WO2019191752A1

WO2019191752A1 - Methods for pre-conditioning patients for t-cell therapy

Info

Publication number: WO2019191752A1
Application number: PCT/US2019/025137
Authority: WO
Inventors: Irvin S.Y. Chen; Sanggu Kim
Original assignee: Ohio State Innovation Foundation
Priority date: 2018-03-30
Filing date: 2019-04-01
Publication date: 2019-10-03

Abstract

Disclosed are methods and compositions related to pre-conditioning a subject for T cell therapy. In one aspect, disclosed herein are methods of treating a cancer or HIV infection in a subject comprising administering to the subject a T cell targeting immunotoxin (such as, for example an anti-CD3 immunotoxin) and therapeutic T cells (such as, for example, CAR T cells). Disclosed herein are methods of pre-conditioning a subject with HIV or a cancer for CAR T cell therapy comprising administering to the subject an anti-CD3 immunotoxin.

Description

METHODS FOR PRE-CONDITIONING PATIENTS FOR T-CELL THERAPY

This application claims the benefit of U.S. Provisional Application No. 62/650,605, filed on March 30, 2018, which is incorporated herein by reference in its entirety. This invention was made with government support under Grant No. R56 HL126544, ROl HL125030, and ROl All 10297, awarded by the National Institutes of Health . The government has certain rights in the invention.

L BACKGROUND

1. Recent advances in T-cell therapy, including CAR T-cell therapy, have generated tremendous hope for the treatment of previously incurable diseases, such as cancers and

HIV/AIDS. Human T-cell therapies rely on enriched or modified human T-cells to target and kill disease cells in a patient, but insufficient repopulation and dysfunction of therapeutic T-cells following transplantation has been a critical limiting factor. Various methods have been developed to enhance the survival and function of the transferred T-cells in patients by depleting lymphoid cells to create a favorable“space” for the transferred cells. These therapies have proven to be effective in improving the efficacy of T-cell therapies, reducing tumor size and improving patient survival. Current lymphodepleting pre-conditioning methods, however, rely on high doses of toxic and non-specific chemotherapies and often result in variable therapeutic efficacy of CAR T cells in patients, sometimes even causing deadly adverse events. Such inconsistencies and toxicity remain a major clinical hurdle. What is needed are new safe and effective pre-conditioning regimens for improved T ceil therapy.

IL SUMMARY

2. Disclosed are methods and compositions related to pre-conditioning a subject for T- celi therapy.

3. In one aspect, disclosed herein are methods of treating a cancer or HIV infection in a subject comprising administering to the subject a T cell targeting immunotoxin (such as, for example an anti-CD3 immunotoxin) and therapeutic T cells (such as, for example, CAR T cells).

4. Also disclosed are method of treating a cancer or HIV of any preceding aspect, wherein the anti-CD3 immunotoxin comprises a diphtheria toxin (DT), ricin toxin, Pseudomonas enterotoxin A (ETA), saporin, Alpha-sarcin, restictocin, human pancreatic ribonuclease A (HPR), eosinophilic cationic protein (ECP), eosinophil-derived neurotoxin (EDN), botulinim toxin, cholera toxin, Clostridium difficile toxin, Clostridium perfringens toxin, Aerolysin, Cereolysin, Listeria listeriolysin, Listeria hemolysin, Shiga toxin, saxitonix, pheumolysin, tetanus toxin, Gelonin, or any mutants or fragments thereof (such as, for example diphtheria toxin fragments and mutants selected from the group consisting of DT483, DT390 (for example RES IMMUNE® (A-dmDT390-bisFv; Angimmune LLC) and C207 (A-dmDT390-scfbDb)), DT389, DT383, DT370. CRM9, CRM107, CRM 103, CRM197, MSPA5, and DTM1)

5. In one aspect, disclosed herein are methods of treating a cancer or HIV of any preceding aspect, wherein the administration of the immunotoxin ceases at least 2 days prior to administration of CAR T cells.

6. Also disclosed are method of treating a cancer or HIV of any preceding aspect, wherein the immunotoxin is administered at least one time per day for at least two, three, or four days.

7. In one aspect, disclosed herein are methods of pre-conditioning a subject with HIV or a cancer for CAR. T cell therapy comprising administering to the subject an anti-CD3

immunotoxin.

8. Also disclosed are methods of preconditioning a subject of any preceding aspect, wherein the anti-CD3 immunotoxin comprises a diphtheria toxin (DT), ricin toxin, Pseudomonas enterotoxin A (ETA), saporin, botulinim toxin, cholera toxin, Clostridium difficile toxin, Clostridium perfringens toxin, Aeroiysin, Cereolysin, Listeria listen oly sin, Listeria hemolysin, Shiga toxin, saxitonix, pheumolysin, tetanus toxin, or any mutants or fragments thereof (such as, for example diphtheria toxin fragments and mutants selected from the group consisting of DT483, DT390, DT389, DT383, DT370. CRM9, CRM107, CRM103, CRM197, MSPA5, and DTM1).

9. In one aspect, disclosed herein are methods of preconditioning a subject of any- preceding aspect, wherein the administration of the immunotoxin occurs at least 2 days prior to administration of CAR T cells.

10. Also disclosed are methods of preconditioning a subject of any preceding aspect, wherein the immunotoxin is administered at least one time per day for at least two days.

