WO2019090355A1 - Cells expressing antibodies and methods of treatment using the same - Google Patents

Abstract

Genetic constructs, compositions and methods related to generating cellular products that will be used for T-cell- and NK cell -based immunotherapy of refractory solid tumors, hematologic malignancies, and brain tumors with a strong immunosuppressive component are provided. The cells are manufactured to target one or a plurality of hyperproliferative cells, made resistant to the immunosuppressive environment, and modified to secrete antibodies that both neutralize and eliminate essential components of the hyperproliferative cell.

Description

[0001] The present application claims priority to U.S. Provisional Patent

Application No. 62/582,292, filed November 6, 2017, the contents of which are hereby incorporated by reference in its entirety.

GOVERNMENT SUPPORT

[0002] This disclosure and its embodiments were funded at least partially by

National Institutes of Health Grant Number R21 AH22391. The United States Government may have rights underlying this disclosure.

FIELD OF INVENTION

[0003] The present disclosure is focused on the fields of immunotherapy and cancer therapeutics. The invention directs a cell, that simultaneously serves as a direct effector targeting tumor cells as well as the vehicle for molecules that target the tumor environment, the method used to generate composition comprising the ceil or plurality of ceils to allow for optimal migration into disease sites, and the genetic constructs necessary for conferring activity to the immune cell and potent antibody or antibody-like secretory function.

BACKGROUND

[0004] The difficulties involved in producing tumor vaccines arise from the complex nature of tumor interactions with the endogenous immune system, and traditional approaches often fail because there are multiple layers of immune dysfunction, immune escape, and immune suppression in cancer patients.

[0005] The tumor is sheltered from immune attack because immune cells are prevented from trafficking to the tumor, as the continued immune dysfunction in the body propagates a scenario where the cells are tolerant to the tumor's presence. Consequently there is no upregulation of chemokine receptors that will allow them to actively traffic to the tumor site.

[0006] T cell-based immunotherapies promise to revolutionize cancer treatment.

They have led to 80-90% response rates in refractory hematologic malignancies and to 30% response rates in metastatic melanoma (Lizee G, et al. Armu Rev Med. 2013;64:71 -90). However, there is little evidence that this success has translated well into the brain tumor and other solid tumor settings [both of which have dismal 5-year survival rates: 33% survival rates for the former (NCI. Brain and Other Nervous System Cancer 2015 [cited 2018 August 28, 2018]. Available from: https :// seer. cancer.gov/statfacts htmi/brain .html), and an average of 19% for some of the common, poor-prognosis solid tumors, including esophageal, liver, lung, pancreatic, and stomach cancers (NCI. Common Cancer Sites 2015 [cited 2018 August 28, 2018], Available from: https://seer.cancer.gov/statfacts/html/common.html. )]. Briefly, the more pronounced complexity of these malignancies limits the applicability of current T cell therapeutics: few targets exclusively expressed by these tumors have been identified (June CH, et ai. Sci Trans! Med. 2015;7(280):280), and cellular components and secreted factors from a dysfunctional innate immune system (Han ah an D, et al. Cell . 201 1; 144(5): 646 -74. PubMed ) lead to an immune suppressive tumor microenvironment (Chen DS, et ai. Nature. 2017;541 (7637):321-30).

[0007] One immunotherapy-resistant tumor illustrates this complexity well.

Glioblastoma multiforme (GBM), a brain tumor with a devastating prognosis, is highly heterogeneous, so that no single antigen can be targeted with any degree of success (Friedmann-Morvinski D. 2014;19(5):327-36.). Moreover, within the microenvironment of this tumor are cells and components that simultaneously provide a protumorigenic and immune suppressive milieu (Hanahan D and Coussens LM. Cancer Cell. 2012;21(3):309-22) these components evade recognition by the immune system through expression of, and interaction with, regulatory receptors such as CD47, which emit a "don't-eat-me" signal (Koh E, et al. Biomaterials. 2017, 121 : 121 -9; Chauhan S, et al. J Proteome Res. 2017;16(l):238-46; Wang M, et al. Clin Transl Oncol. 2018;20(7):906-11; Li CW, et ai. Frontiers of medicine. 2018; Tong B, et al. Future oncology (London, England). 2018).

[0008] Efforts to improve adoptive cell therapy are currently focused on combination treatments (such as combinations with epigenetic-modifying drugs or checkpoint inhibitors) to enhance T ceil specificity, potency, and proliferation (June CH et a!., 2015),

[0009] High-grade GBM affects 4/100,000 individuals and causes a disproportionately high mortality due to the limited efficacy of current therapies. The 5 -year survival rate for this tumor ranges from 15-35%, with a mean survival time of 15 months (Thakkar JP,et al. Cane Epid Biomark Prev. 2014;23(10): 1985-96). The need for alternative therapies is clear, and has led to a resurgence of interest in methods of tumor cell eradication based on immune modulation. Augmenting the antitumor immune response has been a successful strategy for malignancies other than GBM, and some preclinical studies have shown promising results for high-grade gliomas (Kirkin AF, et ai. Nat Commun. 2018;9(1):785; Zhang C, et al. Journal of the National Cancer Institute. 2016; 108(5); Bielamowicz K, et al. Neuro-oncology. 2018;20(4):506-18). There is evidence that immunity is altered in patients with CNS malignancies, and this is thought to contribute to a poor prognosis (Gustafson MP, et al. Neuro-oncology. 2010; 12(7):631-44). Although vaccines have shown promising activity against high-grade gliomas, it is unclear whether they will be successful for all patients - particularly those with altered immune responses (Zhou Q, et al. Hum Vaccin Immunother. 2015; l l(^'l l):2654-8). Importantly, T cell therapies have not yet proved useful against GBM. Clinical trials using HER2 (Ahmed N, et al. JAMA Oncol. 2017;3(8): 1094-101.), IL13Ra2 (Brown CE et al, Molecular therapy : the journal of the American Society of Gene Therapy. 2018;26(1):31 -44), or EGFRvIII CAR T cells (O'Rourke DM et al. Sci Transl Med. 2017;9(399)) elicited immune responses in GBM patients but with little clinical activity,

[0010] The efficiency of immunotherapy correlates with the ability of T cells to persist in vivo (June CH et al. 2015), but unfortunately in the case of GBM and other malignancies with complex microenvironments, the presence of dysfunctional macrophages and antigen-presenting cells means that antitumor responses are not stimulated by any encounter with T cells, as these cells are critical for initiating and maintaining activation and proliferation.

SUMMARY OF THE DISCLOSURE

[0011] The disclosure relates to a composition comprising an vector, nucleic acid sequence or nucleic acid molecule comprising at least one expressible sequence operably linked to a regulatory sequence. In some embodiments, the vector, nucleic acid sequence or nucleic acid molecule comprises an expressible sequence wherein the expressible sequence comprises a nucleic acid sequence encoding IL~6Ra, IL-lORa or functional fragments or salts thereof. In some embodiments, the expressible sequence also comprises a nucleic acid sequence that comprises a secretory signal sequence either 5' and/or 3 ' from the nucleic acid sequence that encodes the IL-6Ra, IL-1 ORa or functional fragment thereof.

[0012] In some embodiments, the composition comprises antigen-presenting cell, a T-cell or an NK cell comprising one or a plurality of vectors, nucleic acid sequences or nucleic acid molecules comprising at least one expressible sequence operably linked to a regulatory sequence. In some embodiments, the T cell or NK cell further comprises an exogenous vector, nucleic acid sequence or nucleic acid molecule comprising a nucleic acid sequence that encodes an antibody or antibody fragment comprising a TGF-β receptor domain and at least one CDR capable of binding one or more tumor associated antigens. In some embodiments, the TGF-β receptor domain comprises extracellular portion of TGFp-RII or a functional fragment thereof that comprises 70% sequence identity to extracellular portion of TGFp-RII.

[0013] The disclosure also relates to a method of treating and/or preventing a hyperproiiferative disorder comprising administering to a subject a pharmaceutical composition comprising: (a) an antigen -presenting cell, a T-cell or an NK cell comprising (i) one or a plurality of vectors, nucleic acid sequences or nucleic acid molecules comprising at least one expressible sequence operably linked to a regulatory sequence, the wherein the expressible sequence comprises a nucleic acid sequence encoding IL-6Ra, IL-! GRa or functional fragments or salts thereof, and (ii) one or a plurality of exogenous vectors, nucleic acid sequences or nucleic acid molecules comprising a nucleic acid sequence that encodes an antibody or antibody fragment comprising a TGF-β receptor domain and at least one CDR capable of binding one or more tumor associated antigens; and (b) a pharmaceutically acceptable carrier. In some embodiments, the cell secretes a pharmaceutically effective amount of one or a combination of: IL-6Ra, IL-l ORa or functional fragments or salts thereof, and an antibody or antibody fragment comprising a CDR capable of binding one or more tumor associated antigens. In some embodiments, the antibody or antibody fragment binds one or a combination of: H3K27M, DNAJB 1 -PRK AC A, bcr-abi, CDK4, MUM I . CTNNBl, CDC27, TRAPPCi, TP!. ASCC3, HHAT, FN1, OS-9, PTPRK, CDKN2A, HLA-A1 1, GAS 7, SIR2, PrdxS, CLPP, PPP1R3B, EF2, ACTN4, MEL NF-YC, HSP70-2, KIAA1440, CASP8, gag, pol, nef, env, survivin, MAGEA4, SSX2, FRAME, NYESOl, Oct4, Sox2, Nanog, WT1, p53, or MYCN. In some embodiments, the hyperproiiferative disorder is a cancer. In some embodiments, the hyperproiiferative disorder is a blood cancer such as leukemia. In some embodiments, the hyperproiiferative disorder is a metastatic cancer.

[0014] The disclosure relates, at least in part, to a method of treating and/or preventing a hyperproiiferative disorder associated with expression of the Cluster of Differentiation 47 protein (CD47). In certain embodiments, the disorder is a cancer, e.g., a breast cancer. In some embodiments, the method comprises administering an antibody or antibody binding fragment that binds CD47 or CD45 in combination with a TGFp Receptor inhibitor or a cell expressing a TGFp Receptor II antagonist or inhibitor, an IL-6 Receptor (IL-6R) antagonist or inhibitor, and /or IL-10 receptor (IL-10R) antagonist or inhibitor. In some embodiments, the disclosure relates to a method of treating and/or preventing a hyperproliferative disorder by administering one or a plurality of cells that express (i) an antibody or antibody binding fragment capable of binding CD47, CD45, and/or other cancer antigens; and (ii) a TGFP Receptor II antagonist or inhibitor, an IL-6 Receptor (IL-6R) antagonist or inhibitor, and /or IL-10 receptor (IL-10R) antagonist or inhibitor.

[0015] The terms "an antibody capable of binding glypican-1 " or "anti-glypican-1 antibody," for example, refer to an antibody, or an antigen binding fragment thereof, that is capable of binding glypican-1 with sufficient affinity such that the antibody is useful as a diagnostic, prophylactic and/or therapeutic agent in targeting glypican-1.

[0016] The terms "an antibody capable of binding CD73" or "anti-CD73 antibody," for example, refer to an antibody, or an antigen binding fragment thereof, that is capable of binding CD73 with sufficient affinity such that the antibody is useful as a diagnostic, prophylactic and/or therapeutic agent in targeting CD73.

[0017] The terms "an antibody capable of binding TGF□ " or "anti- TGF D antibody," for example, refer to an antibody, or an antigen binding fragment thereof, that is capable of binding TGFp with sufficient affinity such that the antibody is useful as a diagnostic, prophylactic and/or therapeutic agent in targeting TGFp.

[0018] The disclosure relates to cells that express an antibody or antibody fragment that binds or has an affinity to CD47 and/or CD45. In some embodiments, the antibody has a modified Fc domain that is capable of eliciting Antibody-Dependent Cell-mediated Cytotoxicity (ADCC), Complement Dependent Cytotoxicity (CDC), Antibody-Dependent Cell Phagocytosis (ADCP), endocytosis activity, cytokine secretion, or a combination of at least two of these activities. In some embodiments, the antibody has a modified Fc domain that is capable of encouraging recruitment of K cells. In some embodiments, the antibody has a modified Fc domain that is capable of eliciting ADCC, Antibody-dependent cell- mediated cytotoxicity (ADCC) is a mechanism of cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell, whose membrane-surface antigens have been bound by specific antibodies. ADCC is often thought of as being mediated by natural killer (NK) cells, but dendritic cells, macrophages, monocytes, and granulocytes can also mediate ADCC.

[0019] Natural killer (NK) cells play a major role in cancer immunotherapies that involve tumor-antigen targeting by monoclonal antibodies (mAbs). In the context of targeting ceils, NK cells can be "specifically activated" through certain Fc receptors that are expressed on their cell surface. NK cells can express ΡαγΚΠΙΑ and/or FcyRIIC, which can bind to the Fc portion of immunoglobulins, transmitting activating signals within NK ceils. Once activated through Fc receptors by antibodies bound to target cells, NK cells are able to lyse target cells without priming, and secrete cytokines like interferon gamma to recruit adaptive immune cells. Likewise, tumor-associated macrophages (TAMs) express surface receptors that bind the Fc fragment of antibodies and enable them to engage in Ab-dependent cellular cytotoxicity/phagocytosis (ADCC/ADCP). Because SIRPa/CD47 signaling induces a "don't eat me" response that reduces ADCC/ADCP, blocking of this signaling, for example by anti- CD47 antibodies with a modified Fc domain that is capable of eliciting ADCC, can enhance ADCC of tumor cells bearing the antigenic determinant to which the therapeutic antibody is directed.

[0020] For example, in some embodiments, the disclosure relates to a method of treating and/or preventing a hyperproliferative disorder by administering to a subject in need thereof a therapeutically effective amount of one or a plurality of clonal cells that expresses one or more (e.g., one, two, three or more) ΤΟΡβ Receptor II polypeptides or a salt thereof that are free of or substantially free of a biologically active signaling domain. In some embodiments, the disclosure relates to a method of treating and/or preventing a hyperproliferative disorder by administering to a subject in need thereof a therapeutically effective amount of one or a plurality of clonal cells that expresses one or more (e.g., one, two, three or more) IL-6R polypeptides or a salt thereof that are free of or substantially free of a biologically active signaling domain. In some embodiments, the disclosure relates to a method of treating and/or preventing a hyperproliferative disorder by administering to a subject in need thereof a therapeutically effective amount of one or a plurality of clonal cells that expresses one or more (e.g., one, two, three or more) IL-10R polypeptides or a salt thereof that are free of or substantially free of a biologically active signaling domain. In some embodiments, the disclosure relates to a method of treating and/or preventing a hyperproliferative disorder by administering to a subject in need thereof a therapeutically effective amount of one or a plurality of cells that express one or a combination of: ΤΟΡβ Receptor II polypeptides or a salt thereof, IL-6R polypeptides or a salt thereof, IL-10R polypeptides or a salt thereof, each of which are free of or substantially free of a biologically active signaling domain.

[0021] In some embodiments, the subject is exposed to one or a combination of polypeptides that modulate the immunosuppression of hyperproliferative cells in the subject. The polypeptides that modulate are chosen from an inhibitor of CD47, CD45, or a combination thereof. In some embodiments, the combination maintains or has better clinical effectiveness as compared to either therapy alone. In some embodiments, the methods herein involve the use of engineered cells, e.g., T cells, to express an antibody molecule or antibody binding fragment that binds CD47, and to express an inhibitor of cancer immunosuppression (e.g., polypeptide) capable of reducing ceil signaling of a humoral immune response in the subject. In some embodiments, the inhibitor of cancer immunosuppression is a polypeptide that binds cytokines or chemokines that that promote a humoral response in a subject (e.g., IL-6, ILIO, TGPP) or a antibody-expressing cell that binds to cytokines or chemokines that that promote a humoral response in a subject to treat a hyperproliferative disorder in a subject associated with expression of CD47 and/or CD45. The disclosure additionally features novel antigen binding domains and antibodies or antibody binding fragments directed to CD47 and/or CD45, and uses, e.g., as monotherapies or in combination therapies. Accordingly, in one aspect, the invention pertains to a method of treating a subject (e.g., a mammal) having a disease associated with expression of CD47 or a hyperproliferative disorder. The method comprises administering to the subject a CD47 inhibitor, e.g., one or a plurality of antibodies or antibody binding fragments that binds CD47 described herein, in combination with a polypeptide that encourages recruitment of NK cells.

[0022] The disclosure relates to administering to the subject an effective number of one or more cells that express: (i) an antibody molecule or antibody binding fragment that binds CD47, e.g., a antibody molecule or antibody binding fragment that binds CD47 described herein (e.g., a wild-type or mutant CD47); and, optionally (ii) one or a plurality of polypeptides that encourage a cell-mediated response or inhibit a humoral immune response in the subject to a hyperproliferative cell such as a cancer cell. In certain embodiments, the polypeptide is chosen from one or a combination of a TGFp Receptor II antagonist or inhibitor, an IL-6 Receptor (IL-6R) antagonist or inhibitor, and /or IL-IO receptor (IL-10R) antagonist or inhibitor. In some embodiments, the antagonist is an antibody or antibody fragment capable of binding to IL-lORalpha and or IL-6Ralpha. In some embodiments, the antibody or antibody fragment comprises at least a first and a second CDR, wherein the first CDR binds 1L-10R alpha and the second CDR binds IL-6Ralpha.

[0023] In a related aspect, the present disclosure provides a method of reducing the proliferation of cancer cells, e.g., by administering to a subject, e.g., a patient in need thereof, a combination therapy as described herein, e.g., a CD47 and/or CD45 inhibitor in combination with a polypeptide inhibitor described above, e.g., one or more one or a plurality of polypeptides that encourage a cell-mediated response or inhibit a humoral immune response in the subject described herein. In some embodinents, the combination therapy is a modified T cell or NK cell that comprises a vector or genetic construct comprising a nucleic acid sequence encoding a polypeptide inhibitor disclosed herein ora functional fragment thereof. In another aspect, the present disclosure provides a method of selectively killing cancer cells expressing CD47 and/or CD45 e.g., by administering to a subject, e.g., a patient in need thereof, a combination therapy as described herein, e.g., a CD47 and/or CD45 inhibitor in combination with a polypeptide inhibitor described above, e.g., one or more one or a plurality of polypeptides that encourage a cell-mediated response or inhibit a humoral immune response in the subject described herein. In certain aspects, the disclosure provides a method of providing an anti-tumor immunity in a subject, e.g., a mammal, comprising administering to the mammal an effective amount of a combination (e.g., one or more antibody or antibody binding fragment-expressing cells) as described herein. In some embodiments, the disclosure relates to a pharmaceutical composition comprising one or a plurality of modified T cells or NK cells comprising at least one, two or three vectors or genetic constructs, each vector or cgenetic construct comprsing at least one expressible sequence operably linked to a regulatory sequence and each expressible sequence comprising a nucleic acid sequence that encodes an antibody or antobdy fragment that binds to a tumor assosicated antigen and a IL-6Ralpha and/or IL-lORalpha amino acid sequence or functional fragment thereof comprising at least 70% sequence identity to the SEQ ID NO:33 or the sequence of IL-10R-alpha..

[0024] In some embodiments, the disclosure provides a method of preventing growth of a hyperproliferative cell in a subject, comprising administering to the subject one or more cells that express an antibody or antibody binding fragment that binds CD47 or a functional fragment thereof, optionally in combination with one or more polypeptides that bind EL-6, IL-10 and/or TGFp. In some embodiments, the subject has been diagnosed with breast cancer. DETAILED DESCRIPTION OF EMBODIM ENTS

[0025] The present disclosure is based, in part, on the development of T cells reconfi gured for antibody effector neutralization of neoplastic environments for use against T cell therapy-refractory malignancies. The cells described in the present disclosure are designed to disrupt the malignant niche, and recruit endogenous immune ceils against tumor cells.

[0026] The present disclosure relates, in part, to a single T cell-based treatment platform that is used to target a sufficient number of immune components in the tumor and its microenvironment to move the impact of T cell therapies beyond hematologic cancers and into frontline strategies for difficult-to-treat solid tumors.

[0027] Many modifications and other embodiments of the inventions set forth herein will easily come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclos ed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Definitions

[0028] Before the present compositions and methods are described, it is to be understood that this disclosure is not limited to the particular molecules, compositions, methodologies or protocols described, as these may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. It is understood that these embodiments are not limited to the particular methodology, protocols, cell lines, vectors, and reagents described, as these may vary. It also is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present embodiments or claims. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the disclosure described herein are capable of operation in other sequences than described or illustrated herein.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated by reference. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior disclosure.

[0030] As used in the specification and the appended claims, the singular forms "a,"

"an" and "the" include plural referents unless the context clearly dictates otherwise.

[0031] The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2% or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0032] The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i .e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and/or B," when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0033] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, "either," "one of," "only one of "

0 or "exactly one of "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0034] As used herein, the phrase "integer from X to Y" means any integer that includes the endpoints. That is, where a range is disclosed, each integer in the range including the endpoints is disclosed. For example, the phrase "integer from X to Y" discloses I, 2, 3, 4, or 5 as well as the range 1 to 5.

[0035] As used herein, the terms "comprising" (and any form of comprising, such as

"comprise", "comprises", and "comprised"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain"), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0036] As used herein, "substantially equal" means within a range known to be correlated to an abnormal or normal range at a given measured metric. For example, if a control sample is from a diseased patient, substantially equal is within an abnormal range. If a control sample is from a patient known not to have the condition being tested, substantially equal is within a normal range for that given metric.

[0037] As used herein, "substantially free of means absent or absent to a degree that its presence does not confer or result in biological activity when in presence of a cell or genetic construct. For example, if a genetic construct is free or substantially free of a signal seqeunce, it means that the genetic construct does not comprise a signal sequence that encodes an amount of signal sequence capable of functioning biologically when exposed in a ceil.

[0038] As used herein, the term "subject," "individual" or "patient," used interchangeably, means any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, such as humans.

[0039] The term "subject" is used throughout the specification to describe an animal from which a cell sample is taken. In some embodiments, the subject is a human. For diagnosis of those conditions which are specific for a specific subject, such as a human being, the tenn "patient" may be interchangeably used. In some instances in the description of the present invention, the term "patient" will refer to human patients suffering from a particular disease or disorder. In some embodiments, the subject may be a human suspected of having or being identified as at risk to develop a hyperproliferative disease. In some embodiments, the subject may be diagnosed as having malignant cancer and of having or being identified as

1 at risk to develop a metastatic hyperproliferative disease. In some embodiments, the subject is suspected of having or has been diagnosed with breast cancer or lung cancer. In some embodiments, the subject may be a human suspected of having or being identified as at risk to develop lung cancer or breast cancer. In some embodiments, the subject may be a mammal which functions as a source of the isolated cell sample. In some embodiments, the subject may be a non-human animal from which a ceil sample is isolated or provided. In some embodiments, the subject may be a human suspected of having or being identified as at risk to develop a type of cancer more severe or invasive than initially diagnosed. In some embodiments, the subject may be diagnosed as having a resistance to one or a plurality of treatments to treat a disease or disorder afflicting the subject. In some embodiments, the subject is suspected of having or has been diagnosed with stage I, II, III or greater stage of cancer. In some embodiments, the subject may be a human suspected of having or being identified as at risk to a terminal condition or disorder. In some embodiments, the subject may be a mammal which functions as a source of the isolated sample of biopsy or bodily fluid. In some embodiments, the subject may be a non-human animal from which a sample of biopsy or bodily fluid is isolated or provided.

|0040| As used herein, the term "animal" includes, but is not limited to, humans and non-human vertebrates such as wild animals, rodents, such as rats, ferrets, and domesticated animals, and farm animals, such as dogs, cats, horses, pigs, cows, sheep, and goats. In some embodiments, the animal is a mammal. In some embodiments, the animal is a human. In some embodiments, the animal is a non-human mammal.

[0041] As used herein, the term "mammal" means any animal in the class

Mammalia such as rodent (i.e., a mouse, a rat, or a guinea pig), a monkey, a cat, a dog, a cow, a horse, a pig, or a human. In some embodiments, the mammal is a human. In some embodiments, the mammal refers to any non-human mammal. The present disclosure relates to any of the methods or compositions of matter disclosed herein wherein the sample is taken from a mammal or non-human mammal. The present disclosure relates to any of the methods or compositions of matter disclosed herein wherein the sample is taken from a human or non- human primate.

|0042| As used herein, the phrase "in need thereof means that the animal or mammal has been identified or suspected as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis or observation. In any of the methods and treatments described herein, the animal or mammal can be in need thereof. In some embodiments, the animal or mammal is in an environment or will be traveling to an environment in which a particular disorder or condition is prevalent or more likely to occur.

[0043] As used herein, the term "cancer" refers to diseases in which abnormal cells divide without control and are able to invade other tissues. There are more than 100 different types of cancer. Most cancers are named for the organ or type of cell in which they start - for example, cancer that begins in the colon is called colon cancer, cancer that begins in melanocytes of the skin is called melanoma. Cancer types can be grouped into broader categories. The main categories of cancer include: carcinoma (meaning a cancer that begins in the skin or in tissues that line or cover internal organs, and its subtypes, including adenocarcinoma, basal cell carcinoma, squamous ceil carcinoma, and transitional cell carcinoma), sarcoma (meaning a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue): leukemia (meaning a cancer that starts in blood-forming tissue (e.g., bone marrow) and causes large numbers of abnormal blood cells to be produced and enter the blood; lymphoma and myeloma (meaning cancers that begin in the cells of the immune system); and central nervous system (CNS) cancers (meaning cancers that begin in the tissues of the brain and spinal cord). The term "myelodysplastic syndrome" refers to a type of cancer in which the bone marrow does not make enough healthy blood ceils (white blood cells, red blood cells, and platelets) and there are abnormal ceils in the blood and/or bone marrow. Myelodysplastic syndrome may become acute myeloid leukemia (AML). In certain embodiments, the cancer is selected from cancers including, but not limited to, ACUTE lymphoblastic leukemia (ALL), ACUTE myeloid leukemia (AML), anal cancer, bile duct cancer, bladder cancer, bone cancer, bowel cancer, brain tumour, breast cancer, cancer of unknown primary, cancer spread to bone, cancer spread to brain, cancer spread to liver, cancer spread to lung, carcinoid, cervical cancer, choriocarcinoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), colon cancer, colorectal cancer, endometrial cancer, eye cancer, gallbladder cancer, gastric cancer, gestational trophoblastic tumour (GTT), hairy cell leukemia, head and neck cancer, Hodgkin lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma skin cancer, mesothelioma, men's cancer, molar pregnancy, mouth and oropharyngeal cancer, myeloma, nasal and sinus cancers, nasopharyngeal cancer, non hodgkin lymphoma (NHL), oesophageal cancer, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, rare cancers, rectal cancer, salivary gland cancer, secondary cancers, skin cancer (non melanoma),

3 soft tissue sarcoma, stomach cancer, testicular cancer, thyroid cancer, unknown primar cancer, uterine cancer, vaginal cancer, and vulval cancer. According to one embodiment, the cancer is glioblastoma multiforme (GBM).

[0044] As used herein, the terms "activate," "stimulate," "enhance" "increase" and/or "induce" (and like terms) are used interchangeably to generally refer to the act of improving or increasing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition. "Activate" refers to a primary response induced by ligation of a cell surface moiety. For example, in the context of receptors, such stimulation entails the ligation of a receptor and a subsequent signal transduction event. Further, the stimulation event may activate a ceil and upregulate or downregulate expression or secretion of a molecule. Thus, ligation of cell surface moieties, even in the absence of a direct signal transduction event, may result in the reorganization of cytoskeletal structures, or in the coalescing of ceil surface moieties, each of which could serve to enhance, modify, or alter subsequent cellular responses.

[0045] As used herein, the terms "activating CD8+ T ceils" or "CD8+ T cell activation" refer to a process (e.g., a signaling event) causing or resulting in one or more cellular responses of a CD8+ T cell (CTL), selected from : proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. As used herein, an "activated CD8+ T cell" refers to a CD8+ T cell that has received an activating signal, and thus demonstrates one or more cellular responses, selected from proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. Suitable assays to measure CD8+ T cel l activation are known in the art and are described herein ,

[0046] As used herein, the terms "expanding a CD8+ T cell" or "CD8+ T cell expansion" refer to a process wherein a CD8+ T cell undergoes a series of cell divisions and thereby expands in cell number. The term "expanded CD8+ T cells" relates to CD8+ T cells obtained through CD8+ T cell expansion. Suitable assays to measure T cell expansion are known in the art and are described herein.

[0047] As used herein, the term "activating an NK cell" or "NK cell activation" refers to a process (e.g., a signaling event) causing or resulting in an NK cell being capable of killing cells with deficiencies in MHC class I expression. As used herein, an "activated NK cell" refers to an NK cell that has received an activating signal, and is thus capable of killing ceils with deficiencies in MHC class I expression. Suitable assays to measure NK cell activation are known in the art and are described herein.

[0048] As used herein, the terms "expanding an K cell" or "NK cell expansion" refer to a process wherein an NK cell undergoes a series of cell divisions and thereby expands in cell number. The term "expanded NK cells" relates to NK cells obtained through NK cell expansion. Suitable assays to measure NK cell expansion are known in the art and are described herein.

[0049] As used herein, the term "cytokine" refers to small soluble protein substances secreted by cells which have a variety of effects on other ceils. Cytokines mediate many important physiological functions including growth, development, wound healing, and the immune response. They act by binding to their cell-specific receptors located in the cell membrane, which allows a distinct signal transduction cascade to start in the cell, which eventually will lead to biochemical and phenotypic changes in target cells. Cytokines can act both locally and distantly from a site of release. They include type I cytokines, which encompass many of the interleukins, as well as several hematopoietic growth factors, type II cytokines, including the interferons and interleukin-10; tumor necrosis factor ("TNF")-related molecules, including TNFa and lymphotoxin; immunoglobulin super-family members, including interieukin 1 ("IL-l "); and the chemokines, a family of molecules that play a critical role in a wide variety of immune and inflammatory functions. The same cytokine can have different effects on a ceil depending on the state of the ceil. Cytokines often regulate the expression of, and trigger cascades of other cytokines. Non limiting examples of cytokines include e.g., IL-l , IL-2, IL-3, IL-4, L-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-l 1 , IL- 12/IL-23 P40, IL13, IL-15, IL-15/IL15-RA, IL-17, IL-l 8, IL-21, IL-23, TGF-β, IFNy, GM- CSF, Groa, MCP-1 and TNF-a.

[0050] As used herein, "cell culture" means growth, maintenance, transfection, transduction, or propagation of cells, tissues, or their products. As used herein, "culture medium" refers to any solution capable of sustaining the growth of the targeted cells either in vitro or in vivo, or any solution with which targeted cells or exogenous nucleic acids are mixed before being applied to ceils in vitro or to a patient in vivo.

[0051] As used herein, the terms "heterologous" and "foreign" with reference to nucleic acids, such as DNA and RNA, are used interchangeably and refer to nucleic acid that does not occur naturally as part of a genome or cell in which it is present or which is found in a location! s) and/or in amounts in a genome or cell that differ from the location(s) and/or amounts in which it occurs in nature, i.e., nucleic acid that is not endogenous to the cell and

1 has been exogenously introduced into the cell. Examples of heterologous DNA include, but are not limited to, DNA that encodes a gene product or gene product(s) of interest introduced into cells, for example, for production of an encoded protein. Other examples of heterologous DNA include, but are not limited to, DNA that encodes an antigen binding domain, a chemokine receptor, or an antibody.

[0052] The terms "immune response" and "immune-mediated" are used interchangeably herein to refer to any functional expression of a subject's immune system, against either foreign or self-antigens, whether the consequences of these reactions are beneficial or harmful to the subject.

[0053] The terms "immunomodulatory", "immune modulator" and "immune modulatory" are used interchangeably herein to refer to a substance, agent, or cell that is capable of augmenting or diminishing immune responses directly or indirectly by expressing chemokines, cytokines and other mediators of immune responses.

[0054] As used herein the term "immunostimulatory amount" of the disclosed compositions refers to an amount of an immunogenic composition that is effective to stimulate an immune response, for example, as measured by ELISPOT assay (cellular immune response), ICS (intracellular cytokine staining assay) and major histocompatibility complex (MHC) tetramer assay to detect and quantify antigen-specific T cells, quantifying the blood population of antigen-specific CD4+ T cells, or quantifying the blood population of antigen specific CD8+ T cells by a measurable amount, or where the increase is 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 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, when compared to a suitable control,

[0055] As used herein, "delivery" refers to the process by which exogenous nucleic acid molecules are transferred into a cell such that they are located inside the cell. Deliver}' of nucleic acids is a distinct process from expression of nucleic acids. Nucleic acid material can be introduced into the cell ex vivo or in vivo by genetic transfer methods, such as transfecti on or transduction, to provide a genetically modified cell. Various expression vectors (i.e., vehicles for facilitating delivery of exogenous nucleic acid into a target cell) are known to one of ordinary skill in the art.

[0056] As used herein, "expression" refers to the process by which nucleic acid is translated into peptides or is transcribed into mRNA and translated into peptides, polypeptides or proteins. If the nucleic acid is derived from genomic DNA, expression may,

6 if an appropriate eukaryotic host cell or organism is selected, include splicing of the niRNA. For heterologous nucleic acid to be expressed in a host cell, it must initially be delivered into the cell and then, once in the cell, ultimately reside in the nucleus.

[0057] As used herein, "transfection of cells" refers to the acquisition by a ceil of new nucleic acid material by incorporation of added DNA. Thus, transfection refers to the insertion of nucleic acid into a cell using physical or chemical methods. Several transfection techniques are known to those of ordinary skill in the art including: calcium phosphate DNA co-precipitation (Methods in Molecular Biology ( 1991)); DEAE- dextran (supra); el ectrop oration (supra); cationic iiposome-medi ated transfection (supra); and tungsten particle-facilitated microparticle bombardment (Johnston (1990)). Strontium phosphate DNA co-precipitation (Brash et ai. (1987)) is also a transfection method.

[0058] In contrast, "transduction of cells" refers to the process of transferring nucleic acid into a cell using a DNA or RNA virus. A RNA virus (i.e., a retrovirus) for transferring a nucleic acid into a cell is also referred to herein as a transducing retrovirus. Exogenous nucleic acid material contained within the retrovirus is incorporated into the genome of the transduced cell, A cell that has been transduced with a DNA virus (e.g., an adenovirus carrying a cDNA encoding a therapeutic agent), will not have the exogenous nucleic acid material incorporated into its genome but will be capable of expressing the exogenous nucleic acid material that is retained extrachromosomally within the cell.

[0059] The exogenous nucleic acid material can include a nucleic acid encoding an antibody together with a promoter to control transcription. The promoter characteristically has a specific nucleotide sequence necessary to initiate transcription. The exogenous nucleic acid material may further include additional sequences (i.e., enhancers) required to obtain the desired gene transcription activity. For the purpose of this discussion an "enhancer" is simply any non -translated DNA sequence that works with the coding sequence (in cis) to change the basal transcription level dictated by the promoter. The exogenous nucleic acid material may be introduced into the cell genome immediately downstream from the promoter so that the promoter and coding sequence are operativelv linked so as to permit transcription of the coding sequence. An expression vector can include an exogenous promoter element to control transcription of the inserted exogenous gene. Such exogenous promoters include both constitutive and regulatabie promoters.

[0060] The term "domain" as used herein applies to a portion or subsequence of amino acids within a peptide or nucleic acids within a nucleotide sequence. In some embodiments, a domain provides a functionality, activity, or benefit. In one embodiment for example a domain may be a receptor or a signaling portion of a receptor. In another embodiment a domain may have a linker function between two other domains. In another embodiment a domain may serve to bind a specific target analyte (target domain), such as an antigen or chemokine.

[0061] An "intracellular signaling domain," as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain can generate a signal that promotes an immune effector function of a cell, e.g., a cancer cell. Examples of immune effector function, such as cytolytic activity and helper activity, including the secretion of cytokines. In embodiments, the intracellular signal domain is the portion of the protein which transduces the effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain suffi cient to transduce the effector function signal.

[0062] An "antigen binding protein" is a protein comprising a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a confirmation that promotes binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include antibodies, antibody fragments (e.g., an antigen binding fragment of an antibody), antibody derivatives, and antibody analogs. The antigen binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR fragments or variants with substantially the same binding affinity as one or more disclosed CDR amino acid sequences Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. See, for example, Korndorfer et a!,, 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1 : 121 -129; Roque et al., 2004, Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics ("PAMs") can be used, as well as scaffolds based on antibody mimetics utilizing fibronection components as a scaffold.

[0063] The term "antibody" as used herein refers to a polypeptide or group of

8 polypeptides that are comprised of at least one binding domain that is formed from the folding of polypeptide chains having three-dimensional binding spaces with internal surface shapes and charge distributions complementary to the features of an antigenic determinant of an antigen. The term "antibody" is synonymous with immunoglobulin (Ig) and is to be understood as commonly known in the art. The basic antibody structural unit is a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). Generally, the amino- terminai portion of each antibody chain includes a variable region that is primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region, e.g., responsible for effector function. Human light chains are classified as kappa or lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 3 or more amino acids. The term antibody may mean an antibody of classes IgG, IgM, IgA, IgD or IgE, or fragments, binding fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies, antigen binding proteins thereof and derivatives thereof The antibody may be an antibody isolated from the serum sample of mammal, a polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom.

[0064] The variable regions of each heavy/light chain pair (VH/VL), respectively, form the antigen binding site. The variable regions of antibody heavy and light chains (VH/VL) exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is known in the art, including, for example, definitions as described in Kabat et al. in Sequences of Proteins of Immunological Interest, 5 Ed., US Dept. of Health and Human Sendees, PHS, NIH, NIH Publication no. 91-3242, 1991 (herein referred to as "Kabat numbering"). For example, the CDR regions of an antibody can be determined according to Kabat numbering.

[0065] The terms "intact antibody" or "full length antibody" refer to an antibody composed of two identical antibody light chains and two identical antibody heavy chains that each contain an Fc region.

[0066] An "antigen binding domain," "antigen binding region," or "antigen binding site" is a portion of an antigen binding protein that contains amino acid residues (or other moieties) that interact with an antigen and contribute to the antigen binding protein's specificity and affinity for the antigen. For an antibody that specifically binds to its antigen, this will include at least part of at least one of its CDR domains.

[0067] An "epitope" is the portion of a molecule that is bound by an antigen binding protein (e.g., by an antibody). An epitope can comprise non-contiguous portions of the molecule (e.g., in a polypeptide, amino acid residues that are not contiguous in the polypeptide's primary sequence but that, in the context of the polypeptide's tertiary and quaternary structure, are near enough to each other to be bound by an antigen binding protein). Generally the variable regions, particularly the CDRs, of an antibody interact with the epitope.

[0068] As used herein, the term "genetic construct" refers to the DNA or RNA molecules that comprise a nucleotide sequence which encodes an amino acid sequence or immunomodulating protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered.

[0069] As used herein, the term "expressible sequence" refers to gene constructs that contain the necessary regulator}' elements operably linked to a coding sequence that encodes an amino acid sequence or an immunomodulating protein, such that when present in the cell of the individual, the coding sequence will be expressed.

[0070] The term "Fc polypeptide" includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization also are included. Fusion proteins comprising Fc moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Protein A or Protein G columns.

[0071] As used herein, the term "variants" is intended to mean substantially similar sequences. For nucleic acid molecules, a variant comprises a nucleic acid molecule having deletions (i.e., truncations) at the 5' and/or 3' end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" nucleic acid molecule or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For nucleic acid molecules, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the disclosure. Variant nucleic acid molecules also include synthetically derived nucleic acid molecules, such as those generated, for example, by using site-directed mutagenesis but which still encode a protein of the disclosure. Generally, variants of a particular nucleic acid molecule of the disclosure will have at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%>, 99%> or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described herein.

[0072] The term "analog" as used herein refers to compounds that are similar but not identical in chemical formula and share the same or substantial function of the compound with the similar chemical formula.

[0073] "Inhibitors" and "antagonists" may be agents that decrease, block, or prevent, signaling (e.g., TGFp Receptor signaling) via a pathway and/or which prevent the formation of protein interactions and complexes between receptors and their natural ligands. The term "antagonist" or "inhibitor" of TGFp signaling, as used herein, is meant to refer to an agent that inhibits, arrests, or otherwise negatively regulates TGFP signaling, due to, in some embodiments, its antagonizing effect on TGFp Receptor and/or its antagonizing effect on natural signaling effects of TGFp signaling pathway. Said TGFp antagonist can be a compound or agent which abrogates the signaling of TGFp in one or a plurality of ceils by competing for extracellular TGFp and thereby abrogates the activity of TGFp Receptors I and/or II on a cell. In some embodiments, the TGF receptor antagonist is a modified TGFP- Receptor I and/or II that is free of or substantially free of its signaling domain such that its presence on a cell competes for bioavailabie TGF thereby preventing signaling through TGFp-reeeptor I and/or II expressed on a cell. In such systems in vivo, the modified cells of the present disclosure can, in some embodiments upon administration, or mimics prevents the binding of TGFp or TGFp analogs to TGFp-reeeptor I and/or II on a hypeiproiiferative ceil.

Cells of the immune System

[0074] There are a large number of cellular interactions thai comprise the immune system. These interactions occur through specific receptor-iigand pairs that signal in both directions so that each cell receives instructions based on the temporal and spatial distribution of those signals.

[0075] Murine models have been highly useful in discovering immunomodulatory pathways, but clinical utility of these pathways does not always translate from an inbred mouse strain to an outbred human population, since an outbred human population may have individuals that rely to varying extents on individual immunomodulatory pathways.

[0076] Cells of the immune system include lymphocytes, monocytes/macrophages, dendritic ceils, the closely related Langerhans cells, natural killer (NK) cells, mast cells, basophils, and other members of the myeloid lineage of cells. In addition, a series of specialized epithelial and stromal cells provide the anatomic environment in which immunity occurs, often by secreting critical factors that regulate growth and/or gene activation in cells of the immune system, which also play direct roles in the induction and effector phases of the response. (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed, Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999), at p. 102).

[0077] The cells of the immune system are found in peripheral organized tissues, such as the spleen, lymph nodes, Peyer's patches of the intestine and tonsils. Lymphocytes also are found in the central lymphoid organs, the thymus, and bone marrow where they undergo developmental steps that equip them to mediate the myriad responses of the mature immune system. A substantial portion of lymphocytes and macrophages comprise a recirculating pool of cells found in the blood and lymph, providing the means to deliver immunocompetent cells to sites where they are needed and to allow immunity that is generated locally to become generalized. (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999), at p. 102).

[0078] The term "lymphocyte" refers to a small white blood ceil formed in lymphatic tissue throughout the body and in normal adults making up about 22-28% of the total number of leukocytes in the circulating blood that plays a large role in defending the body against disease. Individual lymphocytes are specialized in that they are committed to respond to a limited set of structurally related antigens through recombination of their genetic material {e.g. to create a T ceil receptor and a B ceil receptor). This commitment, which exists before the first contact of the immune system with a given antigen, is expressed by the presence of receptors specific for determinants (epitopes) on the antigen on the lymphocyte's surface membrane. Each lymphocyte possesses a unique population of receptors, all of which have identical combining sites. One set, or clone, of lymphocytes differs from another clone in the structure of the combining region of its receptors and thus differs in the epitopes that it can recognize. Lymphocytes differ from each other not only in the specificity of their receptors, but also in their functions, (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999), at p. 102).

[0079] Two broad classes of lymphocytes are recognized: the B-lymphocytes (B- cells), which are precursors of antibody-secreting cells, and T-lymphocytes (T-cells).

