TREATMENT OF CANCERS WITH IMMUNOSTIMULATORY HIV TAT DERIVATIVE POLYPEPTIDES

Abstract

Disclosed herein are compositions comprising a Human Immunodeficiency Virus (HIV) trans-activator of transcription (Tat) derivative polypeptide with increased immunostimulatory properties relative to the native Tat polypeptide, pharmaceutical compositions comprising the Tat derivative polypeptide, and methods of treating cancer using the Tat derivative polypeptide.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation of U.S. patent application Ser. No. 15/587,262, filed May 4, 2017, which is a continuation of U.S. patent application Ser. No. 14/505,977 filed Oct. 3, 2014, now U.S. Pat. No. 9,663,556, which claims the benefit under 35 USC .sctn. 119(e) to U.S. Provisional Patent Application 61/887,166 filed Oct. 4, 2013, the entire contents of each of which are incorporated by reference herein.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of immune-based therapeutic agents for cancer.

BACKGROUND

[0003] Immune checkpoints represent inhibitory molecules that result in the inhibition of an effective immune response towards cancer which can result in tumor evasion. Immune checkpoint molecules such as the cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death 1 (PD-1) along with programmed cell death ligand 1 (PD-L1) are believed to be contributing to the immune dysfunction that accompanies cancer progression and their therapeutic blockade has shown clinical benefit. Specifically, the engagement of tumor PD-L1 with PD-1 on infiltrating Cytotoxic T lymphocytes (CTL) is believed to be an important mechanism underlying tumor evasion and immune resistance by inducing T-cell anergy, exhaustion, and programmed cell death. Understanding the manipulation of immune checkpoint molecules during the immune response is an important strategy for designing effective immunotherapies for human cancers.

[0004] The Human Immunodeficiency Virus (HIV) trans-activator of transcription (Tat) is a variable RNA binding peptide which increases viral RNA transcription and may initiate apoptosis in T4 cells and macrophages and possibly stimulates the over production of alpha interferon. However, the Tat protein isolated from HIV-infected long term non-progressors (LTNP) is different from Tat found in patients who have progressed to Acquired Immunodeficiency Syndrome (AIDS) as a result of their infections. The Tat protein found in LTNP is capable of trans-activating viral RNA; however, this immunostimulatory Tat does not induce apoptosis in T4 cells or macrophages and is not immunosuppressive. Variants of immunostimulatory Tat found in lentiviruses that infect monkey species yet do not result in the development of immunodeficiency and epidemic infection direct monocyte differentiation into dendritic cells (DCs) that stimulate cytotoxic T lymphocyte (CTL) responses. Thus, immunostimulatory Tat may have utility in stimulating an immune response towards human cancers.

[0005] Cancers and chronic infections are the most prominent examples of common human diseases that respond to immune-based treatments. Although infections were the first diseases to be controlled by immunization, clinical trials in humans have established that an immune response, particularly of the CTL arm of the immune system, could regress some human melanomas and renal cancers. These observations were broadened by the discovery that DCs, a specific class of antigen-presenting cells (APC), are particularly effective at initiating CTL activity against cancers and other diseases. Technologies that target and activate DCs have yielded some early successes against human cervical pre-malignancies caused by infection with Human Papilloma Virus (HPV) and human lung cancer. In contrast to chemotherapeutic drugs currently used against cancer, agents that provoke a CTL response against cancer potentially are accompanied by few side effects, owing to the great specificity of the immune response.

[0006] Efforts to develop immunotherapeutic drugs that treat cancer have been hampered by technical difficulties in targeting and activating DCs to deliver and sustain the required entry signals to the CTLs. Antigen targeting for the induction of a CTL response is a challenge, insofar as natural processing requires that the antigen enter the cytoplasm of the cell in order to bind to the immune system's major histocompatibility complex (MHC) Class I antigen, a prerequisite to CTL activation because the ligand for activating the T cell receptor on CTLs is a complex of antigen and MHC Class I. In almost all cases, protein antigens, even when they are coupled with a DC co-activator, enter exclusively into the alternative MHC Class II antigen presentation pathway that excludes CTL stimulation. This can be overcome, in part, by peptide-based technologies, because peptides bind to MHC Class I that is already on the surface of the DC. However, this technology is non-specific, and most peptides are poor DC activators, which limits their efficacy as treatments for human cancer.

[0007] A limited group of biological proteins are known to stimulate a CTL response. Variants and derivatives of the Human Immunodeficiency Virus 1 (HIV-1) trans-activator of transcription (Tat) can stimulate this CTL response. Additional biologics that are currently known to directly trigger a CTL response are based on heat shock proteins (HSP), or on the outer coat protein of certain bacteria. Heat shock proteins have shown limited efficacy in the treatment of certain genital neoplasms related to HPV infection.

SUMMARY OF THE INVENTION

[0008] Disclosed herein are derivatives of the Human Immunodeficiency Virus (HIV) trans-activator of transcription (Tat) protein for use as cancer therapeutic agents. Artificial immunostimulatory Tat derivative polypeptides have the potential to treat cancer.

[0009] In one embodiment, a trans-activator of transcription (Tat) derivative polypeptide is provided having an amino acid sequence comprising, in the following order: (i) a transcription factor (TF) domain sequence from a human immunodeficiency virus (HIV) or a simian immunodeficiency virus (SIV) Tat protein, (ii) a cysteine-rich domain sequence from SIV, HIV, or a defensin, and (iii) a C-terminal domain sequence from a HIV or SIV Tat protein.

[0010] Also disclosed herein is a pharmaceutical composition comprising a Tat derivative polypeptide disclosed herein.

[0011] In one embodiment of the Tat derivative polypeptide, the HIV is HIV-1 or HIV-2. In another embodiment, the HIV-1 Tat is from a long-term non-progressor. In another embodiment, the SIV is from a host selected from Table 2. In another embodiment, the defensin is an .alpha.-defensin or a .beta.-defensin. In yet another embodiment, the Tat derivative polypeptide further comprises an arginine-rich domain from HIV-1 or HIV-2 Tat.

[0012] In another embodiment of the Tat derivative polypeptide, at least one of the amino acids in the TF domain is deleted or substituted with an alanine, an aspartic acid, a glutamic acid, a glycine, a lysine, a glutamine, an arginine, a serine, or a threonine. In another embodiment, the at least one substituted amino acid is a proline.

[0013] In certain embodiments, the TF domain comprises an amino acid sequence of one of SEQ ID NOs:96-123. In other embodiments, the cysteine-rich domain comprises an amino acid sequence of one of SEQ ID NOs:124-132. In other embodiments, the C-terminal domain comprises an amino acid sequence of one of SEQ ID NOs:133-150.

[0014] In another embodiment, the Tat derivative polypeptide has greater than 85% sequence identity to one of SEQ ID NOs 5-95. In another embodiment, the Tat derivative polypeptide is not one of SEQ ID NOs:2, 3, or 4.

[0015] Also disclosed herein is a method of treating cancer comprising administering a therapeutically effective amount of a Tat derivative polypeptide or pharmaceutical composition disclosed herein to a subject in need thereof; and causing cessation of growth of the cancer or regression of the cancer in the subject.

[0016] Also disclosed herein is a method of reducing tumor burden in a subject with cancer, the method comprising administering a therapeutically effective amount of a Tat derivative polypeptide or a pharmaceutical composition disclosed herein, to a subject in need thereof; and causing regression of the cancer in the subject.

[0017] Also disclosed herein is a method of inhibiting the suppression of an anti-tumor immune response in a subject with cancer, the method comprising administering a therapeutically effective amount of a Tat derivative polypeptide or a pharmaceutical composition disclosed herein to the subject; wherein the administration results in reduction or inhibition of growth of the cancer or in regression of the cancer in the subject.

[0018] Also disclosed herein is a method of treating a PD-L1-expressing tumor in a subject with cancer, the method comprising administering a therapeutically effective amount of a Tat derivative polypeptide or a pharmaceutical composition disclosed herein; wherein the administration results in reduction or inhibition of growth of the cancer or in regression of the cancer in the subject.

[0019] In one embodiment of the methods, the Tat derivative polypeptide has greater than 85% sequence identity to one of SEQ ID NOs 5-95.

[0020] In one embodiment of the methods, the Tat derivative polypeptide is administered in a plurality of doses. In another embodiment of the methods or uses, the administration comprises a repetitive administration cycle wherein each cycle comprises administering a plurality of doses of the Tat derivative polypeptide in a defined time period followed by a rest period and wherein the cycle is repeated a plurality of times. In another embodiment of the methods or uses, the administration comprises a repetitive administration cycle wherein each cycle comprises administering a plurality of doses of the Tat derivative polypeptide in a defined time period followed by a administration of one or a plurality of doses of a therapeutic agent in a defined time period and wherein the cycle is repeated a plurality of times. In another embodiment of the methods or uses, the therapeutic agent is cyclophosphamide.

[0021] In another embodiment of the methods, the cancer is adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal-cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, bronchial adenomas/carcinoids, carcinoid tumor, cervical cancer, chronic myeloproliferative disorders, colon cancer, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, eye cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, glioma, gastric carcinoid, head and neck cancer, heart cancer, hepatocellular cancer, Hodgkin's lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi sarcoma, kidney cancer, leukemias, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancer, lymphomas, macroglobulinemia, medulloblastoma, melanoma, merkel cell carcinoma, mesothelioma, mouth cancer, multiple myeloma/plasma cell neoplasm, mycosis fungoides, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma, pituitary adenoma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, throat cancer, thymoma, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, or Wlms tumor.

[0022] In another embodiment of the methods, at least one pre-treatment tumor from the subject contains at least 5% PD-L1-expressing cells, between 5% and 20% PD-L1-expressing cells, between 5% and 15% PD-L1-expressing cells, or between 5% and 10% PD-L1-expressing cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 depicts stimulation of human monocytes with Tat derivatives.

[0024] FIG. 2 depicts a dose-response curve of stimulation of human monocytes with Tat derivatives.

[0025] FIGS. 3A and 3B depict the effect of therapy with Tat derivatives on 4T1 tumor growth in vitro. BALB/c mice injected with 1.times.10.sup.4 4 T1 tumor cells were treated with Nani-P1 or Nani-P2 (400 ng, subcutaneous [SC]) (FIG. 3A) or Nani-P3 (400 ng or 2 .mu.g, SC) (FIG. 3B) on days 0, 7, 14 and 21 after injection of tumor cells. The control group was treated with PBS. Data represents mean tumor volume; bars.+-.SE. Each group contained 10 mice. From day 15, the differences between the control group and groups treated with Nani-P1 or Nani-P2 were significant (p<0.05**). The differences between control and Nani-P2 or Nani-P2 was highly significant starting at day 22 (p<0.01**). There was no difference between Nani-P3 (either dose) and controls.

[0026] FIG. 4 depicts a dose response curve for the effects of purified Nani-P2 on 4T1 breast tumor growth in vivo. Four groups of ten BALB/c mice each were implanted with 1.times.10.sup.4 4 T1 cells. Three groups were given escalating doses of 0.4 ng, 4 ng, and 40 ng per mouse, respectively, in the left flank four times over 21 days. The fourth, control group was injected in the left flank with PBS. Data represent mean tumor volume. The differences between the control group and 0.4 ng dose was significant (p<0.5*), and the difference between control and 4 ng or 40 ng Nani-P2 treated groups was highly significant (p<0.1**, p<0.01**).

[0027] FIGS. 5A and 5B depict a Kaplan-Meier survival curve of Nani-P2 treatment of mice bearing 4T1 breast tumors. Mice were injected SC with 1.times.10.sup.4 4 T1 cells in the mammary pad at day 0. Treatment was started at day 0 with four doses of Nani-P2 (40 ng) administered SC. At day 42, the treatment group had statistically significant better survival over controls (**) (FIG. 5A). In one group, therapy was delayed until day 13, at which time a series of three doses of Nani-P2 (40 ng) were administered weekly either intravenous (IV), SC into the draining lymph nodes, or intratumoral (IT) (FIG. 5B). The survival benefit of IV Nani-P2 was highly statistically significant at day 47 (**), while the survival benefit of SC Nani-P2 was also statistically significant (*).

[0028] FIGS. 6A and 6B depict the anti-tumor activity of Nani-P2 in TS/A and SM1 breast carcinoma models. Mice were implanted SC with 1.times.10.sup.5 TS/A breast cancer cells (FIG. 6A) and treated with escalating doses of SC Nani-P2 (0.4, 4, and 40 ng). Even at the lowest dose, the primary anti-cancer difference was highly significant (p<0.01**), while the 40 ng dose was also highly significant (p<0.01***). FIG. 6B depicts mice implanted SC with 2.times.10.sup.5SM1 breast cancer cells and treated SC with Nani-P2 (40 ng) on days 0, 7, 14, and 21. The difference in primary tumor growth between control and Nani-P2 treated SM1 animals was highly statistically significant (p<0.01***).

[0029] FIG. 7 depicts INF-.gamma. production from spleen cells of mice bearing 4T1 breast tumors. BALB/c mice were injected SC with 1.times.10.sup.4 4 T1 cells. Control animals received weekly injections of PBS, while the Nani-P2 treatment comprised once weekly SC injections (40 ng) initiated at day 0 and continued for 4 weeks. On day 33, when control mice were at end stage, the mice were sacrificed, the spleens harvested and frozen as single cell suspensions until time of assay. Spleen cells (2.times.10.sup.5) and 1.times.10.sup.4 mitomycin C-treated (50 .mu.g/ml for 30 min) 4T1 stimulator cells (S) were plated into 96-well plates. After 72 hr of stimulation, the supernatants were collected, and IFN-.gamma. concentration was determined using a commercial IFN-.gamma. ELISA kit. IFN-.gamma. production was significantly (p<0.05*) higher in cultures of spleen cells from Nani-P2-treated mice under all conditions of in vitro culture. 1: no restimulation; 2: IL-4 (50 ng/ml)/GM-CSF (100 mg/ml); 3: stimulator cells/IL-4/GM-CSF; 4: stimulator cells only. Addition of in vitro agonists IL-4 and GM-CSF (2 and 3) induced highly significant increases in IFN-.gamma. production (p<0.01**).

[0030] FIGS. 8A and 8B depict regression of established 4T1 breast tumors and inhibition of lung metastasis by Nani-P2 treatment. In FIG. 8A, two groups of 10 BALB/c mice were injected with 1.times.10.sup.4 4 T1 cells in the mammary pad on day 0. One group was dosed with Nani-P2 (40 ng) weekly for three weeks beginning at day 14. A second group was PBS-treated and used as control. Tumor burden was highly significant by day 22 and remained so throughout the duration of the trial (p<0.01**). Mice were sacrificed when tumor diameter reached 15 mm, at which time lung metastases were counted (FIG. 8B). Data represent total lung metastases as quantitated by two observers blinded to the treatment protocol (p<0.01**).

[0031] FIG. 9 depicts 4T1 tumor growth and lung metastasis in BALB/c mice. Two groups of 10 BALB/c mice were implanted subcutaneously (SC) with either 1.times.10.sup.4 4 T1 cells, mice injected IV with 40 ng Nani-P2 or PBS. On day 28 of treatment, the mice were killed and the lungs and tumor were removed, and tumor nodules were counted by eye. Photographs of the tumors and lungs, which were representative of 10 mice, are shown. Whitish tumor lesions can be observed on the surface of the lungs. Three experiments yielded similar results.

[0032] FIG. 10 depicts Nani-P2 treatment-induced regression of established 4T1 breast tumors. One of 10 mice underwent a complete remission and remained disease-free over 50 days, at which point the study was terminated. Two groups of 10 BALB/c mice were injected with 1.times.10.sup.44 T1 cells in the mammary pad on day 0. One group was dosed with Nani-P2 (40 ng) per mouse IV weekly over three weeks beginning at day 14 and the other group was treated with PBS and served as control. The difference in primary tumor growth between control and Nani-P2-treated groups was highly significant (p<0.01**).