III. BRIEF DESCRIPTION OF THE DRAWINGS

11. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

12. Figures LA and IB show the specific and effective T-cell depletion with CD3e- immunotoxin. The effects of CD3e-IT (murine anti-CD3e monoclonal antibody-saporin) on local tissue lymphocytes were analyzed in comparison to those of CTX in wild-type C57BL/6 (B6) mice. Diphtheria toxin (DT)-mediated T-cell ablation model (CD4-iDTR B6 mice) was

Ί also used as a control. Figure 1 A shows a schematic view of the experiments. Wild-type

C57BL/6 (B6) mice were treated with CTX (300mg/kg; Day 1) and CD3e-IT (15pg/mouse x 2/day x 4 days; Day 1 to Day 4). PBS was injected as a control. CD4-Cre-inducible diphtheria toxin receptor mice (CD4-iDTR mice: Buch, T., et al. Nat Methods, 2005) - these mice express diphtheria toxin receptors on CD4+ T cell surface --- were treated with Diphtheria Toxin

(200ng/mouse x 2/day x 4 days; Day 1 to Day 4) to specifically ablate T-cells. Organs were collected and analyzed at Day 6 Figure I B shows the relative frequency of blood lineages shown as percentage of CD45+ cells (y-axis), including CD3+ T cells (blue bars), T helper cells, cytotoxic T cells (grey bars), and B cells (orange bars). In CD3 e-IT -treated mice, CD4+ and CD8+ T cells declined almost 14- to 20 fold and 18- to 29-fold, respectively. Bone marrowy thymus, and liver showed similar results.

13. Figures 2A and 2B show the effect of CD3e-immunotoxin on body weight and organ weight. Figure 2A show's the changes in the body weight of test mice over time. CD3e- immunotoxin was administered from Day 1 to Day 4 (n=5). CTX was administered at Day 1 (n=6). Body weights were normalized based on Day 0 data (Day 0 = 100%). Figure 2B shows the impact of CTX and CD3e-IT on body organs. The weight of spleen, liver, thymus, and one rear femur at Day 6 (the second day after the end of CD3e-IT treatment) are shown as a % of body weight. The numbers of recovered mesenchymal lymph nodes (MLN) and Peyer’s Patches per animal were recorded at the same day.

14. Figures 3A and 3B show the survival of transplanted cells in CD3e-immunotoxin- treated mice. Figure 3A shows the changes in the body weight over time PBS (light blue) or

CD3e-IT were administered from Day 1 to Day 4. At Day 6, a different number of donor cells (a

4: 1 mixture of spleen ceils from tdTomato transgenic B6 mice and lymph node cells from eYFP transgenic B6 mice) were infused into CD3e-IT treated mice. Mice infused with 5xl0⁶ (green; n=2), 5x10^s (orange; n=2), 5xl0⁴ (brown; n=2), 5xl0³ (red; n=2), and 0 donor cells (black, n=3) were analyzed. Body weights were normalized based on Day 1 data (Day 1 = 100%). Figure 3B shows the repopulation of tdTomato and eYFP (yellow) cells compared with the repopulation of total cells (including the surviving endogenous cells and transplanted tdTomato and eYFP cells) in 5xl0⁶ and 5x10^s transplanted mice. The left panels show CD3+ T cell repopulation. tdTomato

(red) and eYFP (yellow⁷) cells are shown as a % of total CD3+ T cells (right y-axis). The total

CD3+ T cells (black) are shown as a % of total CD45+ cells (left y-axis) for comparison. The middle panels show CD3+CD4+ T cell repopulation. tdTomato (red) and eYFP (yellow) cells are shown as a % of CD3+CD4+ T cells (right y-axis) and total CD3+CD4+ cells (black) are shown as a % of total CD45 cells (left y-axis). ). The right panels show CD3+CD8+ T cell repopulation. tdTomato (red) and eYFP (yellow') cells are shown as a % of CD3+CD8+ T cells (right y-axis) and total CD3+CD8+ cells (black) are shown as a % of total CD45 cells (left y- axis).

15. Figure 4 shows the specific and effective depletion of CD3+ T cells in rhesus macaques after CD3e-immunotoxin treatment. Two animals (RA1209 and RAI 174) w_'ere treated with primate anti-CD3e-immunotoxin (C207) for four days and tissues were collected 3 days post-CD3e-immunotoxin. CD3+ T cells (red), CD20+ B cells (grey), CD 14+ monocytes (yellow), CD3-CD141owCD20~ (blue) and CD3-CD14-CD20- cells (black) are shown for bone marrow⁷ (BM), four different lymph nodes (LNI, LN13, and LN15 for RA1209; LN1, LN4, and LN7 for RAI 174), and peripheral blood (PB). As a baseline control, bone marrow⁷, inguinal lymph node, and peripheral blood were analyzed two months prior to CD3e-immunotoxin treatment.

16. Figure 5 shows rapid recover)⁷ of CD.3+ T-cell count following CD3e-immunotoxin treatment. Three rhesus macaques (95E132, 2RC003, and RQ5427) were treated with CD3e- immunotoxin (C207) for four days, and peripheral blood was collected over time for flow cytometry analysis. CD3+ T cells (orange), CD2Q+ B cells (grey), CD 14+ monocytes (yellow⁷), and CD16+CD56- Natural Killer cells (NK cells, denoted in blue) were analyzed by flow cytometry over time. Total cell count per microliter was calculated based on both the total lymphocyte complete blood count (CBC) and lymphocyte gates in flow' cytometry analysis. Total CD3+ T-cell counts were recovered within I to 2 months following CD3e-immunotoxin treatment.