B-Lymphocytes

[0080] B-lymphocytes are derived from hematopoietic cells of the bone marrow. A mature B-cell can be activated with an antigen that expresses epitopes that are recognized by its cell surface. The activation process may be direct, dependent on cross-linkage of membrane Ig molecules by the antigen (cross-linkage-dependent B-cell activation), or indirect, via interaction with a helper T-cell, in a process referred to as cognate help. In many physiological situations, receptor cross-linkage stimuli and cognate help synergize to yield more vigorous B-cell responses (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0081] Cross-linkage dependent B-cell activation requires that the antigen express multiple copies of the epitope complementary to the binding site of the cell surface receptors, because each B-cell expresses Ig molecules with identical variable regions. Such a requirement is fulfilled by other antigens with repetitive epitopes, such as capsular polysaccharides of microorganisms or viral envelope proteins. Cross-linkage-dependent B- cell activation is a major protective immune response mounted against these microbes (Paul, W. E., "Chapter 1 : The immune system: an introduction", Fundamental Immunology, 4th Edition, Ed. Paul, W. E„, Lippicott-Raven Publishers, Philadelphia, (1999)).

[0082] Cognate help allows B-cells to mount responses against antigens that cannot cross-link receptors and, at the same time, provides costimulatory signals that rescue B cells from inactivation when they are stimulated by weak cross-linkage events. Cognate help is dependent on the binding of antigen by the B-cell' s membrane immunoglobulin (Ig), the endocytosis of the antigen, and its fragmentation into peptides within the endosomal/lysosomal compartment of the cell. Some of the resultant peptides are loaded into a groove in a specialized set of cell surface proteins known as class II major histocompatibility complex (MHC) molecules. The resultant class Il/peptide complexes are expressed on the ceil surface and act as Iigands for the antigen-specific receptors of a set of T-cells designated as CD4⁺ T-cells. The CD4^" T-ceils bear receptors on their surface specific for the B-cell's class Il/peptide complex. B-cell activation depends not only on the binding of the T cell through its T cell receptor (TCR), but this interaction also allows an activation ligand on the T-cell (CD40 ligand) to bind to its receptor on the B-cell (CD40) signaling B- ceil activation. In addition, T helper cells secrete several cytokines that regulate the growth and differentiation of the stimulated B-cel! by binding to cytokine receptors on the B cell (Paul, W. E., "Chapter 1 : The immune system: an introduction, "Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Li ppicott -Raven Publishers, Philadelphia, (1999)).

[0083] During cognate help for antibody production, the CD40 ligand is transiently expressed on activated CD4⁺ T helper ceils, and it binds to CD40 on the antigen-specific B ceils, thereby transducing a second costimulatory signal. The latter signal is essential for B ceil growth and differentiation and for the generation of memory B ceils by preventing apoptosis of germinal center B cells that have encountered anti gen. Hyperexpression of the CD40 ligand in both B and T cells is implicated in pathogenic autoantibody production in human SLE patients (Desai-Mehta, A. et al, "Hyperexpression of CD40 ligand by B and T ceils in human lupus and its role in pathogenic autoantibody production," J. Clin. Invest. Vol. 97(9), 2063-2073, (1996)).

T-Lymphocytes

[0084] T -lymphocytes derived from precursors in hematopoietic tissue, undergo differentiation in the thymus, and are then seeded to peripheral lymphoid tissue and to the recirculating pool of lymphocytes. T-lymphocytes or T cells mediate a wide range of immunologic functions. These include the capacity to help B cells develop into antibody- producing cells, the capacity to increase the microbicidal action of monocytes/macrophages, the inhibition of certain types of immune responses, direct killing of target cells, and mobilization of the inflammatory response. These effects depend on T cell expression of specific cell surface molecules and the secretion of cytokines (Paul, W. E., "Chapter 1 : The immune system: an introduction", Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0085] T cells differ from B cells in their mechanism of antigen recognition.

Immunoglobulin, the B cell's receptor, binds to individual epitopes on soluble molecules or on particulate surfaces. B-cell receptors see epitopes expressed on the surface of native molecules. While antibody and B-cell receptors evolved to bind to and to protect against microorganisms in extracellular fluids, T cells recognize antigens on the surface of other cells and mediate their functions by interacting with, and altering, the behavior of these antigen- presenting cells (APCs). There are three main types of APCs in peripheral lymphoid organs that can activate T cells: dendritic cells, macrophages and B cells. The most potent of these are the dendritic cells, whose only function is to present foreign antigens to T cells. Immature dendritic cells are located in tissues throughout the body, including the skin, gut, and respiratory tract. When they encounter invading microbes at these sites, they endocytose the pathogens and their products, and cany them via the lymph to local lymph nodes or gut associated lymphoid organs. The encounter with a pathogen induces the dendritic cell to mature from an antigen-capturing cell to an APC that can activate T cells. APCs display three types of protein molecules on their surface that have a role in activating a T cell to become an effector cell : (1) MHC proteins, which present foreign antigen to the T cell receptor; (2) costimulatory proteins which bind to complementary receptors on the T cell surface, and (3) cell-cell adhesion molecules, which enable a T cell to bind to the APC for long enough to become activated ("Chapter 24: The adaptive immune system," Molecular Biology of the Cell, Alberts, B. el ah . Garland Science, NY, (2002)).

[0086] T-cells are subdivided into two distinct classes based on the cell surface receptors they express. The majority of T cells express T cell receptors (TCR) consi sting of a and β-chains. A small group of T cells express receptors made of γ and δ chains. Among the α/β T cells are two sub-lineages: those that express the coreceptor molecule CD4 (CD4⁺ T cells), and those that express CDS (CDS^"" T cells). These cells differ in how they recognize antigen and in their effector and regulator}' functions.

[0087] CD4⁺ T cells are the major regulatory cells of the immune system. Their regulatory function depends both on the expression of their cell-surface molecules, such as CD40 ligand whose expression is induced when the T cells are activated, and the wide array of cytokines they secrete when activated.

[0088] CD8+ (cytotoxic) T cells, like CD4+ Helper T cells, are generated in the thymus and express the T-cell receptor. However, rather than the CD4 molecule, cytotoxic T ceils express a dimeric co-receptor, CDS, usually composed of one CDSa and one ϋ)8β chain. CD8+ T cells recognize peptides presented by MHC Class I molecules, found on all nucleated cells. The CDS heterodimer binds to a conserved portion (the a3 region) of MHC Class I during T cell/antigen presenting cell interactions. CD8+ T cells (often called cytotoxic T lymphocytes, or CTLs) are important for immune defense against intracellular pathogens, including viruses and bacteria, and for tumour surveillance. When a CD8+ T cell recognizes its antigen and becomes activated, it has three major mechanisms to kill infected or malignant cells. The first is secretion of cytokines, primarily TNF-a and ΙΡΝγ, which have anti-tumour and anti-viral microbial effects. The second major function is the production and release of cytotoxic granules. These granules, also found in K cells, contain two families of proteins, perforin, and granzymes. Perforin forms a pore in the membrane of the target cell, similar to the membrane attack complex of complement. This pore allows the granzymes also contained in the cytotoxic granules to enter the infected or malignant ceil. Granzymes are serine proteases which cleave the proteins inside the ceil, shutting down the production of viral proteins and ultimately resulting in apoptosis of the target cell. CD8+ T cells are able to release their granules, kill an infected ceil, then move to a new target and kill again, often referred to as serial killing. The third major function of CD8+ T cell destruction of infected ceils is via Fas/FasL interactions. Activated CD8+ T ceils express FasL on the cell surface, which binds to its receptor, Fas, on the surface of the target cell. This binding causes the Fas molecules on the surface of the target cell to trimerize, which pulls together signaling molecules. These signaling molecules result in the activation of the caspase cascade, which also results in apoptosis of the target cell. Because CD8+ T cells can express both molecules, Fas/FasL interactions are a mechanism by which CD8+ T cells can kill each other, called fratricide, to eliminate immune effector cells during the contraction phase at the end of an immune response.

[0089] T cells also mediate important effector functions, some of which are determined by the patterns of cytokines they secrete. The cytokines can be directly toxic to target cells and can mobilize potent inflammatory mechanisms.

[0090] In addition, T cells, particularly CD8⁺ T cells, can develop into cytotoxic T- lymphocytes (CTLs) capable of effi ciently lysing target cells that express antigens recognized by the CTLs (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0091] T cell receptors (TCRs) recognize a complex consisting of a peptide derived by proteolysis of the antigen bound to a specialized groove of a class II or class I MHC protein. CD4⁺ T cells recognize only peptide/class II complexes while CD8^÷ T cells recognize peptide/class I complexes (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0092] The TCR's ligand (i.e., the peptide/MHC protein complex) is created within

APCs. In general, class II MHC molecules bind peptides derived from proteins that have been taken up by the APC through an endocytic process. These pepti de-loaded class II molecules are then expressed on the surface of the cell, where they are available to be bound by CD4^"r T cells with TCRs capable of recognizing the expressed cell surface complex. Thus, CD4^~r T cells are specialized to react with antigens derived from extracellular sources (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0093] In contrast, class I MHC molecules are mainly loaded with peptides derived from internally synthesized proteins, such as viral proteins. These peptides are produced from cytosolic proteins by proteolysis by the proteosome and are translocated into the rough endoplasmic reticulum. Such peptides, generally composed of nine amino acids in length, are bound into the class I MHC molecules and are brought to the cell surface, where they can be recognized by CDS^"'^" T cells expressing appropriate receptors. This gives the T cell system, particularly CD8⁺ T cells, the ability to detect cells expressing proteins that are different from, or produced in much larger amounts than, those of cells of the remainder of the organism (e.g., viral antigens) or mutant antigens (such as active oncogene products), even if these proteins in their intact form are neither expressed on the cell surface nor secreted (Paul, W. E., "Chapter 1 : The immune system : an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0094] T cells can also be classified based on their function as helper T cells; T cells involved in inducing cellular immunity; suppressor T cells; and cytotoxic T cells. In some embodiments, the compositions or pharmaceutical compositions of the disclosure are free or substantially free of B cells.

Helper T Cells

[0095] Helper T cells are T ceils that stimulate B cells to make antibody responses to proteins and other T cell-dependent antigens. T cell-dependent antigens are immunogens in which individual epitopes appear only once or a limited number of times such that they are unable to cross-link the membrane immunoglobulin (Ig) of B cells or do so inefficiently. B cells bind the antigen through their membrane Ig, and the complex undergoes endocytosis. Within the endosomal and lysosomal compartments, the antigen is fragmented into peptides by proteolytic enzymes, and one or more of the generated peptides are loaded into class II MHC molecules, which traffic through this vesicular compartment. The resulting peptide/class II MHC complex is then exported to the B-cell surface membrane. T cells with receptors specific for the peptide/class II molecular complex recognize this complex on the B-cell surface. (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia ( 1999)).

[0096] B-cell activation depends both on the binding of the T cell through its TCR and on the interaction of the T-cell CD40 ligand (CD40L) with CD40 on the B cell. T cells do not constitutively express CD40L. Rather, CD40L expression is induced as a result of an interaction with an APC that expresses both a cognate antigen recognized by the TCR of the T cell and CD80 or CD86. CD80/CD86 is generally expressed by activated, but not resting, B cells so that the helper interaction involving an activated B cell and a T cell can lead to efficient antibody production. In many cases, however, the initial induction of CD40L on T cells is dependent on their recognition of antigen on the surface of APCs that constitutively express CD80/86, such as dendritic cells. Such activated helper T cells can then efficiently interact with and help B cells. Cross-linkage of membrane Ig on the B cell, even if inefficient, may synergize with the CD40L/CD40 interaction to yield vigorous B-ceil activation. The subsequent events in the B-cell response, including proliferation, Ig secretion, and class switching of the Ig class being expressed, either depend or are enhanced by the actions of T cell-derived cytokines (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0097] CD4⁺ T cells tend to differentiate into cells that principally secrete the cytokines IL-4, IL-5, IL-6, and IL-10 (T_H2 cells) or into cells that mainly produce IL-2, IF y, and lymphotoxin (¾! cells). The ¾2 cells are very effective in helping B-cells develop into antibody-producing cells, whereas the T_H1 cells are effective inducers of cellular immune responses, involving enhancement of microbicidal activity of monocytes and macrophages, and consequent increased efficiency in lysing microorganisms in intracellular vesicular compartments. Although CD4^"r T cells with the phenotype of T_H2 cells (i.e., IL-4, IL-5, IL-6 and IL-10) are efficient helper cells, T_H1 cells also have the capacity to be helpers (Paul, W. E., "Chapter 1 : The immune system: an introduction, "Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, ( 1999)).

Natural Killer (NK) Cells [0098] Natural Killer (NK) Cells are lymphocytes in the same family as T and B cells, coming from a common progenitor. However, as cells of the innate immune system, NK cells are classified as group I Innate Lymphocytes (LLCs) and respond quickly to a wide variety of pathological challenges. NK cells protect against disease, for example killing virally infected cells, and detecting and controlling early signs of cancer. NK cells were first noticed for their ability to kill tumour ceils without any priming or prior activation (in contrast to cytotoxic T cells, which need priming by antigen presenting cells). They are named for this 'natural' killing. Additionally, NK cells secrete cytokines such as ΙΡΝγ and TNFa, which act on other immune cells like Macrophage and Dendritic cells to enhance the immune response.

[0099] While on patrol NK cells constantly contact other ceils. Whether or not the

NK cell kills these cells depends on a balance of signals from activating receptors and inhibitory receptors on the NK cell surface. Activating receptors recognize molecules that are expressed on the surface of cancer cells and infected cells, and 'switch on' the NK cell . Inhibitor}- receptors act as a check on NK cell killing. Most normal healthy ceils express MHC I receptors which mark these cells as 'self . Inhibitory receptors on the surface of the NK cell recognize cognate MHC I, and this ' switches off the NK cell, preventing it from killing. Cancer cells and infected cells often lose their MHC I, leaving them vulnerable to NK ceil killing. Once the decision is made to kill, the NK cell releases cytotoxic granules containing perforin and granzymes, which leads to lysis of the target cell.

T cell Involvement in Cellular Immunity Induction

[00100] T cells also may act to enhance the capacity of monocytes and macrophages to destroy intracellular microorganisms. In particular, interferon-gamma (IFNy) produced by helper T cells enhances several mechanisms through which mononuclear phagocytes destroy intracellular bacteria and parasitism including the generation of nitric oxide and induction of tumor necrosis factor (TNF) production. T_H1 cells are effective in enhancing the microbicidal action, because they produce IFNy. In contrast, two of the major cytokines produced by TH2 ceils, IL-4 and IL- 10, block these activities (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E,, Lippicott-Raven Publishers, Philadelphia, (1999)).

Regulatory T (Treg) Cells

[00101 ] Immune homeostasis is maintained by a controlled balance between initiation and downregulation of the immune response. The mechanisms of both apoptosis and T cell anergy (a tolerance mechanism in which the T cells are intrinsically functionally inactivated following an antigen encounter (Scwartz, R. H., "T cell anergy", Annu. Rev. Immunol., Vol . 21 : 305-334 (2003)) contribute to the downregulation of the immune response. A third mechanism is provided by active suppression of activated T cells by suppressor or regulator}' CD4⁺ T (Treg) ceils (Reviewed in Kronenberg, M. et ah, "Regulation of immunity by self-reactive T cells", Nature, Vol. 435: 598-604 (2005)). CD4^÷ Tregs that constitutively express the IL-2 receptor alpha (IL~2Ra) chain (CD4⁺ CD25^"r) are a naturally occurring T cell subset that are anergic and suppressive (Taams, L. S. et a , "Human anergic/suppressive CD4⁺CD25⁺ T cells: a highly differentiated and apoptosis-prone population", Eur. J. Immunol. Vol. 31 : 122-1131 (2001)). Depletion of CD4⁺CD25⁺ Tregs results in systemic autoimmune disease in mice. Furthermore, transfer of these Tregs prevents development of autoimmune disease. Human CD4⁺CD25^~r Tregs, similar to their murine counterpart, are generated in the thymus and are characterized by the ability to suppress proliferation of responder T cells through a cell-cell contact-dependent mechanism, the inability to produce IL-2, and the anergic phenotype in vitro. Human CD4⁺CD25^" T cells can be split into suppressive (CD25^b¾h) and nonsuppressive (CD25^!ow) cells, according to the level of CD25 expression. A member of the forkhead family of transcription factors, FOXP3, has been shown to be expressed in murine and human CD4 CD25⁺ Tregs and appears to be a master gene controlling CD4^~1~CD25⁺ Treg development (Battaglia, M. et ah, "Rapamycin promotes expansion of functional CD4⁺CD25 ^*Foxp3⁺ regulator T cells of both healthy subjects and type 1 diabetic patients", J. Immunol., Vol. 177: 8338-8347, (2006)).

Cytotoxic T Lymphocytes

[00102] CD8⁺ T cells that recognize peptides from proteins produced within the target cell have cytotoxic properties in that they lead to lysis of the target ceils. The mechanism of CTL-induced lysis involves the production by the CTL of perforin, a molecule that can insert into the membrane of target cells and promote the lysis of that cell. Perforin- mediated lysis is enhanced by granzymes, a series of enzymes produced by activated CTLs. Many active CTLs also express large amounts of fas ligand on their surface. The interaction of fas ligand on the surface of CTL with fas on the surface of the target cell initiates apoptosis in the target cell, leading to the death of these ceils. CTL-mediated lysis appears to be a major mechanism for the destruction of virally infected cells.

Lymphocyte Activation

[00103] The term "activation" or "lymphocyte activation" refers to stimulation of lymphocytes by specific antigens, nonspecific mitogens, or allogeneic cells resulting in synthesis of RNA, protein and DNA and production of lymphokines; it is followed by proliferation and differentiation of various effector and memon' cells. T-cell activation is dependent on the interaction of the TCR/CD3 complex with its cognate ligand, a peptide bound in the groove of a class I or class II MHC molecule. The molecular events set in motion by receptor engagement are complex. Among the earliest steps appears to be the activation of tyrosine kinases leading to the tyrosine phosphorylation of a set of substrates that control several signaling pathways. These include a set of adapter proteins that link the TCR to the ras pathway, phospholipase Cyl, the tyrosine phosphorylation of which increases its catalytic activity and engages the inositol phospholipid metabolic pathway, leading to elevation of intracellular free calcium concentration and activation of protein kinase C, and a series of other enzymes that control cellular growth and differentiation. Full responsiveness of a T cell requires, in addition to receptor engagement, an accessory ceil -delivered costimulatory activity, e.g., engagement of CD28 on the T cell by CD80 and/or CD86 on the APC.

T-memory Cells

[00104] Following the recognition and eradication of pathogens through adaptive immune responses, the vast majority (90-95%) of T cells undergo apoptosis with the remaining cells forming a pool of memory T cells, designated central memory T ceils (TCM), effector memory T cells (TEM), and resident memon,' T cells (TRM) (Clark, R.A., "Resident memory T cells in human health and disease", Sci . Transl . Med,, 7, 269rvl , (2015)).

[00105] Compared to standard T cells, these memory T cells are long-lived with distinct phenotypes such as expression of specific surface markers, rapid production of different cytokine profiles, capability of direct effector cell function, and unique homing distribution patterns. Memory T cells exhibit quick reactions upon re-exposure to their respective antigens in order to eliminate the reinfection of the offender and thereby restore balance of the immune system rapidly. Increasing evidence substantiates that autoimmune memory T ceils hinder most attempts to treat or cure autoimmune diseases (Clark, R.A., "Resident memory T cells in human health and disease", Sci. Transl. Med., Vol. 7, 269rv l , (201 5)).

Tumor Antigens

[00106] In some embodiments, the antibodies, antibody fragments or antigen binding proteins are capable of binding any one or plurality of antigens including tumor antigens and viral antigens. In some embodiments, the tumor antigens are selected from H3K27M, DNAJB 1 -PRK AC A, bcr-abl, CDK4-R24C, CDK4-R24L, MUMl, CTNNB 1, CDC27, TRAPPCl, TPI, ASCC3, HHAT, FN1, OS-9, PTPRK, CDKN2A, HLA-A1 1, GAS7, SIR2, Prdx5, CLPP, PPP1R3B, EF2, ACTN4, ME1, NF-YC, HSP70-2, KIAA1440, CASP8, gag, pol, nef, env, survivm, MAGEA4, SSX2, FRAME, NYESOl, Oct4, Sox2, Nanog, WT1 , p53, and MYCN. In some embodiments, the viral antigens are selected from cytomegalovirus (CMV) antigens including pp65, IE1, IE!, UL40, UL103, UL151 , UL153, UL28, UL32, UL36, UL55, UL40, UL48, UL82, UL94, UL99, us24, us32, and us32; herpes simplex virus (HSV) antigens including glycoprotein G; Epstein-Barr virus (EBV) antigens including BARF1, BMLF 1 , BMRF1, BZLFl, EBNALP, EBNAl, EBNA2, EB A3A, EBNA3B, EBNA3C, gp350/340, LMPl, and LMP2; human herpesvirus-8 (HHV8) antigens including LNA-1 , LANA-1, viral cyclin D, vFLIP, and RTA; human papillomavirus strain 16 (HPV16) antigens including E6, E7, and LI; and human papillomavirus strain 18 (FEPV18) antigens including E6 and E7. In some embodiments, the one or a plurality of antigens have at least about 70% sequence homology (e.g. at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%< 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to one of the sequences of Table 1 :

Table 1. Antigen sequences

Antigen Sequence

ID NO:

H3K27M MARTKOTARKSTGGKAPRKQLATKAAriMSAPSTGGVKKPHRY PGTVALRE I

IRRYQKSTELLIRKLPFQRLVJAEIAQDF TDLRFQSAAIGALQEASEAYLVGLF

EDTNl CAIHAKRVTIMP DIQLARRIRGERA

DNAJB 1- MGKDYYQTT_^GLARGASDEEIKRAYRRQALRYHPDKNKEPGAEEKFKEIAEAY 2 PRKACA DVLSDPRKREIFDRYGEEVKEFLAKA EDFLKKWESPAQNTAHLDQFERiKTL

GTGSFGRVMLVKHKETGNHYAMKILDKQK KLKQ1EHTLNEKR1LQAVNFP

FLVKLEFSFKDNSNLYMV IEYVPGGE IFSHLRRIGJAFSEPHARFYAAQIVLTF

EYLHSLDLIYRDLKPENLLIDQQGYIQVTDFGFAKRVKGRTWTLCGTPEYLAP

EIILSKGYNKAVDWWALG XIYEMAAGYPPFFADQPIOIYEKIVSGKVRFPSHF

SSDLKDLLRNLLOVDLTKRF^'GNLKNGVNDI NHKWFATTDWIAIYQRKVEAP

FIP FKGPGDTSNFDDYEEEEIRVSINEKCGKEFSEF

bcr-abl MVOPVGFAEAWKAOFPDSEPPRMELRSVGDIEQELERCKASIRRLEQEVNQE

RFRMIYLQTLLAKEKKSYDRQRWGFRRAAQAPDGASEPRASASRPOPAPADG

ADPPPAEEPEARPDGEGSPGKARPGTARRPGAAASGERDDRGPPASVAALRSN

FERIRKGHGQPGADAEKPFYVmTFHHERGLVKWDKEVSDRtSSLGSQAMQ ERKKSQHGAGSSVGDASRPPYRGRSSESSCGVDGDYEDAELNPRFLKDNLI

DANGGSRPPWPPLEYQPYQSIYVGGMMEGEGKGPLLRSQSTSEQEKRLTWPR

RSYSPRSFEDCGGGYTPDCSSNENLTSSEEDFSSGQSSRVSPSPTTYRMFRDKS

RSPSQNSQQSFDSSSPPTPQCHKRHRHCPVVVSEATIVGVRKTGQIWPNDGEG

AFHGDADGSFGTPPGYGCAADRAEEQRRHQDGLPYIDDSPSSSPHLSSKGRGS

RDALVSGALESTKASELDLEKGLEMRKWVLSG3LASEETYLSHLQMLTNSCV

KLQTVHSIPLTINKEEALQRPVASDFEPQGLSEAARWNSKENLLAGPSENDPN LFVALYDFVASGDNTLSITKGEKLRVLGY HNGEWCEAQTKNGOGWYPSNY

ITPVNSLEKHSWYHGPVSRNAAEYLLSSGINGSFLVRESESSPGORSISLRYEGR

VYHYRINTASDGKLYVSSESRFOTLAELVHHHSTVADGLITT1.HYPAPKRNKP

TVYGVSPNYDKWEMERTDITMKHKLGGGQYGEVYEGVWKKYSLTVAVKTL

K ! ! ⁾ rM \ l - H!^;! .K i-:A A \-M K I - ! kl l P\ S .\ QI .S ⁽ iYC TRUWYI H | · ! \ Π YGM .i .DY

LRECNRQEVNAVVLLYMATQISSA EYLEKK FIHRDLAARNCLVGENHLV

KVAI)FGLSRLMTGDTTTAHAGAKFPIKWTAPESLAYNKFSIKSDVWAFGVLL

WEIATTGMSPYPGroLSQVYELLEKDYRMERPEGCPEKVYELMRACWQWNP

SDRPSFAEfflOAJ^T QESSISDEVEKELGKQGVRGAVSTLLQAPELPT TRT

SRRAAEHRDTTDVPEMPHSKGQGESDPLDHEPAVSPLLPRKERGPPEGGLNED

ERLLPKDKKTNLFSALIKKKKKTAPTPPKRSSSFREMDGQPERRGAGEEEGRDI

SNGAL AFTPLDT ADP AK SPKP SNG AGVPNGALRES GGSGFRSPHL WKKS STLT

SSRLATGEEEGGGSSSKRFLRSCSASCVPHGAKDTEWRSVTLPRDLQSTGRQF

DSSTTGGHKSEKPALPRKRAGENRSDQVTRGTVTPPPRLVKKNEEAADEVFK

DIMESSPGSSPPNLTPKPLRROVTVAPASGLPHKEEAGKGSALGTPAAAEPVTP

TSKAGSGAPGGTSKGPAEESRVRRHKHSSESPGRDKGKLSRLKPAPPPPPAAS

AGKAGGKPSQSPSQEAAGEAVLGAKTKATSLVDAVNSDAAKPSQPGEGLKK

PVLPA.TPKPQSAKPSGTPISPAPVPSTLPSASSALAGDQPSSTAFIPLTSTRVSLRK

TRQPPERIASGATT GWLDSTEALCLAISRNSEQMASHSAVLEAGKNLYTFCV

SYVDSIQQMRNKFAFREA1NKLEN LRELQTCPATAGSGPAATQDFSKLLSSV

KEISDIVQR

CDK4- MATSRYEPVAEIGVGAYGTVYKACDPHSGHFVALKSVRVPNGGGGGGGLPIS 4 R24C TVTiEVALLRRLEAFFJIPNV\mA/E)VCATSRTDREIKVTI TEH\⁷DQDLRTYL

DKAPPPGLPAETIKDLMRQFLRGLDFLHANCIVHRDLKPENILVTSGGTVKLA DFGLARIYSYQMALTPVVVTLWYRAPEVIXOSTYATPVOMWSVGCIFAE R RKPLFCGNSEADQLGKIFDLIGLPPEDDWPRDVSLPRGAFPPRGPRPVQSVVPE

1S/IEESGAQLLLEM-.TFNPHKRISAFRALQHSYLHKDEGNPE

CDK4- MATSRYEPVAEiGVGAYGTVYKALDPHSGHFVALKSVRVPNGGGGGGGLPIS 5 R24L TVREVALLRRLEAFEHPNVVRLMDVCATSRTDRE1KVTLVFEHVDQDLRTYL

DKAPPPGLPAETKDLMRQFLRGLDFLHANCIVHRDLKPENILVTSGGTVKLA

DFGLARIY SYQMALTPVVWLWYRAPEVLLQSTYATPVDMWSVGCIFAEMFR

RKPLFCGNSEADQLGKIFDLIGLPPEDDWPRDVSLPRGAFPPRGPRPVQSWPE

MEESGAQLLLEMLTFNPHKRISAFRALQHSYLHKDEGNPE

sag MGQTKTKSKYASYLSFIKiLLKRGGVRVSTKNIJKLFQTTEQFCPWFPEOGNL 6

(HERV-K) DLEDWKRIGKELKQAGRKGNITPLTVWNDWPTIKAALEPFQTEDSVSVSDAPG

SCTTDCNEKTRKKSQKETETLHCEYVAEPLMAQSTQNVDYNQLQEVIYPETLK LEGKGPELVGPLESKPRGPSPLSA.GOVTVTLOPQAOVRENKTOLPVAYQYWP PAELQYRPPPESQYGYLGMPPAPQDREPYPQPPTRRQCYGTT

pol MIPKDWPLIIIDLKDCFFTIPLAEODCEKFAFTIPAIN KEPATRFQWKVLPQGM

(HERV-K) LNSPTT_^CQTFVGRALQPVRDKFSDCYIIHYFDDILCAAET DKLIDCYTFLOAE

VANAGLAIASDKIQTSTPFHYLGMQIENRKIKPQKIEIRKDTLKTLNDFQKLLG

DINWIRPTLGIPTTAMSNLFSILRGDSDLNSKRMLTPEATKEIKLVEEKIOSAQI

NRTOPLAPLOLLIFATAHSPTGinQNTDLVEWSFLPHSTVKTFTLYLDOIATLIG

PTRLRIIKLCGNDPDKIV LTKEQVTIQAFINSGAWQIGLAKTVGIIDNI-IYPKT

KIFQFLKLTTWILPKITRREPLENALTVFTDGSSNGKVAYTGPKERVIKTPYQS

AORAELVAVITVLQDFDQPI IISDSAYVVOATRDVETALIKYSMDDOLNOLF

NLLQQTVRKRNFPFYrTHIRAHTNLPGPLTXANEQADLLVSSAFIKAQELHAL^

HVT AAGLKK¾FDVTWKQAKDIVOHCTQCQ\'XDIJTQEAG\^EVCV^M-IY

GKW SHMYLHLGRLSYVHWVDTYSHFMCATCQTGESTSHVKKHLLSCFAV

MG EKTKTDNGPGYCSKAFQKFLSOWKTSHTTGIPYNSQGQAIVERTNRTLK

TQLVKQKE GGDSKECTTPQMOLNL ALY^. FLNIYRNQTTTSAEHLTGKKNS

PHEGKLI

env MVTPVl^\ ffi>NPIE\^VW⁾SVWVPGPTDDRCP_JAKPEEEGlVaiINISIGYHYPPI 8

(HERV-K) CLGRAPGCLMPAVQNWLVEVPTVSPNSRFTYHMVSGMSLRPRV CLQDFSY

QRSLKFRPKGKTCPKEIPKGSKNTEV VWEECV^ANSVVILQNNEFGTIIDWAP

RGQFYHNCSGQTQSCPSAQVSPAVDSDLTESLDKHKHKKLQSFYLWEWEEK

GISTPRPKIISPVSGPEHPELWRLTVASHHIRIWSGNQTLETRYRKPFYTIDLNSI

LTWLQSCVKPPYMLVVGNIVIKPASQTITCENCRLFTCIDSTTT TWQHRILLVR

ARKGN iW iPX ^TDRPtt KA 'Sll i l l ΓΗΙ ί .Κ ϋΥί ΛΊ^ΚΚ ί i l l i i AYiA iGi .i A Vi Vi

AAVAGVALHSSVQSVNFVNYWQKNSTRLW SQSSIDQKLASQINDLRQTVIW MGDRLMILEHHFOLO DWNTSDFCITPQIYNESEHHWDMVRRHLQGREDNL '! i . DiSk i Ki H!^;!-: \SK Ai ii .\! .\^'P i Γ!·:.\ ! ·\(.\ ΛίΧϋ .Λ Λ ΥΊ Ί\ IK/FIRS^'! ILIWCLFCLLLVCRCTQQLRRDSDIENGP

QGNTGQTQGKPTLLGT^'IIVl NFKKGFNGDYRVTMTPGKFRTLCEIDCPALEV 9

(HERV-E) GWPSEGSLGRSLVSKVWHKVTGKSGHSDOFPYroTWLQLMLNPPQWLRGQA

AAVLVA

pol MNHFMSSYSTSPGQAETCFAETPVAPMMSIIFLY GATGAVTrECLAPVSTRK 10

(HERV-E) SMSLPLTVILNIGSFGVLEPGPPQSNNPSARLSRAPFSLSGASCSESPCFLFS env MNHFMSSYSTSPGQAETCFAETPVAPMMSI1FLYKGATGAVTTECLAPVSTRK 11

(HERV-E) SMSLPLTVILNIGSFGVLEPGPPQSNNPSARLSRAPFSLSGASCSESPCFLFS gag VT PLSLTLQHWGDVORIASNQSVDVRKRRWITFCSAEWPTFNVGWPQDGTF 12

(XMRV) M,SIISOVK.SR CPGPHGHPDQ\ IV WEALAYDPPPW PFVSP LPPLPT

APVLPPGPSAQPPSRSAL

survivin MGAPTLPPAWQPFL DHRISTFKNWFLEGCACTPERJVlAEAGFiHCPTENEPD 13

LAQCFFCFKELEGWEPDDDP1EEHKKHSSGCAFLSVKKQFEELTLGEFLKLDR

ERA N IAKET NKKKEFEETAKKVRRAIEQLA^ID

MAGEA4 MSSEOKSQHCKPEEGVEAQEEALGLVGAOAPTTEEQEAAVSSSSPLVPGTLEE 14

VPAAESAGPPOSPQGASALPTTISFTCWRQPNEGSSSQEEEGPSTSPDAESLFRE

ALSNKVDELAHFLLRKYRA ELVTKAEMLERViKNYKRCFPVIFGKASESLK

MFGroVKEVDPASNTTTl-VTCLGLSYDGLLGNNOlFPKTGLLIIVLGTTAMEG

DSASEEEIWEELGV GVYDGREHTVYGEPRKLLTQDWVQENYLEYRQVPGS

NPARYEFLWGPRALAETSYVKVLEHWRVNARVRTAYPSLREAALLEEEEGV

SSX2 MNGDDAFARRPTVGAQIPEKIQKAFDDIAKYFSKEEWEKMKASEKIFYVYM 15

RKYEAMTKLGFKATLPPFMCNKRAEDFOGNDLDMDPNRGNOVERPOMTFGR LQGISPKIMPKKPAEEGMDSEEVPEASGPQNDGKELCPPGKPTTSEKIHERSGP KRGEHAWTHRLRERKQLVIYEEISDPEEDDE

FRAME MERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFP 16

PLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLD

VLLAQEVRPRRWKLQVLDLRKNSHODFWTVWSG RASLYSFPEPEAAQP T

KKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVKR KNVLRLC

CKKLKIFAMPMQDIKA'liL MVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINL

RRLLLSH1HASSYISPEKEEQY1AQFTSQFLSLQCLQALYVDSLFFLRGRLDQLL

RHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQ

ALLERASATLQDLVFDECG1TDDQLLALLPSLSHCSQLTTLSFYGNSISISALQS

LLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSM

VWLSANPCPHCGDRTFYDPEPILCPCFMPN

NYESOl MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAA 17

RASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAE LARRSLAQDAPPLPVPGVLLKEFTVSGN1LTTRLTAADHRQLQLSISSCLQQLSL

LMWiTQCFLPVFLAQPPSGQRR

Oct4 MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVG 18

PGSEVWGIPPCPPPYEFCGGMAYCGPQVGVGLVPQGGLETSOPEGEAGVGVE

SNSDGASPEPCTVTPGAVKLEKEKLEQNPEESODKALOKELEOFAKLLKO R

ITLGYTQADVGLTLGVLFGKWSQTTICRFEALQLSFKNMCKLRPLLQKWVEE

ADNNENLQEICKAETLVOARKLRKRTSffiNRVRGNLENLFLQCPKPTLOQISHIA

OQLGLEKDWRVWFCNRROKGKRSSSDYAQREDFEAAGSPFSGGPVSFPLAP

GPHFGTPGYGSPHFTALYSSVPFPEGEAFPPVSVTTLGSPMHSN

Sox2 MYNMlVffiTELK PGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPM AFMV 19

WSRGQRRKMAQENP MHNSEiSKRLGAEWKLLSETEKRPFiDEAKRLRALH

MKEHPDYKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGA

GV QRMDSYAHMNGWSNGSYSMMQDQLGYPQHPGLNAHGAAQMQPMHR

YDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVVKSEASS

SPPWTSSSHSRAPCQAGDLRDMISMYLPGAEVPEPAAPSRLHMSQHYQSGPV

PGTAiNGTLPLSHM

Nanog MSVDPACPQSLPCFEASDCKESSPMPVICGPEENYPSLOMSSAEMPHTETVSPL 20

PSSMDLLIQDSPDSSTSPKGKQPTSAEKSVAKKEDKVPVKKQKTRTVFSSTQL CVLNDRFQRQKYLSLQQMQELSN1LNLSYKQVKTWFQNQR KSKRWOK WPKNSNGWQKASAPTYPSLYSSYHQGCLV PTGNLPMWSNQTWNNSTWSN

QTQNIQSWSNHSWNTQTWCTQSWNNQA\VNSPFYNCGEESLQSCMQFQPNSP ASDLEAALEAAGEGLNVIQQTTRYFSTPQTMDLFLNYSMNMQPEDV

WTI MGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSL 23

GGPAPPPAPPPPPPPPPHSFTKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGA

CRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAiR QGYSTVTFDGTPSY

GHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGS

Q ALLLRTP YS SDNL YQMTSQLECMT WNQMNLGATLKG V A AG S S S S VKWTEG

QSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASE

TSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSD

QLKRHQRRHTGVKPFQCKTCQRKPSRSDHLKTHTRTHTGKTSEKPFSCRWPS

CQKKEARSDELVRHH MHQRNMTKLQLAL

p53 MEEPQSDPSVEPPLSQETFSDLWKLLPEN VLSPLPSQAVIDDLMLSPDDIEOW 22

FTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSWSQKTYQGS

YGFRLGFLHSGTAKSWCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVR

AMATYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEG LRVEYLDDRN

TFRHSVV YEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNL

LGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQ

PKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAOAGKEPGGSRAHSSHLK

SKKGQSTSRHKKLMFKTEGPDSD

MYCN MPSCSTSTMPGMICKNPDLEFDSLQPCFYPDEDDFYFGGPDSTPPGEDIWKKFE 23

LLPTPPLSPSRGFAEHSSEPPSWVTEMLLENELWGSPAEEDAFGLGGLGGLTPN

PVILQDCMWSGFSAREKLERAVSEKLQHGRGPPTAGSTAOSPGAGAASPAGR

GHGGAAGAGRAGAALPAELAHPAAECVDPAVVFPFPVNKREPAPVPAAPAS

APAAGPAVASGAGIAAPAGAPGVAPPRPGGRQTSGGDHKALSTSGEDTLSDS

DDEDDEEEDEEEEIDVVTVEKRRSSSNTKAVTITTITVRP AALGPGRAQSSE

LILKRCLPIHOQHNYAAPSPYVESEDAPPOKKIKSEASPRPLKSVIPPKAKSLSP

RNSD SED SERRRNHNTLERQRRNDLRS SFLTLRDHVTELVKNEK A AKWILKK

ATEYVHSLOAEEHQLLLEKEKLOARQQQLLKKIEHARTC

[00107] The terms "an antibody capable of binding CD47" or "anti-CD47 antibody," for example, refer to an antibody, or an antigen binding fragment thereof, that is capable of binding CD47 with sufficient affinity such that the antibody is useful as a diagnostic, prophylactic and/or therapeutic agent in targeting CD47. CD47 consists of one extracellular IgV domain, a five times transmembrane-spanning domain, and a short cytoplasmic tail. CD47 functions as a cellular ligand with binding mediated through the NH2-terminal IgV domain of SIRPa.

[00108] Signal -regulatory protein alpha (SIRPa) as used herein is any amino acid sequence comprising, consisting of 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, or 70% sequence identity to the SEQ TD NO: 24, shown below or a fragment of the aforementioned sequences having the capability of binding a CD47 domain. In some embodiments, SIRPa is a nucleic acid sequence encoding any of the aforementioned amino acid sequences. In some embodiments, the SIRPa peptide is one or a plurality of binding fragments identified in Piccione, E.G., et al. SIRPa-Antibody Fusion Proteins Selectively Bind and Eliminate Dual Antigen-Expressing Tumor Ceils. Clin Cancer Res; 22(20), 5109-5119 (2016). Binding of SIRPa to the CD47 receptor relies on a distinctive immunoglobulin superfamily V-like fold. It involves the BC, FG, and DE loops, which distinguishes it from other immunoglobulin superfami ly surface proteins that use the faces of the fold, but resembles antigen receptors.

The extracellular domain of SIR a is shown below:

EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFP

RVTTVSDLTKRNNlViDFSIRIGNITPADAGTYYTY'KFRKGSPDDVEFKSGAGTELSY'R

AKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITL WFKNGNELSDFQTNVDPVG

ESY^rSYSIHSTAKV ⁷LTREDYΉSQY^CEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQ

QPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENE DGTYNWMS

WLLVNVSAHPJDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNE

RNIY (SEQ ID NO:29)

[00110] The terms "an antibody capable of binding ILlORa" or "anti- ILlORa antibody," for example, refer to an antibody, or an antigen binding fragment thereof, that is capable of binding IL10 with sufficient affinity such that the antibody is useful as a ignostic, prophylactic and/or therapeutic agent in targeting ILlORa.

ILlORa as used herein is any amino acid sequence consisting of 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 %, 90%, 85%, 80%, 75%, or 70% sequence identity to the sequence shown below or a fragment of the aforementioned sequences having the capability of binding ILl ORa. In some embodiments, ILIO-Ra is a nucleic acid sequence encoding any of the aforementioned amino acid sequences.

Full Sequence IL-lORa

MLPCLVVLLAALLSLRLGSDAHGTELPSPPSVWFEAEFFFIHILFiWT^IPNQSESTCYE

VALLRY JIESWNSISNCSQTLS YDLTAVTLDLYHSNGYRARVI AVDGSRHS WTV NTRFSYTJEVTLTVGSVNLEIIING GNFTFTHKKVKHENFSLLTSGEVGEFCVQVKPSVASRS KGMWSKEECISLTRQY FT\^rTNVIIFF AF\ -LLS GAL AYCLALQL Y ¾RRKKLP SVXLFKKP SPFIFISQRP SPETQ DTIHPLDEEAFLKVSPEL NLDLHGSTDSGFGST PSLQTEEPQFLLPDPHPQADRTLG NREPPVLGDSCSSGSSNSTDSGICLQEPSLSPSTGPTWEQQVGSNSRGQDDSGIDLVQ NSEGRAGDTQGGSALGHHSPPEPEVPGEEDPAAVAFQGYLRQTRCAEEKAT TGCL EEESPLTDGLGPKFGRCLVDEAGLHPPALAKGYLKQDPLEMTLASSGAPTGQWNQP TEEWSLLALSSCSDLGISDWSFAHDLAPLGCVAAPGGLLGSFNSDLVTLPLISSLQSSE

(SEQ ID NO: 30)

[00112] Extracellular Domain IL 1 ORa is shown below:

HGTELPSPPSVWFEAEFFHHILHWTPIPNQSESTCYEVALLRYGIESWNSISNCSQTLS

YDLTAVTLDLYHS GYRARWA\OGSRHSmVTVTNTRFS\T EVTLTVGS ⁷ LEIHN GFILGKIQLPRPK MAPANDTYESIFSI IFREYEI AIRKVPG FTFTH KVKHE FSLLTS GEVGEFCVQVKPSVASRS KGMWSKEECISLTRQYFTVTN (SEQ ID NO:31)

[00113] The terms "an antibody capable of binding TGFp" or "anti- TGFp antibody," for example, refer to an antibody, or an antigen binding fragment thereof, that is capable of binding TGFP with sufficient affinity such that the antibody is useful as a diagnostic, prophylactic and/or therapeutic agent in targeting TGFp.

[00114] Transforming growth factor β receptor II (TGF])R Π ) as used herein is any amino acid sequence consisting of 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, or 70% sequence identity to the SEQ ID NO: 2 below or a fragment of the aforementioned sequences having the capability of binding ΤΟΡβ. In some embodiments, TGFBRII is a nucleic acid sequence encoding any of the aforementioned amino acid sequences.

MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDV

RFSTCDNQKSCMSNCSITSICEKPQEVCVAWRKNDENTTLETVCHDPKIJYHDFILE

DAASPKCIMKEKKKPGETFFMCSCSSDECNDNI1FSEEYNTSNPDLLLVIFQVTGISLLP

PLGVAISVIIIFYCYRVNRQQKLSSTWΈTGKTRKLMEFSEHCAIILEDDRSDISSTCANJSΓ

ESlHNTCLLPffiLDTI-VGKGRFAEVYKAKLKQNTSEQFE AVKIFPYEEYASWKTE^

Oil SDIN I l 1Ι Ί! .(. Ι Ί ^'ΓΛί'.Ι ^'Κ Κ Γ! GKQYWI JTAI I iAKGXI QKYI . I Rl i\^'f S\Vl f)! RK

LGS SL ARGIAHLHSDHTPCGRPi MPIVHRDLKS SNILVKNDLTCCLCDFGLSLRLDPT

LSVDDLANSGQVGTARYMAPEVLESRMNLENA^SFKQTDVYSMALVLWEMTSRCN

AVGEVKDYEPPFGSKVREHPCVESM DNVLRDRGRPEIPSFWLNHQGIQMVCETLTE

CWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK (SEQ ID NO: 2)

[00115] Extracellular Domain TGFBRII is shown below:

TIPPHV'QKSV' NDMIVTD NGAV^'KFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKP QEVCVAVWRKNDE ITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCS CSSDECNDNIIFSEEYNTSNPDLLLVIFQ (SEQ ID NO:32)

[00116] The terms "an antibody capable of binding IL6" or "anti- IL6 antibody," for example, refer to an antibody, or an antigen binding fragment thereof, that is capable of binding IL6R with sufficient affinity such that the antibody is useful as a diagnostic, prophylactic and/or therapeutic agent in targeting TGFp.

[00117] Interleukin 6 receptor alpha (IL-6Ra) as used herein is any amino acid sequence consisting of 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, or 70% sequence identity to the sequence shown below or a fragment of the aforementioned sequences having the capability of binding IL6. In some embodiments, IL6Ra is a nucleic acid sequence encoding any of the aforementioned amino acid sequences. Full Sequence IL~6Ra

MLAVGCALLAALLAAPGAALAPRRCPAQEVARGVLTSLPGDSVTLTCPGVEPEDNA

T VHW VLRKP AAGSHP SRW AGMGRRLLLRS VQLHD S GNYS C YRAGRP AGT VHLL VD

VPPEEPQLSCFR SPLSNVVCEWGPRSTPSLTTKAVLLVRKFQNSPAEDFQEPCQYSQ

ESQKFSCQLAVPEGDSSFYIVSMC\^rASSVGSKFSKTQTFQGCGILQPDPPANITVTAV

ARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTT MVKDLQHHCVEHDA

WSGLRHVVQLRAQEEFGQGEWSEWSPEAMGTPWTESRSPPAENEVSTP QALTTN

KDDDNILFRD S AN AT SLP VQD S S S VPLPTFL VAGG SLAFGTLLCIAI VLRFKKT WKLR

ALKEGKTSMFiPPYSLGQL ERPRPTPVLWLISPPVSPSSLGSDNTSSH PDARDPR

SPYDISNTDYFFPR

(SEQ ID NO:33)

[00118] Extracellular Domain IL~6Ra is shown below:

LAPRRCPAQEVARGVLTSLPGDSVTLTCPG-VEPEDNATVFIWVLRKPAAGSHPSRWA

GMGRRLLLRSVQLHDSGNYSCYRAGRPAGTVilLLVDVPPEEPQLSCFRKSPLSNVVC

EWGPRSIPSLTTKAVLLVRKFQNSPAEDFQEPCQYSQESQKFSCQLAVPEGDSSFYTV

SMCVASSVGS FSKTQTFQGCGILQPDPPANITVTAVARNPRWLSVTWQDPHSWNSS

FYRLRFELRYRAERSKTFTTWMVKDLQHHCVIHDAWSGLRir QLRAQEEFGQGE

WSEWSPEAMGTPWTESRSPPAENEVSTPMQALTTNKDDDNILFRDSANATSLPVQDS SSVPLP (SEQ ID NO: 34)

[00119] As used herein, "conservative" amino acid substitutions may be defined as set out in Tables A, B, or C below. The polypeptides of the disclosure include those wherein conservative substitutions (from either nucleic acid or amino acid sequences) have been introduced by modification of polynucleotides encoding antibodies, antibodies, antibody fragments thereof, or antigen binding proteins thereof. In some embodiments, these polypeptides comprise CDRs or functional fragments thereof. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. In some embodiments, the conservative substitution is recognized in the art as a substitution of one nucleic acid for another nucleic acid that has similar properties, or, when encoded, has a binding affinity to a target or binding partner similar to the binding affinity of the sequence upon which the conservative substitution is based. Exemplary conservative substitutions are set out in Table A.

Table A ~ Conservative Substitutions I

Side Chain Characteristics Amino Acid

Aliphatic

Non-polar G A P I L V F

Polar - uncharged C S T M N Q

Polar - charged D E K R

Aromatic H F W Y

Other N Q D E

[00120] Alternately, conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp. 71 - 77) as set forth in Table B.

Table B -- Conservative Substitutions II

Side Chain Characteri tic Amino Acid

Non-polar (hydrophobic)

Aliphatic: A L I V P

Aromatic: F W Y

Sulfur-containing: M

Borderline: G Y Uncharged-polar

Hydroxy.: S T Y

Amides; N Q

Sulfhydryi: C

Borderline: G Y

Positively Charged (Basic): K R H

Negatively Charged (Acidic): D E

[00121] Alternately, exemplary conservative substitutions are set out in Table C.

Table C— Conservative Substitutions III

Original Residue Exemplary Sub

Ala (A) Val Leu He Met

Arg (R) Lys His

Asn (N) Gin

Asp (D) Glu

Cys (C) Ser Thr

Gin (Q) Asn

Glu (E) Asp

Giy (G) Ala Val Leu Pro

His (H) Lys Arg

He (I) Leu Val Met Ala Phe

Leu (L) He Val Met Ala Phe

Lys (K) Arg His

Met (M) Leu He Val Ala

Phe (F) Trp Tyr He

Pro (P) Gly Ala Val Leu He

Ser (S) Thr

Thr (T) Ser

Trp (W) Tyr Phe He

Tyr (Y) Tip Phe Thr Ser

Val (V) He Leu Met Ala

[00122] It should be understood that the antibodies or any functional fragments thereof described herein are intended to include amino acid sequences comprising polypeptides bearing one or more insertions, deletions, or substitutions, or any combination thereof, of amino acid residues as well as modifications other than insertions, deletions, or substitutions of amino acid residues, such as but not limited to conservative amino acid substitutions,

[00123] As used herein, "specific for" or "specifically binds to" means that the binding affinity of a substrate to a specified target nucleic acid or amino acid sequence, is statistically higher than the binding affinity of the same substrate to a generally comparable, but non- target nucleic acid or amino acid sequence. Normally, the binding affinity of a substrate to a specified target nucleic acid or amino acid sequence is at least 1.5 fold, and preferably 2 fold or 5 fold, of the binding affinity of the same substrate to a non-target nucleic acid or amino acid sequence. It also refers to binding of a substrate to a specified nucleic acid or amino acid target sequence to a detectably greater degree, e.g., at least 1.5 -fold over background, than its binding to non-target nucleic acid or amino acid sequences and to the substantial exclusion of non-target nucleic acids or amino acids. The substrate's Kd to each nucleotide or amino acid sequence can be compared to assess the binding specificity of the substrate to a particular target nucleotide or amino acid sequence.

[00124] The terms "specific binding", "specifically binds" or "specifically binding", as used herein in the context of an antibody, refer to non-covalent or covalent preferential binding of an antibody to an antigen relative to other molecules or moieties (e.g., an antibody specifically binds to a particular antigen relative to other available antigens). In some embodiments, an antibody specifically binds to an antigen (e.g., a tumor or viral antigen) if it binds with a dissociation constant K_D of from about 1 pM to about 500 niM, In some embodiments, the antibody or antigen binding protein has a dissociation constant K_D in a range from about 1 pM to about 1000 nM. In some embodiments, the antibody or antigen binding protein has a dissociation constant K_D in a range from about 1 pM to about 500 nM. In some embodiments, the antibody or antigen binding protein has a dissociation constant KD in a range from about 1 pM to about 250 nM. In some embodiments, the antibody or antigen binding protein has a dissociation constant D in a range from about 1 pM to about 100 nM. In some embodiments, the antibody or antigen binding protein has a dissociation constant K_D in a range from about 1 pM to about 10 nM. In some embodiments, the antibody or antigen binding protein has a dissociation constant K_D in a range from about 1 pM to about 1 nM. In some embodiments, the antibody or antigen binding protein has a dissociation constant K_D in a range from about 1 pM to about 750 pM , In some embodiments, the antibody or antigen binding protein has a dissociation constant K_D in a range from about 1 pM to about 500 pM. In some embodiments, the antibody or binding protein has a dissociation constant K_D in a range from about 1 nM to about 100 nM.

[00125] The term "human antibody", as used herein, refers to an antibody, or an antigen binding fragment of an antibody, comprising heavy and lights chains derived from human immunoglobulin sequences. Human antibodies may be identified in a variety of ways, examples of which are described below, including through the immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and/or light chain-encoding genes. In some embodiments, a human antibody is made using recombinant methods such that the glycosylation pattern of the antibody is different than an antibody having the same sequence if it were to exist in nature.

[00126] The term "chimeric antibody" refers to an antibody that contains one or more regions derived from a particular source or species, and one or more regions derived from a different source or species.

[00127] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance. In general, the humanized antibody or antibody fragment thereof will compri se substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et ai. Nature, 321 : 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992, which are incorporated by reference in their entireties,

[00128] An "antibody fragment", "antibody portion", "antigen-binding fragment of an antibody", or "antigen -binding portion of an antibody" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')₂; Fd; and Fv fragments, as well as dAb; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide. Antigen binding portions of an antibody may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, and the like, [00129] In some embodiments, the antibody fragment is an scFv. A single-chain antibody (scFv) is an antibody in which a V_L and a V_H region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain (see e.g., Bird et al (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883)).

[00130] A Fab fragment is a monovalent fragment having the V_L, V_H, C_L and C_HI domains; a F(ab')₂ fragment is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment has the V_H and CHI domains; an Fv fragment has the V_L and V_H domains of a single arm of an antibody, and a dAb fragment has a V_H domain, a V_L domain, or an antigen -binding fragment of a V_H or V_L domain (U.S. Patents 6,846,634, 6,696,245, US App Pub 20/0202512, 2004/0202995; 2004/0038291; 2004/0009507; 2003/0039958, and Ward et al., Nature 341 :544-546, 1989).

[00131] "Binding fragments" of an antibody are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain antibodies. An antibody other than a "bi specific" or "bifunctional" antibody is understood to have each of its binding sites identical. An antibody substantially inhibits adhesion of a receptor to a counter-receptor when an excess of antibody reduces the quantity of receptor bound to counter-receptor by at least about 20%, 40%, 60% or 80%, and more usually greater than about 85% (as measured in an in vitro competitive binding assay). An antibody may be oligoclonal, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a multi-specific antibody, a bi-specific antibody, a catalytic antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an anti -idiotypic antibody and antibodies that can be labeled in soluble or bound form as well as fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences provided by known techniques. An antibody may be from any species. The term antibody also includes binding fragments of the antibodies of the invention; exemplary fragments include Fv, Fab, Fab', single stranded antibody (svFC), dimeric variable region (Diabody) and disulphide stabilized variable region (dsFv). As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% sequence identity to the antibodies or immunoglobulin molecules described herein. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that have related side chains. Genetically encoded amino acids are generally divided into families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged poiar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are: serine and threonine are an aliphatic- hydroxy family; asparagine and glutamine are an amide-containing family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family. In some embodiments, the present disclosure relates to one or a plurality of modified cells, such as T cells, that are isolated from a subject and then modified to express one or a plurality of antibodies, antibody binding fragments or salts thereof.

[00132] As used herein, the term "functional fragment" means any portion of a polypeptide that is of a sufficient length to retain at least partial biological function that is similar to or substantially similar to the wild-type polypeptide upon which the fragment is based. In some embodiments, a functional fragment of a polypeptide associated with an antigen expressed on a hyperproliferative cell is a polypeptide that comprises 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity of any polypeptide disclosed in Table I and has sufficient length to retain at least partial binding affinity to one or a plurality of ligands that bind to the polypeptide in Table 1. In some embodiments, the fragment is a fragment of any polypeptide di sclosed in Table 1 and has a length of at least about 10, about 20, about 30, about 40, about 50 , about 60, about 70, about 80, about 90, or about 100 contiguous amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 50 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 100 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table I and has a length of at least about 150 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 200 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table I and has a length of at least about 250 amino acids. In some embodiments, the tragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 300 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 350 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 400 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 450 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 500 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 550 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 600 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 650 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 700 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 750 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 800 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 850 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 900 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 950 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 1000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 1050 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 1250 amino acids, hi some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 1500 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 1750 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 2000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 2250 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 2500 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 2750 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 3000 amino acids.

[00133] As used herein, "more than one" or "two or more" 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. In some embodiments, "more than one" means 2, 3, 4, or 5 of the amino acids or nucleic acids or mutations described herein. In some embodiments, "more than one" means 2, 3, or 4 of the amino acids or nucleic acids or mutations described herein. In some embodiments, "more than one" means 2 or 3 of the amino acids or nucleic acids or mutations described herein. In some embodiments, "more than one" means 2 of the amino acids or nucleic acids or mutations described herein.

[00134] "Sequence homology" or "sequence identity" or "homologous to" are used herein interchangeably for nucleotides and amino acids sequences determined using FASTA, BLAST and Gapped BLAST (Altschul et a!,, Nuc. Acids Res,, 1997, 25, 3389, which is incorporated herein by reference in its entirety) and PAUP* 4.0bIO software (D. L. Swofford, Sinauer Associates, Massachusetts). Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity (Altschul et al., J. Mol. Biol, 1990, 215, 403-410, which is incoiporated herein by reference in its entirety). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered similar to another if the smallest sum probability in comparison of the test nucleic acid to the other nucleic acid is less than about 1 , preferably less than about 0. 1 , more preferably less than about 0.01, and most preferably less than about 0.001. "Percentage of similarity" or percentage of sequence identity" can be calculated using PAUP* 4.0MO software (D. L. Swofford, Sinauer Associates, Massachusetts). The average similarity of the consensus sequence is calculated compared to all sequences in the phylogenic tree. In some embodiments, the compositions disclosed herein comprise nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homologous to any of SEQ ED NOS: 25-28, or amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%*, 99% homologous to any of SEQ ID NOS: 1 -24.

[00135] The "percent identity" or "percent homology" of two polynucleotide or two polypeptide sequences are interchangeable concepts and may be determined by comparing the sequences using the GAP computer program (a part of the GCG Wi sconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. "Identical" or "identity" as used herein in the context of two or more nucleic acids or amino acid sequences, may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[00136] The terms "polynucleotide," "oligonucleotide" and "nucleic acid" are used interchangeably throughout and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturaliy occurring nucleotide analogs), and hybrids thereof. The nucleic acid molecule can be single-stranded or double-stranded. In some embodiments, the nucleic acid molecules of the disclosure comprise a contiguous open reading frame encoding an antibody, or a fragment thereof, as described herein. "Nucleic acid" or "oligonucleotide" or "polynucleotide" as used herein may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions. Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods, A nucleic acid will generally contain phosphodiester bonds, although nucleic acid analogs may be included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, which are incorporated by reference in their entireties. Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included within one definition of nucleic acids. The modified nucleotide analog may be located for example at the 5'-end and/or the 3'-end of the nucleic acid molecule. Representative examples of nucleotide analogs may be selected from sugar- or backbone-modified ribonucleotides. It should be noted, however, that also nucleobase- modified ribonucleotides, i.e. ribonucleotides, containing a non-naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridines or cyti dines modified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosines and guanosines modified at the 8-position, e.g. 8-bromo guanosine, deaza nucleotides, e.g. 7-deaza- adenosine; O- and N-aikyiated nucleotides, e.g. N6-methyl adenosine are suitable. The 2'- OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH.sub.2, NHR, N.sub.2 or CN, wherein R is C.sub. l -C.sub.6 alkyl, alkenyl or alkynyl and halo is F, CI, Br or I. Modified nucleotides also include nucleotides conjugated with cholesterol through, e.g., a hydroxy proli no! linkage as described in Krutzfeldt et al., Nature (Oct. 30, 2005), Soutschek et al., Nature 432: 173-178 (2004), and U.S. Patent Publication No. 20050107325, which are incorporated herein by reference in their entireties. Modified nucleotides and nucleic acids may also include locked nucleic acids (LNA), as described in U.S. Patent No. 20020115080, which is incorporated herein by reference. Additional modified nucleotides and nucleic acids are described in U.S. Patent Publication No. 20050182005, which is incorporated herein by reference in its entirety. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments, to enhance diffusion across ceil membranes, or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs may be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.

[00137] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-natural amino acids or chemical groups that are not amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosyiation, lipidation, acetyl ati on, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein the term "amino acid" includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

[00138] The term "polypeptide", as used herein, generally has its art-recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate that the term "polypeptide" is intended to be sufficiently general as to encompass not only polypeptides having the complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%>, 97%>, 98%>, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term "polypeptide" as used herein.

[00139] Two single-stranded polynucleotides are "the complement" of each other if their sequences can be aligned in an anti-parallel orientation such that every nucleotide in one polynucleotide is opposite its complementan,' nucleotide in the other polynucleotide, without the introduction of gaps, and without unpaired nucleotides at the 5' or the 3' end of either sequence. A polynucleotide is "complementary" to another polynucleotide if the two polynucleotides can hybridize to one another under moderately stringent conditions. Thus, a polynucleotide can be complementary to another polynucleotide without being its complement.

[00140] A "vector" is a nucleic acid that can be used to introduce another nucleic acid linked to it into a ceil. One type of vector is a "plasmid," which refers to a linear or circular double stranded DNA molecule into which additional nucleic acid segments can be iigated. Another type of vector is a viral vector {e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), wherein additional DNA segments can be introduced into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced {e.g., bacterial vectors comprising a bacterial origin of replication and episomai 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. An "expression vector" is a type of vector that can direct the expression of a chosen polynucleotide. The disclosure relates to any one or plurality of vectors that comprise nucleic acid sequences encoding any one or plurality of amino acid sequence disclosed herein.

[00141] The term "operably linked" or "transcriptional control" refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame. A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression {e.g., the level, timing, or location of expression) of the nucleotide sequence. A "regulatory sequence" is a nucleic acid that affects the expression (e.g., the level, timing, or location of expression) of a nucleic acid to which it is operably linked. The regulatory sequence can, for example, exert its effects directly on the regulated nucleic acid, or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and/or the nucleic acid). Examples of regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif, and Baron et al., 1995, Nucleic Acids Res. 23 :3605-06.

[00142] A "host cell" is a cell that can be used to express a nucleic acid, e.g., a nucleic acid of the disclosure, can be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK.293 cells, BHK cells and MDC II cells. Suitable insect cells include, but are not limited to, Sf9 cells. The phrase "recombinant host cell" can be used to denote a host cell that has been transformed or transfected/ transduced with a nucleic acid to be expressed. A host cell also can be a cell that comprises the nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell such that it becomes operably linked with the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell but also to the progeny or potential progeny of such a ceil. Because certain modifications may occur in succeeding generations due to, e.g., mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[00143] The term "recombinant antibody" refers to an antibody that is expressed from a ceil (or cell line) transfected/transduced with an expression vector (or possibly more than one expression vector) comprising the coding sequence of the antibody, or a portion thereof (e.g., a DNA sequence encoding a heavy chain or a light chain variable region as described herein). In some embodiments, said coding sequence is not naturally associated with the ceil. In some embodiments, a recombinant antibody has a glycosylation pattern that is different than the glycosylation pattern of an antibody having the same sequence if it were to exist in nature. In some embodiments, a recombinant antibody is expressed in a mammalian host cell which is not a human host cell. Notably, individual mammalian host ceils have unique glycosylation patterns.

[00144] The term "isolated" refers to a protein (e.g., an antibody) that is substantially free of other cellular material. In some embodiments, an isolated antibody is substantially free of other proteins from the same species. In some embodiments, an isolated antibody is expressed by a cell from a different species and is substantially free of other proteins from the different species. A protein may be rendered substantially free of naturally associated components (or components associated with the cellular expression system used to produce the antibody) by isolation, using protein purification techniques well known in the art. In some embodiments, the antibodies, or antigen binding fragments, of the disclosure are isolated.

[00145] One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an antigen binding protein. An antigen binding protein may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs permit the antigen binding protein to specifically bind to a particular antigen of interest.

[00146] In some embodiments, the substitutions made within a heavy or light chain that is at least 95% identical (or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical) are conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well -known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by- reference in its entirety. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine.

[00147] Antigen-binding fragments of antigen binding proteins of the disclosure may be produced by conventional techniques. Examples of such fragments include, but are not limited to, Fab and F(ab')2 fragments. [00148] Single chain antibodies may be formed by linking heavy and light chain variable domain (Fv region) fragments via an amino acid bridge (short peptide linker), resulting in a single polypeptide chain. Such single-chain Fvs (scFvs) have been prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (VL and VFI), The resulting polypeptides can fold back on themselves to form antigen-binding monomers, or they can form muitimers (e.g., dimers, trimers, or tetramers), depending on the length of a flexible linker between the two variable domains (Kortt et al, 1997, Prot. Eng. 10:423; Kortt et al., 2001 , Biomol. Eng. 18:95-108). By combining different VL and VH-compri sing polypeptides, one can form multimeric scFvs that bind to different epitopes (Kriangkum et al , 2001, Biomol. Eng. 18:31 -40). Techniques developed for the production of single chain antibodies include those described in U. S. Patent 4,946,778; Bird,

1988, Science 242:423, Huston et al , 1988, Proc. Natl . Acad. Sci. USA 85 :5879, Ward et aL,

1989, Nature 334:544, de GraaiV/ al, 2002, Methods Mol. Biol. 178:379-87.

[00149] Techniques are known for deriving an antibody of a different subclass or isotype from an antibody of interest, i.e., subclass switching. Thus, IgG antibodies may be derived from an IgM antibody, for example, and vice versa. Such techniques allow the preparation of new antibodies that possess the antigen-binding properties of a given antibody (the parent antibody), but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody. Recombinant DNA techniques may be employed. Cloned DNA encoding particular antibody polypeptides may be employed in such procedures, e.g. , DNA encoding the constant domain of an antibody of the desired isotype (Lantto et al , 2002, Methods Mol. Biol. 178:303-16). In some embodiments, the antibody of the disclosure is an IgG2a antibody. In some embodiments, the antibody of the disclosure is an IgG2b antibody. In some embodiments, the antibody of the disclosure is an IgGl antibody.

[00150] The present disclosure provides a number of antibodies structurally characteri zed by the amino acid sequences of their variable domain regions. However, the amino acid sequences can undergo some changes while retaining their high degree of binding to their specific targets. More specifically, many amino acids in the variable domain region can be changed with conservative substitutions and it is predictable that the binding characteristics of the resulting antibody will not differ from the binding characteristics of the wild type antibody sequence. There are many amino acids in an antibody variable domain that do not directly interact with the antigen or impact antigen binding and are not critical for determining antibody structure. For example, a predicted nonessential amino acid residue in any of the disclosed antibodies is preferably replaced with another amino acid residue from the same class. Methods of identifying amino acid conservative substitutions which do not eliminate antigen binding are well- known in the art (see, e.g., Brummeli et ai., Biochem. 32: 1 180-1 187 (1993); Kobayashi et al Protein Eng. 12(10):879-884 (1999); and Burks et al Proc. Natl . Acad. Sci. USA 94:412-417 ( 1997)). Near et al Mol. Immunol. 30:369-377, 1993 explains how to impact or not impact binding through site-directed mutagenesis. Near et al only mutated residues that they thought had a high probability of changing antigen binding. Most had a modest or negative effect on binding affinity (Near et al Table 3) and binding to different forms of digoxin (Near et al Table 2).

[00151] Those of ordinary skill in the art will appreciate standard methods known for determining the KD of an antibody, or fragment thereof. For example, In some embodiments, K_D is measured by a radiolabeled antigen binding assay (RIA). In some embodiments, an RTA is performed with the Fab version of an antibody of interest and its antigen. For example, solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of ( ^' I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al ., J. Mol. Biol. 293 :865-881(1999)).

[00152] According to another embodiment, !¾ s measured using a B I AC ORE surface plasmon resonance assay. The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that al lows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ), Surface plasmon resonance can also be used to determine K_off and K_a values.

[00153] The present disclosure further provides multi-specific antigen binding proteins, for example, bispecific antigen binding protein, e.g., antigen binding protein that bind to two different tumor or viral epitopes, or to a tumor or viral epitope and an epitope of another molecule, via two different antigen binding sites or regions. Moreover, bispecific antigen binding protein as disclosed herein can comprise a tumor or viral antigen binding site from one of the herein-described antibodies and a second tumor or viral antigen binding region from another of the herein-described antibodies, including those described herein by reference to other publications. Alternatively, a bispecific antigen binding protein may comprise an antigen binding site from one of the herein described antibodies and a second antigen binding site from another tumor or viral antibody that is known in the art, or from an antibody that is prepared by known methods or the methods described herein. According to one embodiment, the disclosure includes anti-CD47 bispecific and bifunctional antibodies and antigen-binding fragments, having specificity for another antigen such as, for example, CD 19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD117, CD123, c-Met, CEA, EGFR, EpCAM, HER2, HER3, PSMA, P^'S^'I f 2. mesothelin, PD-1, PD-L1, TIMS, and methods of use thereof. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai, et al., (1990) Clin. Exp. Immunol. 79: 315-321, Kosteiny, et al., (1992) J Immunol. 148: 1547-1553. In addition, bispecific antibodies may be formed as "diabodies" (Holliger, et al., (1993) PNAS USA 90:6444-6448) or as "Janusins" (Traunecker, et al., (1991) EMBO J. 10:3655- 3659 and Traunecker, et a! ,, (1992) Int. J, Cancer Suppl . 7:51 -52), Included are "Duobodies," which are bispecific antibodies with normal IgG staictures (Labrijn et al., 2013, Proc. Natl. Acad. Sci. USA 110 (13): 5145-5150).

[00154] Numerous methods of preparing bispecific antibodies are known in the art.

Such methods include the use of hybrid-hybridomas as described by Milstein et al., 1983, Nature 305:537, and chemical coupling of antibody fragments (Brennan et al., 1985, Science 229:81; Glennie et al., 1987, J. Immunol. 139:2367, U. S. Patent 6,010,902). Moreover, bispecific antibodies can be produced via recombinant means, for example by using leucine zipper moieties (i.e., from the Fos and Jun proteins, which preferentially form heterodimers; Kosteiny et al., 1992, J. Immunol. 148: 1547) or other lock and key interactive domain structures as described in U.S. Patent 5,582,996. Additional useful techniques include those described in U.S. Patents 5,959,083, and 5,807,706.

[00155] In another aspect, the antigen binding protein comprises a derivative of an antibody. The derivatized antibody can comprise any molecule or substance that imparts a desired property to the antibody, such as increased half-life in a particular use. The derivatized antibody can comprise, for example, a detectable (or labeling) moiety (e.g., a radioactive, colorimetric, antigenic or enzymatic molecule, a detectable bead (such as a magnetic or electrodense (e.g., gold) bead), or a molecule that binds to another molecule (e.g., biotin or streptavidin), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharmaceutically active moiety), or a molecule that increases the suitability of the antibody for a particular use (e.g., administration to a subject, such as a human subject, or other in vivo or in vitro uses). Examples of molecules that can be used to derivatize an antibody include albumin (e.g., human serum albumin) and polyethylene glycol (PEG), Albumin-linked and PEGylated derivatives of antibodies can be prepared using techniques well known in the art. In some embodiments, the antibody is conjugated or otherwise linked to transthyretin (TTR) or a TTR variant. The TTR or TTR variant can be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly(n-vinyl pyurrolidone), polyethylene glycols, propropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohols.

[00156] Antigen binding proteins, including antibodies and antibody fragments described herein, may be prepared by any of a number of conventional techniques. For example, they may be purified from cells that naturally express them (e.g., an antibody can be purified from a hybridoma that produces it), or produced in recombinant expression systems, using any technique known in the art. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eels.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., (1988).

[00157] Any expression system known in the art can be used to make the recombinant polypeptides, including antibodies and antibody fragments described herein, of the disclosure. In general, host cells are transformed with a recombinant expression vector or egentic construct that comprises DNA encoding a desired polypeptide. Among the host cells that may be employed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli. Higher eukaryotic ceils include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Giuzman et al., 1981, Cell 23 : 175), L cells, 293 cells, C 127 cells, 3T3 ceils (ATCC CCL 163), Chinese hamster ovary (( HO) cells, He! .a cells, BHK (ATCC CRL 10) ceil lines, and the CV1/EBNA cell line derived from the African green monkey kidney ceil line CV1 (ATCC CCL 70) as described by McMahan et al., 1991, EMBO J. 10: 2821 , Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985), When taken up by a cell, the genetic constructs s) may remain present in the cell as a functioning extrachromosomal molecule . DNA may be introduced into cells, where it is present on a transient basis, in the form of a plasmid or plasmids. Alternatively, RNA may be administered to the cell. It is also contemplated to provide the genetic construct as a linear minichromosome including a centromere, telomeres and an origin of replication. Gene constructs may constitute part of the genetic material in attenuated live microorganisms or recombinant microbial vectors which are administered to subjects. Gene constructs may be part of genomes of recombinant viral vaccines where the genetic material remains extrachromosomal. Genetic constructs include regulatory elements necessary for gene expression of a nucleic acid molecule. The elements include: a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, enhancers are often required for gene expression of the sequence that encodes the target protein or the immunomodulating protein. It is necessary that these elements be operably linked to the sequence that encodes the desired proteins and that the regulatory elements are operable in the individual to whom they are administered.

[00158] An initiation codon and a stop codon are generally considered to be part of a nucleotide sequence that encodes the desired protein. However, it is necessary that these elements are functional in the individual to whom the gene construct is administered. The initiation and termination codons must be in frame with the coding sequence.

[00159] Promoters and polyadenylation signals used must be functional within the cells of the individual. Examples of promoters useful to practice the present invention, especially in the production of a genetic vaccine for humans, include but are not limited to promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (MV) such as the BIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Vims (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human Actin, human Myosin, human Hemoglobin, human muscle creatine and human metaiothionein.

Examples of polyadenylation signals useful to practice the present invention, especially in the production of a modified T cell or K cell for humans, include but are not limited to SV40 polyadenylation signals, bovine growth hormone polyadenylation (bgh-PolyA) signal and LTR polyadenylation signals. In particular, the SV40 polyadenylation signal that is in pCEP4 plasmid (Invitrogen, San Diego Calif), referred to as the SV40 polyadenylation signal, is used. In addition to the regulatory elements required for DNA expression, other elements may also be included in the DNA molecule. Such additional elements include enhancers. The enhancer may be selected from the group including but not limited to: human Actin, human Myosin, human Hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV. Genetic constructs can be provided with mammalian origin of replication in order to maintain the construct extrachromosomally and produce multiple copies of the construct in the cell. Plasmids pVAXl, pCEP4 and pREP4 from Invitrogen (San Diego, Calif.) contain the Epstein Barr vims origin of replication and nuclear antigen EBNA-I coding region which produces high copy episomal replication without integration. Examples of expression vectors of the present disclosure include the following:

Table 2. Vector seqiseeces

AGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAAC

CTCCTCGTTCGACCCCGC-CTCGATCCTCCCTTTATCCAGCCCTCACTC

CTTCTCTAGGCGCCCCCATATGGCCATATGAGATCTTATATGGGGCA

CCCCCGCCCCTTGTAAACTTCCCTGACCCTGACATGACAAGAGTTAC

TAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCC

AGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACT

GGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTG

TGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAG

GACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGAC

GGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACC

CCGGGGGTGGACCATCCTCTAGACTGCCATGATGGAGTTTGGACTTT

CTTGGTTGTTTTTGGTGGCAATTCTGAAGGGTGTCCAGTGTACGATC

CC ACCGC ACGTTC A GA AGTCGGTT AA T A A CG A C ATGAT AGTC A CTG

ACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGAT

GTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTG

CAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTG

T ATGG A G A A A G A ATG A CGA G A AC AT A AC ACT A GAGA C A GTTTGCC A

TGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTT

CTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTT

CTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCT

TCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATA

TTTCAACACCATCACCATCACCATGGAAGTGGAACCACTTCAGGTAC

TACCCGTCTTCTATCTGGGCACACGTGTTTCACGTTGACAGGTTTGCT

TGGGACGCTAGTAACCATGGGCTTGCTGACTTGACAACCTCGATCCG

GATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGCTCTA

GTTTTGACTCAACAATATCACCAGCTGAAGCCTATAGAGTACGAGCC

ATAGATAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGA

ATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGC

CATTTTGCAAGGCATGGAAAAATACATAACTGAGAATAGAGAAGTT

CAGATCAAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCAAAC

AGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAA

CAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAG

CAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATG

CGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGG

GTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCA

ATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTC

AATAAAAGAGCCCACAACCCCTCACTCGGGGCGCCAGTCCTCCGAT

TGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAG

TTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGA

GTGATTGACTACCCGTCAGCGGGGGTCTTTCACACATGCAGCATGTA

TCAAAATTAATTTGGTTTTTTTTCTTAAGTATTTACATTAAATGGCCA

TAGTACTTAAAGTTACATTGGCTTCCTTGAAATAAACATGGAGTATT

C AGA ATGTGTC AT A AAT ATTTCT A ATTTT AAGAT ACT ATCTCC ATTG

GCTTTCTACTTTTTCTTTTATTTTTTTTTGTCCTCTGTCTTCCATTTGTT

GTTGTTGTTGTTTGTT GTTTGTTTGTTGGTTGGTTGGTTAATTTTTTT

TTAAAGATCCTACACTATAGTTCAAGCTAGACTATTAGCTACTCTGT

AACCCAGGGTGACCTTGAAGTCATGGGTAGCCTGCTGTTTTAGCCTT

CCCACATCT.AAGATTACAGGTATGAGCTATCATTTTTGGTATATTGA

TTGATTGATTGATTGATGTGTGTGTGTGTGATTGTGTTTGTGTGTGTG

ACTGTGAAAATGTGTGTATGGGTGTGTGTGAATGTGTGTATGTATGT

GTGTGTGTGAGTGTGTGTGTGTGTGTGTGCATGTGTGTGTGTGTGAC

TGTGTCTATGTGTATGACTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG

TGTGTGTGTGTGTGTGTTGTGAAAAAATATTCTATGGTAGTGAGAGC

CAACGCTCCGGCTCAGGTGTCAGGTTGGTTTTTGAGACAGAGTCTTT

CACTTAGCTTGGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTG

GGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCC

CTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCT

TCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGT

ATTTTCTCCITACGCATCTGTGCGGTATTTCACACCGCATATGGTGCA

CTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGA CACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCC

GGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGT

GTCAGAGGTTTTCACCOTCATCACCGAAACGCGCGATGACGAAAGO

GCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATG

GTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAAC

CCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCAT

GAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG

AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCG

GCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGT

AAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAA

CTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGA

ACGTTTTCCAATGATGAGCACTTTT.AAAGTTCTGCTATGTGGCGCGG

TATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATA

CACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAA

GCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCC

ATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGAT

CGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGAT

CATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCAT

ACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACA

ACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCG

GCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCA

CTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCT

GGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGC

CAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGT

CAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTG

CCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATAT

ATACTT AGATTGATTTAAAACTTCATTTTTAATrTAAAAGGATCTAG

GTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGA

GTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT

CTTCT GAGATCCTTTTrTTCTGCGCGTAATCTGCTGCTTGCAAACAA

AAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCT

ACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATAC

CAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG

AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACC

AGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACT

CAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGG

GGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAA

CTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCG

AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA

CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCT

TTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTT

GTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAAC

GCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATG

TTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCC

TTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCA

GCG AGTC A GTGAGCG AGGA AGC GG A AGA GCGC CC AAT A CGCA A A C

CGCCTCTCCCCGCGC GTTGGCCG ATTC ATT A ATGC AGCTGGCACG AC

AGG i " 1¹ i C CCGA CTGGA A AGCGGGC A GTG AGCGC A ACGC A ATT A ATG

TGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACITT^'ATGCTTC

CGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAA.CAATTTCACA.CA

GGA AA C AGCT ATG A C C ATGATT ACGCC

AAGCTTTGCTCTTAGGAGTTTCCTAATACATCCCAAACTCAAATATA 26

TAAAGCATTTGACTTGTTCTATGCCCTAGGGGGCGGGGGGAAGCTA

AGCCAGCT TTTTTAACAT TAAAATGTTAATTCCATTTTAAATGCA

CAGATGTTTTTATTTCATAAGGGTTTCAATGTGCATGAATGCTGCAA

TATTCCTGTTACCAAAGCTAGTATAAATAAAAATAGATAAACGTGG

AAATTACTTAGAGTTTCTGTCATTAACGTTTCCTTCCTCAGTTGACA

ACATAAATGCGCTGCTGAGCAAGCCAGTTTGCATCTGTCAGGATCA

ATTTCCCATTATGCCAGTCAT ATT AATT ACTA GTCAATTAGTTGATT

TTTATTTTTGACATATACATGTGAATGAAAGACCCCACCTGTAGGTT TGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAAT

ACATAACTGAGAATAGAAAAGTTCAGATCAAGGTCAGGAACAGAT

GGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTT

CCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATG

GGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGG

GCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTT

CTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAAT

GACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTC

TGTTCGCGCGCTTATGCTCCCCGAGCTCAATAAAAGAGCCCACAAC

CCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGT

ACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGT

CTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTC

AGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCC

TGCCCAGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGC

AACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGA.CTGATTTTA

TGCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGG

ACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCCTG

GGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTG

AGTCCTAAAATCCCGATCGTTTAGGACTCTTTGGTGCACCCCCCTTA

GAGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACCTAAAACAG

TTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCG

CGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTC

TGACTGTGTTTCTGTATTTGTCTGAAAATATGGGCCCGGGCTAGCCT

GTTACCACTCCCTTAAGTTTGACCTTAGGTCACTGGAAAGATGTCG

AGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTT

GGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATG

GCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAG

ATCAAGGTCTTTTCACCTGGCCCGCATGGACACCCAGACCAGGTGG

GGTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGG

GTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATC

CGCCCCGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCIXGAT

CCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCCCCATATGG

CCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACTTCCC

TGACCCTGACATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCT

CACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTC

TGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCA

CCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACT

AAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGA

CCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACA

CGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCT

AGACTGCCATGATGGAGTTTGGACTTTCTTGGTTGTTTTTGGTGGC

ATTCTGAAGGGTGTCCAGTGTACGATCCCACCGCACGTTCAGAAGT

CGGTTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAA

GTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTG

AC AACCAG A A ATCCTGC ATGAGCAACTGC AGC ATCACCTCC ATCTG

TGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGA

CGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCC

TACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTAT

GAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGT

AGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATA

ACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAgcgagcaccaaag gcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctg gtgaaagattattttccggaaccggtgaccgtgagcfggaacagcggcgcgctgaccagcggcgtgcatacctf tccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctggg cacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaa agctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctggcgggcccggatgtgtttctgtt tccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagc catgaagatccggaagtgaaatttaaciggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcg cgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggc aaagaatataaatgcaaagtgagcaacaaagcgctgccgctgccggaagaaaaaaccattagcaaagcgaaa ggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgafgaacfgaccaaaaaccaggtgag ccfgacctgcctggigaaaggctitfatccgagcgatattgcggtggaatgggaaagcaiicggccagccggaa aacaaclataaaaccaccccgccggigctggatagcgafggcagcttitftciglaiagcaiiacfgaccgtggata aaagccgctggcagcagggcaacgtgtftagcfgcagcgtgatgcatgaagcgctgcataaccattatiicccag aaaagcctgagcctgagcccgggcaaaCACCATCACCATCACCATtatgcgcgcctgggcct gcgcGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATAT

CTGTCATCATCATCTTCTACCAACCTCGATCCGGATTAGTCCAATTT

GTTAAAGACAGGATATCAGTGGTCCAGGCTCTAGTTTTGACTCAAC

AATATCACCAGCTGAAGCCTATAGAGTACGAGCCATAGATAAAATA

AAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCC

ACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGG

CATGGAAAAATACATAACTGAGAATAGAGAAGTTCAGATCAAGGT

CAGGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTG

TGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAAC

AGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGC

CCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCC

CTC AGC AGTTTCT AGAG A A C CATC A GATGTTTCC AGGGTGCCC CA A

GGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTC

GCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAA

GAGCCCACAACCCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGA

GTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATC

CGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATT

GACTACCCGTCAGCGGGGGTCTTTCACACATGCAGCATGTATCAAA

ATTAATTTGGTTTTTTTTCTTAAGTATTTACATTAAATGGCCATAGT

ACTTAAAGTTACATTGGCTTCCTTGAAATAAACATGGAGTATTCAG

AATGTGTCATAAATATTTCTAATTTTAAGATAGTATCTCCATTGGCT

T CTACTT TTCTTTrAT T TTTTTGTCCTCTGTCTTCCATTTGTTGT

TGTTGTTGTTTGTTTGTTTGTTTGTTGGTTGGTTGGTTAATTTTTTTTT

AAAGATCCTACACTATAGTTCAAGCTAGACTATTAGCTACTCTGTA

ACCCAGGGTGACCTTGAAGTCATGGGTAGCCTGCTGTTTTAGCCTTC

CCACATCTAAGATTACAGGTATGAGCTATCATTTTTGGTATATTGAT

TGATTGATTGATTGATGTGTGTGTGTGTGATTGTGTTTGTGTGTGTG

ACTGTGAAAATGTGTGTATGGGTGTGTGTGAATGTGTGTATGTATG

TGTGTGTGTGAGTGTGTGTGTGTGTGTGTGCATGTGTGTGTGTGTGA

CTGTGTCTATGTGTATGACTGTGTGTGTGTGTGTGTGTGTGTGTGTG

TGTGTGTGTGTGTGTGTGTTGTGAAAAAATATTCTATGGTAGTGAG

AGCCAACGCTCCGGCTCAGGTGTCAGGTTGGTTTTTGAGACAGAGT

CTTTCACTTAGCTTGGAATTCACTGGCCGTCGTTTTACAACGTCGTG

ACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACA

TCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGAT

CGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGA

TGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATA

TGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCC

AGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTG

TCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGG

AGCTGC ATGTGTCAG A GGTTTTC ACC GTC ATC A C CGA A A C GCGCG A

TGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCAT

GATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAAT

GTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATAT

GTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAT

TG A A A A A GG A A G AGT A TGAGT ATTC A A C ATTTCCGTGTCGCCCTT A

TTCCCTTTTTTGCGGC ATTTTGCCTTCCTGTTTTTGCTC AC CC AGA A A

CGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAG

TGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAG

TTTTCGCCCCGAAGAACGTTTTCC.AATGATGAGCACTTTTAAAGTTC

TGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCA

ACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACT

CACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAG

AATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAA

CTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTT TTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAAC

CGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA

TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGA

ACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAG

GCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTG

GCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCG

CGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATC

GTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGA

AATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGT

AACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAA

CTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAA

TCTCATGACCAAAATCCCTTAACGTGAGIL !TCGTTCCACTGAGCGT

CAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTT

CTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAG

CGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAG

GT A ACTGGCTTC AGCAGA GCGC AG AT ACC AA AT ACTGTCCTTCT A G

TGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC

TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTG

GCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACC

GGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACA

GCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAG

CGTGAGCAT GAGAAACK:GCCACGCTTCCCGAAGGGAGAAAGGCG

GACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACG

AGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCG

GGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCA

GGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA

CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCG

TTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGC

TGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT

GAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCC

CGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCC

GACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAG

CTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCG

TATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAAC

AGCTATGACCATGATTACGCC

AAGCTTTGCTCTTAGGAGTTTCCTAATACATCCCAAACTCAAATATA 27

TAAAGCAT'iTGACTTGTTCTATGCCCTAGGGGGCGGGGGGAAGCTA

AGCCAGCTTTTTTTAACATTTAAAATGTTAATTCCATTTTAAATGCAC

AGATGTTTTTATTTCATAAGGGTTTCAATGTGCATGAATGCTGCAAT

ATTCCTGTTACCAAAGCTAGTATAAATAAAAATAGATAAACGTGGA

AATTACTTAGAGTTTCTGTCATTAACGTTTCCTTCCTCAGTTGACAAC

ATAAATGCGCTGCTGAGCAAGCCAGTTTGCATCTGTCAGGATCAATT

TCCCATTATGCCAGTCATATTAATTACTAGTCAATTAGTTGATTTTTA

TTTTTGACATATACATGTGAATGAAAGACCCCACCTGTAGGTTTGGC

AAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAATACAT

AACTGAGAATAGAAAAGTTCAGATCAAGGTCAGGAACAGATGGAAC

AGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGC

CCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAA

ACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAG

AACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAG

AACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCT

GTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGC

GCGCTTATGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACT

CGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTA

TCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTC

CTTGG G AGG GTCTCCTCTGAGTG ATTG ACT ACCCGTC AG CGG GGGTC

TTTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCCAGGGAC

CACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTG

TCTGTCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCG

GTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAA CTGACGAGTTCGGAACACCCGGCCGCAACCCTGGGAGACGTCCCAG

GGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCC

GATCGTTTAGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGT

GGTTCTGGTAGGAGACGAGAACCTAAAACAGTTCCCGCCTCCGTCTG

AATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGCCGCGCGTCTTGTC

TGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTGTAT

TTGTCTGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCCTTAAG

TTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCACAACC

AGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCA

GAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTA

ACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGC

CCGCATGGACACCCAGACCAGGTGGGGTACATCGTGACCTGGGAAG

CCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTA

AGCCTCCGCCTC-CTCTTC-CTCCATCCGCCCCGTCTCTCCCCCTTGAAC

CTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTC

CTTCTCTAGGCGCCCCCATATGGCCATATGAGATCTTATATGGGGCA

CCCCCGCCCCTTGTAAACTTCCCTGACCCTGACATGACAAGAGTTAC

TAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCC

AGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACT

GG A CCG ACCGGTGGT ACCTC A CCCTT ACCG A GTCGGCG AC A C AGTG

TGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAG

GACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGAC

GGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACC

CCGGGGGTGGACCATCCTCTAGACTGCCATGATGGCGCGCCCGCTGT

GCACCCTGCTGCTGCTGATGGCGACCCTGGCGGGCGCGCTGGCGCA

GGTGCAGCTGCAGGAAAGCGGCCCGGGCCTGGTGAAACCGAGCGGC

ACCCTGAGCCTGACCTGCGC-GGTGAGCGGCGTGAGCATTCGCAGCA

TTAACTGGTGGAACTGGGTGCGCCAGCCGCCGGGCAAAGGCCTGGA

ATGGATTGGCGAAATTTATCATAGCGGCAGCACCAACTATAACCCG

AGCCTGAAAAGCCGCGTGACCATTAGCGTGGATAAAAGCAAAAACC

AGTTTAGCCTGAAACTGAACAGCGTGACCGCG GCG GATACCGCGGT

GTATTATTGCGCGCGCGATGGCGGCATTGCGGTGACCGATTATTATT

ATTATGGCCTGGATGTGTGGGGCCAGGGCACCACCGTGACCGTGAG

CAGCGCGAGCACCAAAGGCCCGAGCGTGTTTCCGCTGGCGCCGTGC

AGCCGCAGCACCAGCGGCGGCACCGCGGCGCTGGGCTGCCTGGTGA

AAGATTATTTTCCGGAACCGGTGACCGTGAGCTGGAACAGCGGCGC

GCTG ACCAG CGG CGTGC AT ACCTTTCCGGCGGTGCTGC AG AG C AG C

GGCCTGTATAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAGCC

TGGGCACCCAGACCTATACCTGCAACGTGAACCATAAACCGAGCAA

CACCAAAGTGGATAAACGCGTGGAACTGAAAACCCCGCTGGGCGAT

ACCACCCATACCTGCCCGCGCTGCCCGGAACCGAAAAGCTGCGATA

CCCCGCCGCCGTGCCCGCGCTGCCCGGAACCGAAAAGCTGCGATAC

CCCGCCGCCGTGCCCGCGCTGCCCGGAACCGAAAAGCTGCGATACC

CCGCCGCCGTGCCCGCGCTGCCCGGCGCCGGAACTGCTGGGCGGCC

CGAGCGTGTTTCTGTTTCCGCCGAAACCGAAAGATACCCTGATGATT

AGCCGCACCCCGGAAGTGACCTGCGTGGTGGTGGATGTGAGCCATG

AAGATCCGGAAGTGCAGTTTAAATGGTATGTGGATGGCGTGGAAGT

GCATAACGCGAAAACCAAACCGCGCGAAGAACAGTATAACAGCACC

TTTCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTGGCTGAA

CGGCAAAGAATATAAATGCAAAGTGAGCAACAAAGCGCTGCCGGCG

CCGATTGAAAAAACCATTAGCAAAACCAAAGGCCAGCCGCGCGAAC

CGCAGGTGTATACCCTGCCGCCGAGCCGCGAAGAAATGACCAAAAA

CCAGGTGAGCCTGACCTGCCTGGTGAAAGGCTTTTATCCGAGCGATA

TTGCGGTGGAATGGGAAAGCAGCGGCCAGCCGGAAAACAACTATAA

CACCACCCCGCCGATGCTGGATAGCGATGGCAGCTTTTTTCTGTATA

GCAAACTGACCGTGGATAAAAGCCGCTGGCAGCAGGGCAACATTTT

TAGCTGCAGCGTGATGCATGAAGCGCTGCATAACCGCTTTACCCAGA

AAAGCCTGAGCCTGAGCCCGGGCAAAGGCAGCGGCGCGACCAACTT

TAGCCTGCTGAAACAGGCGGGCGATGTGGAAGAAAACCCGGGCCCG

ATGGCGCGCCCGCTGTGCACCCTGCTGCTGCTGATGGCGACCCTGGC GGGCGCGCTGGCGGAAATTGTGCTGACCCAGAGCCCGGCGACCCTG AGCCTGAGCCCGGGCGAACGCGCGACCCTGAGCTGCCGCGCGAGCG

AAAGCGTGAGCAGCAACCTGGCGTGGTATCAGCAGAAACCGGGCCA

GGCGCCGCGCCTGCTGATTTATGGCGCGTTTAACCGCGCGACCGGCA

TTCCGGCGCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTG

ACCATTAGCAGCCTGGAACCGGAAGATTTTGCGGTGTATTATTGCCA

GCAGCGCAGCGATTGGTTTACCTTTGGCGGCGGCACCAAAGTGGAA

ATTAAAACCGTGGCGGCGCCGAGCGTGTTTATTT TCCGCCGAGCGA

TGAACAGCTGAAAAGCGGCACCGCGAGCGTGGTGTGCCTGCTGAAC

AACTTTTATCCGCGCGAAGCGAAAGTGCAGTGGAAAGTGGATAACG

CGCTGCAGAGCGGCAACAGCCAGGAAAGCGTGACCGAACAGGATA

GCAAAGATAGCACCTATAGCCTGAGCAGCACCCTGACCCTGAGCAA

AGCGGATTATGAAAAACATAAAGTGTATGCGTGCGAAGTGACCCAT

CAGGGCCTGAGCAGCCCGGTGACCAAAAGCTTTAACCGCGGCGAAT

GCGGCAGCGGCGCGACCAACTTTAGCCTGCTGAAACAGGCGGGCGA

TGTGGAAGAAAACCCGGGCCCGATGCCACCTCCTCGCCTCCTCTTCT

TCCTCCTCTTCCTCACCCCCATGGAAGTCAGGCCCGAGGAACCTCTA

GTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAGTGCCTCA

AGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGA

GTCCCCGCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCC

TGGGAATCCACATGAGGCCCCTGGCCATCTGGCTTTTCATCTTCAAC

GTCTCTCAACAGATGGGGGGCTTCTACCTGTGCCAGCCGGGGCCCCC

CTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATGTGGAGGGC

AGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGG

GCTGTGGCCTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCC

GGGAAGCTCATGAGCCCCAAGCTGTATGTGTGGGCCAAAGACCGCC

CTGAGATCTGGGAGGGAGAGCCTCCGTGTCTCCCACCGAGGGACAG

CCTGAACCAGAGCCTCAGCCAGGACCTCACCATGGCCCCTGGCTCCA

CACTCTGGCTGTCCTGTGGGGTACCCCCTGACTCTGTGTCCAGGGGC

CCCCTCTCCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATTGCT

GAGCCTAGAGCTGAAGGACGATCGCCCGGCCAGAGATATGTGGGTA

ATGGAGACGGGTCTGTTGTTGCCCCGGGCCACAGCTCAAGACGCTG

GAAAGTATTATTGTCACCGTGGCAACCTGACCATGTCATTCCACCTG

GAGATCACTGCTCGGCCAGTACTATGGCACTGGCTGCTGAGGACTG

GTGGCTGGAAGGTCTCAGCTGTGACTTTGGCTTATCTGATCTTCTGC

CTGTGTTCCCTTGTGGGCATTCTTCATCTTCAAAGAGCCCTGGTCCTG

AGGAGGAAAAGAAAGCGAATGACTGACCCCACCAGGAGATTCCAA

CCTCGATCCGGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGT

CCAGGCTCTAGTTTTGACTCAACAATATCACCAGCTGAAGCCTATAG

AGTACGAGCCATAGATAAAATAAAAGATTTTATTTAGTCTCCAGAA

AAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCT

TAAGTAACGCCATTTTGCAAGGCATGGAAAAATACATAACTGAGAA

TAGAGAAGTTCAGATCAAGGTCAGGAACAGATGGAACAGCTGAATA

TGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAG

GGCCAAGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATC

TGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGT

CCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGAT

GTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTG

AACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTC

CCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGGCGCCAG

TCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACC

CTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGT

CTCCTCTGAGTGATTGACTACCCGTCAGC-GGGGGTCTTTCACA.CATG

CAGCATGTATCAA.AATTAATTTGGTTTTTTTTCTTAAGTATTTACATT

AAATGGCCATAGTACTTAAAGTTACATTGGCTTCCTTG.AAATAAACA

TGGAGTATTCAGAATGTGTCATAAATATTTCTAATTTTAAGATAGTA

TCTCCATTGGCTTTCTACTTTTTCTTTTATTTTTTTTTGTCCTCTGTCTT

CCATT GTTGTTGTTGT GT TGTT GTTTGTTTGTTGGTTGGTTGGTT

AATTTTTTTTTAAAGATCCTACACTATAGTTCAAGCTAGACTATTAGC

TACTCTGTAACCCAGGGTGAC-CTTGAAGTCATGGGTAGCCTGCTGTT TTAGCCTTCCCACATCTAAGATTACAGGTATGAGCTATCATTTTTGGT

ATATTGATTGATTGATTGATTGATGTGTGTGTGTGTGATTGTGTTTGT

GTGTGTGACTGTGAAAATGTGTGTATGGGTGTGTGTGAATGTGTGTA

TGTATGTGTGTGTGTGAGTGTGTGTGTGTGTGTGTGCATGTGTGTGT

GTGTGACTGTGTCTATGTGTATGACTGTGTGTGTGTGTGTGTGTGTGT

GTGTGTGTGTGTGTGTGTGTGTGTTGTGAAAAAATATTCTATGGTAG

TGAGAGCCAACGCTCCGGCTCAGGTGTCAGGTTGGTTTTTGAGACAG

AGTCTTTCACTTAGCTTGGAATTCACTGGCCGTCGTTTTACAACGTCG

TGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCAC

ATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGAT

CGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGAT

GCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATG

GTGC ACTCTC A GT A C A ATCTGCTCTG ATGCCGC ATA GTT A AGCC A GC

CCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTG

CTCCCGGC ATCCGCTT A C AG AC A AGCTGTGACC GTCTCCGGG A GCTG

CATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGATGACGA

AAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAAT

AATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCG

GAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGC

TCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAG

GAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTT

TGCGGCATTTTGCCTTCCTG 1 " 1 'i TGCTCACCCAGAAACGCTGGTGA

AAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACAT

CGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCG

AAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGC

GCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCC

GCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACA

GAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTG

CTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACA

ACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGG

GGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGA

AGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATG

GCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGC

TTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCA

GGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGA

TAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCAC

TGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACG

GGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGA

TAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTAC

TCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGG

ATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTA

ACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCA

AAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGC

AAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCA

AGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGC

AGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCAC

TTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCT

GTTA.CCAGTGGCTGCTGCCAGTGGCGA.TAAGTCGTGTCTTACCGGGT

TGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTG

AACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTAC

ACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGC

TTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGT

CGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTG

GTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCG

ATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCC

AGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCT

CACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATT

ACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCG

AGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATAC

GCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGG CACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAA TTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTT ATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATT TCACACAGGAAACAGCTATGACCATGATTACGCC

AAGCTTTGCTCTTAGGAGTTTCCTAATACATCCCAAACTCAAATATA 28

TAAAGCATTTGACTTGTTCTATGCCCTAGGGGGCGGGGGGAAGCTA

AGCCAGCT TTTT AACATTTAAAATGTTAATTCCATTTTAAATGCA

CAGATGTTTTTATTTCATAAGGGTTTCAATGTGCATGAATGCTGCAA

TATTCCTGTTACCAAAGCTAGTATAAATAAAAATAGATAAACGTGG

AAATTACTTAGAGTTTCTGTCATTAACG^'l"i"i^'CCTTCCTCAGTTGACA

ACATAAATGCGCTGCTGAGCAAGCCAGTTTGCATCTGTCAGGATCA

ATTTCCCATTATGCCAGTCATATTAATTACTAGTCAATTAGTTGATT

TTTATTTTTGACATATACATGTGAATGAAAGACCCCACCTGTAGGTT

TGGCAAGCTAGCTTAAOTAACGCCATT^'ITGCAAGGCATGGAAAAAT

ACATAACTGAGAATAGAAAAGTTCAGATCAAGGTCAGGAACAGAT

GGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTT

CCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATG

GGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGG

GCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTT

CTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAAT

GACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTC

TGTTCGCGCGCTTATGCTCCCCGAGCTCAATAAAAGAGCCCACAAC

CCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGT

ACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGT

CTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTC

AGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCC

TGCCCAGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGC

AACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTA

TGCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGG

ACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCCTG

GGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTG

AGTCCTAAAATCCCGATCG'i'i' i'AGGACTCTTTGGTGCACCCCCCTTA

GAGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACCTAAAACAG

TTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCG

CGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTC

TGACTGTGTTTCTGTATTTGTCTGAAAATATGGGCCCGGGCTAGCCT

GTTACCACTCCCTTAAGTTTGACXTTAGGTCACTGGAAAGATGTCG

AGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTT

GGGTT A C CTTCTGCTCTGC A GA ATGGC C A ACC I " I ' i A ACGTCGG ATG

GCCGCGAGACGGCACCi 'i'iAACCGAGACCTCATCACCCAGGTTAAG

ATCAAGGTCTTTTCACCTGGC-CCGCATGGACACCCAGACCAGGTGG

GGTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCC-CCTCCCTGG

GTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATC

CGCCX:CGTCTC fCCCCCTTGAACCTCCTCGTTCGACCX;CGCCTCGAT

CCTCCC1U ATCCAGCCCTCACTCCTTCTCTAGGCGCCCCCATATGG

CCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACTTCCC

TGACCCTGACATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCT

CACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTC

TGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCA

CCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACT

AAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGA

CCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACA

CGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCT

AGACTGCCATGGACATTGATTATTGACTAGTTATTAATAGTAATCA

ATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTAC

ATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCC

CGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAA

TAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAAC

TGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC

CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC AGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTA

TTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAA

TGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCAC

CCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGG

ACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGG

CGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTA

GTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACC

TCCATAGAAGACACCGGGACCGATCCAGCCTCCATCGGCTCGCATC

TCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCC

GGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACT

GCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCC

TTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCC

ACGCTTTGCCTGACCCTGCTTGCTCAACTCTAGTTAACGGTGGAGG

GCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCGCCACCAG

ACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTT

TCTGCAGTCACCGTCGTCGACACGTGTGATCAGATATCGCGGCCGC

TCTAGACCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCA

ACTGCAACCGGTGTACATTCAATGGA.GTTTGGACTTTCTTGGTTGTT

TTTGGTGGCAATTCTGAAGGGTGTCCAGTGTgaagaagaacfgcaggtgattcag ccggataaaagcgtgctggtggcggcgggcgaaaccgcgaccctgcgctgcaccgcgaccagcctgattcc ggtgggcccgattcagtggttfcgcggcgcgggcccgggccgcgaactgatttataaccagaaagaaggccat tttccgcgcglgaccaccgtgagcgatetgaccaaacgcaacaacatggattttagcattcgcattggcaacatta ccccggcggatgcgggcacctattattgcgtgaaatttcgcaaaggcagcccggatgatgtggaatttaaaagc ggcgcgggcaccgaactgagcgtgcgcgcgaaaccgagcgcgccggtggtgagcggcccggcggcgcgc gcgaccccgcagcataccgtgagctttacctgcgaaagccatggctttagcccgcgcgatattaccctgaaatg gtttaaaaacggcaacgaactgagcgattttcagaccaacgtggatccggtgggcgaaagcgtgagctatagca ttcatagcaccgcgaaagtggtgctgacccgcgaagatgtgcatagccaggtgatttgcgaagtggcgcatgtg acccigcagggcgatccgctgcgcggcaccgcgaaccigagcgaaaccattogcgtgccgccgaccctgga agtgacccagcagccggtgcgcgcggaaaaccaggtgaacgtgacctgccaggtgcgcaaattttatccgca gcgcctgcagctgacctggctggaaaacggcaacgtgagccgcaccgaaaccgcgagcaccgtgaccgaaa acaaagatggcacctataactggatgagctggctgctggtgaacgtgagcgcgcatcgcgatgatgtgaaactg acctgccaggtggaacatgaiggccagccggcggtgagcaaaagccatgafctgaaagtgagcgcgcatccg aaagaacagggcagcaacaccgcggcggaaaacaccggcagcaacgaacgcaacattlatGGTGGTG

GTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGG

TGGATCCgcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggc ggcaccgcggcgctgggcigcctggtgaaagattattttccggaaccggtgaccgigagctggaacagcggc gcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtgg tgaccgtgccgagcagcagcctgggcacccagacciaiatitgcaacgtgaaccaiaaaccgagcaacaccaii agtggatiiaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaact gctggcgggcccggatgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaag tgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaag tgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgt gctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccgctgccggaa gaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcga tgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggctttlatccgagcgatattgcggtggaatg ggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagctttttt ctgtatagcaaartgaccgtggalaaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatga agcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaaCACCATCACCATC

ACCATGGAAGTGGAACCACTTCAGGTACTACCCGTCTTCTATCTGG

GCACACGTGTTTCACGTTGACAGGTTTGCTTGGGACGCTAGTAACC

ATGGGCTTGCTGACTTGACAACCTCGATCCGGATTAGTCCAATTTGT

T.AAAGACAGGATATCAGTGGTCCAGGCTCTAGTTTTGACTCAACAA

T ATC ACC AGCTG A A GCCT AT AG A GT ACG AGCC AT A GAT AAA AT A A A

AGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCAC

CTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCA

TGGAAAAATACATAACTGAGAATAGAGAAGTTCAGATCAAGGTCA

GGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTG

GTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACA

GCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCC

CCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCC TCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAG

GACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCG

CTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAG

AGCCCACAACCCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAG

TCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCC

GACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTG

ACTACCCGTCAGCGGGGGTCTTTCACACATGCAGCATGTATCAAAA

TTAATTTGGTTTTTTTTCTTAAGTATTTACATTAAATGGCCATAGTAC

TTAAAGTTACATTGGCTTCCTTGAAATAAACATGGAGTATTCAGAA

TGTGTC AT A AAT ATTTCT AATTTT AAG AT AGT ATCTCC ATTGGCTTT

CTACTTTTTCTTTTATTTTTTTTTGTCCTCTGTCTTCCATTTGTTGTTG

TTGTTGTTTGTTTGTTTGTTTGTTGGTTGGTTGGTTAATTTTTTTTTAA

AGATCCTACACTATAGTTCAAGCTAGACTATTAGCTACTCTGTAACC

CAGGGTGACCTTGAAGTCATGGGTAGCCTGCTGTTTTAGCCTTCCCA

CATCTAAGATTACAGGTATGAGCTATCATTTTTGGTATATTGATTGA

TTGATTGATTGATGTGTGTGTGTGTGATTGTGTTTGTGTGTGTGACT

GTGAAAATGTGTGTATGGGTGTGTGTGAATGTGTGTATGTATGTGT

GTGTGTGAGTGTGTGTGTGTGTGTGTGCATGTGTGTGTGTGTGACTG

TGTCTATGTGTATGACTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT

GTGTGTGTGTGTGTGTTGTGAAAAAATATTCTATGGTAGTGAGAGC

C A ACGCTCC GGCTC A GGTGTC AGGTTGGTTTTTGAG A C AGAGTCTTT

CACTTAGCTTGGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTG

GGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATC-CC

CCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCC

CTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCG

GTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGT

GCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCC

CCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTG

CTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCT

GCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGATGAC

GAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGAT

AATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTG

CGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTA

TCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGA

AAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTC

CCTTTTT GCGGCAT TTGCCTTCCTGTTTTTGCTCACCCAGAAACGC

TGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGG

GTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTT

TCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGC

TATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACT

CGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCA

CCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAA

TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACT

TACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTT

GCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCG

GAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATG

CCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAAC

TACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGC

GGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGC

TGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCG

GTATCATTGCAGCACTGGGGCCAGATGGT.AAGCCCTCCCGTATCGT

AGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAA

TAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAA

CTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTT.AAAACT

TCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATC

TCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCA

GACCCCGTAGAAAAGATCAAAGGATCTTCT GAGATCCTTTTTTTCT

GCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCG

GTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGT

AACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTG TAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTA

CATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGC

GATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGG

ATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGC

CCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCG

TGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGA

CAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAG

GGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGG

TTTCGCCACCTCTGACTTGAGCGTCGATT TTGTGATGCTCGTCAGG

GGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACG

GTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTT

ATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTG

ATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGA

GCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCG

CGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCC GA

CTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTC

ACTCATTAGGCA.CCCCAGGCTTTA.CACTTTA.TGCTTCCGGCTCGTAT

GTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGC

T ATG ACC ATGATT A CGC C

[00160] SEQ ID NO:25 is an expression vector (SEQ ID NO: 25) encoding TGFpRII extracellular domain. Vectors of the disclosure include vectors that comprise SEQ ID NO:25 but are free of ΤΟΡβΚΙΙ extracellular domain.

[00161] SEQ ID NO: 26 is an expression vector encoding ΤΟΡβΚΙΙ extracellular and transmembrane domains. Vectors of the disclosure include vectors that comprise SEQ ID NO:26 but are free of ΤΟΡβΙ Π extracellular and transmembrane domains.

[00162] SEQ ID NO:27 is an expression vector encoding anti-CD47 antibody domains.

Vectors of the disclosure include vectors that comprise SEQ ID NO:27 but are free of the nucleic acid sequence encoding anti-CD47 antibody domains.

[00163] SEQ ID NO:28 is an expression vector encoding SIRPa domain. Vectors of the disclosure include vectors that comprise SEQ ID NO:28 but are free of the nucleic acid sequence encoding a SIRPa domain.

[00164] The transformed or transduced cells can be cultured under conditions that promote expression of the polypeptide, and the polypeptide recovered by conventional protein purification procedures. One such purification procedure includes the use of affinity chromatography, e.g., over a matrix having all or a portion of antigen bound thereto. Polypeptides contemplated for use herein include substantially homogeneous recombinant mammalian antibody polypeptides capable of binding a tumor or viral antigen, substantially free of contaminating endogenous materials.

[00165] Antigen binding proteins may be prepared, and screened for desired properties, by any of a number of known techniques. Certain of the techniques involve isolating a nucleic acid encoding a polypeptide chain (or portion thereof) of an antigen binding protein of interest, and manipulating the nucleic acid through recombinant DNA technology. The nucleic acid may be fused to another nucleic acid of interest, or altered (e.g., by mutagenesis or other conventional techniques) to add, delete, or substitute one or more amino acid residues, for example.

[00166] Antibodies and fragments thereof of the present disclosure can be produced using any standard methods known in the art. In one example, the polypeptides are produced by recombinant DNA methods by inserting a nucleic acid sequence (a cDNA) encoding the polypeptide into a recombinant expression vector and expressing the DNA sequence under conditions promoting expression. Antigen-binding polypeptides can also be produced by chemical synthesis (such as by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, 111.). Modifications to the protein can also be produced by chemical synthesis,

[00167] The polypeptides of the present disclosure can be purified by i ol ation/purifi cation methods for proteins generally known in the field of protein chemistry. Non-limiting examples include extraction, recrystallization, salting out (e.g., with ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reversed-phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution or any combinations of these. After purification, polypeptides may be exchanged into different buffers and/or concentrated by any of a variety of methods known to the art, including, but not limited to, filtration and dialysis.

[00168] The purified polypeptide is preferably at least 85% pure, more preferably at least 95% pure, and most preferably at least 98% pure. Regardless of the exact numerical value of the purity, the polypeptide is sufficiently pure for use as a pharmaceutical product.

[00169] In certain embodiments, the present disclosure provides monoclonal antibodies that bind to tumor or viral antigens. Monoclonal antibodies may be produced using any technique known in the art, e.g., by immortalizing spleen cells harvested from the transgenic animal after completion of the immunization schedule. The spleen cells can be immortalized using any technique known in the art, e.g., by fusing them with myeloma cells to produce hybridomas. Myeloma cells for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Examples of suitable cell lines for use in mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NSl/l .Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-11, MPC1 1 -X45-GTG 1.7 and S 194/5 XXO Bui; examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F and 48210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6.

[00170] In certain embodiments, a nucleic acid disclosed herein can comprise a nucleotide sequence encoding at least one, two, or three CDRs from a heavy chain variable region having an amino acid sequence as set forth herein, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical thereto, and/or having one or more substitutions, e.g., conserved substitutions). In some embodiments, the nucleic acid can comprise a nucleotide sequence encoding at least one, two, or three CDRs from a light chain variable region having an amino acid sequence as set forth herein, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%>, 96%o, 97%, 98%, 99%> or more identical thereto, and/or having one or more substitutions, e.g., conserved substitutions). In some embodiments, the nucleic acid can comprise a nucleotide sequence encoding at least one, two, three, four, five, or six CDRs from heavy and light chain variable regions having an amino acid sequence as set forth in the tables herein, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical thereto, and/or having one or more substitutions, e.g., conserved substitutions).

[00171] The nucleic acids disclosed herein include deoxyribonucleotides or ribonucleotides, or analogs thereof. The polynucleotide may be either single-stranded or double-stranded, and if single-stranded may be the coding strand or non-coding (anti sense) strand. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The nucleic acid may be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a nonnatural arrangement.

[00172] Nucleic acids encoding any of the various polypeptides disclosed herein may be synthesized chemically. Codon usage may be selected so as to improve expression in a ceil. Such codon usage will depend on the cell type selected. Specialized codon usage patterns have been developed for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells and insect cells.

[00173] General techniques for nucleic acid manipulation are described for example in Sambrook et al., Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F. Ausubel et al., Current Protocols in Molecular Biology (Green Publishing and Wiiey-interseience: New York, 1987) and periodic updates, herein incorporated by reference. The DNA encoding the polypeptide is operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, viral, or insect genes. Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants is additionally incorporated.

[00174] The recombinant DNA can also include any type of protein tag sequence that may be useful for purifying the protein. Examples of protein tags include but are not limited to a histidine tag, a FLAG tag, a myc tag, an HA tag, or a GST tag. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts can be found in Cloning Vectors: A Laboratory Manual, (Elsevier, N.Y., 1985).

[00175] In some aspects, the application features host cells and vectors containing the nucleic acids described herein. The nucleic acids may be present in a single vector or separate vectors present in the same host cell or separate host cell, as described herein.

[00176] Further provided herein are vectors comprising nucleotide sequences encoding an antibody molecule described herein. In some embodiments, the vectors comprise nucleotides encoding an antibody molecule described herein. In some embodiments, the vectors comprise the nucleotide sequences described herein. The vectors include, but are not limited to, a virus, plasmid, cosmid, lambda phage or a yeast artificial chromosome (YAC).

[00177] Numerous vector systems can be employed. For example, one class of vectors utilizes DNA elements which are derived from animal viruses such as, for example, bovine papilloma virus, polyoma virus, adenovirus, vaccinia vims, baculovirus, retroviruses (Rous Sarcoma Virus, MMTV or MOMl.V) or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki Forest virus, Eastern Equine Encephalitis virus and Flaviviruses.

[00178] Additionally, cells which have stably integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow for the selection of transfected/ transduced host cells. The marker may provide, for example, prototropy to an auxotrophic host, biocide resistance, (e.g., antibiotics), or resistance to heavy metals such as copper, or the like. The selectable marker gene can be either directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcriptional promoters, enhancers, and termination signals.

[00179] Once the expression vector or DNA sequence containing the constructs has been prepared for expression, the expression vectors may be transfected/ transduced or introduced into an appropriate host cell. Various techniques may be employed to achieve this, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid based transfection or other conventional techniques. In the case of protoplast fusion, the cells are grown in media and screened for the appropriate activity.

[00180] Methods and conditions for culturing the resulting transfected/ transduced ceils and for recovering the antibody molecule produced are known to those skilled in the art, and may be varied or optimized depending upon the specific expression vector and mammalian host ceil employed, based upon the present description.

[00181] As used herein, the terms "treat," "treated," or "treating" can refer to therapeutic treatment and/or prophylactic or preventative measures wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder or disease, or obtain beneficial or desired clinical results. For purposes of the embodiments described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder or disease. Treatment can also include eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[00182] As used herein, the terms "diagnose," "diagnosing," or variants thereof refer to identifying the nature of a physiological condition, disorder or disease. In some embodiments, diagnosing a subject refers to identifying whether a patient has a hyperproliferative disorder or a tumor.

[00183] In certain aspects, the present disclosure provides a method of neutralizing a hyperproliferative disorder, comprising contacting the hyperproliferative disorder with an antibody, a fragment thereof, a cell comprising an antibody, or a combination, as described herein.

[00184] In some aspects, the present disclosure provides a method for treating a subject having a hyperproliferative disorder, the method comprising administering an effective amount of a cell, the antibody, or antigen-binding fragment thereof, as described herein. The method may further comprise administering a chemotherapeutic agent to the subject. In some embodiments, administration of a cell or antibody molecule is parenteral or intravenous. According to some embodiments, the hyperproliferative disorder is cancer. According to further embodiments, the cancer is glioblastoma multiforme,

[00185] The term "parenteral" administration of an immunogenic composition includes, e.g., subcutaneous (s.c), intravenous (i.v.), intramuscular (i.m.), or intraci sternal injection, intratumoral, or infusion techniques.

[00186] The present disclosure also provides prophylactic methods. In some embodiments, a method of preventing a cancer or hyperproliferative disorder by administering a cell, an antibody, or antigen-binding fragment thereof as disclosed herein to a subject who is not, at the time, infected with a cancer or hyperproliferative disorder. For instance, in certain aspects, the present disclosure provides a method of reducing a patient's risk of cancer or hyperproliferative disorder, comprising administering to a subject in need thereof a cell, an antibody, or antigen-binding fragment thereof, as described herein, in an amount effective to reduce the risk of cancer or hyperproliferative disorder. For example the risk may be reduced by, e.g., at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more. In some embodiments, the cell or antibody molecule is provided to a patient who does not have cancer or hyperproliferative disorder, with the result that if cancer or hyperproliferative disorder occurs, the course of the disease is likely to be milder than the course of disease in a similar patient who has not received the ceil or antibody molecule. Such risk may be reduced, e.g., by at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%>, 99%, or more compared to a patient that did not receive the cell or antibody molecule, [00187] The term "anti-cancer effect" refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of cancer ceils, a decrease in the number of metastases, an increase in life expectancy, decrease in cancer cell proliferation, decrease in cancer cell survival, or amelioration of various physiological symptoms associated with the cancerous condition. An "anti-cancer effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies described herein in prevention of the occurrence of cancer in the first place. The term "anti-tumor effect" refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.

[00188] The term "therapeutically effective amount" means a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, for example, an amount which results in the prevention or amelioration of or a decrease in the symptoms associated with a disease that is being treated, e.g., disorders associated with cancer growth or a hyperproliferative disorder. The amount of compound administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The regimen of administration can affect what constitutes an effective amount. The compound of the invention can be administered to the subject either prior to or after the onset of a ΤΟΤβ signaling-related disorder. Further, several divided dosages, as well as staggered dosages, can be administered daily or sequentially, or the dose can be continuously infused, or can be a bolus injection. Further, the dosages of the compound(s) of the invention can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation. Typically, an effective amount of the compounds of the present invention, sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Preferably, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. The compounds of the present invention can also be administered in combination with each other, or with one or more additional therapeutic compounds. Those skilled in the art will recognize and determine a therapeutically effective amount of any of the antagonists or inhibitors disclosed therein whether calculated when administered alone or part of a therapeutic regimen that includes one or more cells expressing the amino acids dsiclsoed herein individually or in combination and/or one or more one or more other therapeutic agents and/or one or more other therapeutic treatments or interventions. Generally, therapeutically effective amount refers to an amount of a CD47 or CD 5 agonist or antagonist that ameliorates symptoms, or reverses, prevents or reduces the rate of progress of disease, or extends life span of an individual when administered alone or in combination with other therapeutic agents or treatments as compared to the symptoms, rate of progress of disease, or life span of an individual not receiving a therapeutically effective amount an inhibitor disclosed herein. In some embodiments, the therapeutically effective amount refers to an amount of a modified cell that expresses a CD47 or CD45 agonist or antagonist that ameliorates symptoms, or reverses, prevents or reduces the rate of progress of disease, or extends life span of an individual when administered in combination with a cell that expresses a ΤΟΤβ receptor I and/or II agonist or antagonist.

[00189] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[00190] The phrase "pharmaceutically acceptable carrier" is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyi cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes, oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoi and polyethylene glycol; esters, such as ethyl oleate and ethyl iaurate, agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen -free water; isotonic saline; Ringers solution; ethyl alcohol; phosphate buffer solutions; and other nontoxic compatible substances employed in pharmaceutical formulations. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, which is incorporated herein by reference in its entirety.

[00191] The term "salt" refers to acidic salts formed with inorganic and/or organic acids, as well as basic salts formed with inorganic and/or organic bases. Examples of these acids and bases are well known to those of ordinary skill in the art. Such acid addition salts will normally be pharmaceutically acceptable although salts of non-pharmaceutically acceptable acids may be of utility in the preparation and purification of the compound in question. Acid addition salts of the compounds of the invention are most suitably formed from pharmaceutically acceptable acids, and include for example those formed with inorganic acids e.g. hydrochloric, hydrobromic, sulphuric or phosphoric acids and organic acids e.g. succinic, malaeic, acetic or fumaric acid. Other non-pharmaceutically acceptable salts e.g. oxalates can be used for example in the isolation of the compounds of the invention, for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt. Also included within the scope of the invention are solvates and hydrates of the invention.

[00192] The conversion of a given compound salt to a desired compound salt is achieved by applying standard techniques, in which an aqueous solution of the given salt is treated with a solution of base e.g. sodium carbonate or potassium hydroxide, to liberate the free base which is then extracted into an appropriate solvent, such as ether. The free base is then separated from the aqueous portion, dried, and treated with the requisite acid to give the desired salt.

[00193] In vivo hydrolyzable esters or amides of certain compounds of the invention can be formed by treating those compounds having a free hydroxy or amino functionality with the acid chloride of the desired ester in the presence of a base in an inert solvent such as methylene chloride or chloroform. Suitable bases include triethylamine or pyridine. Conversely, compounds of the invention having a free carboxy group can be esterified using standard conditions which can include activation followed by treatment with the desired alcohol in the presence of a suitable base.

[00194] Examples of pharmaceutically acceptable addition salts include, without limitation, the non-toxic inorganic and organic acid addition salts such as the hydrochloride derived from hydrochloric acid, the hydrobromide derived from hvdrobromic acid, the nitrate derived from nitric acid, the perchl orate derived from perchloric acid, the phosphate derived from phosphoric acid, the sulphate derived from sulphuric acid, the formate derived from formic acid, the acetate derived from acetic acid, the aconate derived from aconitic acid, the ascorbate derived from ascorbic acid, the benzenesulphonate derived from benzensulphonic acid, the benzoate derived from benzoic acid, the cinnamate derived from cinnamie acid, the citrate derived from citric acid, the embonate derived from embonic acid, the enantate derived from enanthic acid, the fumarate derived from fumaric acid, the glutamate derived from glutamic acid, the glycolate derived from glycolic acid, the lactate derived from lactic acid, the maleate derived from maleic acid, the malonate derived from malonic acid, the man delate derived from mandelic acid, the methanesulphonate derived from methane suiphonic acid, the naphthalene-2-sulphonate derived from naphtalene-2-sulphonic acid, the phthalate derived from phthalic acid, the salicylate derived from salicylic acid, the sorbate derived from sorbic acid, the stearate derived from stearic acid, the succinate derived from succinic acid, the tartrate derived from tartaric acid, the toluene-p-sulphonate derived from p- toluene suiphonic acid, and the like. Particularly preferred salts are sodium, lysine and arginine salts of the compounds of the invention. Such salts can be formed by procedures well known and described in the art.

[00195] Other acids such as oxalic acid, which cannot be considered pharmaceutically acceptable, can be useful in the preparation of salts useful as intermediates in obtaining a chemical compound of the invention and its pharmaceutically acceptable acid addition salt. Metal salts of a chemical compound of the invention include alkali metal salts, such as the sodium salt of a chemical compound of the invention containing a carboxy group. Mixtures of isomers obtainable according to the invention can be separated in a manner known per se into the individual isomers; diastereoisomers can be separated, for example, by partitioning between polyphasic solvent mixtures, recrystallization and/or chromatographic separation, for example over silica gel or by, e.g., medium pressure liquid chromatography over a reversed phase column, and racemates can be separated, for example, by the formation of salts with optically pure salt-forming reagents and separation of the mixture of diastereoisomers so obtainable, for example by means of fractional crystallization, or by chromatography over optically active column materials,

[00196] As used herein, the term "sample" refers to a biological sample obtained or derived from a source of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood ceils; ascites; tissue or fine needle biopsy samples; cell -containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs, oral swabs; nasal swabs, washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or ceils therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term "sample" refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a "processed sample" may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc. In some embodiments, the cell sample is a tissue sample taken from a subject suspected of having cancer or being diagnosed as having cancer. In some embodiments, the cell sample is a tissue sample taken from a subject with lung cancer or breast cancer. In some embodiments, the cell sample comprises a plurality of lymphocytes or T cells from the subject diagnosed with or suspected of having a hyperproliferative disorder such as cancer of the adrenal gland, bladder, blood, bone, bone marrow, brain, spine, breast, cervix, gall bladder, ganglia, gastrointestinal tract, stomach, colon, heart, kidney, liver, lung, lymph nodes, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, or uterus. In some embodiment, the ceil sample comprises a plurality of T cells derived from the subject's blood. In some embodiments, the cell sample comprises a plurality of cells T cells from a subject diagnosed with or suspected as having breast cancer,

[00197] As used herein, "control sample" or "reference sample" refer to samples with a known presence, absence, or quantity of substance being measured, that is used for comparison against an experimental sample.

[00198] The term "hyperproliferative disorder" refers to a disease or disorder characterized by abnormal proliferation, abnormal growth, abnormal senescence, abnormal quiescence, or abnormal removal of cells in an organism, and includes all forms of hyperplasias, neoplasias, and cancer. In some embodiments, the hyperproliferative disease is a cancer derived from the gastrointestinal tract or urinary system. In some embodiments, a hyperproliferative disease is a cancer of the adrenal gland, bladder, bone, bone marrow, brain, spine, breast, cervix, gall bladder, ganglia, gastrointestinal tract, stomach, colon, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, or uterus. In some embodiments, the term hyperproliferative disease is a cancer chosen from: lung cancer, bone cancer, CMML, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, testicular, gynecologic tumors (e.g., uterine sarcomas, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina or carcinoma of the vulva), Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system (e.g., cancer of the thyroid, parathyroid or adrenal glands), sarcomas of soft tissues, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, solid tumors of childhood, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter (e.g., renal cell carcinoma, carcinoma of the renal pelvis), or neoplasms of the central nervous system (e.g., primary CNS lymphoma, spinal axis tumors, brain stem gliomas or pituitary adenomas).

[00199] The disclosure relates to, among other things, the following embodiments:

A composition or pharmaceutical composition comprising a modified NK cell, derived from any donor source, expanded ex vivo using feeder ceils. The disclosure also relates to one or a plurality of T cells, derived from any source, expanded ex vivo to allow specific recognition of tumor antigens (neoantigens, endogenous retrovimses, cancer/testis antigens, pluripotency factors, overexpressed proteins).

a. Neoantigens comprise H3K27M, DNAJB 1 -PRK AC A, bcr-abl, CDK4-R24C, CDK4-R24L, MUM1, CTNNB1, CDC27, TRAPPC 1, TPI, ASCC3, HHAT, FN1 , OS-9, PTPRK, CDKN2A, HLA-A1 1 , GAS 7, SIR2, PrdxS, CLPP, PPP1R3B, EF2, ACTN4, MEL NF-YC, HSP70-2, KIAA1440, CASP8. b. Endogenous retroviruses comprise gag, pol, nef, and env sequences from HERV-K, HERV-E, XMRV.

c. Cancer/testis antigens include survivin, MAGEA4, SSX2, FRAME, NYESOl . d. Pluripotency factors include Oct4, Sox2 Nanog.

e. Overexpressed proteins include WT1, p53, MYCN.

In some embodiments, the composition comprises any of the cells disclosed herein cell the cells , also expanded against and specific for viral antigens that are associated with malignancies or infections associated with patients with malignancy.

f. Viral antigens comprise CMV antigens (pp65, IE!, IE!, UL40, UL103, UL151, UL153, UL28, UL32, UL36, UL55, UL40, UL48, UL82, UL94, UL99, us24, us32, us32).

g. Viral antigens comprise HSV antigens (glycoprotein G),

h. Viral antigens comprise EBV antigens (BARFl, BMLF1, BMRFl, BZLF1, EBNALP, EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, gp350/340, LMP1, LMP2).

i. Viral antigens comprise HHV8 antigens (LNA-1, LANA-1 , viral cyclin D, vFLIP, RTA).

j . Viral antigens comprise HPV16 antigens (E6, E7, LI),

k. Viral antigens comprise HPV18 antigens (Έ6, E7).

1. A cell product from embodiment 1 or embodiment 2, cultured in certain cytokines and feeder cell conditions to allow for upregulation of at least one specific chemokine receptor (increased expression by more than 25%).

a. The said chemokine receptor being CXCR1 to target tumors that are enriched for CXCL8 expression.

b. The said chemokine receptor being CXCR2 to target tumors that are enriched for CCL2, CXCL5, and CXCL12.

c. The said chemokine receptor being CCR4 to target tumors that are enriched for CCL22 and CCL28.

d. The said chemokine receptor being CCR6 to target tumors enriched for CCL20.

e. The said chemokine receptor being CXCR4 to targettumors enriched for CXCL12 .