[0033] FIG. 11 depicts tumor growth after therapy with repeated doses of Nani-P2 and cyclophosphamide.

[0034] FIG. 12 depicts the survival benefit of repeated doses of Nani-P2 and cyclophosphamide vs. weekly cyclophosphamide.

[0035] FIG. 13A-B depicts immunohistochemical (IHC) staining of CD8+ cells in spleen tissue from a mouse with 4T1 mammary carcinoma treated with PBS (Control, FIG. 13A) or Nani-P2 (FIG. 13B).

[0036] FIGS. 14A-14D depict IHC staining of primary 4T1 breast tumors for PD-L1 and CD8. FIG. 14A depicts IHC staining with PD-L1 antibodies in a PBS control animal. PD-L1 staining was observed in cells with a morphological resemblance to myeloid-derived suppressor cells, tumor-associated macrophage, as well as tumor-associated dendritic cells and fibroblast. FIG. 14B depicts IHC staining in a Nani-P2 treated mouse. FIG. 14C depicts IHC staining of infiltrating CD8+ cytotoxic lymphocytes (CTL) in a PBS control animal. FIG. 14D depicts IHC staining of CD8+ CTL in a Nani-P2 treated mouse.

DETAILED DESCRIPTION

[0037] A series of artificial Human Immunodeficiency Virus (HIV) trans-activator of transcription (Tat) protein derivatives has been designed which are active in cancer. The molecules are referred to herein as "Tat derivative polypeptides," "Tat derivatives," or "Precision Immune Stimulants" (PINS) and comprise Tat molecules having modifications to change Tat from being immunosuppressive to immunostimulatory.

[0038] Despite a relative abundance of tumor-associated antigens, cancer has proven to be a difficult target for immunotherapeutics. Evidence has accumulated that the refractory state of cancer to immunotherapeutics could derive from immune suppression that accompanies established cancers. Epidemiological studies have shown that women with HIV infection, and even Acquired Immunodeficiency Syndrome (AIDS), were paradoxically protected from developing breast cancer, even in late-stage disease when immunodeficiency is pronounced.

[0039] The HIV-Tat protein can repetitvely trigger precursor cells of the innate immune lineage into activated antigen presenting cells (APC). These observations have been confirmed in specific reference to the dendritic cell APC, whose activation initiates rounds of HIV replication even in AIDS. Taken together, these data supported the conclusion that Tat had a counter suppressive activity. It is hypothesized that these observations on Tat could be linked to the epidemiological data on breast cancer through the theory that Tat in HIV-infected individuals was chronically stimulating innate immunity thereby restricting breast cancer progression.

[0040] Tat Derivative Polypeptides

[0041] The HIV Tat protein is a variable RNA binding protein of 86 to 110 amino acids in length that is encoded on two separate exons of the HIV genome. Tat is highly conserved among all human lentiviruses and is essential for viral replication. When lentivirus Tat binds to the TAR (trans-activation responsive) RNA region, transcription (conversion of viral RNA to DNA and then to messenger RNA) levels increase significantly. It has been demonstrated that Tat increases viral RNA transcription, and it has been proposed that Tat may initiate apoptosis (programmed cell death) in T4 cells and macrophages (a key part of the body's immune surveillance system for HIV infection) and may stimulate the over production of .alpha.-interferon (.alpha.-interferon is a well established immunosuppressive cytokine).

[0042] Extracellular Tat's presence early in the course of HIV infection could reduce a patient's immune response, giving the virus an advantage over the host. Furthermore, the direct destruction of T4 cells and induction of .alpha.-interferon production could help explain the lack of a robust cellular immune response seen in AIDS patients, as well as accounting for the initial profound immunosuppression.

[0043] Based on molecular analysis, the Tat protein (SEQ ID NO:1) includes four distinct domains: (1) the transduction (SH3) domain (amino acids 3-19); (2) the cysteine-rich ligand binding domain (amino acids 22-37); (3) the membrane translocation sequence (amino acids 47-57) and (4) a tail portion encoded by the second exon (amino acids 73-101).

[0044] The amino terminal portion of Tat includes a short peptide region from a nuclear transcription factor (TF) typically flanked by proline residues. This region determines, at least in part, how stimulatory or suppressive the Tat polypeptide is for cells of the immune system, particularly innate immune cells such as dendritic cells (DC) and macrophages (antigen-presenting cells or APCs). Consequently, it is predicted that modifications to the TF region can render the polypeptides more active in the therapy of cancer and other chronic diseases.

[0045] HIV-1 Tat SH3 binding domain is identical to the sequence found in another TF protein, hairless (hr), that had previously been shown to have immunosuppressive properties in mice. Mice carrying the hr mutation develop an immune dysregulation, now most commonly called "the TH1 to TH2 shift," that is the sine qua non of HIV-infected individuals who are progressing to AIDS. Further analysis established that SH3 binding sequence derived from the hr gene is a nearly invariant feature of Tat isolated from HIV-1, and a very consistent feature of HIV-2.

[0046] In contrast, primates infected by certain strains of simian immunodeficiency virus (SIV), a lentivirus closely related to HIV, rarely progress to AIDS, or do so unpredictably. This observation, coupled with the discovery of a putatively immunosuppressive hr TF fragment in immunosuppressive HIV-1 Tat, suggested that some primates could have a different (or no) TF fragment at the amino terminus of SIV Tat. Tat from certain SIV-infected sooty mangabeys with an attenuated course of immunodeficiency has at its amino terminus a fragment from the TF TARA instead of the TF hr.

[0047] In general, an immunostimulatory Tat derivative polypeptide for the treatment of cancer comprises at least three regions (domains). The first domain is a derivatized nuclear transcription factor (TF) region of Tat, the second domain is a cysteine-rich region, and the third region is a C-terminal Tat domain. Each of these domains comprises a sequence from a Tat protein from a source including, but not limited to, HIV-1 or HIV-2 infected progressors, long-term non-progressors, long-term survivors, elite controllers, and/or SIV infected non-human primate species. Alternatively, cysteine-rich defensin molecules can be substituted in place for a Tat-derived cysteine-rich domain. In certain embodiments, the cysteine-rich domain from a retrovirus is combined with a TF domain and C-terminal domain from non-human primate Tat sequence. In another embodiment, non-human primate cysteine-rich domain is combined with a TF domain and C-terminal domain from a retrovirus. In yet another embodiment, the sequence comprising a fragment of the region which maintains the immunostimulatory activity of the full length domain. Exemplary retroviruses are SIV, HIV, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Herpes Simplex Virus 1, Herpes Simplex Virus 2, or equine infectious anemia virus (EIAV). In one embodiment, the retrovirus is a lentivirus such as HIV or SIV. In another embodiment the HIV is HIV-1 or HIV-2.

[0048] Thus disclosed herein are Tat derivative polypeptides comprising an amino acid sequence including a transcription factor (TF) domain, a cysteine-rich domain, and a C-terminal domain in that order, wherein each of the TF domain and the C-terminal domain are from a retrovirus Tat protein, and the cysteine-rich domain is from a retrovirus or a defensin, such as .alpha.-defensin or .beta.-defensin. Exemplary non-limiting Tat derivative polypeptides are presented in Table 1. The TF region has a C-terminal proline residue and the cysteine-rich region has a C-terminal phenylalanine. If the native TF sequence does not have a proline residue at the C-terminus, a proline may be inserted at the C-terminus. Exemplary SIV infected non-human primate species are listed in Table 2.

[0049] In another embodiment, the modified Tat polypeptide further comprises an arginine-rich domain from a lentiviral Tat protein. The arginine-rich domain is found within the C-terminal region.

[0050] The TF domain, cysteine-rich domain, and C-terminal domain sequences are arranged in the Tat derivative polypeptide in that order.

[0051] In additional embodiments, one or more amino acids, including but not limited to proline, in the TF domain is deleted or substituted with a conservative amino acid substitution, such as with an alanine, an aspartic acid, a glutamic acid, a glycine, a lysine, a glutamine, an arginine, a serine, or a threonine.

[0052] In one embodiment, the TF domain comprises, consists essentially of, or consists of, an amino acid sequence of one of SEQ ID NOs:96-123. In another embodiment, the cysteine-rich domain comprises, consists essentially of, or consists of, an amino acid sequence of one of SEQ ID NOs:124-132. In another embodiment, the cysteine-rich domain comprises, consists essentially of, or consists of, an amino acid sequence of one of SEQ ID NOs:133-150.