IV. DETAILED DESCRIPTION

17. Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

A. Definitions

18. As used in the specification and the appended claims, the singular forms“a,”“an” and“the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to“a pharmaceutical earner” includes mixtures of two or more such carriers, and the like. 19. Ranges can be expressed herein as from“about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself For example, if the value“10” is disclosed, then“about 10” is also disclosed. It is also understood that when a value is disclosed that“less than or equal to” the value,“greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value“10” is disclosed the“less than or equal to 10” as well as“greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combinati on of the data points. For example, if a particular data point“10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then I I, 12, 13, and 14 are also disclosed.

20. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings;

21.“Optional” or“optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

22. The term“subject” is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In some embodiments, the subject is a human.

23. Administration” to a subject includes any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g , subcutaneous, intravenous, intramuscular, intra-articuJar, intra-synovial, intrastemal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. "Concurrent administration", "administration in combination", "simultaneous administration" or "administered simultaneously" as used herein, means that the compounds are administered at the same point in time or essentially immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time.“Systemic administration” refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g. greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast,“local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area

immediately adjacent to the point of administration and does not introduce the agent

systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration, but are undetectable or detectable at negligible amounts in distal parts of the subject’s body. Administration includes self-administration and the administration by another.

24.“Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is“effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified“effective amount.” However, an appropriate“effective amount” in any subject case may be determined by one of ordinary' skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an“effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An“effective amount” of an agent necessary' to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation

25. A "decrease" can refer to any change that results in a smaller gene expression, protein expression, amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

26. "Inhibit," "inhibiting,” and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

27. The terms“prevent,”“preventing,”“prevention,” and grammatical variations thereof as used herein, refer to a method of partially or completely delaying or precluding the onset or recurrence of a disease and/or one or more of its attendant symptoms or barring a subject from acquiring or reacquiring a disease or reducing a subject’s risk of acquiring or reacquiring a disease or one or more of its attendant symptoms.

28. "Pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a

pharmaceutical formulation of the invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained . When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration

29. "Pharmaceutically acceptable carrier" (sometimes referred to as a“carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein. 30.“Pharmacologically active” (or simply“active”), as in a“pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.

31.“Therapeutic agent” refers to any composition that has a beneficial biological effect.

Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

32.“Therapeutically effective amount” or“therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of type I diabetes. In some embodiments, a desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a. therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.

33. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

34. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

35. Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular immunotoxin is disclosed and discussed and a number of modifications that can be made to a number of molecul es including the immunotoxin are discussed, specifically contemplated is each and every combination and permutation of immunotoxin and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C- D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

B. Methods of treating cancer or HIV infection

36. In one aspect, it is understood and herein contemplated that the disclosed immunotoxins can be used to modify a tumor microenvironment to pre-condition the tumor for T cell therapy in the treatment of a cancer. Cancer immunotherapy has been advanced in recent years; genetically-modified chimeric antigen receptor (CAR) T cells are an excellent example of engineered immune cells successfully deployed in cancer immunotherapy. These cells were recently approved by the FDA for treatment against CD 19 + B cell malignancies, but success has so far been limited to diseases bearing a few targetable antigens, and targeting such limited antigenic repertoires is prone to failure by immune escape. Furthermore, CAR T cells have been focused on the use of autologous T cells because of the risk of graft-versus-host disease caused by allogeneic T cells. Recent advances in Chimeric Antigen Receptor (CAR) T-cell therapy have generated tremendous hope for the treatment of incurable diseases, including HIV/ AIDS.

Cytotoxic lymphodepletion preconditioning improves the efficacy of CAR T-cells by creating a favorable“lymphoid space” for the enhanced survival and function of the transferred T-cells via depletion of T-cells and“cytokine sink” cells. Lymphodepleting preconditioning is now included in most CAR T-cell therapies, but the current chemotherapeutic regimen has yielded inconsistent results among patients, and the risk of premature implementation has been poignantly demonstrated by the recent deaths in CD19 CAR T-cell preclinical trials. It is understood and herein contemplated that the disclosed immunotoxins a pre-conditioning treatment regimens address this need. Accordingly, disclosed herein are methods of treating a cancer and/or treating HIV comprising administering to a subject an immunotoxin (such as for example an anti-CD3e immunotoxin including but not limited to RES IMMUNE®) and a CAR T cell. In one aspect, it is understood that the administration of the immunotoxin creates immunological space that can improve the safety and efficacy of an immunotherapy. Thus, in one aspect, disclosed herein are methods of pre-conditioning a HIV-infected patient for immunotherapy, and/or pre-conditioning a tumor microenvironment for an immunotherapy (such as, for example a T cell therapy including the administration of CAR T cells) comprising administering to a subject an immunotoxin (such as for example an anti-CD3e immunotoxin including but not limited to RESIMMUNE®).

