tumor-specific antigen (TSA) comprises Mesothelin, TSHR, CD19, CD123, CD22, CD30, CD138, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, prostate specific membrane antigen (PSMA), R0R1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, interleukin- 11 receptor a (IL-l lRa), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet- derived growth factor receptor-beta (PDGFR-beta), S SEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1 , epidermal growth factor receptor (EGFR), NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CX0RF61, CD97, CD 179a, ALK, Poly sialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-1AP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, NKG2D, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, RORi, MUC-16, LICAM, CD20, CD23, CD24, CD37, CD47, CA125, CD56, c-Met, EGFR, GD-3, HPV E6, HPV, E7, MUC-1, HER2, folate receptor alpha, CD97, CD171, CD179a, CD44v6, Claudin 18.2, WTI, VEGF a, VEGFR1, IL-3Ral, IL-3Ra2, IL-iRa, PSA, FcRH5, NKG2D ligand, NY- ESO-1, TAG-72, ephnn A2, ephrin B2, Lewis A antigen, Lewis Y antigen, MAGE, MAGE- Ai, GPC3, RAGE-1, folate receptor, TROP2, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GMi, GM3, o-acetyl-GD2, GD2, a fragment thereof, a variant thereof, or any combination thereof. The recombinant polypeptide of Claim 17, wherein the optional third domain comprises one or more antigen binding regions of an anti-TSA antibody. The recombinant polypeptide of Claim 17, wherein the optional third domain comprises a heavy chain variable domain (VH), a light chain variable domain (VL), a Fab, a Fab’, a F(ab’)2, a Fv, a single-chain Fv (scFv), a minibody, a diabody, a single-domain antibody, and or any combination thereof, of the anti-TSA antibody The recombinant polypeptide of Claim 20, wherein the optional third domain comprises the single chain variable region (scFv). The recombinant polypeptide of any one of Claims 1-14, wherein the recombinant polypeptide comprises the optional third domain that binds an immune cell-specific antigen. The recombinant polypeptide of Claim 22, wherein the optional third domain binds a T-cell, a B-cell, a dendritic cell, a tumor infiltrating lymphocyte (TIL), a CD3⁺ T-cell, a genetically modified T-cell, a y8T, a double negative (CD4-CD8-) T cell, natural killer T (NKT) cells, a natural killer (NK) cell, a macrophage, or a variant thereof, and any combination thereof. The recombinant polypeptide of Claim 22, wherein the optional third domain comprises one or more antigen binding regions of an anti-immune cell specific antibody. The recombinant polypeptide of Claim 24, wherein the optional third domain comprises a heavy chain variable domain (VH), a tight chain variable domain (VL), a Fab, a Fab’, a F(ab’)2, a Fv, a single-chain Fv (scFv), a minibody, a diabody, a single-domain antibody, and or any combination thereof, of the anti-immune cell specific antibody The recombinant polypeptide of Claim 25, wherein the optional third domain comprises the single chain variable region (scFv). The recombinant polypeptide of any one of Claims 1-26, wherein the recombinant polypeptide further comprises an immune stimulatory cytokine domain, or a variant thereof, or a fragment thereof. The recombinant polypeptide of Claim 27, wherein the immune stimulatory cytokine domain comprises IL2, IL7, IL 12, IL 15, IL21, or a variant thereof, or a fragment thereof. The recombinant polypeptide of Claim 28, wherein the immune stimulatory cytokine domain comprises an IL15, IL15-IL15R, or a variant thereof, or a fragment thereof. The recombinant polypeptide of any one of Claim 1-29, further compnsing a signal peptide. The recombinant polypeptide of claim 28, wherein the signal peptide is at least about 80% identical to any one of SEQ ID NOS: 163-1 7. The recombinant polypeptide of any one of Claims 1-31, further comprising one or more linker peptides. The recombinant polypeptide of claim 32, wherein the one or more linker peptides comprise a flexible tinker, a rigid tinker, a whitlow tinker, or any combination thereof. The recombinant polypeptide of Claim 32, wherein the tinker peptide is at least about 60% identical to any one of SEQ ID NOS: 168-204. The recombinant polypeptide of Claim 1, wherein the phosphatidylserine (PS) binding domain comprises an amino acid sequence at least about 80% identical to any one of SEQ ID NO:01- SEQ ID NO:41, or SEQ ID NO:55. The recombinant polypeptide of Claim 1, wherein the PS binding domain comprises an amino acid sequence at least about 80% identical to SEQ ID NO: 84, SEQ ID NO: 85, or both. The recombinant polypeptide of Claim 1, wherein the CD3 binding domain comprises an amino acid sequence at least about 80% identical to one or more of SEQ ID NOS: 86-103. The recombinant polypeptide of Claim 1, wherein the CD16A binding domain comprises an amino acid sequence at least about 80% identical to one or more of SEQ ID NOS: 104-125. The recombinant polypeptide of Claim 28 or Claim 29, wherein the immune stimulatory cytokine comprises an amino acid sequence at least about 80% identical to any one of SEQ ID NOS: 126-132. The recombinant polypeptide of Claim 1, wherein the polypeptide comprises an amino acid sequence at least about 60% identical to any one of SEQ ID NOS: 133-162, or SEQ ID NO: 209. The recombinant polypeptide of Claim 1, wherein the recombinant polypeptide is a bispecific T cell engager (BITE). The recombinant polypeptide of Claim 41, wherein the BITE comprises an annexin A5 or a PS-binding fragment, or variant thereof: and an scFv against CD3, or a variant thereof. The recombinant polypeptide of claim 1, wherein the recombinant polypeptide is