TABLE-US-00001 TABLE 1 Exemplary Tat derivative polypeptides SEQ cysteine-rich C-terminal ID NO. Amino Acid Sequence TF domain* domain.sup..dagger-dbl. domain 2 MEPVDANLEAWKHAGSQPRKTACTTCYCKKCCFH HIV-1 HIV-1 HIV-1 CQVCFTRKGLGISYGRKKRRQRRRAPQDSQTHQA SLSKQPASQSRGDPTGPTESKKKVERETETDPFD (Nani-P1, MPM1, PIN-1) 3 MDPKGEEDQDVSHQDLIKQYRKPRTACNNCYCKK SIVagmVer HIV-1 SIVagmVer CCFHCYACFLRKGLGITYHAFRTRRKKIASADRI PVPQQSISIRGRDSQTTQESQKKVEEQAKANLRI SRKNLGDETRGPVGAGN (Nani-P2, ASH4, PIN-2) 4 METPLKEQENSLESCREHSSSISEVDVPTPVSCL SIVsmm Murine HIV-1 RKGGRCWNRCIGNTRQIGSCGVPFLKCCKRKPFT .beta. defensin RKGLGISYGRKKRRQRRRAPQDSQTHQASLSKQP ASQSRGDPTGPTESKKKVERETETDPFD (Nani-P3, TMPD5, PIN-3) 5 METPLKEQESSLESSREHSSSISEVDADTPESAS SIVsmm HIV-2 HIV-2 LEEEILSQLYRPLETCNNTCYCKECCYHCQLCFL NKGLGIWYDRKGRRRRSPKKIKAHSSSASDKSIS TRTRNSQPEEKQKKTLETTLGTDCGPGRSHIYIS 6 MDAGKAVSDKKEGDVTPYDPFRDRTTPLETCNNT SIVmnd HIV-2 HIV-2 CYCKECCYHCQLCFLNKGLGIWYDRKGRRRRSPK KIKAHSSSASDKSISTRTRNSQPEEKQKKTLETT LGTDCGPGRSHIYISA 7 MDVQGVGLEHPEEVILYDPRTACNNCYCKKCCFH SIVdeb HIV-1 SIVdeb CYACFLQKGLGINYASRARRRRSKEENKADKFPV PNHRSISTTRGNRKLQEKKEKTVEKKVATSTTIG 8 MDKGEEERTVLHQDLIRQYKKPRTACNNCYCKKC SIVagmVer HIV-1 SIVagmVer CFHCYACFLRKGLGITYHAFRTRRKKIASADRIP VPQQSISIRGRDSQTTQESQKKVEEQAKANLRIS RKNLGDETRGPVGAGN 9 MDPKGEEDQDVSHQDLIKQYRKPRTACNNCYCKK SIVagmVer HIV-1 SIVagmVer CCFHCYACFHCYACFLQKGLGVTYHAPRTRRKKS VQPNRLSQQDQSISTRGRDGQATQESQKKVERET TTAQILGRKDLERDKREAVGANA 10 MDQEQEARPQVWEELQEELHRPRTACNNCYCKKC SIVagmSab HIV-1 SIVagmVer CFHCYACFLRKGLGITYHAFRTRRKKIASADRIP VPQQSISIRGRDSQTTQESQKKVEEQAKANLRIS RKNLGDETRGPVGAGN 11 MDPKGEEDQDVSHQDLIKQYRKPRTACNNCYCKK SIVagmVer HIV-1 SIVagmSab CCFHCYACFHKKALGIRYYVPRPRRASKKISHNQ VSLHN 12 MESEGDGMAESLLQDLHRPRTACNNCYCKKCCFH SIVagmTan HIV-1 SIVagmVer CYACFLRKGLGITYHAFRTRRKKIASADRIPVPQ QSISIRGRDSQTTQESQKKVEEQAKANLRISRKN LGDETRGPVGAGN 13 MDPKGEEDQDVSHQDLIKQYRKPRTACNNCYCKK SIVagmVer HIV-1 SIVagmTan CCFHCYACFHCYACFLQKGLGITYHVSRIRRPKK NHSNHQNLVSQQSISAWGGNSQTTQEEKTKIPAA AETSRRPQ 14 MDKGEAEQIVSHQDLSEDYQKPRTACNNCYCKKC SIVagmVer HIV-1 SIVagmVer CFHCYACFLRKGLGITYHAFRTRRKKIASADRIP VPQQSISIRGRDSQTTQESQKKVEEQAKANLRIS RKNLGDETRGPVGAGN 15 MDPKGEEDQDVSHQDLIKQYRKPRTACNNCYCKK SIVagmVer HIV-1 SIVagmVer CCFHCYACFLQKGLGVTYHAPRTRRKKIRSLNLA PLQHQSISTKWGRDGQTTPTSQEKVETTAGSN 16 MDKEEEPHPLLQDLHRPLQPRTACNNCYCKKCCF SIVagmGri HIV-1 SIVagmVer HCYACFLRKGLGITYHAFRTRRKKIASADRIPVP QQSISIRGRDSQTTQESQKKVEEQAKANLRISRK NLGDETRGPVGAGN 17 MDPKGEEDQDVSHQDLIKQYRKPRTACNNCYCKK SIVagmVer HIV-1 SIVagmGri CCFHCYACFLQKGLGVRYHVSRKRRKTSTQDNQD PIRQQSISTVQRNGQTTEEGKTEVEKAAAAN 18 MAQEEGLQVWEELQEELQRPRTACNNCYCKKCCF SIVagmSab HIV-1 SIVagmVer HCYACFLRKGLGITYHAFRTRRKKIASADRIPVP QQSISIRGRDSQTTQESQKKVEEQAKANLRISRK NLGDETRGPVGAGN 19 MDPKGEEDQDVSHQDLIKQYRKPRTACNNCYCKK SIVagmVer HIV-1 SIVagmSab CCFHCYACFTQKGLGIAYYVPRTRRTVKKIQNNQ VPIHNQSISTVVTRNSQAEKKSQTKVGQAATADH TPGRKNS 20 MDKGEDEQGAYHQDLIEQLKAPRTACNNCYCKKC SIVagmVer HIV-1 SIVagmVer CFHCYACFLRKGLGITYHAFRTRRKKIASADRIP VPQQSISIRGRDSQTTQESQKKVEEQAKANLRIS RKNLGDETRGPVGAGN 21 MDPKGEEDQDVSHQDLIKQYRKPRTACNNCYCKK SIVagmVer HIV-1 SIVagmVer CCFHCYACFFLQKGLGVTYHAPRIRRKKIAPLDR FPEQKQSISTRGRDSQTTQKGQEKVETSARTAPS LGRKNLAQQSGRATGASD 22 MDVRAVGSERIEEETLYNPRKTACTTCYCKKCCF SIVrcm HIV-1 HIV-1 HCQVCFTRKGLGISYGRKKRRQRRRAPQDSQTHQ ASLSKQPASQSRGDPTGPTESKKKVERETETDPF D 23 MDVRAVGSERIEEETLYNPRTACNNCYCKKCCFH SIVrcm HIV-1 SIVagmVer CYACFLRKGLGITYHAFRTRRKKIASADRIPVPQ QSISIRGRDSQTTQESQKKVEEQAKANLRISRKN LGDETRGPVGAGN 24 MDVRAVGSERIEEETLYNPLETCNNTCYCKECCY SIVrcm HIV-2 HIV-2 HCQLCFLNKGLGIWYDRKGRRRRSPKKIKAHSSS ASDKSISTRTRNSQPEEKQKKTLETTLGTDCGPG RSHIYIS 25 MDVRAVGSERIEEETLYNPTTACSKCYCKMCCWH SIVrcm HIV-1 HIV-1 CQLCFLNKGLGISYGRKKRKRRRGTPQSRQDHQN PVPKQPLPTTRGNPTNPKESKKEVASKTETNQCD 26 MSSTDQICQTQRVPPSFLEGTFLEKGPPTPRKTA SIVsyk HIV-1 HIV-1 CTTCYCKKCCFHCQVCFTRKGLGISYGRKKRRQR RRAPQDSQTHQASLSKQPASQSRGDPTGPTESKK KVERETETDPFD 27 MSSTDQICQTQRVPPSFLEGTFLEKGPPTPRTAC SIVsyk HIV-1 SIVagmVer NNCYCKKCCFHCYACFLRKGLGITYHAFRTRRKK IASADRIPVPQQSISIRGRDSQTTQESQKKVEEQ AKANLRISRKNLGDETRGPVGAGN 28 MSSTDQICQTQRVPPSFLEGTFLEKGPPTPLETC SIVsyk HIV-2 HIV-2 NNTCYCKECCYHCQLCFLNKGLGIWYDRKGRRRR SPKKIKAHSSSASDKSISTRTRNSQPEEKQKKTL ETTLGTDCGPGRSHIYIS 29 MSSTDQICQTQRVPPSFLEGTFLEKGPPTPTTAC SIVsyk HIV-1 HIV-1 SKCYCKMCCWHCQLCFLNKGLGISYGRKKRKRRR GTPQSRQDHQNPVPKQPLPTTRGNPTNPKESKKE VASKTETNQCD 30 MDGQEAGLERQEEETLYNPFQSVETPRKTACTTC SIVagi HIV-1 HIV-1 YCKKCCFHCQVCFTRKGLGISYGRKKRRQRRRAP QDSQTHQASLSKQPASQSRGDPTGPTESKKKVER ETETDPFD 31 MDGQEAGLERQEEETLYNPFQSVETPRTACNNCY SIVagi HIV-1 SIVagmVer CKKCCFHCYACFLRKGLGITYHAFRTRRKKIASA DRIPVPQQSISIRGRDSQTTQESQKKVEEQAKAN LRISRKNLGDETRGPVGAGN 32 MDGQEAGLERQEEETLYNPFQSVETPLETCNNTC SIVagi HIV-2 HIV-2 YCKECCYHCQLCFLNKGLGIWYDRKGRRRRSPKK IKAHSSSASDKSISTRTRNSQPEEKQKKTLETTL GTDCGPGRSHIYIS 33 MDGQEAGLERQEEETLYNPFQSVETPTTACSKCY SIVagi HIV-1 HIV-1 CKMCCWHCQLCFLNKGLGISYGRKKRKRRRGTPQ SRQDHQNPVPKQPLPTTRGNPTNPKESKKEVASK TETNQCD 34 MSTQGHQQDQDQGKGTLEEAYKTNLEAPRKTACT SIVsun HIV-1 HIV-1 TCYCKKCCFHCQVCFTRKGLGISYGRKKRRQRRR APQDSQTHQASLSKQPASQSRGDPTGPTESKKKV ERETETDPFD 35 MSTQGHQQDQDQGKGTLEEAYKTNLEAPRTACNN SIVsun HIV-1 SIVagmVer CYCKKCCFHCYACFLRKGLGITYHAFRTRRKKIA SADRIPVPQQSISIRGRDSQTTQESQKKVEEQAK ANLRISRKNLGDETRGPVGAGN 36 MSTQGHQQDQDQGKGTLEEAYKTNLEAPLETCNN SIVsun HIV-2 HIV-2 TCYCKECCYHCQLCFLNKGLGIWYDRKGRRRRSP KKIKAHSSSASDKSISTRTRNSQPEEKQKKTLET TLGTDCGPGRSHIYIS 37 MSTQGHQQDQDQGKGTLEEAYKTNLEAPTTACSK SIVsun HIV-1 HIV-1 CYCKMCCWHCQLCFLNKGLGISYGRKKRKRRRGT PQSRQDHQNPVPKQPLPTTRGNPTNPKESKKEVA SKTETNQCD 38 MQQPEQEQHTQQKQHLDQLEEIYKEAITDPRKTA SIVIho HIV-1 HIV-1 CTTCYCKKCCFHCQVCFTRKGLGISYGRKKRRQR RRAPQDSQTHQASLSKQPASQSRGDPTGPTESKK KVERETETDPFD 39 MQQPEQEQHTQQKQHLDQLEEIYKEAITDPRTAC SIVIho HIV-1 SIVagmVer NNCYCKKCCFHCYACFLRKGLGITYHAFRTRRKK IASADRIPVPQQSISIRGRDSQTTQESQKKVEEQ AKANLRISRKNLGDETRGPVGAGN 40 MQQPEQEQHTQQKQHLDQLEEIYKEAITDPLETC SIVIho HIV-2 HIV-2 NNTCYCKECCYHCQLCFLNKGLGIWYDRKGRRRR SPKKIKAHSSSASDKSISTRTRNSQPEEKQKKTL ETTLGTDCGPGRSHIYIS 41 MQQPEQEQHTQQKQHLDQLEEIYKEAITDPTTAC SIVIho HIV-1 HIV-1 SKCYCKMCCWHCQLCFLNKGLGISYGRKKRKRRR GTPQSRQDHQNPVPKQPLPTTRGNPTNPKESKKE VASKTETNQCD 42 METPLKEQESSLRSSSEPSSCTSEAVAATPGLAN SIVstm HIV-1 HIV-1 QEEEILWQLYRPRKTACTTCYCKKCCFHCQVCFT RKGLGISYGRKKRRQRRRAPQDSQTHQASLSKQP ASQSRGDPTGPTESKKKVERETETDPFD 43 METPLKEQESSLRSSSEPSSCTSEAVAATPGLAN SIVstm HIV-1 SIVagmVer QEEEILWQLYRPRTACNNCYCKKCCFHCYACFLR KGLGITYHAFRTRRKKIASADRIPVPQQSISIRG RDSQTTQESQKKVEEQAKANLRISRKNLGDETRG PVGAGN 44 METPLKEQESSLRSSSEPSSCTSEAVAATPGLAN SIVstm HIV-2 HIV-2 QEEEILWQLYRPLETCNNTCYCKECCYHCQLCFL NKGLGIWYDRKGRRRRSPKKIKAHSSSASDKSIS TRTRNSQPEEKQKKTLETTLGTDCGPGRSHIYIS 45 METPLKEQESSLRSSSEPSSCTSEAVAATPGLAN SIVstm HIV-1 HIV-1 QEEEILWQLYRPTTACSKCYCKMCCVVHCQLCFL NKGLGISYGRKKRKRRRGTPQSRQDHQNPVPKQP LPTTRGNPTNPKESKKEVASKTETNQCD 46 MDKGEEERTVLHQDLIRQYKKPRKTACTTCYCKK SIVagmVer HIV-1 HIV-1 CCFHCQVCFTRKGLGISYGRKKRRQRRRAPQDSQ THQASLSKQPASQSRGDPTGPTESKKKVERETET DPFD 47 MDKGEEERTVLHQDLIRQYKKPRTACNNCYCKKC SIVagmVer HIV-1 SIVagmVer CFHCYACFLRKGLGITYHAFRTRRKKIASADRIP VPQQSISIRGRDSQTTQESQKKVEEQAKANLRIS RKNLGDETRGPVGAGN 48 MDKGEEERTVLHQDLIRQYKKPLETCNNTCYCKE SIVagmVer HIV-2 HIV-2 CCYHCQLCFLNKGLGIWYDRKGRRRRSPKKIKAH SSSASDKSISTRTRNSQPEEKQKKTLETTLGTDC GPGRSHIYIS 49 MDKGEEERTVLHQDLIRQYKKPTTACSKCYCKMC SIVagmVer HIV-1 HIV-1 CWHCQLCFLNKGLGISYGRKKRKRRRGTPQSRQD HQNPVPKQPLPTTRGNPTNPKESKKEVASKTETN QCD 50 MQPLQNRPDLGEEILSQLYRPRKTACTTCYCKKC SIVmac HIV-1 HIV-1 CFHCQVCFTRKGLGISYGRKKRRQRRRAPQDSQT HQASLSKQPASQSRGDPTGPTESKKKVERETETD PFD 51 MQPLQNRPDLGEEILSQLYRPRTACNNCYCKKCC SIVmac HIV-1 SIVagmVer

FHCYACFLRKGLGITYHAFRTRRKKIASADRIPV PQQSISIRGRDSQTTQESQKKVEEQAKANLRISR KNLGDETRGPVGAGN 52 MQPLQNRPDLGEEILSQLYRPLETCNNTCYCKEC SIVmac HIV-2 HIV-2 CYHCQLCFLNKGLGIWYDRKGRRRRSPKKIKAHS SSASDKSISTRTRNSQPEEKQKKTLETTLGTDCG PGRSHIYIS 53 MQPLQNRPDLGEEILSQLYRPTTACSKCYCKMCC SIVmac HIV-1 HIV-1 WHCQLCFLNKGLGISYGRKKRKRRRGTPQSRQDH QNPVPKQPLPTTRGNPTNPKESKKEVASKTETNQ CD 54 METPLKEQESSLESSREHSSSISEVDADTPESAS SIVsmm HIV-1 HIV-1 LEEEILSQLYRPRKTACTTCYCKKCCFHCQVCFT RKGLGISYGRKKRRQRRRAPQDSQTHQASLSKQP ASQSRGDPTGPTESKKKVERETETDPFD 55 METPLKEQESSLESSREHSSSISEVDADTPESAS SIVsmm HIV-1 SIVagmVer LEEEILSQLYRPRTACNNCYCKKCCFHCYACFLR KGLGITYHAFRTRRKKIASADRIPVPQQSISIRG RDSQTTQESQKKVEEQAKANLRISRKNLGDETRG PVGAGN 56 METPLKEQESSLESSREHSSSISEVDADTPESAS SIVsmm HIV-1 HIV-1 LEEEILSQLYRPTTACSKCYCKMCCVVHCQLCFL NKGLGISYGRKKRKRRRGTPQSRQDHQNPVPKQP LPTTRGNPTNPKESKKEVASKTETNQCD 57 MDAGKAVSDKKEGDVTPYDPFRDRTTPRKTACTT SIVmnd HIV-1 HIV-1 CYCKKCCFHCQVCFTRKGLGISYGRKKRRQRRRA PQDSQTHQASLSKQPASQSRGDPTGPTESKKKVE RETETDPFD 58 MDAGKAVSDKKEGDVTPYDPFRDRTTPRTACNNC SIVmnd HIV-1 HIV-1 YCKKCCFHCYACFLRKGLGITYHAFRTRRKKIAS ADRIPVPQQSISIRGRDSQTTQESQKKVEEQAKA NLRISRKNLGDETRGPVGAGN 59 MDAGKAVSDKKEGDVTPYDPFRDRTTPTTACSKC SIVmnd HIV-1 HIV-1 YCKMCCWHCQLCFLNKGLGISYGRKKRKRRRGTP QSRQDHQNPVPKQPLPTTRGNPTNPKESKKEVAS KTETNQCD 60 MEPSGKEDHNCPPQDSGQEEIDYKQLLEEYYQPR SIVmnd HIV-1 HIV-1 KTACTTCYCKKCCFHCQVCFTRKGLGISYGRKKR RQRRRAPQDSQTHQASLSKQPASQSRGDPTGPTE SKKKVERETETDPFD 61 MEPSGKEDHNCPPQDSGQEEIDYKQLLEEYYQPR SIVmnd HIV-1 SIVagmVer TACNNCYCKKCCFHCYACFLRKGLGITYHAFRTR RKKIASADRIPVPQQSISIRGRDSQTTQESQKKV EEQAKANLRISRKNLGDETRGPVGAGN 62 MEPSGKEDHNCPPQDSGQEEIDYKQLLEEYYQPL SIVmnd HIV-2 HIV-2 ETCNNTCYCKECCYHCQLCFLNKGLGIWYDRKGR RRRSPKKIKAHSSSASDKSISTRTRNSQPEEKQK KTLETTLGTDCGPGRSHIYIS 63 MEPSGKEDHNCPPQDSGQEEIDYKQLLEEYYQPT SIVmnd HIV-1 HIV-1 TACSKCYCKMCCWHCQLCFLNKGLGISYGRKKRK RRRGTPQSRQDHQNPVPKQPLPTTRGNPTNPKES KKEVASKTETNQCD 64 MDVGEVASDKKEEDITHFDPFRARTTPRKTACTT SIVmnd HIV-1 HIV-1 CYCKKCCFHCQVCFTRKGLGISYGRKKRRQRRRA PQDSQTHQASLSKQPASQSRGDPTGPTESKKKVE RETETDPFD 65 MDVGEVASDKKEEDITHFDPFRARTTPRTACNNC SIVmnd HIV-1 SIVagmVer YCKKCCFHCYACFLRKGLGITYHAFRTRRKKIAS ADRIPVPQQSISIRGRDSQTTQESQKKVEEQAKA NLRISRKNLGDETRGPVGAGN 66 MDVGEVASDKKEEDITHFDPFRARTTPLETCNNT SIVmnd HIV-2 HIV-2 CYCKECCYHCQLCFLNKGLGIWYDRKGRRRRSPK KIKAHSSSASDKSISTRTRNSQPEEKQKKTLETT LGTDCGPGRSHIYIS 67 MDVGEVASDKKEEDITHFDPFRARTTPTTACSKC SIVmnd HIV-1 HIV-1 YCKMCCWHCQLCFLNKGLGISYGRKKRKRRRGTP QSRQDHQNPVPKQPLPTTRGNPTNPKESKKEVAS KTETNQCD 68 MDARKVDLDQQDAGTHFEPRKTACTTCYCKKCCF SIVdrl HIV-1 HIV-1 HCQVCFTRKGLGISYGRKKRRQRRRAPQDSQTHQ ASLSKQPASQSRGDPTGPTESKKKVERETETDPF D 69 MDARKVDLDQQDAGTHFEPRTACNNCYCKKCCFH SIVdrl HIV-1 SIVagmVer CYACFLRKGLGITYHAFRTRRKKIASADRIPVPQ QSISIRGRDSQTTQESQKKVEEQAKANLRISRKN LGDETRGPVGAGN 70 MDARKVDLDQQDAGTHFEPLETCNNTCYCKECCY SIVdrl HIV-2 HIV-2 HCQLCFLNKGLGIWYDRKGRRRRSPKKIKAHSSS ASDKSISTRTRNSQPEEKQKKTLETTLGTDCGPG RSHIYIS 71 MDARKVDLDQQDAGTHFEPTTACSKCYCKMCCWH SIVdrl HIV-1 HIV-1 CQLCFLNKGLGISYGRKKRKRRRGTPQSRQDHQN PVPKQPLPTTRGNPTNPKESKKEVASKTETNQCD 72 MSSKEELRTTPISDPFQEEGRGPRKTACTTCYCK SIVtal HIV-1 HIV-1 KCCFHCQVCFTRKGLGISYGRKKRRQRRRAPQDS QTHQASLSKQPASQSRGDPTGPTESKKKVERETE TDPFD 73 MSSKEELRTTPISDPFQEEGRGPRTACNNCYCKK SIVtal HIV-1 SIVagmVer CCFHCYACFLRKGLGITYHAFRTRRKKIASADRI PVPQQSISIRGRDSQTTQESQKKVEEQAKANLRI SRKNLGDETRGPVGAGN 74 MSSKEELRTTPISDPFQEEGRGPLETCNNTCYCK SIVtal HIV-2 HIV-2 ECCYHCQLCFLNKGLGIWYDRKGRRRRSPKKIKA HSSSASDKSISTRTRNSQPEEKQKKTLETTLGTD CGPGRSHIYIS 75 MSSKEELRTTPISDPFQEEGRGPTTACSKCYCKM SIVtal HIV-1 HIV-1 CCWHCQLCFLNKGLGISYGRKKRKRRRGTPQSRQ DHQNPVPKQPLPTTRGNPTNPKESKKEVASKTET NQCD 76 MDPSVEELPKEQRPGAAPATPRKTACTTCYCKKC SIVmus HIV-1 HIV-1 CFHCQVCFTRKGLGISYGRKKRRQRRRAPQDSQT HQASLSKQPASQSRGDPTGPTESKKKVERETETD PFD 77 MDPSVEELPKEQRPGAAPATPRTACNNCYCKKCC SIVmus HIV-1 SIVagmVer FHCYACFLRKGLGITYHAFRTRRKKIASADRIPV PQQSISIRGRDSQTTQESQKKVEEQAKANLRISR KNLGDETRGPVGAGN 78 MDPSVEELPKEQRPGAAPATPLETCNNTCYCKEC SIVmus HIV-2 HIV-2 CYHCQLCFLNKGLGIWYDRKGRRRRSPKKIKAHS SSASDKSISTRTRNSQPEEKQKKTLETTLGTDCG PGRSHIYIS 79 MDPSVEELPKEQRPGAAPATPTTACSKCYCKMCC SIVmus HIV-1 HIV-1 WHCQLCFLKGLGISYGRKKRKRRRGTPQSRQDHQ NPVPKQPLPTTRGNPTNPKESKKEVASKTETNQC D 80 MEEEMDLFQGRGRGEANHPRKTACTTCYCKKCCF SIVdeb HIV-1 HIV-1 HCQVCFTRKGLGISYGRKKRRQRRRAPQDSQTHQ ASLSKQPASQSRGDPTGPTESKKKVERETETDPF D 81 MEEEMDLFQGRGRGEANHPRTACNNCYCKKCCFH SIVdeb HIV-1 SIVagmVer CYACFLRKGLGITYHAFRTRRKKIASADRIPVPQ QSISIRGRDSQTTQESQKKVEEQAKANLRISRKN LGDETRGPVGAGN 82 MEEEMDLFQGRGRGEANHPLETCNNTCYCKECCY SIVdeb HIV-2 HIV-2 HCQLCFLNKGLGIWYDRKGRRRRSPKKIKAHSSS ASDKSISTRTRNSQPEEKQKKTLETTLGTDCGPG RSHIYIS 83 MEEEMDLFQGRGRGEANHPTTACSKCYCKMCCWH SIVdeb HIV-1 HIV-1 CQLCFLNKGLGISYGRKKRKRRRGTPQSRQDHQN PVPKQPLPTTRGNPTNPKESKKEVASKTETNQCD 84 MNADSIDPFAGNKTPRKTACTTCYCKKCCFHCQV SIVden HIV-1 HIV-1 CFTRKGLGISYGRKKRRQRRRAPQDSQTHQASLS KQPASQSRGDPTGPTESKKKVERETETDPFD 85 MNADSIDPFAGNKTPRTACNNCYCKKCCFHCYAC SIVden HIV-1 SIVagmVer FLRKGLGITYHAFRTRRKKIASADRIPVPQQSIS IRGRDSQTTQESQKKVEEQAKANLRISRKNLGDE TRGPVGAGN 86 MNADSIDPFAGNKTPLETCNNTCYCKECCYHCQL SIVden HIV-2 HIV-2 CFLNKGLGIWYDRKGRRRRSPKKIKAHSSSASDK SISTRTRNSQPEEKQKKTLETTLGTDCGPGRSHI YIS 87 MNADSIDPFAGNKTPTTACSKCYCKMCCWHCQLC SIVden HIV-1 HIV-1 FLNKGLGISYGRKKRKRRRGTPQSRQDHQNPVPK QPLPTTRGNPTNPKESKKEVASKTETNQCD 88 MDPKGEEDQDVSHQDLIKQYRKPRTACNNCYCKK SIVagmVer HIV-1 SIVwrc CCFHCYACFLRKGLFLQKGLGISYRSYSKKTKPD TTTAASRBLGRVTLSLYLSRTTSTTWKRDSKTAK KE 89 MDPKGEEDQDVSHQDLIKQYRKPACYCRIPACIA SIVagmVer NADI SIVagmVer GERRYGTCIYQGRLWAFCCFLRKGLGITYHAFRT .alpha.defensin RRKKIASADRIPVPQQSISIRGRDSQTTQESQKK VEEQAKANLRISRKNLGDETRGPVGAGN 90 MDPKGEEDQDVSHQDLIKQYRKPTCLKSGAICHP SIVagmVer HBD2 SIVagmVer VFCPRRYKQIGTCGLPGTKCCFLRKGLGITYHAF .beta.defensin RTRRKKIASADRIPVPQQSISIRGRDSQTTQESQ KKVEEQAKANLRISRKNLGDETRGPVGAGN 91 MDPKGEEDQDVSHQDLIKQYRKPRTACNNCYCKK SIVagmVer HIV-1 SIVgor CCFHCYACFTKKGLGISYGRKKRRRPARTADKDQ DNQDPVSKQSLAGTRSQQE 92 MDPKGEEDQDVSHQDLIKQYRKPRTACNNCYCKK SIVagmVer HIV-1 SIVcpzPts CCFHCYACFTKKALGISYGRKRRGRKSAGDNKTH QDPVRQQSLPKRSRIQSSQEESQKEVETEAGSGG RPRPEDSSASSGRTSGTSSSGSTRPVSTSSGCW GPYSKP 93 MDPKGEEDQDVSHQDLIKQYRKPRTACNNCYCKK SIVagmVer HIV-1 SIVmon CCFHCYACFLTKGLGISYGRKRKRRRATSPVPGL SSSKNPARKQGRDTLFFLLRSLSHPTRDSQRPTE QAQAVATAATPDRQH 94 METPLREQENSLKSSNGRSSCTSEAAAPTLESAN SIVmne HIV-2 HIV-2 LEEEILSQLYRPLETCNNTCYCKECCYHCQLCFL NKGLGIWYDRKGRRRRSPKKIKAHSSSASDKSIS TRTRNSQPEEKQKKTLETTLGTDCGPGRSHIYIS 95 MDPKGEEDQDVSHQDLIKQYRKPRTACNNCYCKK SIVagmVer HIV-1 SIVcpzPtt CCFHCYACFFMKKGLGISYGRKKRRQRRGASKSN QNHQDSIPEQPFSQSRGDQSSPEKQEKKVESKTT SDPFGC *TF region is italicized .sup..dagger-dbl.Cystein-rich region is underlined