37. The methods of treating a cancer, methods of treating HIV, and/or pre-condition treatments disclosed herein utilize immunotoxins to target and lyse endogenous T cells in the tumor microenvironment prior to administration of the immunotherapy (such as, for example, a T cell therapy including CAR T cells). As used herein,“immunotoxin” refers to a toxin moiety operatively linked to a targeting moiety (such as, for example an antibody including but not limited to polyclonal antibodies, monoclonal antibodies, diabodies, triabodies, antibody fragments, or any combination thereof). In one aspect, the targeting moiety can be a T cell, B cell, or NK cell targeting moiety. For example, the targeting moiety' can target T cells by targeting CD2, CDS, CD7, CD4, CDS, ONTAK, and/or CD25 or target T cells and/or NK cells by specifically binding to CDS. In one aspect, the targeting moiety can be an anti-CD3 antibody such as, for example UCHT1. In another aspect, the targeting moiety can be fragment of variant of an anti-CD3 antibody (for example UCHT1 ) such as, for example, a single chain variable fragment (scFv), a diabody, a triabody, a two tandem unit of scFv (biscFv), or a single chain foldback diabody (scfbDb). CD3e-immunotoxins are shown herein as a potential pre conditioning regimen for adoptive T-cell therapy using mouse models. CD3e-immunotoxins can be a safer and more effective option for pre-conditioning showing greater efficiency and precision in killing T-celis than cyclophosphamide (CTX), a chemical preconditioning agent currently used in clinic (Fig. 1).

38. Given that the goal of lymphodepietion pre-conditioning for T-cell therapies is not only the creation of appropriate conditions for engraftment but also the direct elimination of malignant and pathogenic populations in anti-HIV/ AIDS and certain cancer therapies, CD3e- immunotoxin monotherapy can provide optimum pre-conditioning in particular for the treatment of diseases that require killing diseased T cells, such as HIV/AIDS and T-cell lymphomas.

39. It is understood and herein contemplated that in order to lyse the target cells in the disclosed methods of treating a cancer, methods of pre-conditioning a tumor microenvironment, and/or methods of treating HIV; the disclosed immunotoxins comprise toxin moiety linked to the targeting moiety. Examples of suitable toxins include diphtheria toxin (DT), ricin toxin,

Pseudomonas enterotoxin A (ETA), saporin, botulinim toxin, cholera toxin, Clostridium difficile toxin, Clostridium perfringens toxin, Aerolysin, Cereolysin, Listeria listeriolysin, Listeria hemolysin, Shiga toxin, saxitonix, pheumolysin, tetanus toxin, or any mutants or fragments thereof.

40. Protein toxins used in the constructions of immunotoxins have an A and a B subunit.

The A subunit catalyzes the inactivation of protein synthesis, resulting ultimately in ceil death. The B subunit has two functions: it is responsible for toxin binding to the cell surface, and it facilitates the translocation of the A chain across the membrane and into the cytosol, where the A chain acts to kill cells.

41. Previously, two general types of immunotoxins have been used. Immunotoxins made with the complete toxin molecule, both A and B chains, have the complication of non-specific killing mediated by the toxin B chain binding site. This can be avoided by eliminating the B chain and linking only the A chain to the antibody. However, A chain immunotoxins, although more specific, are much less toxic to tumor cells. The B chain, in addition to having a binding function, also has an entry function, which facilitates the translocation of the A chain across the membrane and into the cytosol. Since A-chain immunotoxins lack the entry function of the B chain, they are less toxic than their intact toxin counterparts containing the complete B chain. An ideal toxin for immunotoxin construction would contain the A chain enzymatic function and the B chain translocation function, but not the B chain binding function. 42. Some toxins have been modified to produce a suitable immunotoxin. The two best known are ricin and diphtheria toxin. Antibodies which bind cell surface antigens have been linked to diphtheria toxin and ricin, forming a new pharmacologic class of cell type-specific toxins. Ricin and diphtheria toxin are 60,000 to 65,000 dalton proteins with two subunits: the A- chain inhibits protein synthesis when in the cytosol, and the B-chain binds cell surface receptors and facilitates passage of the A subunit into the cytosol. Two types of antibody -toxin conjugates (immunotoxins) have been shown to kill antigen-positive cells in vitro. Immunotoxins made by binding only the toxin A subunit to an antibody have little non-target cell toxicity, but are often only minimally toxic to antigen-positive cells. Another type of immunotoxin is made by linking the whole toxin, A and B subunits, to the antibody and blocking the binding of the B subunit to prevent toxicity to non-target cells. For ricin, the non-target cell binding and killing can be blocked by adding lactose to the culture media or by steric restraint imposed by linking ricin to the antibody. Intact ricin immunotoxins may have only 30- to 100- fold selectivity between antigen-positive and negative cells, but they are highly toxic, and the best reagents can specifically kill a great many target cells.

43. Intact ricin and ricin A-chain immunotoxins have been found to deplete allogenic bone marrow of T cells, which can cause graft-versus-host diseases (GVHD), or to deplete autologous marrows of tumor cells.