2. A ceil from embodiment I, 2, 3, or 4, rendered resistant to immune suppression by TGF-beta by knocking down signaling from the TGF-beta receptor,

a. The said knockdown procedure, brought about by introduction of "decoy" TGF-beta RII receptors that do not contain signaling moieties; and are therefore linked to either GPI anchors or the stalks of membrane metalloproteinase-sensitive receptors ,

b. The said knockdown procedure, brought about by recognition of relevant mRNA by a complementary RNA molecule, and mediated by R A interference.

c. The said knockdown procedure, brought about by guide DNA recognizing the TGF-beta RII gene, packaged with a clustered regularly interspaced short palindromic repeat cas gene.

d. The said knockdown procedure, brought about by recognition of genomic DNA by engineered transcription activator-like effectors recognizing the TGF- beta RII gene.

e. The said knockdown procedure, brought about by introduction of transgenes coding for TGF-beta Rll-specific intrabodies.

A cell from embodiment 1 , 2, 3, or 4, with the same methodologies described in claim 5, that target alternates to TGF-beta.

a. The said alternate being IL10, and the purported target being IL 1 OR.

b. The said alternate being EL6, and the purported target being IL6R.

A cell from embodiment 1 , 2, 3, 4, 5, or 6 that is modified to secrete a protein sequence capable of binding to CD47 and recruiting other immune cells through engagement of the Fc receptor.

a. The said modification brought about by using the antibody VH and VL domains to target CD47.

b. The said modification brought about by using the extracellular domain of the endogenous ligand (SIRPa) to CD47.

c. The said modification recruiting phagocytes through Fc receptor binding to IgG3.

d. The said modification recruiting phagocytes through Fc receptor binding to anti-CD 16.

e. The said modification recruiting phagocytes and other immune cells through expression of anti-CD45. A cell product from embodiment 1 , 2, 3, 4, 5, or 6, with the same methodologies described in embodiment 7, that target alternates to CD47.

a. The said alternate being GD2.

b. The said alternate being TGFb.

c. The said alternate being Her2.

d. The said alternate being VEGFR.

The method of embodiment 4, comprising:

a. The said expansion uses healthy donors as sources of cells.

b. The said expansion uses alternate feeder cells modified to express different molecules.

c. The said expansions uses any or all of the following cytokines to help proliferation of NK cells: IL2, 11.6, 11.7. 11. 1 2, IL15, IL21 , 11. 1 8

The method of embodiment 4, comprising:

a. The said expansion uses patient samples as sources of cells.

b. The said expansion uses alternate feeder cells modified to express different molecules.

c. The said expansions uses any or all of the following cytokines to help proliferation of NK cells: IL2, IL6, IL7, IL 12, 11,15, IL2I, IL18.

The method of embodiment 4, comprising

a. The said expansion uses umbilical cord blood as sources of cells

b. The said expansion uses alternate feeder cells modified to express different molecules

c. The said expansions uses any or all of the following cytokines to help proliferation of NK cells: 11.2, IL6, IL7, 11. 12. 11. 1 5. 11.2 1 , IL18

The method of embodiment 4, comprising:

a. The said expansion uses healthy donors as sources of cells.

b. The said expansion uses overlapping peptides spanning the tumor antigens as sources of antigen presented by dendritic ceils in the first stimulation.

c. The said expansion uses overlapping peptides spanning the tumor antigens as sources of antigen presented by PHA blasts or alternative antigen presenting ceils in subsequent stimulations.

d. The said expansion uses alternate feeder cells modified to express different molecules. e. The said expansions uses any or all of the following cytokines to help proliferation of T cells: IL2, IL6, IL7, IL12, IL15, IL21, IL18.

The method of embodiment 4, comprising:

a. The said expansion uses patient samples as sources of cells.

b. The said expansion uses overlapping peptides spanning the tumor antigens as sources of antigen presented by dendritic cells in the first stimulation.

c. The said expansion uses overlapping peptides spanning the tumor antigens as sources of antigen presented by PHA blasts or alternative antigen presenting cells in subsequent stimulations.

d. The said expansion uses alternate feeder cells modified to express different molecules.

e. The said expansions uses any or all of the following cytokines to help proliferation of T cells: IL2, IL6, IL7, IL12, IL15, IL21, IL18.

The method of embodiment 4, comprising:

a. The said expansion uses umbilical cord blood as sources of cells.

b. The said expansion uses overlapping peptides spanning the tumor antigens as sources of antigen presented by dendritic cells in the first stimulation.

c. The said expansion uses overlapping peptides spanning the tumor antigens as sources of antigen presented by PHA blasts or alternative antigen presenting cells in subsequent stimulations.

d. The said expansion uses alternate feeder cells modified to express different molecules.

e. The said expansions uses any or all of the following cytokines to help proliferation of T cells: IL2, IL6, IL7, IL12, IL15, IL21, IL18.

The method of embodiment 4, where the cells as described in embodiment 5 are modified to decrease or eliminate expression of the TGFBRII proteins.

a. The said modification includes RNA interference introduced into the cell by retroviral vectors.

b. The said modification includes RNA interference introduced into the cell by ientiviral vectors.

c. The said modification includes RNA interference introduced into the cell by transfection.

d. The said modification includes CRISPR introduced into the cell by retroviral vectors.

e. The said modification includes CRISPR introduced into the cell by lentiviral vectors.

f. The said modification includes CRISPR introduced into the cell by transfection.

g. The said modification includes TALENS introduced into the cell by retroviral vectors.

h. The said modification includes TALENS introduced into the cell by lentiviral vectors.

i. The said modification includes TALENS introduced into the cell by transfection.

j . The said modification includes genes for expressing TGFBRII-specific intrabodies introduced into the cell by retroviral vectors,

k. The said modification includes genes for expressing TGFBRII-specific intrabodies introduced into the cell by lentiviral vectors.

1. The said modification includes genes for expressing TGFBRII-specific intrabodies introduced into the cell by transfection.

The method of embodiment 4, where the cells as desciibed in embodiment 6 are modified similar to embodiment 15 but targeting alternative receptors.

The method of embodiment 4, where the cells as described in embodiment 7 are modified to secrete antibodies directed to CD45.

a. The said modification includes transgenes introduced into the cell by retroviral vectors,

b. The said modification includes transgenes introduced into the cell by lentiviral vectors.

c. The said modification includes transgenes introduced into the cell by transfection.

d. Genes for expressing TGFBRII-specific intrabodies introduced into the cell by transfection.

The method of embodiment 4, where the cells as described in embodiment 8 are modified similar to embodiment 17 but targeting alternative receptors.

The sequence of the construct described in embodiment 5 as well as sequences representing the following elements: TGFbRII binding domain (without the wildtype transmembrane and intracellular signaling domains), IgG4 hinge, GPI, 2A cleavage site, selection marker (truncated CD 19) OR TGFbRII binding domain (without the wildtype transmembrane and intracellular signaling domains), IgG4 hinge, FGF receptor external domain, 2A cleavage site, selection marker (truncated CD 19).

The sequence of the construct described in embodiment 6.

a. Sequences representing the following elements: IL10R binding domain (without the wildtype transmembrane and intracellular signaling domains), IgG4 hinge, GPI, 2 A cleavage site, selection marker (truncated CD 19) or ILIOR binding domain (without the wildtype transmembrane and intracellular signaling domains), IgG4 hinge, FGF receptor external domain, 2A cleavage site, selection marker (truncated CD 19).

b. Sequences representing the following elements: IL6R binding domain (without the wildtype transmembrane and intracellular signaling domains), IgG4 hinge, GPI, 2 A cleavage site, selection marker (truncated CD 19) or IL6R binding domain (without the wildtype transmembrane and intracellular signaling domains), IgG4 hinge, FGF receptor external domain, 2A cleavage site, selection marker (truncated CD 19).

The sequence of the construct described in embodiment 7 as well as sequences representing the following elements:

a. antibody to CD47 (comprising the VH and VL segments) attached to a constant region derived from IgG3, 2A cleavage site, selection marker (e.g. truncated CD 19); or

b. antibody to CD47 (comprising the VH and VL segments) attached to a constant region derived from a GASDALIE mutant of IgGl, 2 A cleavage site, selection marker (e.g. truncated CD 19); or

c. antibody to CD47 (comprising the VH and VL segments) attached to a linker and anti CD 16, 2A cleavage site, selection marker (e.g. truncated CD 19); or d. antibody to CD47 (comprising the VH and VL segments) attached to a linker and anti CD45, 2 A cleavage site, selection marker (e.g. truncated CD 19); or e. SIRPa binding domain attached to a constant region derived from IgG3, 2A cleavage site, selection marker (e.g. truncated CD 19); or

f SIRPa binding domain attached to a constant region derived from a GASDALIE mutant of IgGl, 2A cleavage site, selection marker (e.g. truncated CD 19); or

g. SIRPa binding domain attached to a linker and anti CD 16, 2 A cleavage site, selection marker (e.g. truncated CD 19); or

h. SIRPa binding domain attached to a linker and anti CD45, 2A cleavage site, selection marker (e.g. truncated CD 19).

The sequence of the construct described in embodiment 8 where instead of CD47, the target is GD2:

a. antibody to GD2 (comprising the VH and VL segments) attached to a constant region derived from IgG3, 2A cleavage site, selection marker (e.g. truncated CD 19); or

b. antibody to GD2 (comprising the VH and VL segments) attached to a constant region derived from a GASDALEE mutant of IgGl, 2A cleavage site, selection marker (e.g. truncated CD 19); or

c. antibody to GD2 (comprising the VH and VL segments) attached to a linker and anti CD 16, 2A cleavage site, selection marker (e.g. truncated CD 19); or d. antibody to GD2 (comprising the VH and VL segments) attached to a linker and anti CD45, 2 A cleavage site, selection marker (e.g. truncated CD 19); or e. The sequence of the construct described in embodiment 8 where instead of CD47, the target is TGFB:

f. TGFBRII binding domain attached to a constant region derived from IgG3, 2 A cleavage site, selection marker (e.g. truncated CD19); or

g. TGFBRII binding domain attached to a constant region derived from a GASDALIE mutant of IgGl , 2A cleavage site, selection marker (e.g. truncated CD 19); or

h. TGFBRII binding domain attached to a linker and anti CD 16, 2 A cleavage site, selection marker (e.g. truncated CD 19); or

i. TGFBRII binding domain attached to a linker and anti CD45, 2A cleavage site, selection marker (e.g. truncated CD 19).

The sequence of the constmct described in embodiment 8 where instead of CD47, the target is Her2:

a. antibody to Her2 (comprising the VH and VL segments) attached to a constant region derived from IgG3, 2A cleavage site, selection marker (e.g. truncated CD 19); or b. antibody to Her2 (comprising the VH and VL segments) attached to a constant region derived from a GASDALIE mutant of IgGl, 2 A cleavage site, selection marker (e.g. truncated CD 19), or

c. antibody to Her2 (comprising the VH and VL segments) attached to a linker and anti CD 16, 2A cleavage site, selection marker (e.g. truncated CD 19); or d. antibody to Her2 (comprising the VH and VL segments) attached to a linker and anti CD45, 2 A cleavage site, selection marker (e.g. truncated CD 19).

21. The sequence of the construct described in embodiment 8 where instead of CD47, the target is VEGFR:

a. antibody to CD47 (comprising the VH and VL segments) attached to a constant region derived from IgG3, 2 A cleavage site, selection marker (e.g. truncated CD 19), or

b. antibody to CD47 (comprising the VH and VL segments) attached to a constant region derived from a GASDALIE mutant of IgGl, 2 A cleavage site, selection marker (e.g. truncated CD 19); or

c. antibody to CD47 (comprising the VH and VL segments) attached to a linker and anti CD 16, 2 A cleavage site, selection marker (e.g. tamcated CD 19); or d. antibody to CD47 (comprising the VH and VL segments) attached to a linker and anti CD45, 2A cleavage site, selection marker (e.g. truncated CD 19); or e. VEGF receptor binding domain attached to a constant region derived from IgG3, 2A cleavage site, selection marker (e.g. truncated CD 19); or f. VEGF receptor domain attached to a constant region derived from a GASDALIE mutant of IgGl , 2A cleavage site, selection marker (e.g. truncated CD 19); or

g. VEGF receptor domain attached to a linker and anti CD 16, 2 A cleavage site, selection marker (e.g. tamcated CD 19); or

h. VEGF receptor domain attached to a linker and anti CD45, 2A cleavage site, selection marker (e.g. truncated CD 19).

Immune Effector Cells Expressing an Antibody or Antibody Binding fragment

[00200] In some embodiments, the present invention provides a population of antibody or antibody binding fragment-expressing cells.

[00201] In some embodiments, the population of antibody or antibody binding fragment-expressing comprises a ceil that expresses one or more antibody or antibody fragments described herein. In some embodiments, the population of antibody or antibody binding fragment-expressing cells comprises a mixture of cells expressing different antibodies,

[00202] For example, in one embodiment, the population of antibody or antibody binding fragment-expressing cells can include a first cell expressing an antibody or antibody binding fragment having an antigen binding domain to a tumor antigen described herein, e.g., CD 47 and/or CD45, and a second cell expressing an antibody or antibody binding fragment having a different antigen binding domain, e.g., an antigen binding domain to a different tumor antigen described herein, e.g., an antigen binding domain to a tumor antigen described herein that differs from the tumor antigen bound by the antigen binding domain of the antibody or antibody binding fragment expressed by the first cell.

[00203] As another example, the population of antibody or antibody binding fragment-expressing cells can include a first cell expressing an antibody or antibody binding fragment that includes an antigen binding domain to a tumor antigen described herein, and a second cell expressing an antibody or antibody binding fragment that includes an antigen binding domain to a target other than a tumor antigen as described herein. In one embodiment, the population of antibody or antibody binding fragment-expressing ceils includes, e.g., a first cell expressing an antibody or antibody binding fragment that includes a primary intracellular signaling domain, and a second cell expressing an antibody or antibody binding fragment that includes a secondary signaling domain. Either one or both of the antibody or antibody binding fragment expressing ceils can have a truncated PGK promoter, e.g., as described herein, operably linked to the nucleic acid encoding the antibody or antibody binding fragment. In some embodiments, the second antibody or antibody binding fragment targets a cancer cell expressing any one of plurality of cancer antigens or fragments of antigens of Table 1.

[00204] In some embodiments, the present disclosure provides a population of cells wherein at least one cell in the population expresses an antibody or antibody binding fragment having an antigen binding domain to a tumor antigen described herein, and the same cell and/or a second cell expressing another agent, e.g., an agent which enhances the activity of the antibody or antibody binding fragment-expressing cell. The antibody or antibody binding fragment expressing ceils can have a truncated PGK promoter, e.g., as described herein, operably linked to the nucleic acid encoding the antibody or antibody binding fragment. [00205] In some embodiments, the agent can be an agent which inhibits the effects of cytokines known to stimulate the humoral immune response. Inhibitory molecules, e.g., IL-6, can, in some embodiments, decrease the ability of the antibody or antibody binding fragment- expressing cell to mount an immune effector response. Examples of inhibitory molecules include IL-6, IL-10, PD-1, PD-Ll, PD-L2, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIRl , CD 160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVE (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR (e.g., TGFRbeta). In one embodiment, the agent which inhibits the effects of cytokines known to stimulate the humoral immune response comprises a first polypeptide that binds any one or combination of the aforementioned peptides but fails to have an amino acid sequence capable of propagating the signal caused by the aforementioned peptides binding to its natural receptor.

Co-expression of Antibody or Antibody Binding Fragment with Other Molecules or Agents Co-Expression of A Second Antibody Or Antibody Binding Fragment

[00206] In one aspect, the antibody or antibody binding fragment-expressing cell described herein can further comprise a second antibody or antibody binding fragment, e.g., a second antibody or antibody binding fragment that includes a different antigen binding domain, e.g., to the same target (CD47) or a different target (e.g., CD45 or any of the antigens listed in Table 1.

Co-expression of Antibody Or Antibody Binding Fragment with a Chemokine Receptor [00207] In some embodiments, the antibody or antibody binding fragment- expressing cell described herein further comprises a chemokine receptor molecule. Transgenic expression of chemokine receptors CXCR1 or CXCR2 in T cells enhances trafficking to CXCLl-secreting solid tumors including melanoma and neuroblastoma (Craddock et al, J Immunother. 2010 Oct; 33(8):780-8 and Kershaw et a!,, Hum Gene Ther. 2002 Nov 1; 13(16): 1971-80). Thus, without wishing to be bound by theory, it is believed that chemokine receptors expressed in antibody or antibody binding fragment-expressing ceils that recognize chemokines secreted by tumors, e.g., solid tumors, can improve homing of the antibody or antibody binding fragment-expressing cell to the tumor, facilitate the infiltration of the antibody or antibody binding fragment-expressing cell to the tumor, and enhances antitumor efficacy of the antibody or antibody binding fragment-expressing cell. The chemokine receptor molecule can comprise a naturally occurring or recombinant chemokine receptor or a chemokine-binding fragment thereof. A chemokine receptor molecule suitable for expression in an antibody or antibody binding fragment-expressing cell described herein include a CXC chemokine receptor (e.g., CXCRl, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7), a CC chemokine receptor (e.g., CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11), a CX3C chemokine receptor (e.g., CX3CR1), a XC chemokine receptor (e.g., XCRl ), or a chemokine-binding fragment thereof. In one embodiment, the chemokine receptor molecule to be expressed with a antibody or antibody binding fragment described herein is selected based on the chemokine(s) secreted by the tumor. In one embodiment, the antibody or antibody binding fragment-expressing cell described herein further comprises, e.g., expresses, a CXCRl receptor or a CXCR2 receptor. In an embodiment, the antibody or antibody binding fragment described herein and the chemokine receptor molecule are on the same vector or are on two different vectors. In some embodiments, any cell disclosed herein is treated in hypoxic conditions for a time period sufficient to stimulate expression of CXCRl and/or CXCR2 in the cell. In embodiments where the antibody or antibody binding fragment described herein and the chemokine receptor molecule are on the same vector, the antibody or antibody binding fragment and the chemokine receptor molecule are each under control of two different promoters or are under the control of the same promoter. In such cases, the vectors can be transfected/ transduced into one or more cells for expression of the disclosed antibody or antibody binding fragment and CXCRl and/or CXCR2.

[00208] In some embodiments, the composition or pharmaceutical composition disclosed herein comprises one or a plurality of cells that express CXCRl and/or CXCR2 at levels that are higher than zero expression or basal level of expression in an unmodified T ceil or unmodified K cell.

[00209] In some embodiments, the cells of the present disclosure are exposed to conditions sufficient to induce upregulation and protein expression of one or a plurality of one or more of the following receptors:

Table 3.

HTFGFIVPLF LFCYGFTLRTLFKAHMGQ HRAMRVIFAV VL1FLLCWLPYNLVLLADTLMRTQVIQESCERR IGRALDA TEILGFLHSCL PIIYAFIGQNFRHGFLKILAMHGLVSKEFLAR H RVTSYTSSSVNVSS L

CXCR2 MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPE 36

SLElN YFVVtrYALVFLLSLLGNSLVMLVILYSRVGRSVTDV

YLLNLALADLLFALTLPIWAASKVNGWIFGTFLCKVVSLLKE

V FYSGILLLACISVDRYLAIVHATRTLTQKRYLVKFICLSIW

GLSLLLALPVLLFRRTVYSSNVSPACYEDMGN TANWRMLL

RILPQSFGFIVPLLIMLFCYGFTLRTLF AHMGQ HRAMRVIF

AWLIFLLCWLPYNLVLLADTL

MRTQVIQETCERil HIDRALDATEILGlLHSCL PLIYAFIGQ KFRHGLLK IL AIFIGLISKD SLPKD SRP S F VGS S S GHT S TTL

CCR4 MNPTDIADTTLDESIYSNYYLYESIPKPCTKEGEKAFGELFLPP 37

LYSLW GLLGNSVVVLVLFKYKRLRSMTDVYLL LAISD LLF SLPFWGYYAADQWVFGLGLCKMISWMYLVGFYSGIF FVMLMSLDRYLAIVHA SLRARTLTYGVITSLATWSVAVFA

SLPGFLFSTCYTER HTYCKTKYSLNSTTWKVXSSLEINILGL VIPLGIMLFCYSMIIRTLQHCKNEKK^'NKAVKMIFAVVVLFLG FWTPYNIVLFLETLVELEVLQDCTFERYLDYAIQATETLAFV HCCLNPIIYFFLGEKFR YILQLFKTCRGLFVLCQYCGLLQIY S ADTPS S S YTQSTMDHDLHDAL

CD47 MWPLVAALLLGSACCGSAQLLF TKSVEFTFC DTVV PC 38

FVTNMEAQNTTEVYVKWKFKG-RDIY1TDGALNKSTVPTDFS

SAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREG

ETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYR

SGGMDEKTIALLVAGLVITVTVIVGAILFVPGEYSLKNATGLG

LIVTSTGIL1LLHYY STAIGLTSFVIAILVIOVIAYILAVVGLS

LCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVE

CD45 MTMYLWLKLLAFGFAFLDTEVFVTGQSPTPSPTDAYLNASE 39

TTTLSPSGSAVISTTTIATTPSKPTCDEKYAMTVDYLYNKET LFTA LNVNEN\ CGNNTCTNNEVHNLTEC ASVSISHN

SCTAPDKTLILDVPPGVEKFQI HOC TQ VEK ADTTI

CLKWK IETFTCDTQNITYRFQCG vlIFDNKEIKLE LEPEH

EYKCDSEILYNNH FTNASKIIKTDFGSPGEPOIIFCRSEAAHQ

GVITWNPPORSFFf FTLCYIKETEKDCLNl_^D NLI YDLQNL

KPY KY^SLHAYTIAKVQRNGSAAMCHFTTKSAPPSQVWN

MTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVR

NESHKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFIIJ-fflS

TSY SKALIAFLAFLIIVTSIALLVVLYKIYDLHKKRSC LDE

OQELVERDDEKQLMNVEPIHADILLETYKRKIADEGRLFLAE

FQSIPR\'TSKFPI EARKPFNQNKNRY T3ILPYDYNR.VELSEI

NGDAGSNYINASYIDGFKEPRKYIAAQGPRDETVDDFWRMI

WEQKATVIYMYTRCEEGNRNKCAEYWPSMEEGTRAFGDV

VVKINQHKRCPDYIIQKLNR-^ KKEKATGREV HIQFTSWPD

HG EDPFiLLLKLRRRVNAFSNFFSGPIVVHCSAGVGRTGTY

IGIDAMLEGLEAENKVDVYGYVV LRRQRCT MVQYEAQYI

LIHQ AL VE YNQFGETE VNL SELHP YLHNMKKRDPP SEP SPLE

AEFQRLPSYRSWRTQHIG QFENKSKNRNSNYIPYDYNRVP

LKHELEMSKESEHD SDES SDDD SD SEEP SKYINASFIMS YWK PEV1V1IAAQGPLKETIGDFWQMIFQRKVKVIVMLTELKHGDQ

EICAQYWGEGKQTYGDIEVDLKDTDKSSTYTLRVFELRHSK

RKDSRTVYQYQYTNWSVEQLPAEPKELISMIQVVKQKLPQ

NSSEGNKHHKSTPLLIHCRDGSQQTGIFCALL LLESAETEEV

VDIFQWKALRKARPGMVSTFEQYQFLYDVIASTYPAQNGQ

VKK NHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLPEAK

EQAEGSEPTSGTEGPEHSVNGPASPALNQGS

Sources of Cells

[00210] Prior to expansion and genetic modification or other modification, a source of cells, e.g., T ceils or natural killer (NK) cells, can be obtained from a subject. Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.

[00211] In certain aspects of the present disclosure, immune effector ceils, e.g., T ceils, can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In one aspect, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, the cells collected by apheresis may be washed to remove the plasma fraction and, optionally, to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by- methods known to those in the art, such as by using a semi- automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media. It is recognized that the methods of the application can utilize culture media conditions comprising 5% or less, for example 2%, human AB serum, and employ known culture media conditions and compositions, for example those described in Smith et al., "Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement" Clinical & Trans lat onal Immunology (2015) 4, e31; doi: 10.1038/cti.20^'14.31. In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centritugation through a PERCOLLTM gradient or by counterflow centrifugal elutriation.

[00212] The methods described herein can include, e.g., selection of a specific subpopulation of immune effector cells, e.g., T cells, that are a T regulatory cell-depleted population, CD25+ depleted cells, using, e.g., a negative selection technique, e.g., described herein. In some embodiments, the population of T regulatory depleted ceils contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells,

[00213] In one embodiment, T regulatory cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody, or fragment thereof, or a CD25-binding iigand, IL-2. In one embodiment, the anti-CD25 antibody, or fragment thereof, or CD25 - binding ligand is conjugated to a substrate, e.g., a bead, or is otherwise coated on a substrate, e.g., a bead. In one embodiment, the anti-CD25 antibody, or fragment thereof, is conjugated to a substrate as described herein,

[00214] In one embodiment, the T regulatory cells, e.g., CD25+ T ceils, are removed from the population using CD25 depletion reagent from Miltenyi™. In one embodiment, the ratio of cells to CD25 depletion reagent is le7 cells to 20 uL, or le7 cells to 15 uL, or le7 cells to 10 uL, or le7 ceils to 5 uL, or ie7 cells to 2.5 uL, or le7 cells to 1.25 uL. In one embodiment, e.g., for T regulator}' cells, e.g., CD25+ depletion, greater than 500 million ceils/mi is used. In a further aspect, a concentration of cells of 600, 700, 800, or 900 million cells/ml is used,

[00215] In one embodiment, the population of immune effector cells to be depleted includes about 6 x 109 CD25+ T cells. In other aspects, the population of immune effector ceils to be depleted include about 1 x 109 to Ix 1010 CD25+ T cell, and any integer value in between. In one embodiment, the resulting population T regulatory depleted cells has 2 x 109 T regulatory cells, e.g., CD25+ ceils, or less (e.g., 1 x 109, 5 x 108 , 1 x 108, 5 x 107, 1 x 107, or less CD25+ cells).

[00216] In one embodiment, the T regulatory cells, e.g., CD25+ cells, are removed from the population using the CiiniMAC system with a depletion tubing set, such as, e.g., tubing 162-01.

Fusion Polypeptides

[00217] The disclosure provides chimeric or fusion polypeptides. As used herein, a

"chimeric protein" or "fusion protein" comprises all or part (preferably biologically active) of a polypeptide or compound of the invention operably linked to a heterologous amino acid sequence (i.e., an amino acid sequence other than the compound of the invention). Within the fusion protein, the term "operably linked" is intended to indicate that the polypeptide of the invention and the heterologous polypeptide are fused in frame to each other. The heterologous polypeptide can be fused to the N terminus or C terminus of the polypeptide of the invention.

[00218] One useful fusion protein is a GST fusion protein in which the polypeptide of the invention is fused to the C terminus of GST sequences. Such fusion proteins can facilitate the purification of a recombinant polypeptide of the invention,

[00219] In another embodiment, the fusion protein contains a heterologous signal sequence at its N terminus. For example, the native signal sequence of a polypeptide of the invention can be removed and replaced with a signal sequence from another protein. For example, the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992). Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolia, Calif). In yet another example, useful prokaryotic heterologous signal sequences include the phoA secretory signal (Sambrook et al ., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, N. J.).

[00220] In yet another embodiment, the fusion protein is an immunoglobulin fusion protein in which all or part of a polypeptide of the invention is fused to sequences derived from a member of the immunoglobulin protein family. Nucleic acid sequences encoding the immunoglobulin fusion proteins of the invention can be incorporated into cells in pharmaceutically effective amounts and administered to a subject in pharmaceutical compositions to inhibit an interaction between a ligand (soluble or membrane bound) and a protein on the surface of a cell (receptor), to thereby suppress signal transduction in vivo. The immunoglobulin fusion protein can be used to affect the bioavailability of a cognate ligand of a polypeptide of the invention. Inhibition of iigand/receptor interaction may be useful therapeutically, both for treating hyperproliferative and cancer and for modulating (e.g.. promoting or inhibiting) cell survival.

[00221] Chimeric and fusion proteins of the invention can be produced by standard recombinant DNA techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel et a!,, supra). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in frame to the polypeptide of the invention.

[00222] In some embodiments, the invention provides a nucleic acid sequence that encodes polypeptides that are mutant forms of TGFp Receptor II, IL-6R, IL-IOR. double- stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the ΤίίΡβ Receptor I and or TGFp Receptor II, IL-6 Receptor, IL-10 receptor or other genes responsible for immunosuppression of cancer. The dsRNA comprises at least two sequences that, are complementary to each other. The dsRNA comprises a sense strand comprising a first sequence and an an ti sense strand comprising a second sequence. The antisense strand comprises a nucleotide sequence which is substantially complementary to at least part of an mRNA encoding TGFp Receptor II, IL-6R, IL-10R or genes responsible for cancer immunosuppression, and the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length. The dsRNA, upon contacting with a cell expressing the TGF|3 Receptor II, IL-6R, IL-10R or genes responsible for cancer immunosuppression, inhibits the expression of mRNAs transcribed by said genes by at least 40%, 30%, 25%, 20%, 15%, 10%, or 5%.

[00223] In another embodiment, the invention provides a cell or a vector comprising one of the dsRNAs or cDNAs of the invention or functional fragments thereof. The cell is generally a mammalian cell, such as a human cell.

[00224] In another embodiment, the invention provides a method for inhibiting signaling of TGFp Receptor II, EL-6R, EL-10R in a cancer cell in a subject by exposing the subject to a cell expressing a modified TGFp Receptor II, IL-6R, IL-10R, capable of binding TGFp, IL-6, IL-10 respectively . [00225] In another embodiment, the invention provides vectors for inhibiting the signaling of the TGFp Receptor II, IL-6R, IL-IOR in a cancer cell in a subject by administering a cell comprising a nucleic acid sequence, said nucleic acid sequence comprising a regulatory sequence operably linked to a nucleotide sequence that encodes a polypeptide that is a TGFp Receptor II, IL-6R, and/or IL-IOR polypeptide that is free of all or substantially all of its signaling domain, n another embodiment, the invention provides vectors for inhibiting the signaling of the TGFp Receptor II, IL-6R, IL-10R in a cancer cell in a subject by administering a cell comprising a nucleic acid sequence, said nucleic acid sequence comprising a regulatory sequence operably linked to a nucleotide sequence that encodes a polypeptide that is a TGF Receptor II, EL-6R, and/or IL-IOR polypeptide that is deficient in signaling capability. The recombinant receptors act as "decoy" receptors such that the receptors can bind to native forms of their ligands but fail to propagate a signal, thereby reducing the natural effects of TGFp, IL-6, and/or IL-10 in a subject diagnosed with or suspected of having a hyperproliferative disorder such as cancer.

Pharmaceu tical Compositions

[00226] The disclosure relates to pharmaceutical compositions comprising: (i) one or a therapeutically effective amount of one or a plurality of cells disclosed herein and (ii) a pharmaceutically acceptable carrier. The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art- accepted practices. The dose for a therapeutic, e.g., an antibody, e.g., CAMPATH, for example, may be, e.g., in the range 1 to about 100 mg for an adult patient, e.g., administered daily for a period between 1 and 30 days. A suitable daily dose is 1 to 10 mg per day although in some instances larger doses of up to 40 mg per day may be used (described in U.S. Patent No. 6,120,766).

[00227] In one embodiment, a nucleic acid encoding the antibody or antibody binding fragment is introduced into cells, e.g., T ceils or NK cells, e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of antibody or antibody binding fragment-expressing cells, e.g., antibody or antibody binding fragment T cells of the invention, and one or more subsequent administrations of the antibody or antibody binding fragment-expressing cells, e.g., antibody or antibody binding fragment T ceils of the invention, wherein the one or more subsequent administrations are administered less than about 15 days, e.g., 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, more than one administration of the antibody or antibody binding fragment-expressing cells, e.g., antibody or antibody binding fragment T cells of the invention are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the antibody or antibody binding fragment-expressing cells, e.g., antibody or antibody binding fragment T cells of the invention are administered per week. In one embodiment, the subject (e.g., human subject) receives more than one administration of the antibody or antibody binding fragment-expressing cells, e.g., antibody or antibody binding fragment T ceils per week (e.g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no antibody or antibody binding fragment-expressing cells, e.g., antibody or antibody binding fragment T ceils administrations, and then one or more additional administration of the antibody or antibody binding fragment-expressing ceils, e.g., antibody or antibody binding fragment T cells (e.g., more than one administration of the antibody or antibody binding fragment-expressing cells, e.g., antibody or antibody binding fragment T cells per week) is administered to the subject. In another embodiment, the subject (e.g., human subject) receives more than one cycle of antibody or antibody binding fragment- expressing cells, e.g., antibody or antibody binding fragment T cells, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the antibody or antibody binding fragment-expressing cells, e.g., antibody or antibody binding fragment T ceils are administered every other day for 3 administrations per week. In one embodiment, the antibody or antibody binding fragment-expressing cells, e.g., antibody or antibody binding fragment T cells of the invention are administered for at least two, three, four, five, six, seven, eight or more weeks.

[00228] In some embodiments, subjects may be adult subjects (i.e., 18 years of age and older). In certain embodiments, subjects may be between I and 30 years of age. In some embodiments, the subjects are 16 years of age or older. In certain embodiments, the subjects are between 16 and 30 years of age. In some embodiments, the subjects are child subjects (i.e., between J and 18 years of age). In one aspect, antibody or antibody binding fragment- expressing cells, e.g., antibody or antibody binding fragment T cells are generated using lentiviral viral vectors, such as lentivirus. antibody or antibody binding fragment-expressing ceils, e.g., ceils generated that way will have stable antibody or antibody binding fragment expression.

[00229] In one aspect, antibody or antibody binding fragment-expressing cel ls, e.g., antibody or antibody binding fragment expressing cells are generated using a viral vector such as a gammaretroviral vector, e.g., a gammaretroviral vector described herein. Cells generated using these vectors can have stable antibody or antibody binding fragment expression,

[00230] According to one embodiment, the vector is an MLV retroviral vector. The sequence of an empty MLV retroviral vector is shown below:

TGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAA

GGCATGGAAAAATACATAACTGAGAATAGAAAAGTTCAGATCAAGGTCAGGAAC

AGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTG

CCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAAACAGGAT

ATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCA

GATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGC

CCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTT

CTCGCTTCTGTTCGCGCGCTTATGCTCCCCGAGCTCAATAAAAGAGCCCACAACC

CCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTG^

CCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAG

GGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTC

GTCCGGGATCGGGAGACCCCTGCCCAGGGACCACCGACCCACCACCGGGAGGTA

AGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTT

TATGCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGT

GGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCCTGGGAGACGTCCCAGG

GACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCCGATCGTTTA

GGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGAGACG

AGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCG

AAGCCGCGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGA

CTGTGTTTCTGTATTTGTCTGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCC

TTAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGT

CGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAAC

CTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAG

GTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACACCCAGACCAGGTGGGGT

ACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCCTT

TGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTG

AAC V !X T i m i C L C VTO

CTAGGCGCCCCCATATGGCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTG TAAACTTCCCTGACCCTGACATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGC

TCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCA

GCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGC

GACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAA

GGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCG

CAGCTTGGATACACGCCGCCCACGTGATCGATCCGGATTAGTCCAATTTGTTAAA

GACAGGATATCAGTGGTCCAGGCTCTAGTTTTGACTCAACAATATCACCAGCTGA

AGCCTATAGAGTACGAGCCATAGATAAAATAAAAGATTTTATTTAGTCTCCAGAA

AAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAAC

GCCATTTTGCAAGGCATGGAAAAATACATAACTGAGAATAGAGAAGTTCAGATC

AAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGT

AAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGG

GCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAAC

AGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATG

TTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCA

ATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAG

AGCCCACAACCCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGG

TACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGT

TCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCAC

ACATGCAGCATGTATCAAAATTAATTTGGTTTTTTTTCTTAAGTATTTACATTAAA

TGGCCATAGTACTTAAAGTTACATTGGCTTCCTTGAAATAAACATGGAGTATTCA

GAATGTGTCATAAATATTTCTAATTTTAAGATAGTATCTCCATTGGCTTTCTACTT

TTTCTTTTATTTTTTTTTGTCCTCTGTCTTCCATTTGTTGTTGTTGTTGTTTGTTTGT

TTGTTTGTTGGTTGGTTGGTTAATTTTTTTTTAAAGATCCTACACTATAGTTCAAG

CTAGACTATTAGCTACTCTGTAACCCAGGGTGACCTTGAAGTCATGGGTAGCCTG

CTGTTTTAGCCTTCCCACATCTAAGATTACAGGTATGAGCTATCATTTTTGGTATA

TTGATTGATTGATTGATTGATGTGTGTGTGTGTGATTGTGTTTGTGTGTGTGACTG

TGAAAATGTGTGTATGGGTGTGTGTGAATGTGTGTATGTATGTGTGTGTGTGAGT

GTGTGTGTGTGTGTGTGCATGTGTGTGTGTGTGACTGTGTCTATGTGTATGACTGT

GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGTGAAAAAAT

ATTCTATGGTAGTGAGAGCCAACGCTCCGGCTCAGGTGTCAGGTTGGTTTTTGAG

ACAGAGTCTTTCACTTAGCTTGGAATTCACTGGCCGTCGTTTTACAACGTCGTGA

CTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTC GCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTG

CGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGT

GCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGC

ATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGC

TTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGC

ATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGATGACGAAAGGGCCT

CGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGT

CAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAA

ATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAAT

AATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCC

CTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAG

TAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATC

TCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGAT

GAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGG

CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACT

CACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCA

GTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGAT

CGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAAC

TCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCG

TGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGG

CGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGAT

AAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTG

ATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTC^

C AGATGGT AAGCCC TC CCGT ATC GT AGTT ATCT AC ACGAC GGGGAGTC AGGC AA

CTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGC

ATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTT

CATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCA

AAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGAT

CAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAA

AAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCT

TTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTA

GTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACC

TCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTT ACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGA

ACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTG

AGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAG

GCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA

GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC

TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAA

ACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCAC

ATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGA

GTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAG

CGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCC

GATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA

GCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACAC

TTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACA

CAGGAAACAGCTATGACCATGATTACGCCAAGCTTTGCTCTTAGGAGTTTCCTAA

TACATCCCAAACTCAAATATATAAAGCATTTGACTTGTTCTATGCCCTAGGGGGC

GGGGGGAAGCTAAGCCAGCTTTTTTTAACATTTAAAATGTTAATTCCATTTTAAA

TGCACAGATGTTTTTATTTCATAAGGGTTTCAATGTGCATGAATGCTGCAATATTC

CTGTTACCAAAGCTAGTATAAATAAAAATAGATAAACGTGGAAATTACTTAGAG

TTTCTGTCATTAACGTTTCCTTCCTCAGTTGACAACATAAATGCGCTGCTGAGCAA

GCCAGTTTGCATCTGTCAGGATCAATTTCCCATTATGCCAGTCATATTAATTACTA

GTCAATTAGTTGATTTTTATTTTTGACATATACATGTGAA (SEQ ID NO: 40)

[00231] In some embodiments, antibody or antibody binding fragment-expressing cells, e.g., cells transiently express antibody or antibody binding fragment vectors for about 4,

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction. Transient expression of antibody or antibody binding fragments can be effected by DNA or RNA vector delivery. In one aspect, the antibody or antibody binding fragment DNA or RNA is transduced into the cell, e.g., NK cell or T cell, by electroporation. A potential issue that can arise in patients being treated using transiently expressing antibody or antibody binding fragment T cells (particularly with murine scFv bearing antibody or antibody binding fragment) is anaphylaxis after multiple treatments.

[00232] Without being bound by this theory, it is believed that such an anaphylactic response might be caused by a patient developing humoral anti-cell response, i.e., anti-cell antibodies having an anti-IgE isotype. It is thought that a patient's antibody producing cells undergo a class switch from IgG isotype (that does not cause anaphylaxis) to IgE isotype when there is a ten to fourteen day break in exposure to antigen. If a patient is at high risk of generating an anti-cell antibody response during the course of transient antibody or antibody binding fragment expressing cellular therapy (such as those generated by RNA transductions), cell infusion breaks should not last more than about ten to about fourteen days.

[00233] The preparations of the present invention may be given orally, parenterally, topically, or rectaily. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc., administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. IV administration is preferred.

[00234] The terms "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection, and infusion.

[00235] The terms "systemic administration," "administered systemically,"

"peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

[00236] These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, intravenously, rectaily, intravaginally, intraparenterally, intracisternally and topically, as by liquid dosage forms, ointments or drops, including buccally and sublingually.

[00237] The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. [00238] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[00239] In general, a suitable daily dose of a compound of the inventi on will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day, more preferably from about 0.01 to about 50 mg per kg per day, and still more preferably from about 1.0 to about 100 mg per kg per day. An effective amount is that amount treats a hyperproliferative disorder.

[00240] If desired, the effective daily dose of the modified cells may be administered as two, three, four, five, six or more sub -doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to admini ster the compound as a pharmaceutical composition. In some embodiments, the dose of modified cells disclosed herein is from about 1 x 10^b cells to about 1 x 10²⁰ cells.

[00241] In some embodiments, the cells of the disclsoure comprise one, two, three or more exogenous nucleic acid sequences or nucleic acid molecules or genetic constructs that express one or a combination of the following sequences or amino acid sequence that comprises at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the CDR sequences, heavy chains and/or light chains in the examples. For instance, If the cells express an antibody to CD73, some embodiments of the disclosure comprise one, two, three or more exogenous nucleic acid sequences or nucleic acid molecules or genetic constructs that express one or a combination of the following sequences or amino acid sequence that comprises at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 1 14, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 1 17, SEQ ID NO: 1 17, SEQ ID NO: 1 18, SEQ ID NO: 119, SEQ ID NO: 120, and SEQ ID NO: 121. Embodiments also therefore include the exogenous nucleic acids and cells that express the same of the antibody fragments listed in the Examples as well as those that comprise at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to those sequence identifiers.