TABLE-US-00002 TABLE 2 SIV strain abbreviations useful in Tat derivative peptides SIV host designation SIV Host Species Latin designation SIVagmVer (African Green Monkey) Chlorocebus pygerythrus Vervet SIVagmGri (African Green Monkey) Chlorocebus aethiops Grivet SIVagmTan (African Green Monkey) Chlorocebus tantalus Tantalus SIVagmSab (African Green Monkey) Chlorocebus sabaeus Sabeus SIVrcm Red-capped Mangabey Cercocebus torquatus torquatus SIVsyk Sykes Monkey Cercopithecus albogularis SIVagi Agile Mangabey Cercocebus agilis SIVsun Sun-tailed Monkey Cercopithecus solatus SIVlho L'Hoests Monkey Cercopithecus lhoesti SIVstm Stump-tail Macaque Macaca arctoides SIVmac Macaque Macaca mulatta SIVsmm Sooty mangabey monkey Cercocebus atys atys SIVmnd Mandrill Mandrillus sphinx SIVdrl Drill Monkey Mandrillus leucophaeus SIVtal Talapoin Monkey Miopithecus talapoin SIVmus Mustached Monkey Cercopithecus cephus SIVdeb De Brazza's Monkey Cercopithecus neglectus SIVden Dent's Monkey Cercopithecus denti SIVmon Mona Monkey Cercopithecus mona SIVgor Gorilla Gorilla gorilla SIVwrc Western Red Colobus Procolobus verus SIVcpzPtt Pan Troglodytes Pan troglodytes troglodytes Troglodytes SIVcpzPts Pan Troglodytes Pan troglodytes schweinfurthii Schweinfurthi SIVmne Pig-tail Macaque Macaca nemestrina SIVasc Red-tailed Guenon Cercopithecus ascanius schmidti SIVbab Yellow Baboon Papio spp. SIVblc Bioko Black Colobus Cercopithecus satanas Monkey satanas SIVbkm Black Mangabey Lophocebus aterrimus SIVblu Blue Monkey Cercopithecus mitis SIVcol Colobus Monkey Colobus guereza SIVolc Oilve Colobus Monkey procolobus verus SIVgsn Greater Spot-nosed Cercopithecus nictitans Monkey SIVkrc Kibale Red Colobus Procolobus [Piliocolobus] Moneky rufomitratus tephrosceles SIVpat Patas Monkey Erythrocebus patas SIVpre Preussis Monkey Cercopithecus preussi SIVreg Red-eared Guenon Cercopithecus erythrotis erythrotis SIVtrc Tshuapa Red Colobus Piliocolobus tholloni SIVwcm White-crowned Cercocebus torquatus lunulatus Mangabey SIVwol Wolf's Monkey Cercopithecus wolfi

[0053] In additional embodiments, disclosed herein is the use of conservatively modified variants of the Tat derivative polypeptides. The variants described herein maintain the immunostimulating activity of the parent or source Tat derivative polypeptide.

[0054] As used herein the term "conservatively modified variants" refers to variant peptides which have the same or similar biological activity of the original peptides. For example, conservative amino acid changes may be made, which, although they alter the primary sequence of the protein or peptide, do not alter its function. A conservative variant has at least one amino acid substituted by another amino acid or an amino acid analog that has at least one property similar to that of the original amino acid from an exemplary reference peptide. Examples of properties include, without limitation, similar size, topography, charge, hydrophobicity, hydrophilicity, lipophilicity, covalent-bonding capacity, hydrogen-bonding capacity, a physicochemical property, of the like, or any combination thereof. A conservative substitution can be assessed by a variety of factors, such as, e.g., the physical properties of the amino acid being substituted (Table 3) or how the original amino acid would tolerate a substitution (Table 4). The selections of which amino acid can be substituted for another amino acid in a peptide disclosed herein are known to a person of ordinary skill in the art. A conservative variant can function in substantially the same manner as the exemplary reference peptide, and can be substituted for the exemplary reference peptide in any aspect of the present specification.

TABLE-US-00003 TABLE 3 Amino Acid Properties Property Amino Acids Aliphatic G, A, I, L, M, P, V Aromatic F, H, W, Y C-beta branched I, V, T Hydrophobic C, F, I, L, M, V, W Small polar D, N, P Small non-polar A, C, G, S, T Large polar E, H, K, Q, R, W, Y Large non-polar F, I, L, M, V Charged D, E, H, K, R Uncharged C, S, T Negative D, E Positive H, K, R Acidic D, E Basic K, R Amide N, Q

TABLE-US-00004 TABLE 4 Amino Acid Substitutions Amino Favored Neutral Disfavored Acid Substitution Substitutions substitution A G, S, T C, E, I, K, M, L, P, D, F, H, N, Y, W Q, R, V C F, S, Y, W A, H, I, M, L, T, V D, E, G, K, N, P, Q, R D E, N G, H, K, P, Q, R, S, T A, C, I, L, E D, K, Q A, H, N, P, R, S, T C, F, G, I, L, M, V, W, Y F M, L, W, Y C, I, V A, D, E, G, H, K, N, P, Q, R, S, T G A, S D, K, N, P, Q, R C, E, F, H, I, L, M, T, V, W, Y H N, Y C, D, E, K, Q, R, S, A, F, G, I, L, M, P, T, W V I V, L, M A, C, T, F, Y D, E, G, H, K, N, P, Q, R, S, W K Q, E, R A, D, G, H, M, N, P, C, F, I, L, V, W, Y S, T L F, I, M, V A, C, W, Y D, E, G, H, K, N, P, Q, R, S, T M F, I, L, V A, C, R, Q, K, T, W, Y D, E, G, H, N, P, S N D, H, S E, G, K, Q, R, T A, C, F, I, L, M, P, V, W, Y P -- A, D, E, G, K, Q, R, C, F, H, I, L, M, N, S, T V, W, Y Q E, K, R A, D, G, H, M, N, P, C, F, I, L, V, W, Y S, T R K, Q A, D, E, G, H, M, N, C, F, I, L, V, W, Y P, S, T S A, N, T C, D, E, G, H, K, P, F, I, L, M, V, W, Y Q, R, T T S A, C, D, E, H, I, K, F, G, L, W, Y M, N, P, Q, R, V V I, L, M A, C, F, T, Y D, E, G, H, K, N, P, Q, R, S, W W F, Y H, L, M A, C, D, E, G, I, K, N, P, Q, R, S, T, V Y F, H, W C, I, L, M, V A, D, E, G, K, N, P, Q, R, S, T Matthew J. Betts and Robert, B. Russell, Amino Acid Properties and Consequences of Substitutions, pp. 289-316, In Bioinformatics for Geneticists, (eds Michael R. Barnes, Ian C. Gray, Wiley, 2003).

[0055] In one embodiment, a Tat derivative polypeptide is a peptide disclosed in Table 1. In certain embodiments, the Tat derivative is not one of SEQ ID NOs. 2, 3 or 4. A Tat derivative polypeptide can also comprise conservative variants of a Tat derivative polypeptide. In an embodiment, a conservative variant of a Tat derivative polypeptide is a conservative variant of a Tat derivative polypeptide disclosed herein. In aspects of this embodiment, a conservative variant of a Tat derivative polypeptide can be, for example, an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to a Tat derivative polypeptide. In other aspects of this embodiment, a conservative variant of a Tat derivative polypeptide can be, for example, an amino acid sequence having at most 50%, 55%, 60%, 65%, 70%, 75%, at most 80%, at most 85%, at most 90%, at most 95%, at most 97%, at most 98%, or at most 99% amino acid sequence identity to a Tat derivative polypeptide.

[0056] Therefore, disclosed herein are amino acid sequences 85%, 90%, 95%, 98%, 99% or 100% identical to the Tat derivatives disclosed in SEQ ID NOs. 5-95.

[0057] In other aspects of this embodiment, a conservative variant of a Tat derivative polypeptide can be, for example, a Tat derivative polypeptide having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 or more conservative substitutions in the amino acid sequence of a Tat derivative polypeptide. In other aspects of this embodiment, a conservative variant of a Tat derivative polypeptide can be, for example, an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, or at least 25 conservative substitutions in the amino acid sequence of a Tat derivative polypeptide. In yet other aspects of this embodiment, a conservative variant of a Tat derivative polypeptide can be, for example, an amino acid sequence having at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 20, at most 25, or at most 30 conservative substitutions in the amino acid sequence of a Tat derivative polypeptide.

[0058] Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

[0059] Also included are polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids. The peptides disclosed herein are not limited to products of any of the specific exemplary processes listed herein.

[0060] As used herein, amino acid sequences which are substantially the same typically share more than 95% amino acid identity. It is recognized, however, that proteins (and DNA or mRNA encoding such proteins) containing less than the above-described level of identity arising as splice variants or that are modified by conservative amino acid substitutions (or substitution of degenerate codons) are contemplated to be within the scope of the present disclosure. As readily recognized by those of skill in the art, various ways have been devised to align sequences for comparison, e.g., Blosum 62 scoring matrix, as described by Henikoff and Henikoff in Proc. Natl. Acad Sci. USA 89:10915 (1992). Algorithms conveniently employed for this purpose are widely available (see, for example, Needleman and Wunsch in J. Mol. Bio. 48:443 (1970).

[0061] In addition to substantially full length polypeptides, the present disclosure also provides for biologically active fragments of the Tat derivative polypeptides. The term "biologically active fragment" refers to fragments of the Tat derivative polypeptides which have immunostimulatory activity.

[0062] Furthermore, the peptides disclosed herein can self-associate into multimers, including but not limited to, dimers, trimers, and tetramers, in addition to existing in the monomer form. Multimerization of peptides can occur spontaneously or can be facilitated by subjecting the peptides to conditions conducive to multimerization. These conditions are known to persons of ordinary skill in peptide chemistry. The compositions disclosed herein can include monomers or multimers of the peptides, or a mixture of monomers and multimers.