44. Diphtheria toxin is composed of two disulfide-linked subunits: the 21,000 dalton A- chain inhibits protein synthesis by catalyzing the ADP-ribosylation of elongation factor 2, and the 37, 000-dalton B-chain binds cell surface receptors and facilitate transport of the A-chain to the cytosol. A single molecule of either a diphtheria toxin A-chain or a ricin A-chain in the cytosol is sufficient to kill a cell. The combination of these three activities, binding,

translocation, and catalysis, produces the extreme potency of these proteins. The cell surface- binding domain and the phosphate-binding site are located within the carboxyl-terminal 8-kDa cyanogen bromide peptide of the B-chain. Close to the C-terminus region of the B-chain are several hydrophobic domains that can insert into membranes at low pH and appear to be important for diphtheria toxin entry .

45. Antibodies directed against cell surface antigens have been linked to intact diphtheria toxin or its A subunit to selectively kill antigen-bearing target cells. Antibody-toxin

(immunotoxins) or ligand toxin conjugates containing only the diphtheria A-chain have relatively low cytotoxic activity. Intact diphtheria toxin conjugates can be very potent, but can also have greater toxicity to normal cells. Since the B-chain appears to facilitate entry of the A- chain to the cytosol, it is possible that its presence in whole toxin conjugates renders them more

Iz potent, although less specific. Efforts have been made to construct more potent and specific immunotoxins by separating the toxin B-chain domains involved in cell binding from the domains involved in A-chain entry.

46. Target cell toxicity of immunotoxins can be increased by including the toxin B-chain in the antibody -toxin complex or by adding it separately. To achieve maximal in vitro target-cell selectivity with immunotoxins containing intact ricin, lactose must be added to the mediu to block non-target-cell binding and toxicity of the immunotoxin via the ricin B-chain. This approach is feasible in those clinical settings, such as bone marrow transplantation, where the target cell population can be incubated in vitro in the presence of lactose. Without blockage of the B-chain binding domain, however, whole toxin conjugates have a high degree of non-target- cell toxicity, thereby limiting their usefulness in vivo.

47. Construction of reagents that combine the potency of intact toxin conjugates with the cell-type selectivity of toxin A-chain conjugates may be possible if the binding site on the toxin B-chain could be irreversibly blocked. Covalent and noncovalent chemical modifications that block the binding activity of ricin intraeel!ularly also block its entry function, suggesting that the binding and translocation functions may be inseparable.

48. Previously, domain deletion was unsuccessfully used in an attempt to separate the translocation and the binding functions of diphtheria toxin B-chain. Immunotoxins made with the A-chain, intact diphtheria toxin, and a cloned fragment of diphtheria toxin (MspSA) that lacks the C-terminal 17-kDa region of the B subunit were compared. The intact diphtheria conjugate was 100 times more toxic than the MspSA conjugate was, which, in turn, was 100- fold more toxic than was the diphtheria toxin A-chain conjugate. The C-terminal, 17-kDa region, which contains the ceil surface binding site, therefore potentiates immunotoxin activity 100-fold. It has not been possible to determine whether this C-terminal translocation activity was distinct from the binding activity.

49. Laird and Groman, J. Virol. 19: 220 (1976) mutagenized Corynebacteriurn with nitrosoguanidine and ultraviolet radiation and isolated several classes of mutants within the diphtheria toxin structural gene. Leppla and Laird further characterized several of the mutant proteins and found that three of them, CRM 102, CRM103, and CRM 107, retained full enzymatic activity but had defective receptor binding.

50. Although cleavage of ricin or diphtheria toxin into A and B-chains had been thought to improve the specificity of the immunotoxins produced from the A-chain, cleavage of ricin or diphtheria toxins into A and B-chains removes the portion of the molecule containing residues important for transport into the cytosol of the cell. Specific cytotoxic reagents made by coupling toxin A subunits to antibodies have low systemic toxicity but also very low tumor toxicity. More potent reagents can be made by coupling intact toxins to monoclonal antibodies, as detailed in J Immunol. 136: 93-98 and Proc. Natl. Acad. Sci. USA 77: 5483-5486. These reagents, however, have a high systemic toxicity due to the toxin binding to normal cells, although they can have applications in vitro in bone marrow transplantation (cf. Science 222: 512-515).

51. Examples of DT toxins (such as DTM1) that can be used for the disclosed immunotoxins are not restricted to native DT toxins, but can include mutants and fragments including mutants with point mutations and truncation mutants. The phenotypic designation CRM is used to designate the protein product of a tox gene that is serologically identical with diphtheria toxin, but comprise one or two amino acid substitutions relative to native diphtheria toxin in the C region. For example, CRM 103 comprises a SerlOSPhe mutation, CRM 102 comprises a SerSOBPhe and a Pro308ser. CRM107 comprises Ser525Phe substitution and CRM9 is a binding site mutant of diphtheria toxin. The non-toxic DT mutant CRM 197 comprises a point mutation in the enzymatic chain of DT. DT mutants for use in the disclosed immunotoxins that comprise truncations are indicated by a D or the prefix DT followed by a number, for example, MSPD5 which is a truncation at amino acid 385 and DT390, DT389, DT383, and DT370 which are truncation mutants of DT which each are truncation mutants comprising 390, 389, 383, and 370 residues, respectively, from the N-terminal glycine of mature diphtheria toxin. Additional, substitutions are noted in a parenthecial such as“(Ala)dmDT390,” which has an N-terminal alanine,“dmDT390,” has the N-terminal glycine present in native DT, “(TyrValGluPhe)dmDT390” has a YVEF sequence at its N-terminal.