a bispecific NK cell engager (BIKE). The recombinant polypeptide of Claim 43, wherein the BIKE comprises an annexm A5 or a PS-binding fragment or variant thereof; and an scFv against CD16A, or a variant thereof. The recombinant polypeptide of Claim 1, wherein the recombinant polypeptide comprises a tri-specific NK-cell engager (TRIKE). The recombinant polypeptide of Claim 45, wherein the TRIKE comprises an annexin A5 or a PS-binding fragment or variant thereof; an scFv against CD16A, or a variant thereof; and an IL 15, or IL 15 with a sushi domain, a variant, or fragment thereof. A recombinant polypeptide, comprising: a bispecific T cell engager (BITE), wherein the BITE comprises a phosphatidylserine (PS) binding domain; and an scFv against CD3, or a variant thereof. A recombinant polypeptide, comprising: a bispecific NK cell engager (BIKE), wherein the BIKE comprises: a phosphatidylserine (PS) binding domain; and an scFv against CD16A, or a variant thereof. A recombinant polypeptide, comprising: a tri -specific NK-cell engager (TRIKE), wherein the TRIKE comprises: a phosphatidylserine (PS) binding domain; an scFv against CD16A, or a variant thereof; and an immune stimulatory cytokine domain, a variant, or fragment thereof. The recombinant polypeptide of any one of Claims 47-49, wherein the PS binding domain comprises an amino acid sequence at least about 80% identical to any one of SEQ ID NOS: 1- 41, or SEQ ID NO:55 or a variant and/or fragment thereof. The recombinant polypeptide of any one of Claims 47-49, wherein the PS binding domain comprises Annexin A5 or a variant and/or variant thereof. The recombinant polypeptide of Claim 48, wherein the scFv comprises an amino acid sequence at least about 80% identical to one or more of SEQ ID NOS: 86-103. The recombinant polypeptide of Claim 48 or 49, wherein the scFv comprises an amino acid sequence at least about 80% identical to one or more of SEQ ID NOS: 104-125. The recombinant polypeptide of Claim 49, wherein the immune stimulatory cytokine domain comprises an IL2, IL7, IL12, IL 21, IL15, IL15-IL15R, a variant, or a fragment thereof. The recombinant polypeptide of Claim 50, wherein the immune stimulatory cytokine domain comprises an amino acid sequence at least about 80% identical to any one of SEQ ID NO: 126 or SEQ ID NO: 132. A polynucleotide comprising: a nucleic acid sequence encoding the recombinant polypeptide of any one of claims 1-55. An engineered cell, comprising: the recombinant polypeptide of any one of Claims 1-55, the polynucleotide of Claim 56, or both. The engineered cell of Claim 57, wherein the engineered cell comprises an immune cell. The engineered cell of Claim 58, wherein the engineered cell comprises a tumor infiltrating lymphocyte (TIL). The engineered cell of Claim 59, wherein the TIL comprises an autologous TIL or an allogenic TIL. The engineered cell of any one of Claims 57-60, wherein the engineered cell further expresses a chimeric antigen receptor (CAR), a recombinant TCR, aP-TCR, y5-TCR, or a variant thereof. The engineered cell of Claim 61, wherein the chimeric antigen receptor (CAR) targets CD 138,

CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, RORi, MUC-16, LICAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CD47, CA125, CD56, c-Met, EGFR, GD-3, HPV E6, HPV E7, MUC-1, HER2, folate receptor alpha, CD97, CD171, CD179a, CD44v6, Claudin 18.2, WTI, VEGF a, VEGFR1, IL-3Ral, IL-3Ra2, IL-iRa, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, ephrin A2, ephrin B2, Lewis A antigen, Lewis Y antigen, MAGE, MAGE-Ai, RAGE-1, folate receptor, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GMi, GM3, o-acetyl- GD2, TROP2, GD2, or any combination thereof A pharmaceutical composition, comprising: the recombinant polypeptide of any one of Claims 1-55, or a polynucleotide of Claim 56, or an engineered cell of any one of Claims 57-62, and a suitable carrier. The pharmaceutical composition of claim 63, wherein the pharmaceutical composition comprises adjuvants, excipients, diluents, conjugate, radioactive conjugate, a lipidoid, a liposome, a lipoplex, a nanoparticle, a lipid nanoparticle, a polymeric compound, a cell, a nanoparticle mimic, a nanotube, or any combination thereof. A method of treating a subject, the method comprising: admimstenng to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of any one of Claims 63-64. The method of Claim 65, wherein the subject has been diagnosed with cancer or metastatic cancer. The method of Claim 66, wherein the subject has one or more solid tumors. The method of Claim 67, wherein the cancer comprises colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, non-Hodgkin’s lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary' adenoma, Kaposi’s sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers, or any combination thereof. The method of Claim 68, wherein the subject is diagnosed with a colorectal cancer or nonsmall cell lung cancer (NSCLC). The method of any one of Claims 65-69, further comprising administering to the subject one or more additional anti-cancer therapies. The method of Claim 70, wherein the one or more anti-cancer therapies comprises endocrine therapy, radiotherapy, hormone therapy, gene therapy, thermal therapy, ultrasound therapy, or any combination thereof. A method of treating in a subject in need thereof, the method comprising: obtaining or having obtained autologous TILs from a subject; transforming or transfecting the TILs with a polynucleotide of Claim 56 to obtain a population of engineered autologous TILs; and administering to the subject a pharmaceutical composition comprising the engineered autologous TILs.