[00242] In some embodiments, the cells of the disclsoure comprise one, two, three or more exogenous nucleic acid sequences or nucleic acid molecules or genetic constructs that express one or a combination of the following sequences or amino acid sequence that comprises at least 70%, 80%, 85%, 90%_s, 95%, 96%, 97%, 98% or 99% sequence identity to the following:

Example Antibody Effector to CD47 - 1

MARPLCTLLLLMATLAGALADIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGIYrYLHWYQ

QSKPGKAPKLLIYKVSYRFGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPRTFGQ

GGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCTVSGYTFTiNrYYVFWVRQARGQRLEWIGD

INPVNGDTNFNE FKNRVTISADKSISTAYLQWSSLKASDTA^WYCARGGYTMDYWGQGA^

T GPSVFPI PCSRSTSGGTAALGCIA'KDYFPEPVWS SGALTSGVFITFPAVLQSSGLYSL

SSVVWPSSSLGTQTYTCNVNFiKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPR

CPEPKSCDTTPPCPRCPEPKSCDTPPPCPRCPAPEL iiGPSVFLFPPKPKDTLMISRTPEVTCVV

VDVS iEDPEVQFKWYVDGVEVH AKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCK

VSNKALPAPIE^SKT GQPREPQVYTLPPSREFJVITXNQVSLTCLV GFYPSDIAVEWESSG

QPENlSTYNTTPPMLDSDGSFFLYSKLWDKSRWQQGNIFSCSVMHEALFiNRFTQKSLSLSPGK

DYKDDDDKDYKDDDDKLMDYKDDDDKDYKDDDDKLMDYKDDDDKATNFSLLKQAGDV

EENPGPMPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESP

LKPFLKLSLGLPGLGlHMl l'LAlWLFlFTslVSQQMGGFYLCQPGlH^SEKAVVQPG TVNVEGSG

ELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLN

QSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSW W^IPKGPKSLLSLELKDDRPARD^r VVM

ETGLLLPRATAQDAGKYYCHRGNLTMSFFILEITARPVLWHWLLRTGGW VSAVTLAYLIFC

LCSLVGTLFiLQRALVLRRKRKRMTDPTRRF

(SEQ ID NO:41)

Example Antibody Effector to CD47-2

MARPLCTTLLLLMATLAGALAEEELQVIQPDKSVLVAAGETATLRCTATSL1PVGPIQWFRGA

GPGRELIY QKEG-FIFPRVTWSDLTKR NMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFK

SGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNV

DPVGESVSYS1HSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTA1NLSETIRVPPTLEVTQQ

PVRAENQV1NVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKJ_⁾GTY

AFiRDDVKLTCQVEHDGQPAVSKSFIDLKVSAHPKEQGSNTAAENTGSNERNrYASTKGPSVF

PLAPCSRSTSGGTAAIXrCIA'KDYFPEPVWS SGALTSGVT-lTFPAVLQSSGd-,YSLSSVVWP

SSSI jTQTY CN\ iKPSNTKVDKR ^rELKTPLGDT^lTCPRCPEPKSCDTPPPCPR.CPEPKSC

DTPi ⁱCPRCPEPKSCDTPPPCPRCPAPELLGGPSVF

PEVQFKWYVDGVEVHNAKTTCPREEQYNSTTR SVLTVI^QDWl.NGKEYKCKVSN ALPA IEKTISKTKGQPRE QVYTLPPSllEEMTKNQVSLTCLVKGFY^PSDL· ΈWESSGQ ENNYTslT

TPPMLDSDGSFFLYSKLWDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKDYKDDDDK

DYKDDDDKLMDYKDDDDKDYKDDDDKLMDYKDDDDKATNFSLLKQAGDVEENPGPMPP

PRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSL

GI . i l .G U I \ I R 1 Λ f \\ U · f I Λ V S Q Q N f G G i ·^' \ I .CQPGP SHK AW yPGWTYWhGSGhl .HiW \ YS

DLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLT IAPGSTLWLSCGVPPDSVSRGPLSWTHViiPKGPKSLLSLELKDDRPARDMWVMETGLLLPR

ATAQDAGKYYCFD GNLTIVISFFiLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGIL

HLQRALVLRRKRKRMTDPTRRF (SEQ I D NO:42) Example Antibody Effector to glypican 1

MARPLCTLLLLMATLAGALADIQMTQSPASLSASVGETVT1TCRASGNVH YLAWYQQKQG

KSPQLLVYTA TLADGVPSRFSGSGSGTQYSLKINSLQPEDFGTYYCQHFWSNPWTFG JKJTT

LEKGGGGSGGGGSQIQLVQSGPELK PGETVKISCKASGYATTDYSM WVKQAPG

MG-\VTNTE GEFT^TDDFKGRFAFSLETSASTAFLQINNLRNEDT FCARFfYDYGGFPYWG

QGTLV SAASTKGPSVFPLAPCSRSTSG jTAALGCLVKDYFPEPVTVS\\nSiSGALTSGVFrrFP

AVLQSSGLYSLSSVA''TVPSSSLGTQlYTCNVNHKPSNTKVDKRVELKTPLGDTraTCPRCPEP

KSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLM1

SRTTEVTCV DVSHEDPEVQFKWYVDGVEVHNAKTXPREEQYNSTFRVVSVLTVLHQDW

LNGKEY CKVSNKALPAPIE^SKTXGQPREPQWTXPPSREEMT NQVSLTCLVKGFYPSD

TAVEWESSGQPENNYNTTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALFINRFTQ

KSLSLSPGKDYKDDDDKDYKDDDDKLMDYKDDDDKDYKDDDDKLMDYKDDDDKATNFS

LLKQAGDVEENPGPMPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQ

QLTWSRESPLKPFLKLSLGLPGLGliiMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPG

WT ^fNVEGSGELFRWNVSDLGGLG(X}LKNRSSEGPSSPSGKLMSPKLY\AVAKDRPEiWEGEP

PCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDR

PARDNWA^METGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVIAVI-WLLRTGGWKVS

AVTLAYLJFCLCSLVGILHLQRALVLRRKRKRMTDPTRRF (SEQ U.) NO : ·! .·■)

Example Antibody Effector to CD73

MARPLCTLLLLMATLAGALAEIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQKPGQ

APi LLlYDASNRATGlPAllFSGSGPGTDFTLTISSLEPEDFAVYYCQQRSNWHLTFGGGTKVEl GGGGSGGGGSQVQLVESGGG QPGRSLRLSCATSGFTFSNYGMHWVRQAPGKGLEWVA VILYDGSNKYYPDSVKGRFTISRDNSKNTLYLQ^i SLRAEDTAVYYCARGGSSWYPDSFDI WGQGTMVTV S SA STKGPS VFPLAPC SRSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGV HTFPAVLQSSGLYSLSSVV!VPSSSLG

CPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKD

TLMIS TPEVTCVVVDVSHEDPEVQFK )G ΈVHNAKTKPί EEQYNSTF A^rS XTVLH

QDWLNGKEYKCK ¾NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL ^rKGF

YPSDIAVEWE-SSGQPENNYNTTPPMLDSDGSFFLYSKLWDKSRWQQGNIFSCSVMHEALFiN

RFTQKSLSLSPGKDYKDDDDKDYKDDDDKLMDYKDDDDKDYKDDDDKLMDYKDDDDKA

TNFSLLKQAGD^ENPGPMPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSD

GPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMG JFYLCQPGPPSEKAW

QPGWTVNVEGSGELFRVV^VSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVVVAKDl l'EIVVE

GEPPCLPPRDSLNQSLSQDLTTS4APGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELK

DDRPAPJ⁾MWVMETGLLLPRATAQDAGKYYCFfl¾GNLTMSFHLEITARPVLWTr\VLLRTGGW

KVSAVTLAYLIFCLCSLVGILFiLQRALVLRRKRKRMTDPTRRF (SEQ ID NO:44)

Example Antibody Effector to TGF-beta

MAP PLCTLLLLMATLAGALAΉPPFΓ QKSVN DMIVTDNNGAVKFPQLCKFCDVRFSTCDN

QKSCMSNCSITSICEKPQEVCVAVWI KNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEK

KKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQASTKGPSVFPLAPCSRSTSGGTAAL

GCLVKDYFPEPVWSW SGALTSG\TiT PAVLQSSGLYSLSSVVWPSSSLGTQTYTCNVNH PSNTKVDKRVELKTPLGDTTOTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCD

TPPPCPRCPAPEIJ..GGPS TLFPPKPKDTIAlISRTPF^TCVVVDVSHEDPEVQFK ^rDGVE ^r

FiNAKTKPREEQYNSTFRVVSVLWLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREP

QVYTLPPSllEEMTKNQVSLTCLVKGFYPSDL· ΈWESSGQ EN^Y^'NTT PMLDSDGSFFLYS

KLWDKSRWQQGNIFSCSVMHEALI-INRFTQKSLSLSPGKDYKDDDDKDYKDDDDKLMDYK

DDDDKDYKDDDDKLMDYKDDDD AT FSLLKQAGDVEENPGPMPPPRLLFFLLFLTPMEV

RPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHNIRPLAPW^'

LFIF TVSQQMGGFYLCQPGPPSEKAWQPGWT\TWEGSGELFR\\nWSDLGGLGCGLKNRSSE

GPSSPSGkLMSPKLYYWAKDPvPEPW^"EGEPPCLPPPJ⁾SLNQSLSQDLTAlAPGSTLWLSCGVPP

DSVSRGPLSWTHV iPKGPKSLLSLELKDDRPARDMV ^METGLLLPPvATAQDAGKYYCHRG NLTMSFHLEITARPVIA\T:TVVLLRTGGWKVSAVTLAYLIFCLCSIA'OIIiILQRAIA'^'LRRKRKR MTDPTRRF (SEQ ID NO:45)

Example Antibody Effector to IL6

MARPLCTLLLLMATLAGALALAPRRCPAQEVARGVLTSLPGDSVTLTCPGVEPEDNATV⁷FIW VLR PAAGSHPSRWAGMGRRLLLRSVQLHDSGNYSCYRAGRPAGTVHLLVDVPPEEPQLSC FRKSPLSNWCEWGPRSTPSLTTKAVLLVRKFQNSPAEDFQEPCQYSQESQKFSCQLAVPEGD SSFYIVSMCVASSVGSKFSKTQTFQGCGILQPDPPANITYTAVARNPRWLSVTWQDPHSWNSS

FYRLRFELRYRAERSKTFT N KDLQI-fflCVTHDAWSGLRiWVQLRAQEEFGQGEWSEWS

PEAMGTPWTESRSPPAENEVSTPMQALTTNKDDD ILFRDSA ATSLPVQDSSSVPLPASTKG

PSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALT GVHTFPAVLQSSGLYSLSSV

VWPSSSIXJTQTYTCNV HKPSNTKVDKRVELKTPIXJDTTHTCPRCPEPKSCDTPPPCPRCPE

P SCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

SFIEDPEVQFK\ VDGVEVH AKTKPREEQYT\^TSTFRV ¾VLTVLHQDWLNGKEY C VSNK

ALPAPIEKTISKTKGQPREPQVY LPPSREEMTK QVSI CLVKGFYPSDIAVEWESSGQPE N

YNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKDYKDD

DDKDYKDDDDKLMDYKDDDDKDYKDDDDKLMDYKDDDDKATNFSLLKQAGDVEENPGP

MPPPRLLFFLLFLTPMEVRPEEPL KVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLK

LSLGLPGLGIHMRPLArVVLFIF VSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWN

VSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWA DRPEIWEGEPPCLPPRDSLNQSLSQDL

TMAPGSTIAVLSCG TPDSVSRGPLSWTIWHPKGPKSLLSLELKDDRPARD i METGLLLP

RATAQDAGKYYCHRGNLTMSFFiLEITARPVL\\¾WLLRTGrGWKVSAVTLAYLIFCLCSLVGI

LHLQRALVLRRKRKRMTDPTRRF (SEQ ID NO: 46)

Example Antibody Effector to IL10

1ARPLCTLLLL 1ATLAGALAHGTELPSPPSVWFEAEFFHHJLHWTP1PNQSESTCYEVALLRY

GIESWNSISNCSQTLSYDLTAVTLDLYHSNGYRARVRAVDGSRHSNWTV^TNTRFSVDEVTLT

VGSV ILEIHNGFILGKIQLPRP MAPANDTYESIFSHFREYEIAIRKVP^

SLLTSGEVGEFCVQVKPSVASRSNKG IWSKEECJSLTRQYFTVTNAST GPSVFPLAPCSRST

SGGTAAmCL\¾DYFPEP\OVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

TCNV^NHKPSNTKVDKRVELKTPLGDTraTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP

EPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVStEEDPEVQFKWY

VDGVE\¾NA TKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKT

KGQPREPQ\nnrLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSD

GSFFLYSKLTVDKSRWQQGNIFSCSVMHEALFiNRFTQKSLSLSPGKDYKDDDDKDYKDDDD

KLMDYKDDDDKDYKDDDDKLMDYKDDDDKATNFSLLKQAGDVEENPGPMPPPRLLFFLLF

LTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIH

MRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCG

LKNRSSEGPSSPSGKLMSPKLY AKDRPErVVEGEPPCLPPRDSLNQSLSQDLTMAPGSTLW

LSCG PDSVSRGPLS\VTTWHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGK

YYCHRGNLTMSFHLErTARPVLWHWLL^^

RKRKRMTDPTRRF (SEQ ID NO: 47)

Genetic constructs of the disclosure can comprise any nucleic acid seqeince that encodes one of the above mentioned antibody frgaments (e.g. effector) molecules or a sequence that comprises at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the above-identified antibody fragments. In some genetic constructs, the genetic constructs comprise any one or plurality of signal secretory sequences placed before or after the anibody effector molecules, cytoine receptors or other coding sequences disclosed herein including variants thereof. In some embodiments, the secretory sequence is a Cystatin S signal peptide MARPLCTLLLLMATLAGALA (SEQ ID NO: 48) or fuinctionai fragment thereof.

Flag Tag

DYKDDDDKDYKDDDDKLMDYKDDDDKDYKDDDDKLMDYKDDDDK

(SEQ ID NO:49)

GASDALIE

ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYEPEPVTVSWNSGALTSGVi-ITFPAVL

QSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCP

EPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPP

K PK D Ti . iSRT EY i C Y V YDYSi j EDfn -.YQFK lV Y V DGYHY i ΙΝ ΛΚ i K R KEQY N S^'IT^' R

VVSVLTVLHQDWLNGi EYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEM

TKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKS

RWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO:50)

P2A

ATNFSLLKQAGDVEENPGP (SEQ ID NO:51) Methods

The disclosure relates to methods of treating a subject diagnosed with or suspected of having hyperproliferative disorder or in need thereof comprising administering to the subject one or a plurality of cells disclosed herein. In some embodiments, the one or plurality of cells are T cells or NK comprising one or more exogenous nucleic acid molecules, each nucleic acid molecule comprising at least one expressible sequence operably linked to a regulatory sequence. In some embodiments, the disclosure relates to a method in which the one or more exogenous nucleic acid sequences comprises an expressible sequence comprising a nucleic acid sequence encoding IL-6Ralpha, IL-lORalpha, both IL-6Ralpha, IL-lORalpha, and/or any nucleic acid sequence that encodes an amino acid sequenceat least 70% sequence identity to SEQ ID NO: 3, SEQ ID NO:46, SEQ ID NO.47. In some embodiments, the disclosure relates to a method of treating cancer in a subject by administering to the subject in need of the treatment a pharmaceutical composition comprising one or a plurality of cells disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the cells disclosed herein comprises at least one exogenous vector or genetic construct comprising a nucleic acid sequence that compri ses at least 70% sequence identity to (or a nucleic acid sequence that encodes an amino acid sequence comprising at least 70% sequence identity to any of) SEQ ID NO: 33, 34, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 and/or 51.

The disclosure relates to methods of mnufacturing modified antigen presenting cells, T cell s, and NK cells isolating mononuclear cells from te sample of a subject, stimulating the ceils with one or more cytokines for a time sufficient to induce an NK cell or T cell population, further isolating NK cells or T cells from the cells, culturing the cells in viteo for about two to about three days, transducing or transfecting the cells with one or more vectors or genetic constructs disclosed herein, stunmlating the cells with mature antigen presenting ceils and further stimulating the culture with a cytokine cocktail comprising IL6, IL7, IL12, IL15, IL 18, and/or IL21. In some embodiments, the method is performed in about 23 to about 26 days. In some embodiments, the method comprises culturing one or more modified cells for about 23 days, 24 days, 25 days or 26 days.

EXAMPLES

Example 1

Generation of Gesie Modified Natural Killer Cells or T eells

[00243] For expanding NK cells the base protocol described below will be modified to allow for activation of HIF-1 or NFkB, at different time points (initiation, co-culture with feeder cells, expansion) of the protocol.

1. Mononuclear cells are obtained from blood source using density gradient centrifugation.

2. Cells are grown in the presence of cytokines (IL.2, and additional cytokines meant to increase chemokine expression).

3. The NK cell fraction in the population is selected out using magnetic separation.

4. Cells are activated by co-culture with cytokines, or irradiated feeder cells.

5. Cells are fed every 2-3 days with fresh media and cytokines, and restimulated with irradiated feeder cells weekly.

6. At different time points following stimulation, cells are transduced with retroviral vectors containing the transgene of interest.

[00244] For expanding T cells the base protocol described below will be modified to al low for activation of HIF-1 or NFkB, at different time points (initiation, co-culture with feeder cells, expansion) of the protocol. 1. Antigen presenting cell s are first selected from mononuclear cells using adherence or magnetic selection.

2. Antigen presenting cells are exposed to the antigen of interest (in the form of pepmixes, whole protein, or mRNA) and are then activated/matured using cytokines.

3. Antigen presenting cell s are used to prime and stimulate T cells (derived from autologous mononuclear cells), in the presence of various cytokines.

4. Cells are fed every 2-3 days with fresh media and cytokines, and restimulated with irradiated feeder cells and/or antigen presenting cells/weekly.

5. At different time points following stimulation, cells are transduced with retroviral vectors containing the transgene of interest.

[00245] Further in-depth protocols are described below:

Table 4: Generation of T cells

Feed DC or PHA blast; Cytokine cocktail of GMC-SF & IL4

Transduction PHA blast feed with IL2

19-21

Retroviral or Lenti viral transduction may use fresh or previously frozen producer line supernatant

Mature DC or Feed PHA blast Cytokine cocktail of GMC-SF, TNFa, PGE2, Η . ί β,

21-23 IL6, IL4, LPS

May include pepmix addition

APC;T Cell ratio between 1 : 10 and 1 :4 OR. between 1 : 1 :2 and 1 : 1 :4 of PHA blast:! Cell: Artificial ARC Cytokine cocktail may include IL6, IL7, EL12, IL15,

23-26 T Cell Stimulation 3

1L18, IL21

Third stimulation may use the following APCs: DC, PHAb, artificial APC^"

Cell Separation Protocol - Day 1

CD14 Positive Selection; MACs Separation Beads

• Count PBMC

• Centrifuge at 400 G for 5 minutes, RT

® Aspirate media leaving -200 uL

• Add 20 ul CD 14 microbead and 80 ul MACS buffer per 20e6 cells

® Incubate at room temp 20 minutes

® Agitate pellet e e ' 5 mins

• Wash pellet in 10 raL MACS buffer

® Centrifuge at 400 G for 5 minutes, RT

• Prewet an LS column with 3 ml MACS buffer, discard effluent

• Resuspend pellet in 3 ml MACS buffer, pass through column into CD14

nil . conical

® Wash column twice with 4.5 ml MACS buffer

• Replace catch tube with 15 mL conical labeled CD 14+

• Apply 5 mL Macs buffer to LS column, push through with plunger

Plating DCs - CD 14+ Fraction

® Centrifuge at 400 G for 5 minutes, RT

® Count Cells, Aspirate supernatant

• Resuspend le6 cells/mL of DC media

• Add GMC-SF/mL and IL-4/ml

• Plate l e6 cells/well in a 24 well plate surrounded by PBS

Freezing CD 14- Cells

• Thaw Freeze Media, transfer to ice, use cold

® Centrifuge at 400 G for 5 minutes, RT

® Count, Aspirate Supernatant

• Resuspend at max 25e6 cells/mL FM

® Store in -80°C freezer until transfer to LN2 until use in T ceil stimulation Monocyte Adherence

• Count PBMC

® Resuspend PBMCs at 10e6 cells/mi

® Plate PBMCs in 6-well plate at 10e6 cells/well (1 nil cells + 1 ml DC media)

® Incubate for 2 hours at 37°C - minimum incubation of J hour, maximum incubation of 16 hours (overnight)

® Remove 2 ml media/well; collect in 50 ml conical tube

• Wash wells with 1 ml IX PBS, add to 50 ml conical tube

® Add J ml DC media to wells, don't allow to dry

• Add an additional 1 ml DC media + GMC-SF and EL 4 cytokine cocktail/well (2 ml/well)

• Spin 50 ml conical with Non-Adherent Cells (NACs) in DC Media/PBS

® Store in -8Q°C freezer until transfer to LN2 until use in T cell stimulation 1

® Remove 1 mL of Media/well into 50 ml conical tube

® Centrifuge at 400 G for 5 minutes, RT

® Aspirate supernatant

® Resuspend any cells in DC Media

® Add cytokines cocktail to DC media

• Add 1 mL/well of DC Media/Cytokme Cocktail to DCs

PC Maturation - Day 4-6

*DC Maturation may include 4-6 hour incubation of cells with pepmix before maturation cytokine cocktail addition

® Remove 1 mL media

• Add optimal volume of pepmix

® Incubate 4-6 hours at 37°C

DC Maturation Cytokine Cocktail

® Prepare new DC Media/cytokine cocktail

o Cytokine cocktail may include GMC-SF, TNFa, PGE2, IL p, IL6, IL4, LPS ® Add 1 mL DC Media/cytokine cocktail to each well

T Cell Stimulation - Day 7

*T Ceil Stimulation I may include 30 minute incubation of DCs with pepmix before addition to T cells

DCs

® Harvest Dendritic cells by gently scraping bottom of well with transfer pipette

• Transfer cells to 50 mL tube

® Count cells

• Centrifuge at 400 G for 5 minutes, RT

® Add 1-2 ui corresponding pepmix to pellet and incubate -15-30 mins

® Resuspend DCs in le5 cells/mL CTL Media

• Plate 1 mL/well in a 24 well plate

NACs/T cells

• Thaw NACs or PBMC * Resuspend in CTL media

® Count Cells

* Centrifuge at 400 G for 5 minutes, RT

* Resuspend T Cells in le6 cells/mL CTL Media

® Add cytokine cocktail

o Cytokine cocktail may include IL6, IL7, IL12, IL15

* Plate 1 n L NACs+cytokines/well on top of DCs (le6 NACs/weli)

* Final ratio DC:NAC/T Cell should be le5 : l e6 cells per well

Initiation of Antigen Presenting Cells for T Cell Stimulation 2 - Day 9-11

*T Cell Stimulation 2 may use dendritic cells or PHA blast as antigen presentin ratio between 1 : 10 and 1 :4 APC:T Cell

Dendritic Cell Selection - see Cell Selection Protocol Day 1

Initiation of PHA blast

* Start PHA lines from PMBCs of same BC

* Thaw PBMC vial

* Count cells

* Centrifuge at 400 G for 5 minutes, RT

® Resuspend in CTL media at leo cell s/ml

* Add the following cytokines: PHA and IL2

* Plate PBMC at le6 cells/ml or 1 mi/well

® PHA blast will need to be fed even- other day

• If coating plates 24 hrs before transduction, store at 4°C. If coating plates 4-6 hours before transduction, incubate at 37°C.

• Use non tissue culture-treated 24 well plates

• Coat ~4 wells with retronectin per well of Stimulation J T Cells

® Add 300uL of 50 ug/mL retronectin per well

• Seal plates with Parafilm

Transduction - Day 10-11

*Protocol may include transduction after T Cell Stimulation I or T Cell Stimulation 2

• Acquire 4.0mL of retroviral vector per le6 cells

• Transport retroviral vector on ice

• Aspirate retronectin from each well

® Wash with 0.5 mL complete media

• Add 2.0 mL of retroviral supernatant to each well

• Centrifuge plates at 2000G for 2 hours at 30°C

During last 30 minutes of centrifugation:

® Harvest CTL to be transduced in 50 mL tube - leave at least 1 well as untransduced control

® Resuspend at 0.25e6 CTL/mL in CTL media

• Add JU2

® Aspirate retroviral supernatant from plates * Plate 2 mL of cell suspension per retroneetin-coated well for a total of 0.5e6 CTL/well

® Centrifuge plates at 1000G for 5 mins at room temp

*See DC Feed Protocol Day 3

PHA blast Feed

® Remove 1 mL of CTL Media

• Centrifuge at 400 G for 5 minutes, RT

® Resuspend cells in CTL media

• Add IL2

® Plate 1 mL CTL Media/ii.2 cocktail per well of PHA blast

DC Maturation or PHA blast Feed - Day 14-16

*See DC Maturation Protocol Day 4-6

*See PHA Blast Feed Protocol Day 11-13

T Cell Stimulation 2 - Day 16-18

*T Ceil Stimulation 1 may include 30 minute incubation of APC with pepmix before addition to T cells

APC

® Harvest APCs by gently scraping bottom of well with transfer pipette

• Transfer cells to 50 mL tube

• Count cells

® Centrifuge at 400 G for 5 minutes, RT

• Add 1-2 ui corresponding pepmix to pellet and incubate -15-30 mins

® Resuspend APC in correct concentration of CTL Media

® Plate 1 mL/well in a 24 well plate

NACs/T cells

• Thaw NACs or PBMC

• Resuspend in CTL media

® Count Cells

• Centrifuge at 400 G for 5 minutes, RT

® Resuspend T Cells in le6 cells/mL CTL Media

® Add cytokine cocktail

o Cytokine cocktail may include IL6, IL7, IL12, IL15

• Plate 1 mL NACs+cytokines/well on top of APCs (le6 NACs/weli)

® Final ratio APC:NAC/T Cell may be between 1 : 10 and 1 :4

Preparation of Retronectin Coatee! Plate -- Day 16-18

• If coating plates 24 hrs before transduction, store at 4°C. If coating plates 4-6 hours before transduction, incubate at 37°C.

• Use non tissue culture-treated 24 well plates

® Coat ~4 wells with retronectin per well of Stimulation 1 T Cells

• Add 300uL of 50 ug/mL retronectin per well • Seal plates with Parafilm

Initiation of Antigen Presenting Cells for T Cell Stimulation 2 - Day 16-18

*T Cell Stimulation 3 may use dendritic ceils AND/OR PHA blast AND/OR artificial APCs as antigen presenting ceils at a ratio between 1 : 10 and 1 :4 APC :T Cell OR 1 : 1 :4 T CelhPHA blast: Artif cial APC

*See Dendritic Cell Selection Protocol Day 1

*See PHA Blast Initiation Protocol Day 9-1 1

Transduction - Day 19-21

*Protocoi may include transduction after T Cell Stimulation 1 or T Cell Stimulation 2

• Acquire 4.0mL of retroviral vector per le6 cells

® Transport retroviral vector on ice

• Aspirate retronectin from each well

• Wash with 0.5 mL complete media

® Add 2,0 mL of retroviral supernatant to each well

• Centrifuge plates at 2000G for 2 hours at 30°C

During last 30 minutes of centrifugation:

• Harvest CTL to be transduced in 50 mL tube - leave at least 1 well as untransduced control

• Resuspend at 0.25e6 CTL/mL in CTL media

• Add IL2

• Aspirate retroviral supernatant from plates

® Plate 2 mL of cell suspension per retronectin-coated well for a total of 0.5e6 CTL/well

® Centrifuge plates at 1000G for 5 mins at room temp

DC or PHA blast Feed - Day 19-21

*See DC Feed Protocol Day 3

*See PHA Blast Feed Protocol Day 1 1 -13

*See DC Maturation Protocol Day 4-6

*See PHA Blast Feed Protocol Day 1 1 -13

T Cell Stimulation 3 - Day 23-26

*T Cell Stimulation I may include 30 minute incubation of APC with pepmix before addition to T cells

*T Cell Stimulation 3 may use dendritic cells AND/OR PHA blast AND/OR artificial APCs as antigen presenting ceils at a ratio between 1 : 10 and 1 :4 APC:T Cell OR 1 : 1 :4 T Cell:PHA blast: Artificial APC

APC

® Harvest APCs by gently scraping bottom of well with transfer pipette

• Transfer cells to 50 mL tube * Count cells

® Centrifuge at 400 G for 5 minutes, RT

• Add 1-2 ul corresponding pepmix to pellet and incubate - 5-30 mins

* Resuspend APC in correct concentration of CTL Media

® Plate 1 mL/well in a 24 well plate

NACs/T cells

• Thaw NACs or PBMC

® Resuspend in CTL medi a

* Count Cells

• Centrifuge at 400 G for 5 minutes, RT

• Resuspend T Cells in le6 cells/mL CTL Media

® Add cytokine cocktail

o Cytokine cocktail may include IL6, IL7, IL 2, IL15

® Plate 1 mL NACs+cytokines/well on top of APCs (le6 NACs/well)

* Final ratio APC:NAC/T Cell may be between 1 : 10 and 1 :4

Example 2. Engineering Antibody Effectors

For purposes of this patent, "antibody effectors" refer to the novel transgenes described herein that are designed to recognize the tumor and/or components of its microenvironment and disrupt this ecosystem (through elimination of cell elements, neutralization of suppressive cytokines, or neutralization of tumor/TME-derived exosomes). The disruption is meant to encourage infiltration of the tumor site by dendritic cells and other professional antigen presenting cells, phagocytes, and innate immune cell effectors like K cells. The general parameters that are used will be based on the guide published by Welch et al. (20 1. Methods Enzymol 498, 43-66). Genes corresponding to these proteins will be obtained by onlne backtranslation software programs, including GeneDesigner (https://www.atum.bio/resources/genedesigner), Biolnformatics' Sequence Manipulation Suite Reverse Tranlsate Tool (http://www.bioinformatics.org/sms2/rev_trans.html), or Protein to DNA Reverse Translation from BioPHP

(http://www.biophp.org/minitools/protein_to_dna/demo.php) We will design antibody effectors according to the methods described below.

Components of antibody effectors

Promoter and UTR sequences For T cells, we will use the 5' untranslated region of the protein tyrosine kinase lck.^y This sequence is derived from GenBank, Accession M36881 (https://www.ncbi.nlm , nih.gov/nuccore/M36881.1 ).

The sequence for this UTR is CGCCTGGACC ATGTGAATGG

GGCCAGAGGG CTCCCGGGCT GGGCAGGGAC C (SEQ ID NO:52) Alternative 5' UTR will be derived from the most common cell-specific genes for T cells, described by Palmer et al. 2006.¹ These are 5' UTR sequences from CD3G, LEFl, TCF7, CD3D, MAL, CD2, NRFA2, SNPH, TCRIM, CD28, LAT, ITGA6, FBLN5, BCL11B, IL7R, SHFM1 , FTS, ZAP70, TCR Vb, APBA2, CCND2, ITK, FYB, TNFRSF25, TXK, NK4, WNT10B, LEPROTL1, KLRK 1, NGFRAP1, PLEKHK1, BUB IB, INPP4B, GALT, PLXDC1, APEG1, ΪΝΡΡ4Α, CD6, DPP4, TRERF1, AAKl, EVER1, MLLT3, A XA1, PCSK5, ADA, MEN1, PRKCQ, MARLINl, CAMK4, IL6ST, NELL2, FLT3LG, STAT4, P2RY5, PDE9A, PAG, GFI1, FLJ20152, RORA, GAT A3, NPTXR, CDR2, TACTILE, SYNE2, UPP1 , ATP1A1 , SH2D1 A FHIT, LTBP4, RNF144, RAB43, TNFSF8, CD 5, DUSP16, BAG3, KIAA0748, BIN2, RGS 19IPL RBMS 1 , ITPKB, LDHA, PKM2, ID2, MAPKAPK5, ARL7, LCP2, MATN2, CISH, CCL5, SATB1, DOCK9, SEN54L, ACVR2B, GZMK, AQP3L, MAST4, DNASE1 L3, HSPA1 L, EPLIN, PCYT2, GBP2, CD3E, WINS I, CDC 14 A,. CD3Z, TNIK, HOXB2, THEDCI, PTGER2, RGS10, FYN, ACTN1, EEIG1, LDHB, APOE, GBP I, ITM2A, CDC25B, MYBL1, PRKCA, TACC3, MANIC 1, VIPRl, SYTl , DNAJB1, RUNX2, TIAM1, RASGRPI, PRKCI, PXN, WWP1, PDE4D, KLRBl, SEMA4D, GPSM3, TARP, RARRES3, KLRG1, SLC35D2, EDG4, TOB l , NPDC1, GABARAPLl, TM4SFI4, SORL1, MPP7, GIMAP4, GIMAP2, SOCS3, IL6R, LPIN2, IL18R1, PIM1, IFTTMl , PIK3R1, DRLM, SLC03A1, SELPLG, TNFATP3, OPTN, S100A8, DUSP2, SPOCK2, CTSW, DRILL TSGA14, or LRIG1

Lck and alternative UTR's will be appended immediately before the start codon (AUG) of the antibody effector constructs.

We will also be using the following constitutive promoters for expressing our antibody effector gene constructs. These will be appended immediately before the start codon (AUG) of the antibody effector constructs.

- in many cases, we will rely on expression from the promoter embedded the 5' LTR of retroviral or lentiviral vectors.

The MLV LTR (Lund et al„ Brief report. Arch Virol 144, 2207-2212 ( 1999)) is derived from the Moloney murine leukemia vims, a commonly used retroviral gene backbone. This is one of the main vectors we will use for this work. The sequence for this is:

GCGCCAGTCC TCCGATAGAC TGAGTCGCCC GCiGTACCCGT GTATCCAATA AAGCCTTTTG CTGTTGCATC CGAATCGTGG TCTCGCTGAT CCTTGGGAGG GTCTCCTCAG AGTGATTGAC TGCCCAGCCT GGGGGTCTTT CATT (SEQ ID NO:53). We will be using all, or majority, or a segment of this promoter, and will be appending it before the start codon AUG.

® The HIV LTR is derived from HIV1 , which forms the basis for the lentiviral vectors. This is an alternative viral vector we will be using, and the sequence for this is:

TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATAT

CCTTGATCTGTGGATCTACCACACACAAGGCTACTTCC

CTGATTAGCAGAACTACACACCAGGGCCAGGGATCAG

ATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTA

CCAGTTGAGCCAGAGAAGTTAGAAGAAGCCAACAAA

GGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGC

ATGGAATGGATGACCCGGAGAGAGAAGTGTTAGAGT

GGAGGTTTGACAGCCGCCTAGCATTTCATCACATGGC

CCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGA

CATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACT

TTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGT

GGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTT

TTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGA

GCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGC

TTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGT AGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGA

GATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCT AGCA (SEQ ID NO:54). We will be using all, or majority, or a segment of this promoter, and will be appending it before the start codon AUG.

CMV. The IE! CMV promoter is one of the most commonly used constitutive promoters. The sequence for which is:

AGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCG CCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAA (SEQ ID N():55) (Juven-Gershon, T., et al., Nat Methods 3, 917-922,

doi: 10.1038/nmeth937 (2006))

Adenovirus major late core promoter, another strongly constitutive promoter is the adenovirus major late core promoter. The sequence for this is: GGGGCTATAAAAGGGGGTGGGGGCGCGTTCGTCCTCACTCTC TTCCGCATCGCTGTCTGCGAGGGCCAGCTGTTGGGGTGA¹² (SEQ ID NO:56)

NFicP promoter. Upon activation, T cells transcribe genes controlled by the ΝΡ β promoter. One example for T_RA_ENNE involves cells secreting their antibody effectors only when stimulated (i.e. recognizing the tumor at the disease site). The use of ΝΡκβ allows temporal coordination between T cells recognizing their targets and release of antibody effectors. The sequence for NFKP-responsive promoter region is:

CGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGACTAGTGGGA ATTTCCGGGAATTTCCGGGAATTTCCGGGAATTTCCAGATCTAGAC TCTAGAGGGTATATAATCKJAAGCTCGAATTCC GCTTGGC TTCCG GTACTGTTGGTAAAAAGCTTGGCATTCCGGTACTGTTGGTAAAGCC

ACC (SEQ ID NO:57). We will be using all, or majority, or a segment of this promoter, and will be appending it before the start codon AUG.

HIFlot promoter (Minet et al ., Biochem Biophys Res Commun 261 , 534- 540, 1999). The hypoxic conditions in the tumor niicroenvironment encourage expression of genes like HTFlcc which One example for T_RA_ENNE involves ceils secreting their antibody effectors only within the tumor

niicroenvironment. The use of HTFlcx allows spatial coordination between T cells in the microenvironment and release of antibody effectors. The sequence tbr HIFla promoter is: GTCGCTCGCC ATTGGATCTC GAGGAACCCG CCTCCACCTC AGGTGAGGCG GGCTTGCGGG AGCGCGCGCC.

GGCCTGGGCA GGCGAGCGGG CGCGCTCCCG CCCCCTCTCC CCTCCCCGCCG CGCCCGAGCG CGCCTCCGCC CTTGCCCGCC CCCTGACGCT GCCTC AGCTC CTCAGTGCAC (SEQ ID O:58), We will be using all, or majority, or a segment of this promoter, and will be appending it before the start codon AUG.

If there are difficulties in transcript expression, as measured by rtPCR of gene modified cells, the super core promoters (SCP1 and SCP2) can be used, described by Juven-Gershon et al. (Nat Methods 3, 917-922. 2006). ® The sequence for SCP1 is:

GTACTTATATAAGGGGGTGGGGGCGCGTTCGTCCTCAGTCG C GATCGAACACTCGAGCCGAGCAGACGTGCCTACGGACCG

(SEQ ID NC):59)

® The sequence for SCP2 is:

AGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGAT CGC

CTGGAGACGTCGAGCCGAGTGGTTGTGCCTCCATAGAA

(SEQ ID NO:60)

Sigsial peptides

Membrane signal peptides

® Immunoglobulin Heavy¹⁴ - the amino acid sequence is

MDWTWR iXLAVTPGAHP (SEQ ID NO:61), and the reverse- translated nucleic acid sequence takes the form

ATGGAYTGGACNTGGMGNGTNTTYTGYYTNYTNGCNGTN ACNCCNGGNGCNCAYCCN (SEQ ID NO:62), which may be codon-optimized to allow expression of the amino acid sequence above for maximal translation in human T cells.

Immunoglobulin Heavy from Chimeric Antigen Receptor constructs¹⁵ - the amino acid sequence is

MEFGLSWLFLVAILKGVQC (SEQ ID NO:63), and the reverse- translated nucleic acid sequence is ATGG-A(:^TT(X}ACTTTCTTGGTTGTTTTT(K}T(XK AATTCT

GAAGGGTGTCCAGTGT (SEQ ID NO:64), which may be codon- optimized to allow expression of the amino acid sequence above for maximal translation in human T ceils.

Secretory signal peptides

® Cystatin S - the amino acid sequence is

MARPLCTLLLLMATLAGALA (SEQ ID NO:65), and the reverse translated nucleic acid sequence takes the form

ATGGCNMGNCCNYTNTGYACNYTNYTNYTNYTNATGGCN ACNYTNGCNGGNGCNYTNGCN (SEQ ID NO:66), which may be codon-optimized to allow expression of the amino acid sequence above for maximal translation in human T cells,

* IL2 - the sequence i s M YRMQLLSCI ALSL AL VTNS (SEQ ID

NO:67), and a possible reverse translation of the sequence is ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCT TGCACTTGTCACAAACAGT (SEQ ID NO:68), which may be codon-optimized to allow expression of the amino acid sequence above for maximal translation in human T cells (an alternative, optimal IL2 signal sequence is MRRMQLLLLIALSLALVTNS (SEQ ID NO:69), and a possible reverse translated sequence for this is ATGCGGCGGATGCAGCTACTACTCCTGATTGCCTTGAGTCT GGCCTTGGTGACGAATTCT (SEQ ID NO:70), which may be codon-optimized to allow expression of the amino acid sequence above for maximal translation in human T cells).

• IFNy - the sequence is M YTSY ILAFQLCIVLGSLGCYC (SEQ ID NO:71), and a sample reverse-translation is

ATGAAGTACACCAGCTACATCCTGGCCTTCCAGCTGTGCA TCGTGCTGGGCAGCCTGGGCTGCTACTGC (SEQ ID NO:72), which may be codon-optimized to allow expression of the amino acid sequence above for maximal translation in human T cells.