52. In one aspect, it is understood and herein contemplated that the immunotoxin can comprise known immunotoxins such as, for example, RESIMMUNE® (A-dmDT390-bisFv; Angimrnune LLC) and C207 (A-dmDT390-scfbDb).

53. In one aspect, the therapeutic T cells can be any engineered T cell and/or adoptively transferred T cells, including, but not limited to chimeric antigen receptor (CAR) T cell, tumor infiltrating lymphocyte (TIL), and/or engineered T cell. As used herein“chimeric antigen receptor” refers to a chimeric receptor that targets a cancer antigen and brings to bring the ceil expressing the receptor to a cancer cell expressing the target antigen. Typically, the CAR comprises a natural ligand of the tumor antigen a molecule that recognizes peptides derived from the tumor antigen presented by MHC molecul es, or an antibody or fragment thereof (such as for example, a F(ab’)2, Fab’, Fab, Fv, scFv) expressed on the surface of the CAR cell that targets a cancer antigen. The receptor is fused to a signaling domain (such as, for example the CD3 domain for T cells) via a linker. Tumor antigen targets are proteins that are produced by tumor cells that elicit an immune response, particularly B-cell, NK cell, and T-cell mediated immune responses. The selection of the antigen binding domain will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-llRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-i, MUC1, BCMA, bcr-abl, HER2, b-human chorionic gonadotropin, alphafetoprotein (AFP), ALK, CD 19, CD 123, cyclin Bl, lectin-reactive AFP, Fos- r elated antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RU1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAX5, SART3, CLL-1, fucosyl GM1, Globoli, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, plysialic acid, PLAC1, RU1, RU2 (AS), intestinal carboxyl esterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1 , L AGE-1 a, LMP2, NCAM, p53, p53 mutant, Ras mutant, gplOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2/neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPV E6,E7, sperm protein 17, S SEA-4, tyrosinase, TARP, WT1, prostate-carcinoma tumor antigen- 1 (PCT A-1), ML-IAP, MAGE, MAGE-A1,MAD-CT-1, MAD-CT-2, Mel an A/M ART 1, XAGEl , ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-l, ephnnB2, CD20, CD22,

CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61 , folate receptor (FRa), folate receptor beta, ROR1 , Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. Non-limiting examples of tumor antigens include the following: Differentiation antigens such as tyrosinase, TRP-1 , TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5, overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP- 180, MAGE -4, MAGE-5, MAGE-6, RAGE, NY-ESO, p!85erbB2, p!80erhB-3, c-rnet, nm- 23H1, PSA, IL13Ra2, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15- 3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250,

Ga733 VEpC AM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG1 6, TA-90\Mac-2 binding protein\cyclophilm C-associatecl protein, TAAL6, TAG72, TIP, TPS, GPC3, MI X^' ] 6. LMP1, EBMA-i, BARF-1, CS1, CD319, HER1, B7H6, LI CAM, JL6, and MET.

54. The disclosed compositions can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers A non-limiting list of different types of cancers is as follows: lymphomas (Hodgkins and non-Hodgkins), leukemias, carcinomas, carcinomas of solid tissues, squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas, high grade gliomas, blastomas, neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas, hypoxic tumours, myelomas, AIDS-related lymphomas or sarcomas, metastatic cancers, or cancers in general.

55. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary_' cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon cancer, rectal cancer, prostatic cancer, or pancreatic cancer.

56. In one aspect, the disclosed methods of treating, preventing, inhibiting, or reducing a cancer or metastasis; methods of treating, inhibiting, or reducing HIV; methods of pre conditioning a tumor microenvironment for immunotherapy, and/or methods of preconditioning a subject with HIV for immunotherapy comprise administering to a subject any of the immunotoxins disclosed herein . Said immunotoxins can comprise be administered at any frequency appropriate for the reduction of the target immune cells (such as T cells) in the subject. For example, the immunotoxins can be administered to the patient at least once every 2,

4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 hours, once every 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 days, once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, or 12 months. In one aspect, the immunotoxin is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per day or 1, 2, 3, 4,

5, 6, 7, 8, 9, 10, 11, 12, 13, 14 times per week. For example the immunotoxin can be

administered 2 times per day for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

57. While the immunotoxin can be administered concurrently with any immunotherapy (such as, for example CAR T ceil therapy); it is understood and herein contemplated that the immunotoxin reduces the number of targeted immune cells (such as, for example, T cells) and that time can be needed for the immunotoxin to reduce endogenous immune cells prior to administration of immunotherapy. In one aspect, disclosed herein are methods of treating, preventing, inhibiting, or reducing a cancer or metastasis; methods of treating, inhibiting, or reducing HIV; methods of pre-conditioning a tumor microenvironment for immunotherapy, and/or methods of preconditioning a subject with HIV for immunotherapy comprising concluding the administration of an immunotoxin to the subject at least 4, 6, 8, 10, 12, 14, 16,

18, 20, 22, 24, 30, 36, 42, 48 hours, 3, 4, 5, 6, 7, 8, 9 10, 1 1, 12, 13, 14, 15, 16 ,17 ,18, 19, 20,

21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days prior to any immunotherapy. Thus, in one aspect, disclosed herein are methods of treating, preventing, inhibiting, or reducing a cancer or metastasis; methods of treating, inhibiting, or reducing HIV comprising administering to the subject an immunotoxin and an immunotherapy (such as, for example a CAR T cell) wherein the immunotoxin is administered prior to the administration of the immunotherapy.