[0063] The following expression systems are suitable for use in expressing the disclosed Tat derivatives: mammalian cell expression systems such as, but not limited to, Chinese Hamster Ovary (CHO), COS cells (fibroblast-like cells from African green monkey kidney tissue), bovine cells, murine cells, human embryonic kidney cells, or baby hamster kidney cells; insect cell expression systems such as, but not limited to, Bac-to-Bac expression system, baculovirus expression system, and DES expression systems; yeast expression systems: and E. coli expression systems including, but not limited to, pET, pSUMO and GST expression systems. In another embodiment, the Tat derivatives are expressed with a histadine (poly histidine) tag useful for isolation of the polypeptide. Histidine tag purification systems are known to persons of ordinary skill in the art.

[0064] "Therapeutically effective amount" is intended to qualify the amount required to achieve a therapeutic effect. As used herein, the term "therapeutically effective amount" is synonymous with "therapeutically effective dose" and when used in reference to treating cancer means the most beneficial dose of a composition disclosed herein necessary to achieve the desired therapeutic effect and includes a dose sufficient to reduce tumor size, inhibit growth of a tumor, or cause regression of a tumor.

[0065] Override of Immune Checkpoints

[0066] Immune checkpoints, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1) expressed on tumor-specific T cells, lead to compromised activation and suppressed effector functions such as proliferation, cytokine secretion, and tumor cell lysis. Specifically modulating these receptors with immune checkpoint inhibitors is a new approach in cancer immunotherapy.

[0067] An important negative co-stimulatory signal regulating T cell activation is provided by PD-1 (also known as CD279), and its ligand binding partners PD-L1 (also known as B7-H1 and CD274) and PD-L2 (also known as B7-DC and CD273). PD-1 is related to CD28 and CTLA-4, but lacks the membrane proximal cysteine that allows homodimerization. The cytoplasmic domain of PD-1 contains an immunoreceptor tyrosine-based inhibition motif (ITIM, V/IxYxxL/V). Thus far, the only identified ligands for PD-1 are PD-L1 and PD-L2.

[0068] The immunosuppressive nature of the tumor microenvironment is helpful to explain the immune dysfunction that accompanies cancer progression. The PD-1/PD-L1 signaling pathway is one emerging model for immune evasion at the tumor site and represents an important checkpoint and barrier for an effective immune response.

[0069] The presence of PD-L1 in the tumor site is considered to facilitate immune evasion as a result of an active tumor-mediated process for reprogramming host cells present in the tumor microenvironment. The engagement of PD-L1 with its PD-1 receptor on the surface infiltrating T-cells may induce their programmed cell death, anergy, and exhaustion. Induction of PD-L1 in the tumor microenvironment may serve as a "molecular shield" to protect the tumor from a cell-mediated immune response.

[0070] The refractory state of cancers to immunotherapeutics may be a consequence of immunosuppression that accompanies disease progression in established cancers. The Tat derivative polypeptides disclosed herein elicit antitumor immune responses by triggering monocyte-derived dendritic cells to stimulate the CD8+ CTL and override PD-L1 immunosuppression. Thus, the PD-1/PD-L1 immunosuppressive signaling pathway may provide a potential mechanism by which breast tumors evade host tumor immunity and therefore Tat derivative polypeptides can impact solid tumor progression by induction of tumor infiltrating CD8+ CTLs in the face of PD-L1 immunosuppression.

[0071] Modulating of signaling through PD-L1, thereby preventing PD-L1 from sending a negative co-stimulatory signal to T-cells is likely to enhance immunity in response to infection (e.g., acute and chronic) and tumor immunity. In addition, the Tat derivative polypeptides disclosed herein may be combined with antagonists of other components of PD-1:PD-L1 signaling, for example, antagonist anti-PD-1 and anti-PD-L2 antibodies.

[0072] Additionally, agents that modulate immune checkpoints that can be used for immunotherapeutic treatment regimens for cancer in combination with the disclosed Tat derivative polypeptides include, but are not limited to, CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, LAG-3, TIM-3, and GITR, and their respective ligands.

[0073] Use of Tat Derivative Polypeptides

[0074] The disclosed Tat derivatives are immune-stimulating polypeptides which are useful in many types of cancers. In one embodiment, the Tat derivatives are useful in treating a type of cancer including, but not limited to, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal-cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, bronchial adenomas/carcinoids, carcinoid tumor, cervical cancer, chronic myeloproliferative disorders, colon cancer, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, eye cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, glioma, gastric carcinoid, head and neck cancer, heart cancer, hepatocellular cancer, Hodgkin's lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi sarcoma, kidney cancer, leukemias, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancer, lymphomas, macroglobulinemia, medulloblastoma, melanoma, merkel cell carcinoma, mesothelioma, mouth cancer, multiple myeloma/plasma cell neoplasm, mycosis fungoides, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma, pituitary adenoma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, throat cancer, thymoma, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

[0075] In another embodiment, the cancer is breast cancer. In yet another embodiment, the cancer is ovarian cancer. In yet another embodiment, the cancer is prostate cancer. In yet another embodiment, the cancer is lung cancer. In yet another embodiment, the cancer is malignant melanoma.

[0076] While the disclosed Tat derivatives are countersuppressive agents with "stand alone" efficacy in cancer, these observations moreover support the prospect that the Tat derivatives can synergize with other countersuppressive anti-cancer therapeutics currently in clinical development that may have a restricted effect in the face of advanced tumor burden and accompanying severe immunosuppression.

[0077] Expression and presence of PD-L1 by tumors and invading immune cells may be used to predict response to therapy and/or prognosis of disease. Therefore, in one embodiment disclosed herein, a subject is selected for treatment with a Tat derivative polypeptide based on expression of PD-L1 in their tumor tissue. In certain embodiments, the tumor tissue is evaluated for PD-L1 expression before the subject is treated with any cancer therapy. In another embodiment, the tumor tissue is evaluated for PD-L1 expression before the subject is treated with a Tat derivative polypeptide disclosed herein.

[0078] Expression of PD-L1 may be determined by an immunological analysis of tumor tissue such as, but not limited to, immunohistochemistry, immunoassay (ELISA, ELISPOT, radioimmunoassay), protein microarrays, flow cytometry, quantitative immunofluoresence, and surface plasmon resonance. Non immunological assays such as quantitative polymerase chain reaction (qPCR), and determination of messenger RNA can also be used.

[0079] Thus, in some embodiments, a patient is selected for treatment with the Tat derivative polypeptide if the pre-treatment tumor contains more than 5% PD-L1-expressing cells, more than 6% PD-L1-expressing cells, more than 7% PD-L1-expressing cells, more than 8% PD-L1-expressing cells, more than 9% PD-L1-expressing cells, more than 10% PD-L1-expressing cells, more than 11% PD-L1-expressing cells, more than 12% PD-L1-expressing cells, more than 13% PD-L1-expressing cells, more than 14% PD-L1-expressing cells, more than 16% PD-L1-expressing cells, more than 18% PD-L1-expressing cells, or more than 20% PD-L1-expressing cells.

[0080] Pharmaceutical Compositions

[0081] The present disclosure is also directed to pharmaceutical compositions comprising the above-described Tat derivative polypeptides. Dosages and desired drug concentrations of the disclosed pharmaceutical compositions may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mardenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al, Eds., Pergamon Press, New York 1989, pp. 42-96. In one embodiment, the disease is present. In another embodiment, the life of a cell or an individual is prolonged due to the methods described herein.

[0082] The above-described Tat derivative polypeptides can be formulated without undue experimentation for administration to a mammal, including humans, as appropriate for the particular application. Additionally, proper dosages of the compositions can be determined without undue experimentation using standard dose-response protocols.

[0083] Accordingly, the compositions designed for oral, nasal, lingual, sublingual, buccal, intrabuccal, intravenous, subcutaneous, intramuscular and pulmonary administration can be made without undue experimentation by means well known in the art, for example with an inert diluent or with an pharmaceutically acceptable carrier. For the purpose of therapeutic administration, the pharmaceutical compositions may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, solutions, syrups, and the like. A "pharmaceutically acceptable carrier" means any of the standard pharmaceutical carriers. Examples of suitable carriers are well known in the art and may include but are not limited to any of the standard pharmaceutical carriers like phosphate buffered saline solutions, phosphate buffered saline containing polysorbate 80, water, emulsions such as oil/water emulsion, and various types of wetting agents. Other carriers may also include sterile solutions, tablets, coated tablets, and capsules. Typically such carriers contain excipients like starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Compositions comprising such carriers are formulated by well known conventional methods.

[0084] The Tat derivative polypeptide compositions can easily be administered parenterally such as for example, by intravenous, intramuscular, intrathecal, or subcutaneous injection. Parenteral administration can be accomplished by incorporating the compounds into a solution or suspension. Such solutions or suspensions may also include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Parenteral formulations may also include antibacterial agents such as for example, benzyl alcohol or methyl parabens, antioxidants such as for example, ascorbic acid or sodium bisulfite and chelating agents such as EDTA. Buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be added. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

[0085] Transdermal administration includes percutaneous absorption of the composition through the skin. Transdermal formulations include patches, iontophoresis devices, ointments, creams, gels, salves and the like.

[0086] The composition may include various materials which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials which form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule or cachet.

[0087] The Tat derivative polypeptide compositions of the present disclosure may be administered in a therapeutically effective amount, according to an appropriate dosing regimen. As understood by a skilled artisan, the exact amount required may vary from subject to subject, depending on the subject's species, age and general condition, the severity of the infection, the particular agent(s) and the mode of administration. In some embodiments, about 0.001 mg/kg to about 50 mg/kg, of the composition based on the subject's body weight is administered, one or more times a day, to obtain the desired therapeutic effect. In other embodiments, about 1 mg/kg to about 25 mg/kg, of the composition based on the subject's body weight is administered, one or more times a day, to obtain the desired therapeutic effect.

[0088] The total daily dosage of the compositions will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient or subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient or subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed, and other factors well known in the medical arts.

[0089] The disclosed compositions may also be employed in combination therapies. That is, the compositions presently disclosed can be administered concurrently with, prior to, or subsequent to, one or more other desired compositions, therapeutics, treatments or medical procedures. The particular combination of therapies administered will be determined by the attending physician and will take into account compatibility of the treatments and the desired therapeutic effect to be achieved. It will be appreciated that therapeutically active agents utilized in combination may be administered together in a single composition, treatment or procedure, or alternatively may be administered separately.

[0090] In another embodiment, repetitive, or frequent, dosing of the disclosed Tat derivatives is contemplated that could run ahead of tachyphylaxis, as well as reverse the immunosuppressive tide established during cancer progression. Frequent dosing is one procedure used for example in allergy therapy that can support immunological tolerance to an agent. Once the Tat derivative can be used to regain immunoreactivity to a tumor, then other immunotherapeutics that have lost benefit due to advanced disease could potentially regain efficacy. In a second protocol, chemotherapeutic regimens are used that could release a shower of tumor antigens in alternation with Tat derivative immunotherapy. As advanced stage human cancers are often multiply drug resistant, radiotherapy could be a practical alternative in human trials.

[0091] The number of repeated doses of the Tat derivative polypeptides can be established by the medical professional based on the response of the patient to the doses. In one embodiment, the Tat derivative polypeptides is administered once every three days for 3 doses in a ten day period. This administration scheme is then repeated for a plurality of cycles. The present disclosure envisions a variety of different administration schemes wherein the Tat derivative polypeptides is administered multiple times within a selected time frame and then the administration scheme is repeated for a plurality of cycles. In another embodiment, administration of the Tat derivative polypeptides can be alternated with administration of one or more other anti-cancer, immunomodulatory, or immunosuppressive agents. In one embodiment, the immunosuppressive agent is cyclophosphamide.

[0092] Furthermore, treatment with the Tat derivative polypeptides can be combined with other cancer therapies such as surgery, radiation therapy, or chemotherapy. Chemotherapeutic agents include alkylating agents such nitrogen mustards, nitrosoureas, tetrazines, aziridines, cisplatins, and derivatives; anti-metabolites such as anti-folates, fluoropyrimidines, deoxynucleoside analogues, and thiopurines; antimicrotubule agents such as vinca alkaloids and taxanes; topoisomerase inhibitors such as camptothecin, irinotecan, topotecan, novobiocin, merbarone, and aclarubicin; cytotoxic antibiotics such as anthracyclines, actinomycin, bleomycin, plicamycin, and mitomycin.

[0093] Effects of Tat Derivative Polypeptides in Breast Cancer

[0094] Animal trials with recombinantly-produced Tat protein derivatives in three different widely accepted murine models of breast cancer, 4T1, SM1, and TS/A, provided support that Tat derivatives are active in suppressing primary breast cancer growth in mice. Moreover, one derivative, Nani-P2, significantly inhibited the development of spontaneous 4T1 lung metastases and increased survival compared with control mice. Significantly, increased levels of IFN-.gamma. production accompanied treatment of murine breast cancers with Tat derivatives. In studies when 4T1 breast cancers were seeded for fourteen days prior to the initiation of treatment, the Tat derivatives were equally as effective as when given at the time of tumor implantation when assessed by primary tumor growth, survival, and reduction in metastatic lung burden when compared to PBS-treated controls.

[0095] Synthetic Tat derivatives are immunostimulatory to APCs, have substantial activity against primary as well as established cancers in three widely-used murine mammary carcinoma models. In particular, one of the derivatives, Nani-P2, produced a dose- and route-dependant impact on primary tumor growth, lung metastasis formation, and survival in the aggressive Her2(-) 4T1 breast cancer model. Decreased lung metastases correlate with improved survival, because lung metastasis is the leading cause of mortality in advanced breast cancer. Importantly, mice bearing established 4T1 breast tumors treated intravenously with Nani-P2 protein had highly significant tumor growth inhibition and survival benefits that extended out at least 36 days past the last dosing. In limited cases, total remissions were apparently observed that were more frequent with the less aggressive (SM1) and/or somewhat more immunogenic (TS/A) breast tumors. Delaying the administration of Nani-P2 post tumor implant had little negative effect on 4T1 tumor growth suppression, insofar as therapy (SC) initiated on day 0 after tumor cell injection shrank tumor burden on average 53%, while SC therapy begun on day 13, when tumor growth already averaged about 5 mm in diameter, decreased tumor burden on average 52% at its maximal effect. Taken together, these observations indicated that the Tat derivatives can favorably impact advanced and Her2(-) human breast cancers in humans.

[0096] The studies reported here used a protocol of three or four approximately weekly doses of Tat derivative given either IV or SC, with IV administration proving most efficacious for increasing survival and for reducing metastases. No toxicity was observed in over 250 mice given these compositions. The sensitivity of breast cancer to the Tat derivatives contrasts favorably when compared to the dose response curve of HERCEPTIN.RTM. (Genentech), where 4-8 mg/kg is standard therapy. It is estimated that Tat derivatives will be up to 100-fold more bioactive in humans than mice, meaning that even lower doses associated with even less risk of toxicity could likely prove successful.

[0097] Established herein is that the Tat derivatives activate the INF-.gamma. arm of the anti-cancer T cell immune response (FIG. 5). Baseline levels of INF-.gamma. secreted by splenocytes from mice treated with Nani-P2 are 8-fold higher than that from control mice treated with PBS. IFN-.gamma. secretion in response to Tat derivative treatment in vivo could be additionally augmented (up to 53.times.) in vitro by innate immune agonists GM-CSF and IL-4, while splenocytes from control mice remain suppressed even after attempts to co-stimulate with high-dose GM-CSF and/or IL4.

[0098] A more immunogenic breast cancer model (SM1) and/or a breast tumor with an immunodominant epitope (TS/A) have a relatively high regression rate after Tat derivative therapy, while the "non-immunogenic" 4T1 model is more refractory. This is consistent with a model that immune suppression is a dominant factor in breast cancer progression, and in fact may be contributory to breast cancer invasiveness. This model is supported by the observation that 4T1 expresses several common breast cancer antigens, including lactadherin and androgen binding protein, at high levels against which the immune response is apparently fully suppressed absent Tat derivative-induced countersuppression.