Recognition domains

Domains recognizing CD47 Antibody to CD47. Murine antibody sequences were derived from US Patent Application 20140363442 and analyzed using EVIGT/V-Quest

(http://www.imgt.org/IMGT_vquest/vquest)

® Light Chain:

DWMTQTPLSLSVSLGDQASISCRSSQSLVHSNGNTYLH WYLQKPGQSPKLLIYKVSYRFSGVPDRFSGSGSGTDFTL KISRVEAEDLGVYFCSQNTHVPRTFGQG (SEQ ID NO:73) nucleotide sequence for light chain

:GATGTTGTTATGACCCAAACTCCACTCTCCCTGTCTGTCAGTC

TTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCT

TGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAG

AAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCT

ACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATC

AGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGA

GGATCTGGGAGTTTATTTCTGCTCTCAAAATACACATGTTCCTC

GGACGTTCGGCCAAGGAG) (SEQ ID NO:74)

® Heavy Chain:

EVQLQQFGAELVKPGASMKLSCKASGYTFTNYYVFWV KQRPGQGLEWIGDL PVNGDTNF EKFK ATLTVTJKS STTTYLQLSSLTSEDSAVYYCTRGGYTMDYWGQG (SEQ ID NC):75)

(nucleotide sequence encoding SEQ ID NO:75:

GAGGTCCAGCTGCAGCAGTTTGGGGCTGAACTGGTGA

AGCCTGGGGCTTCAATGAAGTTGTCCTGCAAGGCTTC

TGGCTACACCTTCACCAACTACTATGTATTCTGGGTGA

AACAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAG

ACATTAATCCTGTCAATGGTGATACTAACTTCAATGA

GAAATTCAAGAACAAGGCCACACTGACTGTAGACAA

GTCCTCCACCACAACATACTTGCAACTCAGCAGCCTG

ACATCTGAGGACTCTGCGGTCTATTACTGTACAAGAG

GGGGTTATACTATGGACTACTGGGGTCAAGGA) (SEQ

ID NO: 76)

® The complementarity-determining regions of this antibody are CDR1 GYTFTNYYVF (SEQ ID NO:77)

CDR2 DINPVNGDTNFNEKFKN (SEQ ID NO: 78)

CDR3 GGYTMDY (SEQ ID NO:79)

Light Chain

CDR1 RSSQSLVHSNG TY (SEQ ID NO:80)

CDR2 KVSYRFS (SEQ ID NO:81)

CDR3 SQ THVPRT (SEQ ID NO: 82)

One or all of the framework regions may be replaced with the framework regions for human broadly neutralizing antibody 10-1074 for HIV (heavy chain

QVQLQESGPGLVKPSETLSVTCSVSGDSMNNYYWTWIRQSP

GKGLEWIGYISDRESATYNPSLNSRVVISRDTSKNQLSLKL-NS

VTPADTAVYYCATARRGQRIYGVVSFGEFFYYYSMDVWGK

GTTVTVSSASTK (SEQ ID NO:83) and light chain

SYVRPLSVALGETARISCGRQALGSRAVQWYQHRPGQAPILL IY NQDRP S GIPERF S GTPDINF GTR ATLTI S G VE AGDE AD Y Y CHMWDSRSGFSWSFGGATRLTVL) (SEQ ID NO:84), which are:

For the Heavy Chain:

FR1 QVQLQESGPGLVKPSETLSVTCSVS (SEQ ID NO: 85) (nucleotide sequence

CAGGTGCAGCTGCAGGAATCTGGGCCTGGACTGG

TCAAACCCTCCGAGACTCTGAGCGTCACTTGTTCT

GTGAGC (SEQ ID NO: 85)),

FR2 WTWIRQSPGKGLEWIGY (SEQ ID NO:86

) (nucl eoti de sequence :

TGGACATGGATCCGACAGAGCCCAGGCAAGGGG

CTGGAGTGGATCGGCTAC (SEQ ID NO: 87)), FR3

T YNP SLNSRWISRDT SKNQLSLKLNS VTP ADT AVY

YC

(SEQ ID NO: 88) (nucleotide sequence

ACTTATAACCCTAGCCTGAATTCCAGGGTGGTCA TTTCACGCGACACCAGCAAGAACCAGCTGTCCCT

GAAAC TGAATTC TGTGACCCC CGC AGAT AC AGC C

GTCTACTATTGC (SEQ ID NO: 89)),

FR4 WGKGTTVTVSS (SEQ ID NO:90) (nucleotide sequence

TGGGGGAAGGGGACTACAGTGACCGTCTCAAGC

(SEQ ID NO:9!)),

For the light chain

FR1 SYVRPLSVALGETARISCGRQ (SEQ ID NO: 92) (nucleotide sequence

TCCTATGTCAGGCCACTGTCCGTCGCACTGGGGG AGACCGCAAGAATTAGCTGTGGGAGGCAg (SEQ ID NQ:93))

FR2 VQWYQHRPGQAPILLIY (SEQ ID NO:94)

(nucleotide sequence

GTCCAGTGGTACCAGCACCGACCAGGACAGGCA

CCAATCCTGCTGATCTAC (SEQ ID NO:95)j

FR3

DRP S GIPERF S GTPD INF GTRATLTI S GVE AGDE AD

(SEQ ID NO:96) (nucleotide sequence

GACCGGCCTTCAGGCATCCCCGAGAGATTCAGCG

GAACACCCGATATTAACTTTGGCACTAGAGCTAC

CCTGACAATCAGCGGAGTGGAGGCAGGCGACGA

AGCCGAT (SEQ ID NO:97))

Alternatively, the fully humanized CD47 antibody will be used, described by US Patent Application 20140363442

Light Chain:

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTY

LHWYQQSKPGKAP LLIYKVSYRFGVPDRFSGSGS

GTDFTLKISRVEAEDVGVYYCSQ THVPRTFGQG

(SEQ ID NO:98) (nucleotide sequence

GATATTGTGATGACCCAGACTCCACTCTCCCTGC

CCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTG CAGATCTAGTCAGAGCCTTGTACACAGTAATGGA AACACCTATTTACATTGGTATCAGCAGAAACCAG GGAAAGCTCCTAAGCTCCTGATCTATAAAGTTTC

CTACCGATTTTCTGGGGTCCCAGACAGGTTCAGT

GGCAGTGGGTCAGGCACTGATTTCACACTGAAAA

TCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTA

TTACTGTTCTCAAAATACACATGTTCCTCGGACGT TCGGCCAAGGG (SEQ ID N():99)

Heavy Chain:

QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVF WVRQARGQRLEWIGDINPXnsrGDTNFNEKFKNRVTI SADKSISTAYLQWSSLKASDTAMYYCARGGYTMD YWGQG (SEQ ID NO: 100) (nucleotide sequence CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTG GTGAAGCCTTCACAGACCCTGTCCCTCACCTGCA CTGTCTCTGGCTACACCTTCACCAACTACTATGTA TTCTGGGTGCGACAGGCTCGTGGACAACGCCTTG AGTGGATAGGTGACATTAATCCTGTCAATGGTGA TACTAACTTCAATGAGAAATTCAAGAACAGAGTC ACCATCTCAGCCGACAAGTCCATCAGCACCGCCT ACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACAC CGCCATGTATTACTGTGCGAGAGGGGGTTATACT ATGGACTACTGGGGCCAGGGA (SEQ ID NO: 101)) Ligand to CD47: SIRPa is the natural ligand to CD47, and an alternative form of the antibody effector construct uses the extracellular domain of SIRPa as the antibody-like recognition domain. This sequence is:

EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAG

PGRELIYNQKEGHFPRVTTVSDLTKR^'NNMDFSIRIGNITPADA

GTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPWSGPA

ARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTN T3

PVGESVSYSIHSTAKVVLTREDVHSQVICEVAFiVTLQGDPLR

GTA LSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRL QLTWLE G^'NVSRTETASTVTE^'NKDGTYNWMSWLLVNVSAH RDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGS TAAE NTGSNERNIY (SEQ ID NO: 102)

Antibody to glypicari-1

® The antibody sequence (a murine antibody) was obtained from US Patent Application 20180072803, analyzed using IMGT V Quest (http://www.imgt.org/IMGT_vquest vquest)

® The heavy chain sequence is:

QIQLVQSGPELKKPGETVKISCKASGYAFTDYSMNWVKQAP GKGLRWMGWINTETGEPTYTDDFKGRFAFSLETSASTAFLQI NNLR.NEDTATYFCARHYDYGGFPYWGQGTLVTVSA (SEQ ID NO: 103) (nucleotide sequence

GAGGTCCAGCTGCAGCAGTTTGGGGCTGAACTGGTGAAGC

CTGGGGCTI AAI JAAGTTGI CTGCAAGGCTTCTGGCTA

CACCTTCACCAACTACTATGTATTCTGGGTGAAACAGAGG

CCTGGACAAGGCCTTGAGTGGATTGGAGACATTAATCCTG

TCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAA

GGCCACACTGACTGTAGACAAGTCCTCCACCACAACATAC

TTGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCT

ATTACTGTACAAGAGGGGGTTATACTATGGACTACTGGGG

TCAAGGA (SEQ ID NO: 104))

® The light chain sequence is:

DIQMTQSPASLSASVGETVTITCRASGNVHNYLAWYQQKQG KSPQLLVYTAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFG

TYYCQHFWSNPWTFGGGTKLEIK (SEQ ID NO: 105) (nucleotide sequence

(GATATTCAGATGACCCAGAGCCCGGCGAGCCTGAGCGCG

AGC GTGGGC GAA ACCGTGAC C ATT ACC TGCC GCGCGAGC G GCAACGTGCATAACTATCTGGCGTGGTATCAGCAGAAACA GGGCAAAAGCCCGCAGCTGCTGGTGTATACCGCGAAAACC CTGGCGGATGGCGTGCCGAGCCGCTTTAGCGGCAGCGGCA GCGGCACCCAGTATAGCCTGAAAATTAACAGCCTGCAGCC GGAAGATTTTGGCACCTATTATTGCCAGCATTTTTGGAGCA ACCCGTGGACCTTTGGCGGCGGCACCAAACTGGAAATTAA

AC (SEQ 1D NO: 106))

® The complementarity-determining regions of this antibody are

® Heavy Chain

CDR1 -^■ GYTFTNYY (SEQ ID NO: 107) CDR2 - F PVNGDT (SEQ ID NO: 108) CDR3 - TRGGYTMDY (SEQ ID NO: 109)

• Light Chain

CDR1 - GNVHNY (SEQ ID NO: 1 10)

CDR2 - TAK

CDR3 - QHFWSNPWT (SEQ ID NO: 1 1 1)

Antibody to CD73

The humanized antibody sequence is disclosued in US Patent 9605080, incorporated by reference in its entirety herein, analyzed by IMGT V Quest (http://mvwimgt.org/IMGT __vquest/vquest). ® The heavy chain sequence is

QVQLVESGGGVVQPGRSLRLSCATSGFTFSNYGMFWVR QAPGKGLEWVAVILYOGSNKYYPDSVKGRFTISRDNSK NTLYLQMNSLRAEDTAVYYCARGGSSWYPDSFDIWGQ GTMVTVSS (SEQ ID NO: 1 12)

• (nucleotide sequence)

cAGGTGCAGCTGGTGGAAAGCGGCGGCGGCGTGGTGC AGCCGGGCCGCAGCCTGCGCCTGAGCTGCGCGACCAG CGGCTTTACCTTTAGCAACTATGGCATGCATTGGGTGC GCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGCGG TGATTCTGTATGATGGCAGCAACAAATATTATCCGGA T AGCGTGAAAGGCC GCTTT ACC ATT AGC CGC GAT A AC AGCAAAAACACCCTGTATCTGCAGATGAACAGCCTGC GCGCGGAAGATACCGCGGTGTATTATTGCGCGCGCGG CGGCAGCAGCTGGTATCCGGATAGCTTTGATATTTGG GGCCAGGGCACCATGGTGACCGTGAGCAGC (SEQ ID NO: 113)) The light chain sequence is

EIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQK PGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEP EDFAVYYCQQRSNWHLTFGGGTKVEIK (SEQ ID NO: 114) (nucleotide sequence

GAAATTGTGCTGACCCAGAGCCCGGCGACCCTGAGCC TGAGCCCGGGCGAACGCGCGACCCTGAGCTGCCGCGC GAGCCAGGGCGTGAGCAGCTATCTGGCGTGGTATCAG CAGAAACCGGGCCAGGCGCCGCGCCTGCTGATTTATG ATGCGAGCAACCGCGCGACCGGCATTCCGGCGCGCTT TAGCGGCAGCGGCCCGGGCACCGATTTTACCCTGACC ATTAGCAGCCTGGAACCGGAAGATTTTGCGGTGTATT ATTGCCAGCAGCGCAGCAACTGGCATCTGACCTTTGG CGGCGGCACCA AAGTGGAAATTA AA (SEQ ID NO: 115)), The complementarity-determining regions of this antibody are

• Heavy Chain

CDR1 - NYGMH (SEQ ID NO: 116)

CDR2 - VELYDGSNKYYPDSVKG (SEQ ID NO: 117)

CDR3 - GGSSWYPDSFDI (SEQ ID NO: 118)

• Light Chain

CDR1 - RASQGVS S YLA (SEQ ID NO: 119) CDR2 -^■ DASNRAT (SEQ ID NO: 120)

CDR 3 - QQRS WHLT (SEQ ID NO: 121) Domains recognizing ΤΟΡβ

Antibody to ΤΟΤβ

® The complementarity-determining regions of this antibody are from US Patent Application 20050276802, incorporated by reference in its entirety herein; these are:

Heavy Chain

CDR1 - G Y AF TN YLIE (SEQ ID NO: 122)

CDR2 - VNNPGSGGSNYNEKFKG (SEQ ID NO: 123)

CDR3 - SGGFYFDY (SEQ ID NO: 124) Light Chain

CDR1 - RASQ S VL YS SNQKNYL A (SEQ ID NO: 125)

CDR2 - WASTRES (SEQ ID NO: 126)

CDR3 - HQYLSSDT (SEQ ID NO: 127)

• The CDRs in the broadly neutralizing antibody for 10-1074

(see above) will be replaced with the CDRs from the antibody described above.

TGF RII - the extracellular domain for TGF(3RII will be used, shown below:

TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDN

QKSCMSNCSrrSICEKPQEVCVAVWRKNDENITLETVCHDPK

LPYHDFILEDAASPKCEMKEKKKPGETFFMCSCSSDECNDNII

F SEE YNT SNPDLLL VIF Q (SEQ ID NO: 128)

Domains recognizing IL6

Antibody to IL6 was obtained from antibody HaglT-3-lQ, from US Patent 9234035, incorporated by reference in its entirety herein.

® The complementarity-determining regions of this antibody are

Heavy Chain

CDR1 - TGGMSVS (SEQ ID NO: 129)

CDR2 - RID WDDDKF YTP SLKT (SEQ ID NO: 130)

CDR3 - MHIDDSNGYFSDAFHI (SEQ ID NO: 131)

Light Chain

CDR1 - RDSQSVSSTSLA (SEQ ID NO: 1332) CDR2 - DTS RAT (SEQ ID NO: 133)

CDR3 - SFVSRPYPRFT (SEQ ID NO: 134)

The CDRs in the broadly neutralizing antibody for 10-1074 (see above) will be replaced with the CDRs from the antibody described above.

IL6Ra - we will use the extracellular domain for IL6Ra, which binds to IL6 but does not transduce a signal. The sequence is

LAPRRCPAQEVARGVLTSLPGDSVTLTCPGVEPEDNATVHW

VLRKPAAGSHPSRWAGMGRRLLLRSVQLHDSG YSCYRAG RPAGTVHLLVDVPPEEPQLSCFRKSPLSNVVCEWGPRSTPSLT

TKAVLLVRKFQNSPAEDFQEPCQYSQESQKFSCQLAVPEGDS

SFYIVSMCVASSVGSKFSKTQTFQGCGILQPDPPANITVTA.VA

RNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWM

VKDLQHHCVIHDAWSGLRFiVVQLRAQEEFGQGEWSEWSPE

AMGTPWTESRSPPAENEVSTPMQALTTNKDDD ILFRDSAN

ATSLPVQDSSSVPLP (SEQ ID NO: 135}

Linkers

(see, e.g., Chen et al., 2013 Adv Drug Deliv Rev 65, 1357-1369, incorporated by reference in its entirety herein)

Flexible linkers

• G4S - the amino acid sequence is GGGGS (SEQ ID NO: 136), and a potential reverse-translation takes the form

GGNGGNGGNGGNWSN (SEQ ID NO: 137), which we may codon- optimize to allow expression of the amino acid sequence above for maximal translation in human T cells.

• (G4S)3 - the amino acid sequence is GGGGSGGGGSGGGGS (SEQ ID NO: 138), and a potential reverse-translation takes the form GGNGGNGGNGGNW SNGGNGGNGGNGGNW SNGGNGGNGG NGGNWSN (SEQ ID NO: 138), which we may codon-optimize to allow expression of the amino acid sequence above for maximal translation in human T cells.

® G8 - the amino acid sequence is GGGGGGGG (SEQ ID NO: 139), and a potential reverse-translation takes the form

GGNGGNGGNGGNGGNGGNGGNGGN (SEQ ID NO: 140), which we may codon-optimize to allow expression of the amino acid sequence above for maximal translation in human T cells.

• G6 - the amino acid sequence is GGGGGG (SEQ ID NO: 141), and a potential reverse-translation takes the form

GGNGGNGGNGGNGGNGGN (SEQ ID NO: 141), which we may codon-optimize to allow expression of the amino acid sequence above for maximal translation in human T cells.

Rigid linkers (EA3K)3, where the amino acid sequence is

EAAAKEAAAKEAAAK (SEQ ID NO: 142), and a potential reverse- translation takes the form

GARGCNGCNGCNAARGARGCNGCNGCNAARGARGCNGCN

GC AAR (SEQ ID NO: 143), which we may codon-optimize to allow expression of the amino acid sequence above for maximal translation in human T cells.

A(E A3 K)4 ALE A(E A3 K)4 A (SEQ ID NO: 144), where the amino acid sequence is

AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAA KEAAAKA (SEQ ID NO: 145), and a potential reverse-translation takes the form

GCNGARGCNGCNGCNAARGARGCNGCNGCNAARGARGCN

GCNGCNAARGARGCNGCNGCNAARGCNYTNGA GC GAR GCNGCNGCNAARGARGCNGCNGCNAARGARGCNGCNGCN AARGARGCNGCNGCNAARGCN (SEQ ID NO: 146), which we may codon-optimize to allow expression of the amino acid sequence above for maximal translation in human T cells,

PA AP (SEQ ID NO: 147), where a potential reverse-translation takes the form CCNGCNCCNGCNCCN (SEQ ID NO: 148), which we may codon-optimize to allow expression of the amino acid sequence above for maximal translation in human T cells.

AEAAAKEAAAKA (SEQ ID NO: 149), where a potential reverse- translation takes the form

GCNGARGCNGCNGCNAARGARGCNGCNGCNAARGCN (SEQ ID NO: 150), which we may codon-optimize to allow expression of the amino acid sequence above for maximal translation in human T cells.

(AP)N - where N=10-34 amino acids; the amino acid sequence is APAPAPAP to

APAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAP (SEQ ID NO: 151), with a potential reverse-translation taking the form

GCNCCNGCNCCNGCNCCNGCNCCNGCNCCNi NCCNGCN CCNGCNCCNGCNCCNGCNCCNGCNCCNGCNCCNGCNCCN

GCNCCNGCNCCNGCNCCNGCNCCN (SEQ ID NO: 152), which we may codon-optimize to allow expression of the amino acid sequence above for maximal translation in human T cells. Cellular recruitment domains

CD 16 scFv - The sequence is derived from clone NM3E2, with the following complementary determining regions:

• Heavy Chain

CDR1 - DYGMS (SEQ ID NO: 153)

CDR2 - GTNWNGGSTGYADSVKG (SEQ ID NO: 154) CDR3 - GRSLLFDY (SEQ ID NO: 155)

® Light Chain

CDR1 - QGDSLRSYY AS (SEQ ID NO: 156)

CDR2 - GKNNRPS (SEQ ID NO: 157)

CDR3 - NSRDSSGNHW (SEQ ID NO: 157)

• The CDRs in the broadly neutralizing antibody for 10-1074 (see above) will be replaced with the CDRs from the antibody described above.

Fc - Immunoglobulin Constant Region

• IgGl -

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALT SGVHTFP AVLQ S SGLYSLS SWT VP S S SLGTQT YICN V NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP P DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS K ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMi-IEALHNHYTQKSLSLSPGK (SEQ ID NO: 158)

• IgG3 -

ASTKGPS PLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWN

SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYTCNV

NHKPSNTKVD RVELKTPLGDTTHTCPRCPEPKSCDTPPPCP RCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFKWYVDGV EViiNAKTKPREEQYNSTFRWSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESSGQPE NYNTTPPMLDSDGSFFLY SKLTVDKSRVVQQGNIFSCSVMHEALHNRF^'TQ SLSLSPGK (SEQ ID NO: 159)

Mutated Fc: GASDALIE

ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSN T VDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDT PPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVWD VSHEDPEVQFK W YVDGVEVHN AKTKPREEQ YNST FRWSVLTVLHQDWLNGKEYKCKVSNKALPAPffiKTISKT GQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPEN NYNTTPPMLD SD G S FF L Y SKLT VDK S R WQQGNIF S C S VMHE ALH NRFTQKSLSLSPGK (SEQ ID NO: 160)

Internal cleavage sites -Both 2A and furin cleavage sites will be used to allow simultaneous production of the antibody effectors and the surface markers. Membrane cleavage sites - One version of our antibody effector constructs would allow their expression on the membrane and subsequent cleavage upon (a) T cell activation or (b) encounter with matrix metalloproteinases, which are enriched in the tumor microenvironment (Kessenbrock et al., Cell 141, 52-67 (2010)).

GPI - the domain comprises the linker and the transmembrane domain. There is some evidence that suggest GPI anchors release their proteins following stress events (Muller et al., Arch Biochem Biophys 656, 1-18 (2018)) - which may occur in T ceils once they reach the tumor site. The amino acid sequence is

MPPPRLLFFLLFLTP\1EVRPEEPLVVKVEEGDNAVLQCLKGTSDG PTQQLTWSRESPLKPFLKLSLGLPGLG-IHMRPLAIWLFIFNVSQQM GGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGC GLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSL NQSLSQDLTMAPGSTLWLSCGVPPDSVS GPLSWTHVHP GPKSL LSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRG LTM SFHLEETARPVLWHWLLRTGGWKVSAVTLAYLEFCLCSLVGELHL QRALVLRRE RKRMTDPTR F (SEQ ID NO: 161), and the nucleotide sequence is

GGAAGTGGAACCACTTCAGGTACTACCCGTCTTCTATCTGGGC ACACGTGTTTCACGTTGACAGGTTTGCTTGGGACGCTAGTAACC ATGGGCTTGCTGACTTGA (SEQ ID NO: 162), which we may codon- optimize to allow expression of the amino acid sequence above for maximal translation in human T cells.

Matrix Metalloproteinase - the cleavage site for MMP /9, which feature prominently in tumor microenvironments is defined by the amino acid sequence PLGLWA; the reverse-translated nucleotide sequence takes the form CCNYTNGGNYTNTGGGCN (SEQ ID NO: 163), which we may codon-optimize to allow expression of the amino acid sequence above for maximal translation in human T cells.

Surface markers: CD19 (DiStasi et al., N Engl J Med 365, 1673- 1683(201 1 )) A truncated CD19 will be used, featuring the signal sequence, the extracellular domain, and the transmembrane domain as a surface marker for detecting cells and purifying cells. The amino acid sequence is

MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDG

PTQQLTWSRESPLKPFLKLSLGLPGLGEFEMRPLAEWLFEFNVSQQM

GGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGC

GLKNRSSEGPSSPSG LMSP LYVWAKDRPEEWEGEPPCLPPRDSL

NQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTFEVFEPKGPKSL

LSLELE^DDRPAIUDMWVMETGLLLPRATAQDAGKYYCHRGNLTM

SFEiLEETA PVLWE^WLLRTGGWKVSAVTLAYLEFCLCSLVGELFEL

QRALVLRRKRKRMTDPTRRF (SEQ ID NO: 164), and the nucleotide sequence is

ATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCC

ATGGAAGTCAGGCCCGAGGAACCTCTAGTGGTGAAGGTGGAA

GAGGGAGATAACGCTGTGCTGCAGTGCCTCAAGGGGACCTCAG ATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCCCGCT

TAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGA

ATCCACATGAGGCCCCTGGCCATCTGGCTTTTCATCTTCAACGT

CTCTCAACAGATGGGGGGCTTCTACCTGTGCCAGCCGGGGCCC

CCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATGTGG

AGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGG

TGGCCTGGGCTGTGGCCTGAAGAACAGGTCCTCAGAGGGCCCC

AGCTCCCCTTCCGGGAAGCTCATGAGCCCCAAGCTGTATGTGT

GGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCGT

GTCTCCCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAGGA

CCTCACCATGGCCCCTGGCTCCACACTCTGGCTGTCCTGTGGGG

TACCCCCTGACTCTGTGTCCAGGGGCCCCCTCTCCTGGACCCAT

GTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCCTAGAGCTGA

AGGACGATCGCCCGGCCAGAGATATGTGGGTAATGGAGACGG

GTCTGTTGTTGCCCCGGGCCACAGCTCAAGACGCTGGAAAGTA

TTATTGTCACCGTGGCAACCTGACCATGTCATTCCACCTGGAGA

TCACTGCTCGGCCAGTACTATGGCACTGGCTGCTGAGGACTGG

TGGCTGGAAGGTCTCAGCTGTGACTTTGGCTTATCTGATCTTCT

GCCTGTGTTCCCTTGTGGGCATTCTTCATCTTCAAAGAGCCCTG

GTCCTGAGGAGGAAAAGAAAGCGAATGACTGACCCCACCAGG

AGATTC (SEQ ID NO: 165), which we may codon-optiniize to allow expression of the amino acid sequence above for maximal translation in human T cells.

Protein markers - We will add protein markers to our antibody effector constructs to aid in both purification and identification. We will begin by using the following, with the corresponding sequences:

Myc - the amino acid sequence is EQKLISEEDL (SEQ ID NO: 166), and the reverse-translated sequence is

GAGCAAAAGCTCATTTCTGAAGAGGACTTG (SEQ ID NO: 167), which we may codon-optimize to allow expression of the amino acid sequence above for maximal translation in human T cells.

FLAG - the amino acid sequence is

DYKDDDDKDYKDDDDKLMDYKDDDDKDYKDDDDKLMDYKDD DDK (SEQ ID NO: 168), and the reverse-translated sequence is

G AC T AC AAGG ACGAC GATGAC A AGGATT AC A A AGATGAC GAC

GATAAGCTTATGGACTACAAGGACGACGATGACAAGGATTACA

AAGATGACGACGATAAGCTTATGGACTACAAGGACGACGATG ACAAG (SEQ ID NO: 169), which we may codon-optimize to allow expression of the amino acid sequence above for maximal translation in human T cells.

6X HIS - the amino acid sequence is HHHHHH (SEQ ID NO: 170), and the reverse-translated sequence is CACCATCACCATCACCAT (SEQ ID NO: 171 ), which we may codon-optimize to allow expression of the amino acid sequence above for maximal translation in human T cells.

Cainexin - If we find difficulties with expressing our antibody effector constructs, we will co-transduce T cells with a construct comprised of cainexin and a surface marker (e.g. truncated CD 19). The sequence for this is

MEGKWLLCMLLVLGTAIVEAHDGHDDDVIDIEDDLDDVIEEVEDSKPD TTAPPSSP VTYKAPVPTGEVYFADSFDRGTLSGWILSKAKKDDTDDEI AKYDGKWEV EMKESKLPGDKGLVLMSRAKHHAISAKLNKPFLFDTK PLI VQ YE VNF QNGIEC GG A Y VK.LL SK TPELNLD QF HD K TP Y TIMFGPDK CGEDYKLHFIFRHKNPKTGIYEEKHAKRPDADLKTYFTDKKTHLYTLIL NPDNSFEILAT3QSVVNSGNIXNDMTPP\T PSREIEDPEDRKPEDWDERP KIPDPEAVKPDDWDEDAPAKIPDEEATKPEGWLDDEPEYVPDPDAEKP EDWDEDMDGEWEAPQIANPRCESAPGCGVWQRPVIDNPNY GKWKP

PMIDNPSYQGIWKPI KIPNPDFFEDLEPFRM:TPFSAIGLELWSMTSDIFF

DNTIICADRRI\T DWANDGWGLKKAADGAAEPGVVGQMIEAAEERP WLWVVYILTVALPVFLVILFCCSGK QTSGMEYKKTDAPQPDV EEEE EKEEEKDKGDEEEEGEEKLEEKQKSDAEEDGGTVSQEEEDRKPKAEED EILNRSPRNRKPRRE (SEQ ID NO: 172), and a potential reverse translation takes the form

ATGGARGGNAARTGGYTNYTNTGYATGYTOYT GTNYTNGG-NACN

GCNATHGTNGARGCNCAYGAYGGNCAYGAYGAYGAYGTNATHGAY

ATHGARGAYGAYYTNGAYGAYGTNATHGARGARGTNGARGAYWSN AARCCNGAYACNACNGCNCCNCCNW ^'SNW SNCCN AARGTNAC TAY

AARGCNCCNGTNCCNACNGGNGARGTNTAYTTYGCNGAYWSNTTY GAYMGNGGNACNYTNWSNGGNTGGATHYTNWS^'NAARGCNAARAA

RGAYGAYACNGAYGAYGARATHGCNAARTAYGAYGGNAARTGGGA

RGTNGARGARATGAARGARWSNAARYTNCCNGGNGAYAARGGNYT

NGTNYTTSiATGWSNMGNGCNAARCAYCAYGCNATHWSNGCNAARYT

NAAYAARCCNTTYYTNTTYGAYACNAARCCNYTNATHGTNCARTAY

GARGTNAAYTTYCARAAYGGNATHGARTGYGGNGGNGCNTAYGTN

AARYTNYTNWSNAARACNCCNGARYT AAYYTNGAYCARTTYCAY

GAYAARACNCCNTAYACNATHATGTTYGGNCCNGAYAARTGYGGN

G-ARGAYTAYAARYTNCAYTTYATHTTYMGNCAYAARAAYCCNAAR

ACNGGNATHTAYGARGARAARCAYGCNAAR1VIGNCCNGAYGCNGAY

YTNAARACNTAYTTYACNGAYAARAARACNCAYYTNTAYACNYTN

ATHYTNAAYCCNGAYAAYWS^'NTTYGARATHYTNGTNGAYCARWSN

GTNGTNAAYWSNGGNAAYYTNYTNAAYGAYATGACNCCNCCNGTN

AAYCCNWS MGNGARATHGARGAYCCNGARGAYMGNAARCCNGA

RGAYTGGGAYGARMGNCCNAARATHCCNGAYCCNGARGCNGTNAA

RCCNGAYGAYTGGGAYGARGAYGCNCCNGCNAARATHCCNGAYGA

RGARGCNACNAARCCNGARGG TGGYTNGAYGAYGARCCNGARTA

YGTNCCNGAYCCNGAYGCNGARAARCCNGARGAYTGGGAYGARGA

YATGGAYGGNGARTGGGARGCNCCNCARATHGCNAAYCCNMGNTG

YGARWSNGCNCCNGGNTGYGGNGTNTGGCARMGNCCNGTNATHGA

YAAYCCNAAYTAYAARGGNAARTGGAARCCNCCNATGATHGAYAA

YCCNWSNTAYCARGGNATHTGGAARCC MGNAARATHCCNAAYCC

NGAYTTYTTYGARGAYYTNGARCCNTTYMGNATGACNCCNTTYWS

NGCNATHGGNYTNGARYTNTGGWSNATGACNWSNGAYATHTTYTT

YGAYAAYTTYATHATHTGYGCNGAYMGNMGNATHGTNGAYGAYTG

GGCNAAYGAYGG TGGGGNYTNAARAARGCNGCNGAYGGNGCNGC

NGARCCNGGNGTNGTNGGNCARATGATHGARGCNGCNGARGARMG

NCCNTGGYTNTGGGTTSTGTNTAYATHYTNACNGTNGCNYTNCCNGTN

TTYYT GTNATHYTNTTYTGYTGWSNGGNAARAARCARACNWSN

GGNATGGARTAYAARAARACNGAYGCNCCNCARCCNGAYGTNAAR

GARGARGARGARGARAARGARGARGARAARGAYAARGGNGAYGAR

GARGARGARGGNGARGARAARYTNGARGARAARCARAARWSNGAY

GCNGARGARGAYGGNGGNACNGTNWSNCARGARGARGARGAYMG NAARCCNAARGCNGARGARGAYGARATHYTNAAYMGNWSNCCNM GNAAYMGNAARCC MGNMGNGAR (SEQ ID NO: 173), which we may codon-optimize to allow expression of the amino acid sequence above for maximal translation in human T cells.

3 ' UTR - We will use the 3 ' untranslated region of the protein tyrosine kinase lck This sequence is derived from GenBank, Accession M36881

(https://www.ncbi.nlm.ni h.gov/nuccore/M36881 . 1 ).

The sequence for this is as follows: GAGGAGGCC TTGAGAGGCC CTGGGGTTCT CCCCCTTTCT CTCCAGCCTG ACTTGGGGAG ATGGAGTTCT TGTGCCATAG TCACATGGCC TATGCACATA TGGACTCTGC ACATGAATCC CACCCACATG TGACACATAT GCACCTTGTG TCTGTACACG TGTCCTGTAG TTGCGTGGAC TCTGCACATG TCTTGTGCAT GTGTAGCCTG TGCATGTATG TCTTGGAC AC TGT AC A AGGT ACCCCTTTCT GGCTCTCCCA TTTCCTGAGA CCACCAGAGA GAGGGGAGAA GCCTGGGATT GAC AGAAGCT TCTGCCCACC T ACTTTTCTT TCCTC AGATC ATCCAGAAGT TCCTGAAGGG CCAGGACTTT ATCTAATACC TCTGTGTGCT CCTCCTTGGT GCCTGGCCTG GCACACATCA GGAGTTCAAT AAATGTCTGT TGATGACTGC CG (SEQ ID NO: 174) Alternative 3' UTR will be derived from the most common cell-specific genes for T cells, described by Palmer et ai. 2006.¹⁰ These are 5' UTR sequences from CD3G, LEF1, TCI 7. CD3D, MAL, CD2, NRFA2, SNPH, TCRIM, CD28, LAT, ITGA6, FBLN5, BCL11B, 11.7R. SHFM1 , FTS, ZAP70, TCR Vb, APBA2, CCND2, ITK, FYB, TNFRSF25, TXK, NK4, WNT10B, LEPROTL1, KLR 1, NGFRAP1, PLEKHK1, BUB IB, ΓΝΡΡ4Β, GALT, PLXDC 1, APEG1, INPP4A, CD6, DPP4, TRERF 1, AAK1, EVER1, MLLT3, A XA1, PCSK5, ADA, MEN1, PRKCQ, MARLTNl, CAMK4, IL6ST, ELL2, FLT3LG, STAT4, P2RY5, PDE9A, PAG, GFIl, FLJ20152, RORA, GAT A3, NPTXR, CDR2, TACTILE, SYNE2, UPPl, ATP1A1, SH2D1A FHIT, LTBP4, RNF144, RAB43, TNFSF8, CDS, DUSP16, BAG3, KLAA0748, ΒΓΝ2, RGS19IP1, RBMS , ITPKB, LDHA, PKM2, 1D2, MAPKAPK5, A 1.7, LCP2, MATN2, CISH, CCL5, SATB1, DOCK9, SEN54L, ACVR2B, GZMK, AQP3L, MAST4, DNASE1 L3, HSPA1 L, EPLIN, PCYT2, GBP2, CD3E, WINSL CDC 14 A,. CD3Z, TNIK, HOXB2, THEDCL PTGER2, RGS10, FYN, ACTN1 , EEIG1, LDHB, APOE, GBP1, ITM2A, CDC25B, MYBL1, PRKCA, TACC3, MANIC 1, VTPRl, SYTL DNAJB 1, RUNX2, TIAMl, RASGRPL PRKCI, PXN, WWP1, PDE4D, KLRBI, SEMA4D, GPSM3, TARP, RARRES3, KLRG1, SLC35D2, EDG4, TOB 1 , NPDC1, GABARAPLl, TM4SF14, SORL1, MPP7, GIMAP4, GIMAP2, SOCS3, IL6R, LPIN2, IL18R1, PIM1, IFITMl , PIK3R1, DRLM, SLC03A1, SELPLG, TNFAIP3, OPT , S100A8, DUSP2, SPOCK2, CTSW, DRIL1, TSGA14, or LRIGl

Lck and alternative UTR will be appended immediately after the termination codoi of the antibody effector constructs.

In other instances, we will use the 3' LTR of the Moloney murine leukemia vims.¹' The sequence is: AATG AAAGACCCCT

TCATAAGGCT TAGCCAGCTA ACTGCAGTAA CGCCATTTTG CAAGGCATGG GAAAAATACC AGAGCTGATG TTCTCAGAAA AACAAGAACA AGGAAGTACA GAGAGGCTAA AAAGTACCCG GCCCAGGGCC AAGAACAGAT GGTCCCCAGA CCGCTAACGA CAGGATATCT GTGGTTAAGC ACTAGGGCCC CGGCCCAGGG CCAAGAACAG ATGGTCCCCA GACCGCTAAC GACAGGATAT CTGTGGTTAA GCACTAGGGC CCCGGCCCAG GGCCAAGAAC AGATGGTCCC CAGAAATAGC TAAAACAACA ACAGTTTCAA GAG AC C C AG A AACTGTCTCA AGGTTCCCCA GATGACCGGG GATCAACCCC AAGCCTCATT TAAACTAACC AATCAGCTCG CTTCTCGCTT CTGTACCCGC GCTTATTGCT GCCCAGCTCT ATAAAAAGGG TAAGAACCCC ACACTCGGCG CGCCAGTCCT CCGATAGACT GAGTCGCCCG GGTACCCGTG TATCCAATAA AGCCTTTTGC TGTTGCA. (SEQ ID NO: 175) We will use all, or majority, or a segment of this LTR after the termination codon.

In still other instances, we will use the 3' LTR of the HIV-1 virus in lentiviral vectors. The sequence is as follows:

TGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGA TCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAG AACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTG

GATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGATAGA

AGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTG

AGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAG

TGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAG

AGCTGCATCCGGAGTACTTCAAGAACTGCTGACATCGAGCTTG

CTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGC

CTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATAT

AAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCA

GATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTG

CTT AAGCC TC A AT AAAGC TTGCCTTGAGTGC TTC AAGT AGTGTG

TGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGAC

CCTTTTAGTCAGTGTGGAAAATCTCTAGCA (SEQ ID NO: 176). We will use all, or majority, or a segment of this LTR after the termination codon.

Combinations of the antibody effector components

• The following table illustrates the combinations of the components describe above, which will be used for specific applications below

• Alternative components and combinations of the above can be substituted for this specific strategy, as described in the table below

The expression of the antibody constructs will be tested using the following immune assays

• Western Blot

® Flow Cytometry

® Immunohistochemistry

• ELISA

The functionality of the antibody constructs will be tested using the following assays

• Flow Cytometry

• ADCC

Chromium Release Assay

Europium Assay

• Neutralization Example 3.

Generation of T Cells reconfigured for Antibody Effectod Neutralization of Neoplastic Environments (TRAE F.)

Cell Source

Peripheral blood draw from healthy donors, umbilical cord blood samples, peripheral blood draw from patients, commercially available buffy coats and ieukopaks, and

ieukopheresis samples from healthy donors will be used.

Generation of Antigen Presenting Cells

Three kinds of antigen presenting cells (APCs) will be used: monocytes, dendriti c ceils, and PHA blasts. Monocytes will be isolated by adhering mononuclear ceil s obtained from different cell sources in plastic, using X-Vivo media, for 12-18 hours. After incubation, nonadherent cells are removed and frozen, and the isolated monocytes are used as APCs.

Dendritic cells will be generated by first adhering monocytes (as above, but just for two hours) or by CD14 selection. Following separation of either nonadherent or CD 14- negative fraction, dendritic ceils are grown in GMCSF and 11.4 for 3-5 days, after which they receive peptides (25mers, 20mers, or 15mers). They are then matured using combinations of Π.1 β, IL6, TNFa, LPS, and IFNy

PHA blasts are generated by growing mononuclear cells in the presence of phytohem agglutinin (PHA) and subsequent culture with IL2 or JLI 5.

All APCs are irradiated prior to use in stimulating T ceils, with the exception of CD 14-selected, monocyte derived dendritic cells.

T Cell Expansion

The following is a sample protocol that uses CD14-selected dendritic cells as APCs. The protocol below can be modified by substituting monocytes or PHA blasts for each step of DC generation

DAY 1 AM

• After obtaining peripheral blood, carefully transfer samples into a sterile bottle. Dilute samples with sterile I X PBS of at least the same volume

• You will need enough 50 mL tubes to distribute the diluted blood into 25 raL aliquots

• Add 15 mL iymphoprep in each 50 mL tube

® Carefully layer 25 mL diluted blood in each tube Centrifuge at 3 acceleration and 3 deceleration at 400 G x 40 min at room temperature

Thaw freze media, store on ice until use

Remove samples from centrifuge

With transfer pipette, obtain white layer above iymphoprep and transfer to new 50 mL tube

Dilute collected layer with an equal volume of IX PBS

Centrifuge at 9 acceleration and 9 deceleration at 400 G x 10 min

Remove supernatant

Combine pellets by initially resuspending in 5 mL PBS, Resuspend combined pellets with 35 mL PBS

Obtain 18 uL to count with Luna

Centrifuge at 9 acceleration and 9 deceleration at 400 G x 5 min

Resuspend 20 million cells in 10 mL MACS buffer, spin down, aspirate media

Freeze rest of PBMCs

Add 40 uL CD14 microbeads and 60 uL MACS buffer (PBS + 0,5% BSA

+ 2 fflM EDRTA.) to pellet

Incubate at room temperature 20 minutes

Agitate pellet every 5 minutes

Add 10 mL MACS buffer, spin at 400 g x 5 minutes

Prewet LS column with 3 mL MACS buffer; discard effluent

Resuspend pellet in 3 ml MACS buffer, pass through column into CD14- labeied 15 mL conical

Wash column twice with 4.5 ml MACS buffer

Replace catch tube with 15 mL conical labeled CD 14+

Apply 5 mL Macs buffer to LS column, push through with plunger

Spin, count, aspirate supernatant

Resuspend 1 milliion cells/mL of DC media (cell genix + 5 mL glutamax) with cytokines: 1 uL/mL GMCSF and 0,6 uL/mL IL4 in 12 well plate Freeze CD 14 negative cells

Incubate DC in 37 degrees DAY 2 PM

• Add J rnL DC media on top of J mL (with 2 uL/mL GMCSF and 1.2 uL/mL IL4)

DAY 4 AM

® Remove 1 mL media

® Add 1 ,uL pepmix

DAY 4 PM

• Add 3 mL DC media with 2 uL/mL GMCSF, TNFa, ILlb, IL6, LPS and IFNg

DAY 5

® Prepare T cells - Thaw a vial of PBMC by incubating in 37°C water bath for <1 min 30 sec or until you see a core of ice surrounded by liquid (the PBMC should not be completely thawed)

® Resuspend the ceils in 9 mL prewarmed DC media, mix well but gently, get an aliquot for counting

® Spin cells at 400 g x 5 min

® Count ceils

® After spinning, discard supernatant

® Resuspend in Ix O⁶ cells/mL T cell media (45% RPMI 45% Click's 5% human serum 2 raM glutamax) with 10 ng mL DL7 and 60 ng/mL IL21 ® Plate 1 mL/weil in a 12 well plate

• Harvest DC - Use transfer pipette to gently harvest DC

® Mix well and get an aliquot for counting

® Spin cells at 400 g x 5 min

® Count ceils

® After spinning, discard supernatant

• Resuspend in 2.5xl0⁵ cells/mL T cell media (45% RPMI 45% Click' s 5% human serum 2 niM glutamax)

® Plate 1 mL/weil over the T cells in a 12 well plate

DAY 8 Feed T cells

® Check if ceils are confluent. If cells are >80% confluent, split into two wells (1 mL each). Add 1 m L T cell media + 5 ng/mL IL7 and 5 ng/mL

IL15 ® If cells are nto confluent, do ½ media change - take out 1 mL of media

• Replace media with 1 mL T cell media + 5 ng/mL IL7 and 5 ng mL IL15 DAY 11 - Generation of monocyte-derived dendritic cells from frozen PBMC

• Thaw two vials of PBMC by incubating in 37°C water bath for <1 min 30 sec or until you see a core of ice surrounded by liquid (the PBMC should not be completely thawed); Resuspend the cells in 9 mL prewarmed DC media, mix well but gently, get an aliquot for counting; Spin cells at 400 g x 5 min

• Count cells

® After spinning, discard supernatant

• If you have less than 20 million cells, add 40 uL of CD 14 beads to the cell pellet and 60 uL of MAC S buffer; mix

• Incubate at room temperature for 15-20 minutes

• Add 10 mL of MACS buffwer to wash the cells

• Spin cells at 400 g x 5 min

® While spinning, prep the magnet stand. Attach the magnet to the stand, and attach an LS column to the magnet. Put a waste tube underneath to collect effluent. Pre-wet the column by running 3 mL MACS buffer

® After spin, discard supernatant

• Resuspend cells in 3 mL MACS buffer ane allow to run through column ® Once all liquid has flown through, rinse column twice with 4.5 mL

MACS buffer

® To get the ceils, use a new collection tube (15 mL tube), add 5 mL MACS buffer to column, remove the column from the magnet, and use plunger to expel cells from column into the collection tube (1 5 mL tube)

• Mix the cells. Get an aliquot for counting

® After counting, resuspend cells at IxloVmL of DC media + 1 uL/mL

GMCSF and 0,6 uL/mL IL4, Dispense 1 mL/well in a 24 well plate,

® Incubate at 37°C. Make sure you label these cells as DCs (to distinguish from your T cells)

DAY 12 PM

• Add 1 mL DC media on top of 1 mL (with 2 uL/mL GMCSF and 1 .2 uL/mL IL4) DAY 14 AM

® Remove I mL media

® Add 1 ₍uL pepmix

DAY 14 PM

® Add 1 mL DC media with 2 uL/mL GMCSF, TNFa, IL b, IL6, LPS and IFNg

DAY 15

• Prepare T cells

• Harvest your T ceils using a transfer pipette - take note of cell numbers

• Spin cells at 400 g x 5 min

• Count ceils

® After spinning, discard supernatant

• Resuspend in lxlO⁶ eel is/ml . T cell media (45% RPMI 45% Click's 5% human serum 2 mM glutamax) with 10 ng mL DL7 and 200 U/mL IL2

® Plate 1 mL/weil in a 12 well plate

• Harvest DC - Use transfer pipette to gently harvest DC

® Mix well and get an aliquot for counting

• Spin cells at 400 g x 5 min

• Count cells

® After spinning, discard supernatant

• Resuspend in 2.5xl0⁵ cells/mL T cell media (45% RPMI 45% Click's 5% human serum 2 mM glutamax)

• Plate 1 mL/well over the T cells in a 12 well plate

DAY 18

• Feed T cells - Check if cells are confluent. If cells are >80% confluent, split into two wells (1 mL each). Add 1 mL T cell media plus 5 ng/mL IL15

• If cells are nto confluent, do ½ media change - take out 1 mL of media

• Replace media with 1 mL T cell media 5 ng/mL IL15

• Generation of monocyte-derived dendritic ceils from frozen PBMC - Thaw two vials of PBMC by incubating in 37°C water bath for <1 min 30 sec or until you see a core of ice surrounded by liquid (the PBMC should not be completely thawed); Resuspend the cells in 9 mL prewarmed DC media, mix well but gently, get an aliquot for counting; Spin cells at 400 g x 5 min

® Count cells

® After spinning, discard supernatant

® If you have less than 20 million cells, add 40 uL of CD 14 beads to the cell pellet and 60 uL of MACS buffer; mix

® Incubate at room temperature for 15-20 minutes

• Add 10 mL of MACS buffwer to wash the cells

® Spin cells at 400 g x 5 min

® While spinning, prep the magnet stand. Attach the magnet to the stand, and attach an LS column to the magnet. Put a waste tube underneath to collect effluent. Pre-wet the column by running 3 mL MACS buffer

® After spin, discard supernatant

• Resuspend cells in 3 mL MACS buffer ane allow to mn through column ® Once all liquid has flown through, rinse column twice with 4.5 mL

MACS buffer

® To get the ceils, use a new collection tube (15 mL tube), add 5 mL MACS buffer to column, remove the coiumn from the magnet, and use plunger to expel cells from column into the collection tube (1 5 mL tube)

® Mix the cells. Get an aliquot for counting

® After counting, resuspend cells at lxl0⁶/mL of DC media + 1 uL/mL

GMCSF and 0,6 uL/mL IL4, Dispense 1 mL/well in a 24 well plate,

® Incubate at 37°C. Make sure you label these cells as DCs (to distinguish from your T cells)

DAY 19 PM

® Add 1 mL DC media on top of 1 mL (with 2 uL/mL GMCSF and 1.2 uL/mL IL4)

DAY 20 AM:

® Remove 1 mL media

® Add 1 μί. pepmix

DAY 20 PM

® Add 1 mL DC media with 2 uL/mL GMCSF, TNFa, ( L i b, FL6, LPS and IF g DAY 21

® Prepare T cells

® Spin cells at 400 g x 5 min

® Count cells

® After spinning, discard supernatant

* Resuspend in 1 x i 0" cells/ml, T ceil media (45% RPMI 45% Click's 5% human serum 2 mM glutamax) with 200 U/mL IL2

® Plate 1 niL/wel] in a 12 well plate

® Harvest DC - Use transfer pipette to gently harvest DC

® Mix well and get an aliquot for counting

® Spin cells at 400 g x 5 min

® Count cells

® After spinning, discard supernatant

® Resuspend in 2.5x 10⁵ cells/mL T cell media (45% RPMI 45% Click' s 5% human serum 2 mM glutamax)

® Plate 1 mL/well over the T cells in a 12 well plate

DAY 25

® Feed T cells

® Check if cells are confluent. If cells are >80% confluent, split into two wells (1 mL each). Add 1 mL T cell media plus 5 ng/mL IL15

® If cells are nto confluent, do ½ media change - take out 1 mL of media ® Replace media with 1 mL T cell media 5 ng/mL 11.15

® Cells are ready for testing

T Cell Transduction

The following is an example of a transduction protocol.