58. In one aspect administration of the immunotoxin can commence before or concurrent with administration of the immunotherapy and continue during immunotherapy. In one aspect, administration of the immunotoxin can occur for at least , 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48 hours, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16 ,17 ,18, 19, 20, 21, 22, 23, 24, 25,

26, 27, 28, 29, 30, or 31 days following the commencement of immunotherapy.

1. Antibodies

(1) Antibodies Generally

59. The term“antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term“antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin mol ecules or fragments thereof, as long as they are chosen for their ability to interact with CD 3 or other immune cell marker. The antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods. There are five major classes of human

immunoglobulins; IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. 60. The term“monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived fro a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived fro another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.

61. The disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Mil stein. Nature , 256:495 (1975). In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

62. The monoclonal antibodies may also be made by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al.

63. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.

64. As used herein, the term“antibody or fragments thereof’ encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab’)2, Fab’, Fab, Fv, scFv, and the like, including hybrid fragments. Thus, fragments of the antibodies that retain the ability to bind their specific antigens are provided.

For example, fragments of antibodies which maintain CD3 binding activity are included within the meaning of the term“antibody or fragment thereof.” Such an tibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies , A

Laboratory Manual . Cold Spring Harbor Publications, New York, (1988)).

65. Also included within the meaning of“antibody or fragments thereof’ are conjugates of antibody fragments and antigen binding proteins (single chain antibodies).

66. The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory

characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed poly peptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment (Zoller, M.J. Carr. Opin.

Biotechnol. 3:348-354, 1992).

67. As used herein, the term“antibody” or“antibodies” can also refer to a human antibody and/or a humanized antibody. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.

(2) Human antibodies

68. The disclosed human antibodies can be prepared using any technique. The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al ., Proc. Natl Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et ah, Nature, 362:255-258 (1993); Bruggerrnann et al., Year in Immunol , 7:33 (1993)). Specifically, the homozygous deletion of the antibody heavy chain joining region {1(H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human genu-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. Antibodies having the desired activity are selected using Env-CD4-co-receptor complexes as described herein.

(3) Humanized antibodies

69. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Accordingly, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or antibody chain (or a fragment thereof, such as an sFv, Fv, Fab, Fab’, F(ab’)2, or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody

70. To generate a humanized antibody, residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen ). In some instances, Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321 :522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol, 2:593-596 (1992)).

71. Methods for humanizing non-human antibodies are well known in the art. For example, humani zed antibodies can be generated according to the methods of Winter and co-workers (Jones et al., Nature, 321 :522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et ah, Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Methods that can be used to produce humanized antibodies are also described in U.S. Patent No. 4,816,567 (Cabilly et al.), U.S. Patent No. 5,565,332 (Hoogenboom et al.), U.S. Patent No. 5,721,367 (Kay et al.), U.S. Patent No. 5,837,243 (Deo et al.), U.S. Patent No. 5, 939,598 (Kucherlapati et al.), U.S. Patent No 6,130,364 (Jakobovits et al.), and U.S. Patent No. 6, 180,377 (Morgan et al.).

2. Pharmaceutical carriers/Delivery of pharmaceutical products

72. As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be w^?eil known to one of skill in the art.

73. The compositions may be administered orally, parenteral!y (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdennal!y, extracorporeal ly, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery- by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector.

Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory- system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

74. Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein. 75. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem ., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer , 60:275-281, (1989); Bagshawe, et al., Br. J.

Cancer, 58:700-703, (1988); Senter, et al , Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother 35:421-425, (1992); Pietersz and McKenzie, Immunolog.

Reviews, 129:57-80, (1992), and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al. Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104: 179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosorne in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

a) Pharmaceutically Acceptable Carriers

76. The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.

77. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA

1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer’s solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about zz 7 5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

78. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

79. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.

Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

80. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasal ly), orally, by inhalation, or parenteraJly, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

81. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti -oxidants, chelating agents, and inert gases and the like

82. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. 83. Compositions for oral administration include powders or granules, suspensions or solutions in water or o -aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable..

84. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and sub stituted ethan ol ami nes .

b) Therapeutic Uses

85. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary_', and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et ah, eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy , Baber et al., eds.. Raven Press, New York (1977) pp. 365-389.

A typical daily dosage of the antibody used alone might range from about 1 pg/kg to up to 100 mg/kg of body rveight or more per day, depending on the factors mentioned above.

C. Examples

86. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc ), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

1. Example 1: CDSe-imsmmotoxin is a potentially safe and effective pre- conditioning regimen

87. Current chemotherapies, including Cyclophosphamide (CTX) and Fludarabine, have non-specific cytotoxic effects and a high level of variability in therapeutic efficacy among patients, both between and during clinical trials, sometimes even with deadly consequences. By contrast, the murine and nonhuman primate studies have shown that CD3e-immunotoxin (CD3e- IT) is highly specific in killing T cells, and consistently and effectively deplete T-cells in all test animals without causing notable damages to lymphoid organs. Cellular transplant following CD3e-IT treatment is well tolerated and feasible in these test animals (Figs 1-4, see below for more details).