Example 1

In Vitro Activity of Tat Derivatives

[0099] Human monocytes were cultured for 24-48 hours with a Tat derivative (Nani-P2), an immunostimulatory sequence (ISS) of a toll-like receptor (TLR) (FIG. 1), or lipopolysaccharide (LPS) (FIG. 2) and the cells were then washed and stained with fluorescent-labeled CD86. The Tat derivative stimulated higher expression of CD86 than either ISS (TLR) or LPS.

Example 2

Evaluation of Tat Derivatives in Mouse Models of Breast Cancer

[0100] Materials and Methods

[0101] Animals.

[0102] Female BALB/c mice 6 to 8 weeks old were purchased from the Jackson Laboratory (Bar Harbor, Nebr.). Mice were acclimated for at least 1 week before use. Mice were kept in pathogen-free conditions at the Animal Maintenance Facility of the Columbia University of Medical Center and all experiments were approved by the Institutional Animal Care and Use Committee of Columbia University of Medical Center.

[0103] Cell Lines.

[0104] 4T1 cells, a 6-thioguanine-resistant cell line derived from a BALB/c spontaneous mammary carcinoma was obtained from ATCC; TS/A, a murine adenocarcinoma cell line was provided by Dr. Sandra Demaria (Demaria S. et al. Clin Cancer Res. 11:728-34, 2005); and SM1, the BALB/C-derived mammary carcinoma was kindly was provided by Dr. James Allison, University of California, Berkeley. All tumor cell lines were cultured in DMEM, supplemented with 2 mM L-glutamine, 10 mM HEPES, 150 units/ml penicillin/streptomycin, 10% heat-inactivated FCS (Invitrogen), 50 .mu.M 2-mercaptoethanol (Sigma), and 50 mg/L gentamicin (Lanza).

[0105] Tumor Challenge and Treatment.

[0106] BALB/c mice were injected (SC) with 1.times.10.sup.4 4 T1, 1.times.10.sup.5 TS/A or 2.times.10.sup.5 SM1 cells, respectively, in the left mammary pad on day 0. Immunotherapy was performed by directly injecting a Tat derivative into the right flank at 0, 7, 12, and 17 days after establishment of tumors. The control group received PBS injection. In some experiments, when all of the mice had an established measurable tumor (3-5 mm diameter at 14 days after tumor injection), the animals were randomly assigned to various treatment groups as indicated. Tumor burden (tumor volume) was measured and recorded three times weekly. Animals were sacrificed when tumors reached a volume of 15 mm in diameter and the tumors harvested and weighed.

[0107] Detection of Lung Metastases.

[0108] Lungs were examined for 4T1 metastases as previously described (Pulaski B. et al. Cancer Res. 60:2710-2715, 2000). Primary 4T1 tumors that have been established for 2-3 weeks in BALB/c mice metastasize to the lungs in a very large majority of animals. Briefly, mice were sacrificed according to IACUC guidelines established at the start of the trials, the lungs were removed, and tumor nodules on the surface of the lungs were enumerated with the naked eye by two independent investigators blinded to the treatment protocols.

[0109] ELISA Analysis of IFN-.gamma. Production by Immune Spleen Cells.

[0110] Splenocyte secretion of IFN-.gamma. was assessed by an OptEIA.TM. ELISA kit (BD Biosciences). Briefly, spleen cells (1.times.10.sup.5/well) from 4T1 tumor-bearing mice were cultured with or without 5.times.10.sup.3/well mitomycin C (50 .mu.g/ml)-treated 4T1 cells (used to provide tumor antigens) in DMEM at a 20:1 E:T (effector:tumor) ratio with IL-2 (50 ng/mL) and GM-CSF (100 ng/ml) in 96-well plates. Supernatants were collected after 72 hr and kept frozen at -80.degree. C. until analysis without loss of activity. IFN-.gamma. was measured in cell-free supernatants of duplicate wells by ELISA according to the manufacturer's instructions. Tumor-specific IFN-.gamma. production was calculated by subtracting the background values measured in supernatants of spleen cells cultured with medium alone and optical density (OD) values were converted to pg/ml amounts of IFN-.gamma. using a recombinant IFN-.gamma. standard curve. Stimulation index (SI) was calculated as the ratio of IFN-.gamma. in stimulated versus control cultures.

[0111] Statistical Analysis.

[0112] Data were statistically analyzed using Student's t-test (Graph Pad Prism version 5; GraphPad). Data from animal survival experiments were statistically analyzed using log-rank test (Graph Pad Prism version 5).

[0113] Results

[0114] The therapeutic effect of systemic administration of synthetic, Tat-derived compositions in murine models of breast cancer was investigated. To compare the relative protection conferred by a small panel of different derivatives against primary breast tumor growth, female BALB/c mice were injected with 1.times.10.sup.4 4 T1 breast tumor cells SC into the mammary pad, and then treated with 400 ng partially-purified Tat derivatives at day 0, 7, 14, and 21 (SC injection in PBS) into the draining axillary lymph nodes.

[0115] Two of the derivatives, Nani-P1 and Nani-P2, significantly reduced tumor burden when compared to control mice receiving PBS injections alone, with this difference first becoming apparent at 15 days after tumor implantation (FIG. 3A, day 15 p<0.05). By contrast a third derivative, Nani-P3, produced and partially purified with the same protocol as the others, was less effective at suppressing 4T1 primary tumor growth even at five-fold higher doses (2 .mu.g, FIG. 3B) or for extending survival (not shown). These results effectively ruled out that contaminants in preparation contributed to anti-tumor efficacy, particularly insofar as subsequent trials were performed with highly purified (>95% pure) materials at much lower doses. The efficacy of Nani-P2 was significantly more sustained than Nani-P1, so that at day 21 (the final dosing), the difference in primary tumor burden between Nani-P2 and Nani-P1-treated tumors became 18 mm.sup.3 and was highly statistically significant (p<0.01). This effect persisted throughout the remainder of this trial despite no further therapy.

[0116] The breast tumor growth inhibitory effect of highly-purified Nani-P2 on 4T1 tumors was dose-dependent, with significant effects apparent following the SC administration of as little as 0.4 ng of compound (FIG. 4). Increasing the dose of Nani-P2, administered SC in the draining axillary flank, by logarithmic increments from 0.4 ng to 40 ng per dose progressively inhibited 4T1 breast tumor growth. The more robust 4T1 growth inhibition at higher doses of Nani-P2 between 0.4 ng to 40 ng was statistically significant (p<0.01), while increasing the dose to 400 ng and even 2 .mu.g resulted in no further anti-tumor efficacy (data not shown). Importantly, no toxicity was observed following the SC or IV administration of 40 ng of Nani-P2 in multiple trials using multiple dosing schedules. A dose of 40 ng Nani-P2 was selected for further study.

[0117] To determine whether Nani-P2 treatment could extend survival in addition to shrinking primary tumors in mice, treatment protocols using various dosing schedules and routes (SC, IV or IT) of administration of 40 ng Nani-P2 were performed. Cohorts of ten mice per group were followed for length of survival, as assessed by use of the Kaplan-Meier product limit method. As per Columbia University Medical Center Animal Facility regulations, each mouse was euthanized at a mean tumor diameter of approximately 15 mm, or earlier if the mouse became moribund, making one of these two outcomes the defining criteria for fatality.

[0118] In the first trial evaluating Nani-P2, SC treatment was initiated simultaneously to tumor implant. The median survival time for control (PBS treated) mice was 30 days and 100% fatality occurred by day 36. Wth Nani-P2 administration (4 doses over 21 days), 35% of treated mice were still alive at day 48 (p<0.001, FIG. 5A) at which point all of the mice were sacrificed due to primary tumor burden.

[0119] In a second survival trial, the tumors were allowed to become established for fourteen days to better assess efficacy in metastatic disease, after which three cycles of Nani-P2 therapy were administered weekly by one of several routes (SC, IV or IT) to compare relative efficacy for each route of dosing (FIG. 5B). Similar to the previous trial, median survival of control (PBS-treated SC) mice was 32 days, with 100% fatality by day 36. Survival was extended by the IV administration of Nani-P2 (p<0.005, FIG. 5B) with 60% survival at day 47, compared with 20% survival of SC treated mice at day 47 (p<0.05). Intratumoral administration of compound was slightly inferior to SC administration.

[0120] The 4T1 murine mammary tumor model was chosen for study because it is an aggressive and rapidly invasive tumor; it is routinely metastatic at fourteen days post-implant by which time it is difficult to treat. To learn whether the efficacy of Nani-P2 could extend to other murine breast tumor models, two additional mammary tumors, TS/A and SM1 were studied (FIG. 6). TS/A primary mammary tumors were approximately as aggressive as 4T1, reaching a tumor volume of 15 mm at 30 days (FIG. 6A). However, the TS/A tumors were considerably more responsive to Nani-P2 treatment, with an approximate 50% suppression of growth after treatment with 0.4 ng Nani-P2, and a 40% total remission rate at 30 days.

[0121] The SM1 mammary carcinoma model (FIG. 6B) is initially less aggressive as a primary tumor, and deaths appear to be through mechanisms other than metastatic disease. By day 30 of treatment, SM1 tumors reached a mean volume approximately 33% smaller than either TS/A or 4T1. This indicated a heightened sensitivity of the SM1 tumor to Nani-P2 immunotherapy as compared to 4T1, such that tumor growth was suppressed in 100% of animals for 16 days, and 40% of animals remained in remission even at 28 days following implant and fully one week after termination of the regimen.

[0122] To determine whether cytotoxic T-lymphocytes play a role in tumor rejection induced by Nani-P2 therapy, an IFN-.gamma. ELISA assay (FIG. 7) was performed to compare spleen cells of 4T1 tumor-bearing mice treated either without (Control) or with Nani-P2 (FIG. 7). Spleens were removed under sterile conditions and prepared as described elsewhere (duPre'S. et al. Exp. Mol. Path. 85:174-188, 2008). Briefly, spleens were homogenized and splenocytes, as a rich source of systemic cytolytic T cells and APCs, were co-cultured with mitomycin C-treated 4T1 stimulator cells to induce recall immune responses. Control wells were cultured with medium alone.

[0123] IFN-.gamma. concentrations, a standard surrogate for CTL activation, were quantitated by commercial ELISA (BD Biosciences). IFN-.gamma. production was significantly higher (p<0.01**) in cultures of spleen cells taken from Nani-P2-treated BALB/c mice under all conditions of assay. IFN-.gamma. activity in Nani-P2-treated, but not in control, animals could be enhanced by the addition of IL-4 and GM-CSF (p<0.05) under conditions shown to promote DC differentiation, and could be even further augmented if tumor stimulators were added back at the initiation of culture (stimulation index=53 vs control, 3S+IL4+GM-CSF) demonstrating the potency of Nani-P2 in synergy with other CTL agonists.

[0124] To further investigate the efficacy of Nani-P2 against established and metastatic breast cancer, 4T1 cells were injected SC in the abdominal mammary gland of mice and treatment was delayed until such time that the tumors had metastasized to the lungs and averaged 3.5 mm in size (FIG. 8A, day 13), corresponding to a 2.4 cm or stage T2 human breast tumor. Mice were followed for tumor growth (FIG. 8A) and lung metastases (FIG. 8B). At necropsy, animals that had received Nani-P2 treatment showed a dramatic reduction in the visible number of lung metastases when compared against controls (FIG. 9). The average number of grossly visible tumor nodules in the lungs of mice treated IV with Nani-P2 was seven, compared to the PBS-control group, which had an average of 35.3 (p<0.01**). This corresponded to a less aggressive appearance of primary tumor, as well as lung metastases that were on average much smaller in size (FIG. 8B).

[0125] Nani-P2 efficacy in the setting of pre-established, aggressive 4T1 breast cancer is clearly and significantly proven by comparing primary tumor burden in intravenously-treated animals (40 ng IV Nani-P2) against control (PBS-treated) animals (at day 18 p<0.01**, FIG. 10). This statistically significant benefit in primary tumor suppression (FIG. 10) remained throughout the duration of the trial lasting 50 days (p<0.01**) even though only three weekly doses of Tat derivative polypeptide were administered between days 14 and 28. Moreover 7/10 mice demonstrated regression of tumor at the initial treatment of tumor on day 14. This translated into a very highly statistically significant benefit to survival (p<0.005**, and see FIG. 5B). Remarkably, one animal underwent a complete remission and remained disease-free at 50 days, at which point the study was terminated, supporting the inference that this animal had been rendered apparently tumor-free.

Example 3

Repeated Dosing Therapy of Tat Derivatives and Cyclophosphamide

[0126] Four groups of 10 BALB/c mice were implanted with 1.times.10.sup.4 4 T1 cells SC into the mammary fat pad. Treatment was initiated when tumor diameters reached 4-5 mm, on day 10. Control mice were injected IV with PBS at 3 days intervals, while alternating treatment mice received 3 doses of drug every 3 days in rotating 10 day cycles. Tumor burden (tumor size mm.sup.3) was calculated using a standard formula. CY (cyclophosphamide alone) mice were injected IP weekly with 80 mg/kg per mouse beginning on day 10. Cy/Nani-P2 (cyclophosphamide first followed by Nani-P2) mice were first injected IP with cyclophosphamide (80 mg/kg) at 3 days intervals for three doses starting at day 10 and then injected IV with Nani-P2 (40 ng) at 3 days intervals for three doses in rotation. The cycle of 3 doses of CY followed by 3 doses of Nani-P2 was repeated until day 50. Nani-P2/CY (Nani-P2 first followed by cyclophosphamide) mice were first injected IV with Nani-P2 (40 ng) at 3 day intervals for 3 doses starting on day 10 and then injected i.p. with cyclophosphamide at 3 day intervals in rotation. The cycle of 3 doses of Nani-P2 followed by 3 doses of CY was repeated until day 50.

[0127] The decreased tumor burden in the Nani-P2/CY group compared to the CY group is very highly statistically significant (FIG. 11, p=0.003077).

[0128] The survival benefit of Nani-P2 bolus treatment alternating with cyclophosphamide vs. weekly cyclophosphamide is highly statistically significant (FIG. 12, p=0.0001). The Nani-P2 cohort has 3/10 mice in total remission and 9/10 mice in partial remission at day 50 (not shown), while 10/10 cyclophosphamide treated mice were dead by day 42.

Example 4

Presence of Splenic CD8+ CTL in Mice Receiving Nani-P2

[0129] The spleen is a major lymphoid organ and site where antigen presenting cells display captured tumor associated antigens to stimulate cytotoxic T-cell responses. Tumor specific CTLs will migrate to the site of infection and lyse the target cell.

[0130] Female BALB/c mice were inoculated in the mammary fat pad with syngeneic and highly metastatic 4T1 breast cancer cells to model Stage IV human breast cancer. Nani-P2 immunotherapy was initiated 7 days after tumor cell inoculation. Tumors were assessed by caliper measurements throughout the study and resected on Day 29/30. Immunohistochemical staining (IHC) and CD8 was performed on formalin-fixed, paraffin embedded specimens of resected spleen tissue.

[0131] As depicted in FIG. 13, IHC staining reveals increased populations of splenic mouse CD8+ cells following treatment with Tat derivatives (FIG. 13B) versus no treatment (PBS, FIG. 13A)).

Example 5

Induction of 4T1 Breast Tumor Infiltrating CD8+ Cytotoxic T-Lymphocytes by Nani-P2 in the Presence of PD-L1

[0132] The refractory state of cancers to immunotherapeutics may be a consequence of immunosuppression that accompanies disease progression in established cancers. In the tumor microenvironment, expression of a programmed cell death receptor-ligand-1 (PD-L1) has been implicated as a marker of disease progression, poor prognosis, and impairment of host tumor immunity by suppressing the function of tumor infiltrating CD8+ cytotoxic T-lymphocytes (CTL). Therefore, the presence of PD-L1 in various tumor types represents a major barrier for developing effective immunotherapeutics.