• Preparation of Retronectin Coated Plate - If coating plates 24 hrs before

transduction, store at 4°C. If coating plates 4-6 hours before transduction, incubate at 37°C.

• Use non tissue culture-treated 24 well plates

• Coat ~4 wells with retronectin per well of T cells to be stimulated

® Add 300uL of 50 ug/mL retronectin per well

® Seal plates with Parafiim ® Acquire 4.0mL of retroviral vector per 1x10° cells

• Transport retroviral vector on ice

® Aspirate retronectin from each well

• Wash with 0,5 mL complete media

® Add 2.0 mL of retrovi.ral supernatant to each well

® Centrifuge plates at 2000G for 2 hours at 30°C

• During last 30 minutes of centrifugation:

® Harvest CTL to he transduced in 50 mL tube - leave at least 1 well as

untransduced control

® Resuspend at 0.25e6 CTL/mL in CTL media .

® Add IL-2

® Aspirate retroviral supernatant from plates

• Plate 2 mL of cell suspension per retronectin-coated well for a total of 0.5e6

CTL/weli

• Centrifuge plates at 1000G for 5 mins at room temp

Functional T esting of T_RA_ENN_E Cells

Migration: Transwell Assays

® Tumor cells will be plated on the base of a 24 well plate.

• Untransduced and antibody-effector expressing T cells will added to the top of the transwell insert.

• Tumor cells and T cells will be plated in OptiMEM media at lxlO⁶ and 3x10^J cells, respectively. After 3 hours of incubation, inserts will be removed and cells were collected from the bottom of the plate. Absolute cell count and phenotype will be determined using CountBright Absolute Counting Beads (Therm oFischer, Waltham, MA), CD3, and the relevant transduction marker (e.g. CD 19). Absolute ceil count will be calculated by the difference of T cell number and CountBright Absolute Counting Beads multiplied by the concentration of beads per volume of sample and the volume of sample collected.

® Positive controls will include media with the relevant chemokines

® Chemokine receptors will be measured in unmodified and modified T cells by flow cytometry ® The said chemokine receptor being CXCRi to target tumors that are

enriched for CXCL8 expression

® The said chemokine receptor being CXCR2 to target tumors that are enriched for CCL2, CXCL5, and CXCL12.

® The said chemokine receptor being CCR4 to target tumors that are

enriched for CCL22 and CCL28.

® The said chemokine receptor being CCR6 to target tumors enriched for

CCL20.

• The said chemokine receptor being CXCR4 to target tumors enriched for CXCL12 ,

® The results expected include enhanced expression of different chemokine

receptors on modified T cells, and corresponding migration towards the tumors.

Tumor Killing

The ability of transduced T cells to kill tumors will be determined by chronium-51 release cvtoxicitv assay. Both tumor cell lines and primary tumor lines were used as targets and incubated with chromium 51 for 1 hour. Targets will be cocultured with modified and unmodified T cells for 4 hours, in 37°C, at effector to target ratios of 40: 1, 20: 1, 10: 1, 5: 1, and 2.5: 1. After the 4 hour coincubation, plates will be spun to allow cells to settle at the bottom and 100 uL of supernatant will be collected onto a Luma plate (Perkin-Elmer, Waltham, MA). The plate will be incubated overnight at room temperature to allow for the supernatant to dry. Cr51 release will be measured on a MicroBeta2 counter. Specific lysis will be calculated as the difference of experimental and spontaneous release divided by the difference of the maximum and spontaneous release times 100. We expect modified T cells to specifically lyse tumors,

Treg Kitting

The ability of transduced T cells to kill tumors will be determined by chronium-51 release cvtoxicitv assay. Both tumor cell lines and primary tumor lines will be used as targets and incubated with chromium 51 for 1 hour. Targets will be cocultured with modified and unmodified T cells for 4 hours, in 37°C, at effector to target ratios of 40: 1, 20: 1, 10: 1, 5: 1, and 2.5: 1. After the 4 hour coincubation, plates will be spun to allow cells to settle at the bottom and 100 uL of supernatant will be collected onto a Luma plate (Perkin-Elmer, Waltham, MA). The plate will be incubated overnight at room temperature to allow for the supernatant to dry. Cr51 release will be measured on a MicroBeta2 counter. Specific lysis will be calculated as the difference of experimental and spontaneous release divided by the difference of the niaxiraura and spontaneous release times 100. We expect modified T cells to specifically lyse Tregs.

TAM Killing

The ability of transduced T cells to kill tumor-associated macrophages (T AM) will be determined by ehronium-51 release cytoxicity assay. TAM will be used used as targets and incubated with chromium 51 for 1 hour. Targets will be cocultured with modified and unmodified T cells for 4 hours, in 37°C, at effector to target ratios of 40: 1, 20: 1, 10: 1, 5: 1, and 2,5: 1. After the 4 hour coincubation, plates will be spun to allow cells to settle at the bottom and 100 uL of supernatant will be collected onto a Luma plate (Perkin-Elmer, Waltham, MA). The plate will be incubated overnight at room temperature to allow for the supernatant to dry. Cr51 release will be measured on a MicroBeta2 counter. Specific lysis will be calculated as the difference of experimental and spontaneous release divided by the difference of the niaxiraura and spontaneous release times 100. We expect modified T cells to specifically lyse TAMs.

MDSC Killing

The ability of transduced T cells to kill myeloid derived suppressor cells (MDSC) will be determined by chronium-51 release cytoxicity assay, MDSC will be used as targets and incubated with chromium 51 for 1 hour. Targets will be cocultured with modified and unmodified T cells for 4 hours, in 37°C, at effector to target ratios of 40: 1, 20: 1, 10: 1, 5: 1, and 2,5: 1. After the 4 hour coincubation, plates will be spun to allow cells to settle at the bottom and 100 uL of supernatant will be collected onto a Luma plate (Perkin-Elmer, Waltham, MA). The plate will be incubated overnight at room temperature to allow for the supernatant to dry. Cr51 release will be measured on a MicroBeta2 counter. Specific lysis will be calculated as the difference of experimental and spontaneous release divided by the difference of the niaxiraura and spontaneous release times 100. We expect modified T cells to specifically lyse MDSC.

Neutralization of Tumor Environments

® To assess the ability for antibody effectors to neutralize either cytokines or exosomes from the cell supernatant, secreted antibody effectors will be cocultured with tumors secreting ΤΟΡβ, IL6, CD47-expressing exosomes, glypican-1 -expressing exosomes, and CD73 -expressing exosomes. Supernatants will be collected before co culture with secreted antibody effectors, and 24, 48, 96, 120, and 240 hours after. The amount of remaining/free cytokines will be measured using iuminex, and the amount of remaining/free exosomes will be measured using flow cytometry.

• To test whether neutralization has a functional impact on tumor cells, tumors will be grown on cytokine-conditioned media (media enriched by cytokines secreted by tumors +/- co-culture with antibody effectors) or exosome- conditioned media (media enriched by tumor-derived exosomes secreted by tumors +/- co-culture with antibody effectors). Tumors will be monitored for proliferartion by CFSE assays, expression of anti-apoptotic molecules by flow cytometry, and expression of markers for EMT. We expect a decreased ability for cytokine-conditioned media and exosome-conditioned media to increase proliferation, expression of anti-apoptotic molecules and EMT expression in tumor cells grown in conditioned media in the presence of our secreted antibody effectors.

• To test whether neutralization has a functional impact on immune cells,

mononuclear cells be grown on cytokine-conditioned media (media enriched by cytokines secreted by tumors +/- co-culture with antibody effectors) or exosome-conditioned media (media enriched by tumor-derived exosomes secreted by tumors +/- co-culture with antibody effectors). Mononuclear cells will be activated with PMA/I, and will be tested for proliferation, expression of activation markers by flow cytometry, and cytokine secretion. We expect a decreased ability for cytokine-conditioned media and exosome-conditioned media to abrogate proliferation, expression of activation markers, and cytokine secretion in mononuclear cells grown in conditioned media in the presence of our secreted antibody effectors.

Stimulation of Endogenous Immune Cells

The same TRAE NEs described in the previous section will be used, and the ability of their secreted products to bind to CD47, glypican-1 , CD 73, and membrane-bound

TGFP,and membrane-bound IL6, expressed in tumors will be tested. We will then measure whether these bound tumors are (1) more susceptible to -mediated attack using chromium release assays, (2) whether cocultured M2 macrophages revert to Ml macrophages and mediate phagocytosis of tumor cells (labeled with GFP) using flow cytometry - checking for markers of M2 to Ml conversion and uptake of GFP signal, and (3) whether dendritic cells cocultured with tumor cells (in the presence or absence of our antibody effector-secreting T cell products) display increased maturation markers, increased secretion of IL12, and an increased ability to expand tumor antige -specific T ceils that can subsequently lyse our tumors.

In addition to the experiments outlined above, which will test each T ceil component individually, T_RA_ENNE will be tested as an entire platform. Its ability to simultaneously target neoantigens and the microenvironment would effectively allow it to target both of the enabling characteristics (genomic instability and pro-tumorigenic innate immune-mediated inflammation) that together underlie the hallmarks of cancer (5). Hence, we propose to ask whether TRAENNE_S can: (1) alter tumor metabolic activity (by XTT assay) and its synthesis of DNA (by flow cytometry), both indicators of sustained proliferative signaling, (2) affect the ability of tumors to form colonies (by colony forming unit assays), a tactic indicative of growth suppressor evasion, (3) improve or maintain expression of Bcl2 and caspase activity (by flow cytometry), a measure of resistance to ceil death; (4) decrease telomere lengths (by PGR telomere length analysis), to estimate replicative immortality; (5) decrease secretion of VEGF (by ELISA) and recruit endothelial cells (HUVEC, through a Boyden chamber assay), both measures of angiogenesis; and (6) decrease tumor cell invasion (by transwell migration assays) to assess metastatic ability (71). While we expect our T_RA_ENNES to demonstrate tripartite immune responses in vitro, a potential pitfall would be that T cells lose their specificity/function after tra sduction. Although our previous experience with genetically modified T ceils suggests otherwise (37), if this flaw is common to ail constructs, we would predict that the loss of antigen specificity results from the overgrowth of nonspecifically activated T ceils. As such, we will optimize conditions by increasing stimulations before transduction or by selecting for antigen- specific populations using an IFNy capture assay- prior to transduction.

/// Vivo Testing

After determining tumor engraftment with IVIS imaging, TRAENNEs will be injected via tail vein. The Fc portion of IgGl is also recognized by murine Fc receptors on NK ceils and dendritic cells, both of which are present in SCID animals. We will determine whether TRAENNEs (1) traffic to tumor sites, (2) confer a survival advantage over their nontransduced counterparts, and (3) attract murine NK and dendritic cells. We will also test increased activation of NK cells ex vivo by isolating NK cells systemically from animals and mmeeaassuurriinngg tthheeiirr aabbiilliittyy ttoo kkiillll UU8877MMGG cceellllss.. AAlltthhoouugghh tthhiiss mmeeaassuurreess aallllooggeenneeiicc kkiilllliinngg,, mmuurriinnee NK cceellllss aarree aabbllee ttoo llyyssee cceeiillss iimmmmeeddiiaatteellyy aafftteerr iissoollaattiioonn oonnllyy iiff tthheeyy hhaavvee bbeeeenn ssttiimmuullaatteedd iinn vviivvoo.. WWee wwiillll aallssoo tteesstt ssttiimmuullaattiioonn ooff eennddooggeennoouuss mmuurriinnee ddeennddrriittiicc cceellllss bbyy mmeeaassuurriinngg eexxpprreessssiioonn ooff mmaattuurraattiioonn mmaarrkkeerrss aanndd ccyyttookkiinnee sseeccrreettiioonn eexx vviivvoo..

Anti-GBM efficacy of neoantigen-specific T cells modified to secrete CD47 antibodies

Genomic instability leads to the production of unique proteins in tumor cells, so called neoantigens that arise from mutations, alternatively spliced isotorms, translocations, and alternatively translated transcripts. Emerging evidence suggests that these novel proteins get preferentially shuttled for MHC presentation, making them prime targets for T cells. The muitiantigen T cell strategy (recognizing tumor-associated antigens such as cancer testis antigens) can be used as a means to recognize neoantigens. This approach has the advantage of being HLA-agnostie (increasing applicability to more patients), and can be used to simultaneously target multiple antigens to prevent tumor escape. In the case of GBM, mutant PTEN, EGFRvIII, and IDHl, will initially be studied, obtaining 1 5 mers overlapping by 1 1 aa that span the mutations.

Experimental Design

Antigen-specific T cells form the backbone of the immunotherapeutic approach. The ex vz^'voexpanded antigen-specific T ceils are generated by repeated stimulations of peripheral blood mononuclear cells (PBMCs) with antigen-presenting cells that express a mix of overlapping peptides from several GBM neoantigens (PTEN, EGFRvIII, and IDHl). A good manufacturing practices (GMP)-compliant method has previously been optimized for generating different antigen-specific T cells (Cruz, CR et al ,, Clinical cancer research : an official journal of the American Association for Cancer Research. 2011; 17(22):7058-66; McCormack SE, et al. Cytotherapy. 2018;20(3):385-93), and this protocol has been successfully modified to allow expansion of neoantigen-specific T cells. The ability to generate GBM neoantigen-specific T cells is tested by determining specificity using IFNg/IL2/TNFa/perforin ELISPOT assays, tetramer analysis by flow cytometry, and cytotoxicity against partially HLA-matched tumor targets. For this study, the HLA-A2+ (one of the most common HLA types, to facilitate partial HLA-matching of tumors with T ceils) GBM cell line U87MG is used, which harbors a mutation in PTEN; lines with mutations in EGFRvIII and IDH1 are also available from ATCC. T cells are obtained from commercially available ieukopheresis samples or from healthy donors. It is expected that T cells recognizing PTEN, EGFRvIII, and IDHl mutations from different individuals representing different HLA types will be generated. Further, HLA-A2+ neoantigen-specific T cells will be generated that recognize and kill U87MG cells.

Modifications of the methods include adding or substituting additional targets, including previously identified targets WT1, FRAME, and survivin, or alternatively spliced isoforms like CDKN2B, KLF6-SV1, and FGFR1, which are also present in GBM.

The experiments described herein can be expanded to targeting other validated GBM cell lines, for example M069K and U3082MG. Partial HLA matching of targets and effectors may not yield optimal killing. Thus, this approach can be used in the autologous setting - obtaining study samples from patients with available cells after biopsy.

Example 5

Role of the CD47 axis in the maintenance of tumor-associated macrophages, tumor- derived exosomes, and an immune suppressive microenvironment in GBM

An antibody to CD47 will be designed that is capable of recruiting NK and phagocytic cells and that will be released at the site of the tumor. The construct will have the following components: (1) the ectodomain of SIRP-a, which binds to CD47 but which does not have the downstream signaling components, will serve as the variable region of the antibody, a strategy that has been successfully used for other chimeric constructs in lieu of scFv; (2) a hinge region based off IgGl will allow coupling of the antibody construct' s recognition domain to its effector domain; (3) the constant Fc region of IgGl will allow for recognition by NK cells, phagocytes, and dendritic cells, (4) a matrix metalloproteinase 9 MMP9) recognition

domain adjacent to the transmembrane, will allow the antibody to be released only in the tumor microenvironment, where there is a high concentration of MMP9; (5) the stalk and transmembrane region of CDS will anchor the construct to the plasma membrane; and (6) a 2A peptide cleavage sequence followed by a reporter (CD34 binding site) that would allow measurement of transduction efficiency. Retrovirus-mediated transduction will be used to modify the antigen-specific T cells, similar to the approach used to introduce CD 19 chimeric antigen receptors onto our virus-specific T cells, which have shown success in a phase I clinical trial. For the first iteration, we will be modifying nonspecificallv activated T cells (T cells stimulated with CD3/CD28 beads). First, genetically modified cells will be compared with their unmodified counterparts in terms of phenotype and cytokine secretion, to ensure that transgene expression does not alter the T cells' native functions. Next, whether the antibody products of the T cells are functional will be tested by evaluating their effects on tumor-associated macrophages (TAMs) and tumor-derived exosomes (IT⁾Es).

To test their effects on TAMs, these cells will be derived from macrophages by growing them in the presence of M2 -polarizing conditions and co-culturing them with the GBM cells. Interaction of TAMs and GBMs should lead to secretion of TGF-β, making the supernatant highly immune suppressive. TAM/GBM cocultures grown in the presence or absence of the T cell-secreted products will be compared, and the following will be determined: (a) TGF-13 levels in the supernatant; and (b) immune suppressive ability of supernatant against PBMCs. Assays described in the art will be used (Yvon ES, et al. Cytotherapy. 2017; 19(3):408-18; Foster AE, et al. J Immunother. 2008;31(5):500-5). To test T_RA_ENNE effects on TDEs, exosomes from GBM ceils will be isolated and their ability to alter the growth of immortalized but otherwise healthy astroglia (SVGpl 2) will be confirmed. Previous studies have established that TDE from GBM transfer enhanced the proliferative capacity of other cells (Skog et al. Nat Cell Biol. 2008, 10(12): 1470-6). The neutralizing ability of the constructs will be assessed by evaluating the effects of coculturing astroglia and TDEs of the anti-CD47 T cell products - using abrogation in proliferative enhancement, measured by CFSE, as the endpoint.

Other considerations in these experiments include altering the different components of the constructs: signal peptides by using anti-CD47 scFv in lieu of SIRP- ectodomain, or designing an actual antibody based on current CD47 antibodies. Further, secreted antibodies do not disrupt TAMs or bind to TDEs. If secreted antibodies have significantly lower binding affinity than expected, it is possible that antibody folding or glycosylation may have been altered when the T ceils generated these proteins. This explanation will be pursued by introducing several modifications into the antibody sequence in an effort to negate any T cell- mediated abnormalities (e.g. insertion point mutations, especially in the heavy chain and residues that are modified b glycosylation).

The role of endogenous immune responses in mediating clinical antitumor activity has been demonstrated in studies using third-party EBV-specific T cells. Despite poor persistence in vivo, these cells were nevertheless able to stimulate an endogenous immune response, which led to tumor clearance and measurable clinical responses (Rouce RH, et al. ISCT Annual Conference, 2017; London, United Kingdom: Cytotherapy.). In addition, the use of immune-modulating agents (IL2 and, more recently, checkpoint inhibitors) showed antitumor efficacy, albeit with severe immune-related adverse effects, as these agents nonspecificaily activate the immune system (1). Cancer vaccines provide more specific stimulation, but so far, they have not shown promising effects against complex, established malignancies (Lizee G, et al. Annu Rev Med. 2013;64:71-90). The TRAENNE platform described herein effectively provides more specific stimulation: essentially an in situ vaccination that restimulates a specific segment of immunity. Because the cells actively home to the site of the disease and secrete their anti-CD47 cargo there, immunity is initiated only against tumor targets.

The same TRAE ES described in the previous section will be used, and the ability of their secreted products to bind to CD47 expressed in GBM will be measured. Whether these bound tumors are (1) more susceptible to K-mediated attack using chromium release assays, (2) whether cocultured M2 macrophages revert to Ml macrophages and mediate phagocytosis of GBM (labeled with GFP) using flow cytometry - checking for markers of M2 to Ml conversion and uptake of GFP signal, and (3) whether dendritic cells cocultured with GBM (in the presence or absence of our anti-CD47 T cell products) display increased maturation markers, increased secretion of IL12, and an increased ability to expand GBM- specific T cells that can subsequently lyse our tumors will be assessed.

It is expected that the anti-CD47 construct secreted by the modified T cells will be able to bind to CD47-expressing tumors and recruit NK cells, phagocytes, and dendritic cells, however, it is possible that if the secreted antibodies do not demonstrate ADCC, mutations will be introduced in Fc that encourage cytotoxicity (GASDALIE variants) (Ahmed AA, et al. J Struct Biol. 2016; 194(l):78-89; Davis ZB, et al. Semin Immunol. 2017;31 :64-75). If this fails, it is a possibility that T cell processing of the protein introduces some alterations in the Fc region that prevent optimal ADCC activity. If so, the entire Fc region may be changed into one more ideal for ADCC (e.g., IgG3 subclass) (Wren LH, Set al. Vaccine, 2013;31(47):55G6-17), or a designed will be used that has been developed for bispecific killer cell engagers (BiKEs - basically scFv to CD 16) (Davis ZB, et al. Semin Immunol. 201 7;31 :64-75). Because polyclonal antibodies recognizing multiple epitopes also enhance ADCC, it is possible that two antibody constructs (in two separate vectors) will be introduced into the T cells.

Example 7 Although each of the three antitumor immunotherapy strategies described above (targeting neoantigens, neutralizing the microenvironment, enhancing endogenous immunity) is a novel approach that has been used individually to target resistant tumors, the ultimate goal of this proposal is to incorporate all three into a single therapeutic cell . Therefore, TRAENMES will be tested as an entire platform. Its ability to simultaneously target neoantigens and the microenvironment would effectively allow it to target both of the enabling characteristics (genomic instability and pro-tumorigenic innate immune-mediated inflammation) that together underlie the hallmarks of cancer. Hence, the following will be tested: whether TRAENNES can: (1) alter tumor metabolic activity (by XTT assay) and its synthesis of DNA (by flow cytometry), both indicators of sustained proliferative signaling; (2) affect the ability of tumors to form colonies (by colony forming unit assays), a tactic indicative of growth suppressor evasion; (3) improve or maintain expression of Bcl2 and caspase activity (b flow cytometry), a measure of resistance to cel l death, (4) decrease telomere lengths (by PGR telomere length analysis), to estimate replicative immortality; (5) decrease secretion of VEGF (by ELISA) and recruit endothelial cells (HUVEC, through a Boy den chamber assay), both measures of angiogenesis; and (6) decrease tumor cell invasion (by transweil migration assays) to assess metastatic ability (71).

Example 8

Once the TRAENNE platform has shown promise in in vitro experiments, its efficacy will be tested in vivo, in an orthotopic xenogeneic murine model of GBM. Briefly, U87MG GBM cell lines are co-injected with TAMs orthotopically in SCID mice (which have intact NK, phagocytes, and other innate immune populations). TRAENNES will be inj ected via tail vein. The Fc portion of IgGl is also recognized by murine Fc receptors on NK cells and dendritic cells, both of which are present in SCID animals.

The following will be determined: whether TRAENNES (0 traffic to tumor sites, (2) confer a survival advantage over their nontransduced counterparts, and (3) attract murine NK and dendritic cells. Activation of NK cells will be tested ex vivo by isolating NK cells systemicaily from animals and measuring their ability to kill U87MG cells. Although this measures allogeneic killing, murine NK cells are able to lyse cells immediately after isolation only if they have been stimulated in vivo. Stimulation of endogenous murine dendritic cells will be tested by measuring expression of maturation markers and cytokine secretion ex vivo. Statistical A nalysis

T_RA_EN _ES will be compared with their non- transduced counterparts. Phenotype, as defined by T cell subsets and expression of activation and memory markers, will be measured by flow cytometry and expressed as a percentage of CD3+ cells. Comparisons will be analyzed for each pair (unmodified vs altered according to each module) using chi -square analysis. In tests of specificity and function, determined by ΙΡΝγ ELISPOT and ELISA, paired t tests will be used to analyze unmodified vs, modified T cells. Finally, the results of chromium release assays (percent killing at a 20: 1 Effector: Target ratio) to determine cytotoxic potency will be compared by chi-square analysis. The modifications proposed herein should not significantly alter T cell phenotype, specificity, function, and cytotoxicity. A p value of less than 0.05 will be considered significant. For gene modification experiments, comparisons of constructs will be made with each other and with a protein expressed by CHO (or similar cells) as controls. Two-way analysis of variance followed by t test analysis across different pairs (to determine if a superior attribute in one construct is statistically significant) with a Bonferroni correction will be performed for ELISA values. For functional analysis of transgene products, chi~square analysis will be used to compare % killing at a 20: 1 effector: target ratio for ADCC and % neutralization of TAMs and TDEs. A p<0.05 will be considered significant. Paired t tests will be used to compare dendritic cell secretion of IL12 and stimulation of GBM-specific T cells. The optimal construct will be the one allowing the least change in T cell phenotype and function, while demonstrating the most significant binding/neutralization of TAMs and TDEs, and most significant recruitment of endogenous immunity. For other in vitro assays, paired t tests will be used to analyze unmodified vs. modified T cells.

For in vivo assays, the survival times among T_RA_ENNE and non-TRAE E-treated animals will be compared with a log-rank test. Differences in T cell migration will be assessed by comparing the percentages of migrating T cells at tumor sites using McNemar's test. Tumor lysis as measured by IVIS imaging as well as cytokine secretion profile will be assessed with paired t tests. A p value <0.05 will be considered statistically significant. We hypothesize that the median survival times of animals bearing transduced cells will exceed those of control animals by at least 25% upon Kaplan-Meier analysis. We will use 16 animals/ roup to ensure enough observations to detect a significant difference in survival.

[00246] Patients who have been selected to participate and have fulfilled all criteria

(including health screening and informed consent) will be eligible to receive T cell or NK cell products. At the first visit (when the first dose of T cells or NK. cells are administered), complete history and physical exam will be performed. Blood will also be drawn for serum chemistries, CBC, and immune reconstitution studies, and other research studies. A review of labs and clinical findings will be performed by the study PI and members of the patient's primary care team. Depending on the trial, up to six additional doses of cell products may be administered.

1. Study evaluation is performed, which includes reviews of the patient, the protocol, the consent, the potential adverse events, and the research studies to be performed.

2. Blood will be collected to test for CBC, serum chemistries, pregnancy tests for women of childbearing age, tumor and immunological studies, other research studies.

3. Patient is premedicated with diphenhydramine and paracetamol, both according to the institution's guidelines and after careful review by attending physicians and the primary care team.

4. Cells will be administered according to the relevant Standard Operating Procedures (SOPs), depending on the exact product and the disease.

5. Providers will verify that the product to administer matches with the patient, according to SOPs established by the program.

6. Cell products are given by intravenous infusion over 1-10 minutes through a peripheral line. Monitoring will be performed,

7. Infusion site will be assessed, and monitoring will depend on patient's disease status at the time. Outpatients will be monitored a minimum of an hour after infusion. Any averse events will be documented.

8. Study coordinator will keep in contact with the patient and their primary care team.

[00247] All of the references, patent applications, or other documents listed in this application and the Examples section are herein incorporated by reference in their entireties.

REFERENCES

1. Foster, A.E., el al. Antitumor activity of EBV-specific T lymphocytes transduced with a dominant negative TGF-beta receptor. J Im unother 31, 500-505 (2008).

2. Orfanos, C.E., Husak, R., Wolfer, U. & Garbe, C. Kaposi's sarcoma: a reevaluation. Recent Results Cancer R.es 139, 275-296 (1995). 3. Goto, H., et al. Efficacy of anti-CD47 antibody-mediated phagocytosis with macrophages against primary effusion lymphoma. Eur J Cancer 50, 1836-1846 (2014).

4. Chauhan, S., et al. Surface Glycoproteins of Exosomes Shed by Myeloid-Derived Suppressor Cells Contribute to Function. J Proteome Res 16, 238-246 (2017).

5. Alvey, C. & Discher, D.E. Engineering macrophages to eat cancer: from "marker of self CD47 and phagocytosis to differentiation. J Letikoc Biol 102, 31-40 (2017).

6. Colak, S. & Ten Dijke, P. Targeting TGF-beta Signaling in Cancer. Trends Cancer 3, 56-71 (2017).

7. Matlung, H.L., Szilagyi, K., Barclay, N.A. & van den Berg, T.K. The CD47- SIRPalpha signaling axis as an innate immune checkpoint in cancer. Immunol Rev 276, 145- 164 (2017).

8. Weiskopf, K. Cancer immunotherapy targeting the CD47/S1RP alpha axis, Eur J Cancer 76, 100-109 (2017).

9. Bollard, CM., et al. In vivo expansion of LMP 1- and 2-specific T-cells in a patient who received donor-derived EBV-specific T-cells after allogeneic stem ceil transplantation. Leukemia & lymphoma 47, 837-842 (2006).

10. Bollard, CM., et al. Sustained complete responses in patients with lymphoma receiving autologous cytotoxic T lymphocytes targeting Epstein-Barr virus latent membrane proteins. Journal of clinical oncology) : official journal of the American Society of Clinical Oncology 32, 798-808 (2014).

1 1 . Leen, AM., et al. Multicenter study of banked third-party virus-specific T cells to treat severe viral infections after hematopoietic stem cell transplantation. Blood 121, 5113- 5123 (2013).

12. Leen, A.M., et al. Cytotoxic T lymphocyte therapy with donor T cells prevents and treats adenovirus and Epstein-Barr virus infections after haploidentical and matched unrelated stem cell transplantation. Blood 114, 4283-4292 (2009).

13. Leen, A.M., et al. Monoculture-derived T lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals. Nature medicine 12, 1160-1 166 (2006),

14. Piccione, E.G., et al. SIRPa-Antibody Fusion Proteins Selectively Bind and Eliminate Dual Antigen-Expressing Tumor Cells. Cli Cancer Res; 22(20), 5109-5119 (2016).

Claims

A cell comprising a modified TGF-β receptor I or modified TGF-β receptor II (TGFpRI or TGF- RII), a modified IL-10 receptor (IL1.0-R), or a modified IL-6 receptor (IL6R).

The cell of claim 1, wherein the cell is a primary antigen -presenting cell, T-cell, or NK cell from a subject.

The cell of claim 1, wherein the cell is a primary T-cell harvested from a subject or a cell derived from a lymphocyte, T ceil, or NK cell of a subject.

The cell of claim 1, wherein the cell comprises a modified TGF-β receptor I or II that is free of or substantially free of a signaling domain.

The cell of claim 1, wherein the cell comprises a modified IL-10 receptor that is free of or substantially free of a signaling domain.

The cell of claim 1, wherein the cell comprises a modified IL-6 receptor that is free of or substantially free of a signaling domain.

The ceil of claim 1, wherein the cell comprises an exogenous nucleic acid sequence operably linked to a regulatory sequence, such exogenous nucleic acid sequence encoding one or more polypeptide domains capable of binding one or a combination of: TGF-β, IL-10, and IL-6.

The ceil of claim 1, wherein the cell comprises an exogenous nucleic acid sequence operably linked to a regulatory sequence, such exogenous nucleic acid sequence encoding a polypeptide domain capable of binding TGF|3, wherein the polypeptide domain comprises the extracellular portion of ΤΟΡβ^Ι or a functional fragment thereof that is at least 70% homologous to extracellular portion of TGFP-RII.

The ceil of claim 1, wherein the cell comprises an exogenous nucleic acid sequence operably linked to a regulatory sequence, such exogenous nucleic acid sequence encoding a polypeptide domain capable of binding ΤΟΡβ, wherein the polypeptide domain is free of or substantially free of a signaling domain of ΤΟΡβ^Π.

A cell comprising one or a combination of:

(i) an exogenous nucleic acid sequence encoding an amino acid domain capable of binding CD47;

(ii) an exogenous nucleic acid sequence encoding an amino acid domain capable of binding one or a combination of IgG3 and anti-CD16;

(iii) the amino acid domain of (i); and/or (iv) the amino acid domain of (ii).

11. The cell of claim 10, wherein the amino acid domain capable of binding CD47 is an amino acid sequence that comprises at least about 70% sequence identity to SIRPa or 70% sequence identity to a functional fragment of SIRPa.

12. The cell of claim 10, wherein the amino acid domain capable of binding CD47 is an antibody or antibody binding fragment that comprises at least about 70% sequence identity to one or a combination of variable light chains and variable heavy chains encoded by the nucleic acid sequence disclosed herein.

13. The cell of claim 12, wherein the antibody or antibody binding fragment further comprises an amino acid domain capable of binding one or a combination of IgG3 and anti-CD 16.

14. The cell of claim 12, wherein the antibody or antibody binding fragment further comprises an Fc domain capable of recruiting NK ceils in a subject.

15. The cell of claim 10, wherein the cell further comprises:

(i) an exogenous nucleic acid sequence encoding an amino acid sequence that is at least 70% homologous to one or a combination of: CXCRl, CXCR2, CCR4, CCR6, and CXCR4; and/or

(ii) an exogenous amino acid sequence that is at least 70% homologous to the amino acid sequence of CXCR l , CXCR2, CCR4, CCR6, or CXCR4.

16. The cell of claim 10, wherein the cell exhibits expression of one or a plurality of endogenous nucleic acid sequences that encode one or a combination of chemokine receptor chosen from: CXCRL CXCR2, CCR4, CCR6, and CXCR4, said expression being higher than expression of normal CD25+ T-cells isolated from a human sample.

17. The ceil of claim 10, further comprising a modified TGF-β receptor I or modified TGF-β receptor II (ΤΟΡβΜ or TGF-BRH ), a modified IL-10 receptor (ILIO-R), or a modified IL-6 receptor (IL6R).

18. The cell of claim 17, wherein the cell is a primary antigenic-presenting cell, T-cell, or NK cell from a subject.

19. The cell of claim 17, wherein the cell is a primary T-cell harvested from a subject or a cell cionaliy expanded from a lymphocyte of a subject.

20. The cell of claim 17, wherein the ceil comprises a modified TGF-β receptor I or II that is free of or substantially free of a signaling domain.

21. The cell of claim 17, wherein the cell comprises a modified IL-10 receptor that is free of or substantially free of a signaling domain.

22. The cell of claim 17, wherein the cell comprises a modified IL-6 receptor that is free of or substantially free of a signaling domain.

23. The cell of claim 17, wherein the cell comprises an exogenous nucleic acid sequence operably linked to a regulatory sequence, such exogenous nucleic acid sequence encoding one or more polypeptide domains capable of binding one or a combination of: TGF-β, IL-10, and IL-6.

24. The cell of claim 17, wherein the cell comprises an exogenous nucleic acid sequence operably linked to a regulatory sequence, such exogenous nucleic acid sequence encoding a polypeptide domain capable of binding ΤΟΡβ, wherein the polypeptide comprises an extracellular portion of TGFP-RII or a functional fragment thereof that is at least 70% homologous to the extracellular portion of TGFp-RII.

25. The cell of claim 17, wherein the cell comprises an exogenous nucleic acid sequence operably linked to a regulatory sequence, such exogenous nucleic acid sequence encoding a polypeptide domain capable of binding ΤΟΡβ, wherein the polypeptide is free of or substantially free of a si gnaling domain of TGFp-RII.

26. The cell of claim 17, wherein the cell exhibits at least about 25% more expression of CXCR1, CXCR2, CCR4, CCR6, or CXCR4 as compared to a normal CD25+ T-cell isolated from a human sample.

27. A cell exhibiting modified expression of one or a combination of chemokine receptor chosen from: CXCR1, CXCR2, CCR4, CCR6, and CXCR4, as compared to an normal immune effector cell.

28. The cell of claim 27, wherein the cell exhibits at least about 25%> more expression of CXCRl, CXCR2, CCR4, CCR6, and CXCR4 as compared to a normal CD4+ T-cell isolated from a human sample.

29. The cell of claim 28, wherein the expression level is measured by flow cytometry.

30. A cell comprising:

(i) a nucleic acid sequence encoding an amino acid domain capable of binding CD47; and

(ii) a nucleic acid sequence encoding an amino acid domain capable of binding one or a combination of IgG3 and anti-CD46;

wherein the cell exhibits expression of a modified TGF-β receptor domain capable of binding to TGF-β; and wherein the cell exhibits modified expression of a chemokine receptor domain capable of binding one or a combination of ligands.

31 . The cell of any of claims 1 through 30, wherein the cell is a T-cell or NK cell .

32. The cell of any of claims 1 through 30, wherein the cell is a T-cell or NK cell from a donor subject.

33. The cell of any of claims 1 through 30, wherein the cell is capable of secreting an antibody or antibody binding fragment that comprises at least about 70% sequence identity to one or a combination of variable light chains and variable heavy chains encoded by the nucleic acid sequence disclosed herein that binds CD47.

34. The ceil of claim 33, further comprising a receptor on its surface capable of binding one or a combination of tumor or viral antigens from Table 1.

35. The cell of claim 34, wherein the tumor antigens are chosen from:

H3K27M, DNAJB 1 -PRKAC A, bcr-abl, CDK4, MUMl, CTNNB 1, CDC27, TRAPPC1 , TPI, ASCC3, HHAT, FN1 , OS-9, PTPRK, CDKN2A, HLA-A11, GAS7, SIR2, PrdxS, CLPP, PPP1R3B, EF2, ACTN4, ME1, NF-YC, HSP70-2, KIAA1440, CASP8, gag, pol, nef, env, survivin, MAGEA4, SSX2, FRAME, NYESOl, Oct4, Sox2, Nanog, WT1, p53, MYCN, and combinations thereof. The cell of claim 34, wherein the viral antigen comprises one or a combination of:

(i) Cytomegalovirus (CMV) antigens comprising pp65, IE1, IE1, UL40, UL103, UL151, UL153, UL28, UL32, UL36, UL55, 1 40, UL48, UL82, UL94, UL99, us24, us32, and us32;

(ii) Helves simplex virus (HSV) antigens comprising glycoprotein G;

(iii) Epstein-Barr virus (EBV) antigens comprising BARF1, BMLF1, BMRF1, BZLF1, EBNALP, EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, gp350/340, I M P ! , and LMP2;

(iv) Human herpesvirus-8 (HHV8) antigens comprising LNA-1, LANA-1, viral cyclin D, vFLIP, and RTA;

(v) Human papillomavirus strain 16 (HP VI 6) antigens comprising E6, E7, and

(vi) Human papillomavirus strain 18 (HPV18) antigens comprising E6 and E7;

(vii) vIL6, kaposin B, and ORF74.

37, A composition comprising one or a plurality of cells of claims 1 through 36.

38. A pharmaceutical composition comprising: (i) a pharmaceutically effective amount of one or a plurality of cells of any of claims 1 through 36; and

(ii) a pharmaceutically acceptable carrier.

39. An isolated nucleic acid sequence comprising any one of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28.

40. A method for inducing an antigen-specific immune response in a T-celi, the method comprising:

(a) culturing one or a plurality of isolated T-cells;

(b) stimulating the one or plurality of T-cells with at least one cytokine;

(c) exposing the one or plurality of T-cells to antigen-presenting cells exposed to one or a combination of tumor antigens;

(d) transducing the one or plurality of T-cells with one or a plurality of vectors comprising one or more nucleic acid sequences encoding an antibody or antibody binding fragment disclosed herein.

41. The method of claim 40, further comprising transducing the one or plurality of T-cells with one or a plurality of vectors comprising one or more nucleic acid sequences encoding one or more polypeptide domains capable of binding one or a combination of: TGFp, 11.- 10, and 11.-6.

42. The method of claim 40, further comprising exposing the one or plurality of cells to a hypoxic environment at an oxygen concentration and for a time sufficient to induce expression of HIF-1 and/or NFkB.

43. The method of claim 40, wherein the antibody or antibody binding fragment comprises at least one variable heavy chain domain or at least one variable light chain domain comprising a CDR capable of binding one or a combination of CD47 and SIRPa,

44. The method of claim 40, wherein the one or combination of tumor antigens is an amino acid sequence at least 70% homologous to H3K27M, DNAJB1-PRKACA, bcr- abl, CDK4, MIJM , CTNNB1, CDC27, TRAPPC1, TPI, ASCC3, HHAT, FN1, OS-9, PTPRK, CDKN2A, HLA-A11, GAS7, SIR2, Prdx5, CI . PP. PPP1R3B, EF2, ACTN4, ME! , NF-YC, HSP70-2, KIAA1440, CASP8, gag, pol, nef, env, survivin, MAGEA4, SSX2, FRAME, NYESOl, Oct4, Sox2, Nanog, WT1, p53, MYCN, or combinations thereof.

45. A method of treating a hyperproliferative disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of claim 37 or the pharmaceutical composition of claim 38.

46. A method of preventing progression of cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of claim 37 or the pharmaceutical composition of claim 38.

47. A method of preventing the onset of a hyperproiiferative disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of claim 37 or the pharmaceutical composition of claim 38.

48. A method of targeting and/or killing a hyperproiiferative cell in a subject, the method comprising administering to the subject a therapeutically effective amount of the composition of claim 37 or the pharmaceutical composition of claim 38,

49. The method of any of claims 45 through 48, wherein the step of administering comprises administering the composition or pharmaceutical composition intravenously, intraparentally, topically, irrigation of wounds either as wound dressing or in sterile solution, intradermally, intramucosally, subcutaneously, sublingually, orally, intravaginally, intramuscularly, intracavernously, intraoculariy, intranasally, into a sinus, intrarectally, intracranially, gastrointestinally, intraductally, intrathecally, subdurally, extradural!}', intraventricular, intrapulmonary, into an abscess, intra articularly, into a bursa, subpericardially, into an axilla, intrauterine, into the pleural space, or intraperitoneally.

50. A method of manufacturing a modified T-cell or modifying primary human lymphocytes, the method comprising:

(a) cuituring one or a plurality of isolated T-cells;

(b) stimulating the one or plurality of T-cells with at least one cytokine;

(c) exposing the one or plurality of T-celis to antigen-presenting cells exposed to one or a combination of tumor antigens;

(d) transducing the one or plurality of T-cells with one or a plurality of vectors comprising one or more nucleic acid sequences encoding an antibody or antibody binding fragment disclosed herein.

51. The method of claim 50, further comprising transducing the one or plurality of T-ceils with one or a plurality of vectors comprising one or more nucleic acid sequences encoding one or more polypeptide domains capable of binding one or a combination of: TGFp, IL-10, and IL-6.

52. The method of claim 50, wherein the method further comprises isolating a sample from a subject prior to step (a) and isolating the one or plurality of T-eells.

53. The method of any of claims 50 through 52, wherein steps (a) through (d) are performed ex vivo in a sterile chamber.

54. The method of claim 40, further comprising administering the one or plurality of T- cells to a subject comprising a hyperprol iterative cell.

55. The method of claim 44, further comprising administering the one or plurality of T- cells to a subject comprising a hyperproliferative cell expressing one or a combination of tumor antigens chosen from amino acid sequences at least 70% homologous to H3K27M, DNAJB 1 -PRKAC A, bcr-abl, CDK4, MUM1, CTNNB 1, CDC27, TRAPPCl, TPI, ASCC3, HHAT, FN1, OS-9, PTPRK, CDKN2A, HLA-A1 1 , GAS7, SIR2, PrdxS, CLPP, PPP1R3B, EF2, ACTN4, ME1, NF-YC, HSP7Q-2, KIAA1440, CASP8, gag, pol, nef, env, survivin, MAGEA4, SSX2, FRAME, NYESOl , Oct4, Sox2, Nanog, WT1, p53, or MYCN.

56. A method of manufacturing a modified NK cell or modifying a mononuclear cell, the method comprising:

(a) culturing one or a plurality of mononuclear cells;

(b) stimulating the one or plurality of mononuclear cells with at least one cytokine;

(c) isolating the one or plurality of NK cells;

(d) expanding the NK cells in culture;

(e) transducing the one or plurality of NK cells with one or a plurality of vectors comprising one or more nucleic acid sequences encoding an antibody or antibody binding fragment disclosed herein.

57. The method of claim 56, further comprising transducing the one or plurality of NK cells with one or a plurality of vectors comprising one or more nucleic acid sequences encoding one or more polypeptide domains capable of binding one or a combination of: TGFp, IL-10, and IL-6.

58. The method of claim 50, wherein the method further comprises isolating a sample from a subject prior to step (a) and isolating the one or plurality of mononuclear ceils.

59. The method of any of claims 56 through 58, wherein steps (a) through (d) are performed ex vivo in a sterile chamber.