2. Example 2: CD3e-immunotoxin is superior in depleting T cells in various body organs compared to CTX, and well tolerated in mice.

88. Murine version CD3e-IT was prepared by conjugating biotinylated anti-CD3e monoclonal antibody (145-201 clone from Absoluteantibody Ltd) with streptavidin-saporin (Advanced Targeting Systems). Like Diphtheria Toxin, saporin (a ribosome inactivating protein) is an extremely potent toxin and saporin-immunotoxins have been clinically and preclinically evaluated (Shapira A., Toxins, 2010). Four-day CD3e-IT (saporin-immunotoxin) treatment in mice resulted in specific ablation of T cells in all organs tested, including peripheral blood, spleen, bone marrow, thymus, lymph nodes, Peyer’s Patches and liver (Fig. 1). The specificity and effectiveness of T-cell depletion were similar to those of the Diphtheria Toxin (DT)~ mediated T-cell ablation mouse model (Fig. 1). The results were also similar to C207 (primate CD3e~IT) treatment in nonhuman primates (see Fig. 4). CTX, by contrast, significantly reduced B cells (Fig.1). There was no significant difference between the body weights of CTX and CD3e-IT treated animals over the 7-day monitoring period (Fig. 2A). In CTX treated animals, Peyer’s Patches were undetectable and the relative weights of thymus were notably reduced. The impact of CD3e-IT on body organs were relatively less severe compared to CTX (Fig. 2B). Lymphocytes were fully recovered in CD3e-IT treated animals by 12 weeks post-CD3e-IT- treatment in all test organs. As expected, naive T cell populations remained a major pool throughout the 12-week monitoring period, indicating that thymic production played a key role in T-cell homeostasis in CD3e-IT treated mice. 3. Transplantation of spleen and lymph node cells in CD3e~IT treated animals resulted in a T-cei! repopulation patern typical for adoptive T-cell transplant in mice.

89. All mice showed full recovery of body weights in 3-6 weeks (Fig.3A). All transplanted mice showed a transient but sharp increase in T cells for the first 2 weeks, especially in effector CD4+ and CD8+ lymphocytes, followed by a gradual decrease in transferred T-cells (Fig.3B). All naive, effector memory, and central memory cells showed transient expansion within about 2 weeks. The transferred T-cells remained detectable until the endpoint (12 weeks) in all tested organs, including spleen, lymph nodes, and Peyer’s Patches. The results support the use of CD3e-IT as a lymphodepletion preconditioning to promote the survival and repopulation of T cells after adoptive transplant.

4. T cell ablation in nonhuman primates

90. Applying the treatment regimen to nonhuman primates, the nonhuman primate study also showed specific ablation of T-cells in various organs, and fast recovery in T-cell numbers following CD3e-IT (C207) treatment (Figs. 4 and 5). In the nonhuman primates, however, the T-cell population recovered primarily through peripheral homeostatic T-cell expansion, due to the reduced thymic functions in these aged animals (15-17 year old). All test animals remained healthy until the end-point (12 to 19 months post-CD3e-IT), showing no notable adverse effects from the CD3e-IT treatment.

Claims

What is claimed is:

1. A method of treating a cancer or HIV infection in a subject comprising administering to the subject an anti-CD3 immunotoxin and administering to the subject CAR T cells.

2. The method of claim 1, wherein the anti-CD3 immunotoxin comprises a diphtheria toxin (DT), ricin toxin. Pseudomonas enterotoxin A (ETA), saporin, botulinim toxin, cholera toxin, Clostridium difficile toxin, Clostridium perfringens toxin, Aerolysin, Cereolysin, Listeria listeriolysin, Listeria hemolysin, Shiga toxin, saxitonix, pheumolysin, tetanus toxin, or any mutants or fragments thereof.

3. The method of claim 2, wherein the immunotoxin comprises a mutant or fragment of DT selected from the group consisting of DT483, DT390, DT389, DT383, DT370. CRM9, CRM 107, CRM 103, CRM197, MSPA5, and DTM1.

4. The method of claim 1, wherein the administration of the immunotoxin occurs at least 2 days prior to administration of CAR T cells

5. The method of claim 4, wherein the immunotoxin is administered at least one time per day for at least two days.

6. A method of pre-conditioning a subject for CAR T ceil therapy comprising administering to the subject an anti-CD3 immunotoxin.

7. The method of claim 6, wherein the anti~CD3 immunotoxin compri ses a diphtheria toxin

(DT), ricin toxin, Pseudomonas enterotoxin A (PE), saporin, botulinim toxin, cholera toxin, Clostridium difficile toxin, Clostridium perfringens toxin, Aerolysin, Cereolysin, Listeria listeriolysin, Listeria hemolysin, Shiga toxin, saxitonix, pheumolysin, tetanus toxin, or any mutants or fragments thereof.

8. The method of claim 7, wherein the immunotoxin comprises a mutant or fragment of DT selected from the group consisting of DT483, DT390, DT389, DT383, DT370 CRM 9, CRM 107, CRM 103, CRM197, MSPA5, and DTM1.

9. The method of claim 6, wherein the administration of the immunotoxin occurs at least 2 days prior to administration of CAR T cells. The method of claim 9, wherein the immunotoxin is administered at least one time per day for at least two days.