[0133] The Tat derivative polypeptides disclosed herein elicit antitumor immune responses by triggering monocyte-derived dendritic cells to stimulate the CD8+ CTL and override PD-L1 immunosuppression. Thus, the PD-1/PD-L1 immunosuppressive signaling pathway may provide a potential mechanism by which 4T1 tumors evade host tumor immunity and therefore Tat derivative polypeptides can impact solid tumor progression by induction of tumor infiltrating CD8+ CTLs in the face of PD-L1 immunosuppression.

[0134] Female BALB/c mice were inoculated in the mammary fat pad with syngeneic and highly metastatic 4T1 breast cancer cells to model Stage IV human breast cancer. Nani-P2 immunotherapy was initiated 7 days after tumor cell inoculation. Tumors were assessed by caliper measurements throughout the study and resected on Day 29/30. Immunohistochemical staining (IHC) for PD-L1 and CD8 was performed on formalin-fixed, paraffin embedded specimens of primary 4T1 tumors.

[0135] As depicted in FIG. 14, PD-L1 expression is reduced in animals receiving Nani-P2 treatment (FIG. 14B) versus controls (FIG. 14A). PD-L1 staining was observed in cells with a morphological resemblance to myeloid-derived suppressor cells, tumor-associated macrophage, as well as tumor-associated dendritic cells and fibroblast. PD-L1 reduction is based on in vivo tumor measurement data in Nani-P2 treated vs. control, combined with less PD-L1 staining intensity. Tumor edge containing majority of PD-L1 staining is largely absent in Nani-P2 treated as compared to control. Very few cells stained positive for CD8+ CTLs in the PBS control (FIG. 14C) while infiltrating CD8+ CTL advancing around tumor edge in PIN-2 treated mice (FIG. 14D).

[0136] Immunostaining of established primary 4T1 breast tumors in mice administered PIN-2 as compared to PBS control, revealed a significant increase in the population of tumor infiltrating CD8+ CTL. The presence of PD-L1 at the tumor edge may contribute to tumor malignancy and escape from immune surveillance by acting as a molecular shield to inhibit CTL-activity by engaging in the PD-1/PD-L1 signaling pathway. Tumor-infiltrating CD8+ CTLs appear to localize near the tumor edge in Nani-P2 treated mice, where as these CTLs are largely absent in tumor edges of PBS control. Since PD-L1 is a marker associated with disease progression, malignancy, and poor prognosis, the inverse correlation of tumor PD-L1 and CD8+ CTL can be explained based on the antitumor CTL response observed with PIN-2 treatment.

[0137] In conclusion, (i) reduced PD-L1 presence near the tumor edge was observed with PIN-2 treatment; (ii) CD8+ CTLs contribute to anti-tumor immune response observed PIN-2 treated mice; (iii) CD8+ CTL infiltration of PD-L1+ primary breast tumors suggests PINS override immunosuppressive mechanisms used by cancer as a barrier (immune checkpoint) to a successful antitumor immune response; (iv) positive detection of PD-L1 by IHC in established 4T1 primary breast tumors suggests a role exerted by the immunosuppressive PD-1/PD-L1 axis as an important mechanism for tumor evasion; (v) the Tat derivative polypeptides disclosed herein have the capability to override the PD-1/PD-L1 pathway in breast tumors expressing PD-L1; and (vi) administration of at derivative polypeptides disclosed herein reverses the immunosuppressive tide established during tumor progression and re-establishes immunoreactivity.

[0138] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0139] The terms "a," "an," "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0140] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0141] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0142] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term "consisting of" excludes any element, step, or ingredient not specified in the claims. The transition term "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0143] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

[0144] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Sequence CWU 1

1

1501101PRTHuman immunodeficiency virus type 1 1Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser 1 5 10 15 Gln Pro Lys Thr Ala Cys Thr Thr Cys Tyr Cys Lys Lys Cys Cys Phe 20 25 30 His Cys Gln Val Cys Phe Thr Lys Lys Ala Leu Gly Ile Ser Tyr Gly 35 40 45 Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Glu Asp Ser Gln Thr 50 55 60 His Gln Val Ser Pro Pro Lys Gln Pro Ala Pro Gln Phe Arg Gly Asp 65 70 75 80 Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu 85 90 95 Thr His Pro Val Asp 100 2102PRTArtificial SequenceTat derivative polypeptide 2Met Glu Pro Val Asp Ala Asn Leu Glu Ala Trp Lys His Ala Gly Ser 1 5 10 15 Gln Pro Arg Lys Thr Ala Cys Thr Thr Cys Tyr Cys Lys Lys Cys Cys 20 25 30 Phe His Cys Gln Val Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser Tyr 35 40 45 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln 50 55 60 Thr His Gln Ala Ser Leu Ser Lys Gln Pro Ala Ser Gln Ser Arg Gly 65 70 75 80 Asp Pro Thr Gly Pro Thr Glu Ser Lys Lys Lys Val Glu Arg Glu Thr 85 90 95 Glu Thr Asp Pro Phe Asp 100 3119PRTArtificial SequenceTat derivative polypeptide 3Met Asp Pro Lys Gly Glu Glu Asp Gln Asp Val Ser His Gln Asp Leu 1 5 10 15 Ile Lys Gln Tyr Arg Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys 20 25 30 Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu 35 40 45 Gly Ile Thr Tyr His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser 50 55 60 Ala Asp Arg Ile Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg 65 70 75 80 Asp Ser Gln Thr Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala 85 90 95 Lys Ala Asn Leu Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg 100 105 110 Gly Pro Val Gly Ala Gly Asn 115 4130PRTArtificial SequenceTat derivative polypeptide 4Met Glu Thr Pro Leu Lys Glu Gln Glu Asn Ser Leu Glu Ser Cys Arg 1 5 10 15 Glu His Ser Ser Ser Ile Ser Glu Val Asp Val Pro Thr Pro Val Ser 20 25 30 Cys Leu Arg Lys Gly Gly Arg Cys Trp Asn Arg Cys Ile Gly Asn Thr 35 40 45 Arg Gln Ile Gly Ser Cys Gly Val Pro Phe Leu Lys Cys Cys Lys Arg 50 55 60 Lys Pro Phe Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys 65 70 75 80 Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr His Gln Ala 85 90 95 Ser Leu Ser Lys Gln Pro Ala Ser Gln Ser Arg Gly Asp Pro Thr Gly 100 105 110 Pro Thr Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu Thr Asp Pro 115 120 125 Phe Asp 130 5136PRTArtificial SequenceTat derivative polypeptide 5Met Glu Thr Pro Leu Lys Glu Gln Glu Ser Ser Leu Glu Ser Ser Arg 1 5 10 15 Glu His Ser Ser Ser Ile Ser Glu Val Asp Ala Asp Thr Pro Glu Ser 20 25 30 Ala Ser Leu Glu Glu Glu Ile Leu Ser Gln Leu Tyr Arg Pro Leu Glu 35 40 45 Thr Cys Asn Asn Thr Cys Tyr Cys Lys Glu Cys Cys Tyr His Cys Gln 50 55 60 Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile Trp Tyr Asp Arg Lys Gly 65 70 75 80 Arg Arg Arg Arg Ser Pro Lys Lys Ile Lys Ala His Ser Ser Ser Ala 85 90 95 Ser Asp Lys Ser Ile Ser Thr Arg Thr Arg Asn Ser Gln Pro Glu Glu 100 105 110 Lys Gln Lys Lys Thr Leu Glu Thr Thr Leu Gly Thr Asp Cys Gly Pro 115 120 125 Gly Arg Ser His Ile Tyr Ile Ser 130 135 6118PRTArtificial SequenceTat derivative polypeptide 6Met Asp Ala Gly Lys Ala Val Ser Asp Lys Lys Glu Gly Asp Val Thr 1 5 10 15 Pro Tyr Asp Pro Phe Arg Asp Arg Thr Thr Pro Leu Glu Thr Cys Asn 20 25 30 Asn Thr Cys Tyr Cys Lys Glu Cys Cys Tyr His Cys Gln Leu Cys Phe 35 40 45 Leu Asn Lys Gly Leu Gly Ile Trp Tyr Asp Arg Lys Gly Arg Arg Arg 50 55 60 Arg Ser Pro Lys Lys Ile Lys Ala His Ser Ser Ser Ala Ser Asp Lys 65 70 75 80 Ser Ile Ser Thr Arg Thr Arg Asn Ser Gln Pro Glu Glu Lys Gln Lys 85 90 95 Lys Thr Leu Glu Thr Thr Leu Gly Thr Asp Cys Gly Pro Gly Arg Ser 100 105 110 His Ile Tyr Ile Ser Ala 115 7118PRTArtificial SequenceTat derivative polypeptide 7Met Asp Lys Gly Glu Glu Glu Arg Thr Val Leu His Gln Asp Leu Ile 1 5 10 15 Arg Gln Tyr Lys Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys 20 25 30 Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu Gly 35 40 45 Ile Thr Tyr His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala 50 55 60 Asp Arg Ile Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp 65 70 75 80 Ser Gln Thr Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys 85 90 95 Ala Asn Leu Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly 100 105 110 Pro Val Gly Ala Gly Asn 115 8118PRTArtificial SequenceTat derivative polypeptide 8Met Asp Lys Gly Glu Glu Glu Arg Thr Val Leu His Gln Asp Leu Ile 1 5 10 15 Arg Gln Tyr Lys Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys 20 25 30 Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu Gly 35 40 45 Ile Thr Tyr His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala 50 55 60 Asp Arg Ile Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp 65 70 75 80 Ser Gln Thr Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys 85 90 95 Ala Asn Leu Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly 100 105 110 Pro Val Gly Ala Gly Asn 115 9125PRTArtificial SequenceTat derivative polypeptide 9Met Asp Pro Lys Gly Glu Glu Asp Gln Asp Val Ser His Gln Asp Leu 1 5 10 15 Ile Lys Gln Tyr Arg Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys 20 25 30 Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe His Cys Tyr Ala Cys 35 40 45 Phe Leu Gln Lys Gly Leu Gly Val Thr Tyr His Ala Pro Arg Thr Arg 50 55 60 Arg Lys Lys Ser Val Gln Pro Asn Arg Leu Ser Gln Gln Asp Gln Ser 65 70 75 80 Ile Ser Thr Arg Gly Arg Asp Gly Gln Ala Thr Gln Glu Ser Gln Lys 85 90 95 Lys Val Glu Arg Glu Thr Thr Thr Ala Gln Ile Leu Gly Arg Lys Asp 100 105 110 Leu Glu Arg Asp Lys Arg Glu Ala Val Gly Ala Asn Ala 115 120 125 10118PRTArtificial SequenceTat derivative polypeptide 10Met Asp Gln Glu Gln Glu Ala Arg Pro Gln Val Trp Glu Glu Leu Gln 1 5 10 15 Glu Glu Leu His Arg Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys 20 25 30 Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu Gly 35 40 45 Ile Thr Tyr His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala 50 55 60 Asp Arg Ile Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp 65 70 75 80 Ser Gln Thr Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys 85 90 95 Ala Asn Leu Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly 100 105 110 Pro Val Gly Ala Gly Asn 115 1173PRTArtificial SequenceTat derivative polypeptide 11Met Asp Pro Lys Gly Glu Glu Asp Gln Asp Val Ser His Gln Asp Leu 1 5 10 15 Ile Lys Gln Tyr Arg Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys 20 25 30 Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe His Lys Lys Ala Leu 35 40 45 Gly Ile Arg Tyr Tyr Val Pro Arg Pro Arg Arg Ala Ser Lys Lys Ile 50 55 60 Ser His Asn Gln Val Ser Leu His Asn 65 70 12115PRTArtificial SequenceTat derivative polypeptide 12Met Glu Ser Glu Gly Asp Gly Met Ala Glu Ser Leu Leu Gln Asp Leu 1 5 10 15 His Arg Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys 20 25 30 Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu Gly Ile Thr Tyr 35 40 45 His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala Asp Arg Ile 50 55 60 Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp Ser Gln Thr 65 70 75 80 Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys Ala Asn Leu 85 90 95 Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly Pro Val Gly 100 105 110 Ala Gly Asn 115 13110PRTArtificial SequenceTat derivative polypeptide 13Met Asp Pro Lys Gly Glu Glu Asp Gln Asp Val Ser His Gln Asp Leu 1 5 10 15 Ile Lys Gln Tyr Arg Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys 20 25 30 Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe His Cys Tyr Ala Cys 35 40 45 Phe Leu Gln Lys Gly Leu Gly Ile Thr Tyr His Val Ser Arg Ile Arg 50 55 60 Arg Pro Lys Lys Asn His Ser Asn His Gln Asn Leu Val Ser Gln Gln 65 70 75 80 Ser Ile Ser Ala Trp Gly Gly Asn Ser Gln Thr Thr Gln Glu Glu Lys 85 90 95 Thr Lys Ile Pro Ala Ala Ala Glu Thr Ser Arg Arg Pro Gln 100 105 110 14118PRTArtificial SequenceTat derivative polypeptide 14Met Asp Lys Gly Glu Ala Glu Gln Ile Val Ser His Gln Asp Leu Ser 1 5 10 15 Glu Asp Tyr Gln Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys 20 25 30 Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu Gly 35 40 45 Ile Thr Tyr His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala 50 55 60 Asp Arg Ile Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp 65 70 75 80 Ser Gln Thr Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys 85 90 95 Ala Asn Leu Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly 100 105 110 Pro Val Gly Ala Gly Asn 115 15100PRTArtificial SequenceTat derivative polypeptide 15Met Asp Pro Lys Gly Glu Glu Asp Gln Asp Val Ser His Gln Asp Leu 1 5 10 15 Ile Lys Gln Tyr Arg Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys 20 25 30 Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Leu Gln Lys Gly Leu 35 40 45 Gly Val Thr Tyr His Ala Pro Arg Thr Arg Arg Lys Lys Ile Arg Ser 50 55 60 Leu Asn Leu Ala Pro Leu Gln His Gln Ser Ile Ser Thr Lys Trp Gly 65 70 75 80 Arg Asp Gly Gln Thr Thr Pro Thr Ser Gln Glu Lys Val Glu Thr Thr 85 90 95 Ala Gly Ser Asn 100 16116PRTArtificial SequenceTat derivative polypeptide 16Met Asp Lys Glu Glu Glu Pro His Pro Leu Leu Gln Asp Leu His Arg 1 5 10 15 Pro Leu Gln Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys 20 25 30 Cys Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu Gly Ile Thr 35 40 45 Tyr His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala Asp Arg 50 55 60 Ile Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp Ser Gln 65 70 75 80 Thr Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys Ala Asn 85 90 95 Leu Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly Pro Val 100 105 110 Gly Ala Gly Asn 115 1799PRTArtificial SequenceTat derivative polypeptide 17Met Asp Pro Lys Gly Glu Glu Asp Gln Asp Val Ser His Gln Asp Leu 1 5 10 15 Ile Lys Gln Tyr Arg Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys 20 25 30 Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Leu Gln Lys Gly Leu 35 40 45 Gly Val Arg Tyr His Val Ser Arg Lys Arg Arg Lys Thr Ser Thr Gln 50 55 60 Asp Asn Gln Asp Pro Ile Arg Gln Gln Ser Ile Ser Thr Val Gln Arg 65 70 75 80 Asn Gly Gln Thr Thr Glu Glu Gly Lys Thr Glu Val Glu Lys Ala Ala 85 90 95 Ala Ala Asn 18116PRTArtificial SequenceTat derivative polypeptide 18Met Ala Gln Glu Glu Gly Leu Gln Val Trp Glu Glu Leu Gln Glu Glu 1 5 10 15 Leu Gln Arg Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys 20 25 30 Cys Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu Gly Ile Thr 35 40 45 Tyr His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala Asp Arg 50 55 60 Ile Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp Ser Gln 65 70 75 80 Thr Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys Ala Asn 85 90 95 Leu Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly Pro Val 100 105 110 Gly Ala Gly Asn 115 19108PRTArtificial SequenceTat derivative polypeptide 19Met Asp Pro Lys Gly Glu Glu Asp Gln Asp Val Ser His Gln Asp Leu 1 5 10 15 Ile Lys Gln Tyr Arg Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys 20 25 30 Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Thr Gln Lys Gly Leu 35 40 45 Gly Ile Ala Tyr Tyr Val Pro Arg Thr Arg Arg Thr Val Lys Lys Ile 50 55 60 Gln Asn Asn Gln Val Pro Ile His Asn Gln Ser Ile Ser Thr Trp Thr 65 70 75 80 Arg Asn Ser Gln Ala Glu Lys Lys Ser Gln Thr Lys Val Gly Gln Ala 85 90 95 Ala Thr Ala Asp His Thr Pro Gly Arg Lys Asn Ser 100 105 20118PRTArtificial SequenceTat derivative polypeptide 20Met Asp Lys Gly Glu Asp Glu Gln Gly Ala Tyr His Gln Asp Leu Ile 1 5

10 15 Glu Gln Leu Lys Ala Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys 20 25 30 Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu Gly 35 40 45 Ile Thr Tyr His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala 50 55 60 Asp Arg Ile Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp 65 70 75 80 Ser Gln Thr Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys 85 90 95 Ala Asn Leu Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly 100 105 110 Pro Val Gly Ala Gly Asn 115 21120PRTArtificial SequenceTat derivative polypeptide 21Met Asp Pro Lys Gly Glu Glu Asp Gln Asp Val Ser His Gln Asp Leu 1 5 10 15 Ile Lys Gln Tyr Arg Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys 20 25 30 Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Phe Leu Gln Lys Gly 35 40 45 Leu Gly Val Thr Tyr His Ala Pro Arg Ile Arg Arg Lys Lys Ile Ala 50 55 60 Pro Leu Asp Arg Phe Pro Glu Gln Lys Gln Ser Ile Ser Thr Arg Gly 65 70 75 80 Arg Asp Ser Gln Thr Thr Gln Lys Gly Gln Glu Lys Val Glu Thr Ser 85 90 95 Ala Arg Thr Ala Pro Ser Leu Gly Arg Lys Asn Leu Ala Gln Gln Ser 100 105 110 Gly Arg Ala Thr Gly Ala Ser Asp 115 120 22103PRTArtificial SequenceTat derivative polypeptide 22Met Asp Val Arg Ala Val Gly Ser Glu Arg Ile Glu Glu Glu Thr Leu 1 5 10 15 Tyr Asn Pro Arg Lys Thr Ala Cys Thr Thr Cys Tyr Cys Lys Lys Cys 20 25 30 Cys Phe His Cys Gln Val Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser 35 40 45 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser 50 55 60 Gln Thr His Gln Ala Ser Leu Ser Lys Gln Pro Ala Ser Gln Ser Arg 65 70 75 80 Gly Asp Pro Thr Gly Pro Thr Glu Ser Lys Lys Lys Val Glu Arg Glu 85 90 95 Thr Glu Thr Asp Pro Phe Asp 100 23115PRTArtificial SequenceTat derivative polypeptide 23Met Asp Val Arg Ala Val Gly Ser Glu Arg Ile Glu Glu Glu Thr Leu 1 5 10 15 Tyr Asn Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys 20 25 30 Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu Gly Ile Thr Tyr 35 40 45 His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala Asp Arg Ile 50 55 60 Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp Ser Gln Thr 65 70 75 80 Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys Ala Asn Leu 85 90 95 Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly Pro Val Gly 100 105 110 Ala Gly Asn 115 24109PRTArtificial SequenceTat derivative polypeptide 24Met Asp Val Arg Ala Val Gly Ser Glu Arg Ile Glu Glu Glu Thr Leu 1 5 10 15 Tyr Asn Pro Leu Glu Thr Cys Asn Asn Thr Cys Tyr Cys Lys Glu Cys 20 25 30 Cys Tyr His Cys Gln Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile Trp 35 40 45 Tyr Asp Arg Lys Gly Arg Arg Arg Arg Ser Pro Lys Lys Ile Lys Ala 50 55 60 His Ser Ser Ser Ala Ser Asp Lys Ser Ile Ser Thr Arg Thr Arg Asn 65 70 75 80 Ser Gln Pro Glu Glu Lys Gln Lys Lys Thr Leu Glu Thr Thr Leu Gly 85 90 95 Thr Asp Cys Gly Pro Gly Arg Ser His Ile Tyr Ile Ser 100 105 25102PRTArtificial SequenceTat derivative polypeptide 25Met Asp Val Arg Ala Val Gly Ser Glu Arg Ile Glu Glu Glu Thr Leu 1 5 10 15 Tyr Asn Pro Thr Thr Ala Cys Ser Lys Cys Tyr Cys Lys Met Cys Cys 20 25 30 Trp His Cys Gln Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile Ser Tyr 35 40 45 Gly Arg Lys Lys Arg Lys Arg Arg Arg Gly Thr Pro Gln Ser Arg Gln 50 55 60 Asp His Gln Asn Pro Val Pro Lys Gln Pro Leu Pro Thr Thr Arg Gly 65 70 75 80 Asn Pro Thr Asn Pro Lys Glu Ser Lys Lys Glu Val Ala Ser Lys Thr 85 90 95 Glu Thr Asn Gln Cys Asp 100 26114PRTArtificial SequenceTat derivative polypeptide 26Met Ser Ser Thr Asp Gln Ile Cys Gln Thr Gln Arg Val Pro Pro Ser 1 5 10 15 Phe Leu Glu Gly Thr Phe Leu Glu Lys Gly Pro Pro Thr Pro Arg Lys 20 25 30 Thr Ala Cys Thr Thr Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gln 35 40 45 Val Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys 50 55 60 Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr His Gln Ala 65 70 75 80 Ser Leu Ser Lys Gln Pro Ala Ser Gln Ser Arg Gly Asp Pro Thr Gly 85 90 95 Pro Thr Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu Thr Asp Pro 100 105 110 Phe Asp 27126PRTArtificial SequenceTat derivative polypeptide 27Met Ser Ser Thr Asp Gln Ile Cys Gln Thr Gln Arg Val Pro Pro Ser 1 5 10 15 Phe Leu Glu Gly Thr Phe Leu Glu Lys Gly Pro Pro Thr Pro Arg Thr 20 25 30 Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Tyr Ala 35 40 45 Cys Phe Leu Arg Lys Gly Leu Gly Ile Thr Tyr His Ala Phe Arg Thr 50 55 60 Arg Arg Lys Lys Ile Ala Ser Ala Asp Arg Ile Pro Val Pro Gln Gln 65 70 75 80 Ser Ile Ser Ile Arg Gly Arg Asp Ser Gln Thr Thr Gln Glu Ser Gln 85 90 95 Lys Lys Val Glu Glu Gln Ala Lys Ala Asn Leu Arg Ile Ser Arg Lys 100 105 110 Asn Leu Gly Asp Glu Thr Arg Gly Pro Val Gly Ala Gly Asn 115 120 125 28120PRTArtificial SequenceTat derivative polypeptide 28Met Ser Ser Thr Asp Gln Ile Cys Gln Thr Gln Arg Val Pro Pro Ser 1 5 10 15 Phe Leu Glu Gly Thr Phe Leu Glu Lys Gly Pro Pro Thr Pro Leu Glu 20 25 30 Thr Cys Asn Asn Thr Cys Tyr Cys Lys Glu Cys Cys Tyr His Cys Gln 35 40 45 Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile Trp Tyr Asp Arg Lys Gly 50 55 60 Arg Arg Arg Arg Ser Pro Lys Lys Ile Lys Ala His Ser Ser Ser Ala 65 70 75 80 Ser Asp Lys Ser Ile Ser Thr Arg Thr Arg Asn Ser Gln Pro Glu Glu 85 90 95 Lys Gln Lys Lys Thr Leu Glu Thr Thr Leu Gly Thr Asp Cys Gly Pro 100 105 110 Gly Arg Ser His Ile Tyr Ile Ser 115 120 29113PRTArtificial SequenceTat derivative polypeptide 29Met Ser Ser Thr Asp Gln Ile Cys Gln Thr Gln Arg Val Pro Pro Ser 1 5 10 15 Phe Leu Glu Gly Thr Phe Leu Glu Lys Gly Pro Pro Thr Pro Thr Thr 20 25 30 Ala Cys Ser Lys Cys Tyr Cys Lys Met Cys Cys Trp His Cys Gln Leu 35 40 45 Cys Phe Leu Asn Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg 50 55 60 Lys Arg Arg Arg Gly Thr Pro Gln Ser Arg Gln Asp His Gln Asn Pro 65 70 75 80 Val Pro Lys Gln Pro Leu Pro Thr Thr Arg Gly Asn Pro Thr Asn Pro 85 90 95 Lys Glu Ser Lys Lys Glu Val Ala Ser Lys Thr Glu Thr Asn Gln Cys 100 105 110 Asp 30110PRTArtificial SequenceTat derivative polypeptide 30Met Asp Gly Gln Glu Ala Gly Leu Glu Arg Gln Glu Glu Glu Thr Leu 1 5 10 15 Tyr Asn Pro Phe Gln Ser Val Glu Thr Pro Arg Lys Thr Ala Cys Thr 20 25 30 Thr Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gln Val Cys Phe Thr 35 40 45 Arg Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg 50 55 60 Arg Arg Ala Pro Gln Asp Ser Gln Thr His Gln Ala Ser Leu Ser Lys 65 70 75 80 Gln Pro Ala Ser Gln Ser Arg Gly Asp Pro Thr Gly Pro Thr Glu Ser 85 90 95 Lys Lys Lys Val Glu Arg Glu Thr Glu Thr Asp Pro Phe Asp 100 105 110 31122PRTArtificial SequenceTat derivative polypeptide 31Met Asp Gly Gln Glu Ala Gly Leu Glu Arg Gln Glu Glu Glu Thr Leu 1 5 10 15 Tyr Asn Pro Phe Gln Ser Val Glu Thr Pro Arg Thr Ala Cys Asn Asn 20 25 30 Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Leu Arg 35 40 45 Lys Gly Leu Gly Ile Thr Tyr His Ala Phe Arg Thr Arg Arg Lys Lys 50 55 60 Ile Ala Ser Ala Asp Arg Ile Pro Val Pro Gln Gln Ser Ile Ser Ile 65 70 75 80 Arg Gly Arg Asp Ser Gln Thr Thr Gln Glu Ser Gln Lys Lys Val Glu 85 90 95 Glu Gln Ala Lys Ala Asn Leu Arg Ile Ser Arg Lys Asn Leu Gly Asp 100 105 110 Glu Thr Arg Gly Pro Val Gly Ala Gly Asn 115 120 32116PRTArtificial SequenceTat derivative polypeptide 32Met Asp Gly Gln Glu Ala Gly Leu Glu Arg Gln Glu Glu Glu Thr Leu 1 5 10 15 Tyr Asn Pro Phe Gln Ser Val Glu Thr Pro Leu Glu Thr Cys Asn Asn 20 25 30 Thr Cys Tyr Cys Lys Glu Cys Cys Tyr His Cys Gln Leu Cys Phe Leu 35 40 45 Asn Lys Gly Leu Gly Ile Trp Tyr Asp Arg Lys Gly Arg Arg Arg Arg 50 55 60 Ser Pro Lys Lys Ile Lys Ala His Ser Ser Ser Ala Ser Asp Lys Ser 65 70 75 80 Ile Ser Thr Arg Thr Arg Asn Ser Gln Pro Glu Glu Lys Gln Lys Lys 85 90 95 Thr Leu Glu Thr Thr Leu Gly Thr Asp Cys Gly Pro Gly Arg Ser His 100 105 110 Ile Tyr Ile Ser 115 33109PRTArtificial SequenceTat derivative polypeptide 33Met Asp Gly Gln Glu Ala Gly Leu Glu Arg Gln Glu Glu Glu Thr Leu 1 5 10 15 Tyr Asn Pro Phe Gln Ser Val Glu Thr Pro Thr Thr Ala Cys Ser Lys 20 25 30 Cys Tyr Cys Lys Met Cys Cys Trp His Cys Gln Leu Cys Phe Leu Asn 35 40 45 Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Lys Arg Arg Arg 50 55 60 Gly Thr Pro Gln Ser Arg Gln Asp His Gln Asn Pro Val Pro Lys Gln 65 70 75 80 Pro Leu Pro Thr Thr Arg Gly Asn Pro Thr Asn Pro Lys Glu Ser Lys 85 90 95 Lys Glu Val Ala Ser Lys Thr Glu Thr Asn Gln Cys Asp 100 105 34112PRTArtificial SequenceTat derivative polypeptide 34Met Ser Thr Gln Gly His Gln Gln Asp Gln Asp Gln Gly Lys Gly Thr 1 5 10 15 Leu Glu Glu Ala Tyr Lys Thr Asn Leu Glu Ala Pro Arg Lys Thr Ala 20 25 30 Cys Thr Thr Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gln Val Cys 35 40 45 Phe Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg 50 55 60 Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr His Gln Ala Ser Leu 65 70 75 80 Ser Lys Gln Pro Ala Ser Gln Ser Arg Gly Asp Pro Thr Gly Pro Thr 85 90 95 Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu Thr Asp Pro Phe Asp 100 105 110 35124PRTArtificial SequenceTat derivative polypeptide 35Met Ser Thr Gln Gly His Gln Gln Asp Gln Asp Gln Gly Lys Gly Thr 1 5 10 15 Leu Glu Glu Ala Tyr Lys Thr Asn Leu Glu Ala Pro Arg Thr Ala Cys 20 25 30 Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe 35 40 45 Leu Arg Lys Gly Leu Gly Ile Thr Tyr His Ala Phe Arg Thr Arg Arg 50 55 60 Lys Lys Ile Ala Ser Ala Asp Arg Ile Pro Val Pro Gln Gln Ser Ile 65 70 75 80 Ser Ile Arg Gly Arg Asp Ser Gln Thr Thr Gln Glu Ser Gln Lys Lys 85 90 95 Val Glu Glu Gln Ala Lys Ala Asn Leu Arg Ile Ser Arg Lys Asn Leu 100 105 110 Gly Asp Glu Thr Arg Gly Pro Val Gly Ala Gly Asn 115 120 36118PRTArtificial SequenceTat derivative polypeptide 36Met Ser Thr Gln Gly His Gln Gln Asp Gln Asp Gln Gly Lys Gly Thr 1 5 10 15 Leu Glu Glu Ala Tyr Lys Thr Asn Leu Glu Ala Pro Leu Glu Thr Cys 20 25 30 Asn Asn Thr Cys Tyr Cys Lys Glu Cys Cys Tyr His Cys Gln Leu Cys 35 40 45 Phe Leu Asn Lys Gly Leu Gly Ile Trp Tyr Asp Arg Lys Gly Arg Arg 50 55 60 Arg Arg Ser Pro Lys Lys Ile Lys Ala His Ser Ser Ser Ala Ser Asp 65 70 75 80 Lys Ser Ile Ser Thr Arg Thr Arg Asn Ser Gln Pro Glu Glu Lys Gln 85 90 95 Lys Lys Thr Leu Glu Thr Thr Leu Gly Thr Asp Cys Gly Pro Gly Arg 100 105 110 Ser His Ile Tyr Ile Ser 115 37111PRTArtificial SequenceTat derivative polypeptide 37Met Ser Thr Gln Gly His Gln Gln Asp Gln Asp Gln Gly Lys Gly Thr 1 5 10 15 Leu Glu Glu Ala Tyr Lys Thr Asn Leu Glu Ala Pro Thr Thr Ala Cys 20 25 30 Ser Lys Cys Tyr Cys Lys Met Cys Cys Trp His Cys Gln Leu Cys Phe 35 40 45 Leu Asn Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Lys Arg 50 55 60 Arg Arg Gly Thr Pro Gln Ser Arg Gln Asp His Gln Asn Pro Val Pro 65 70 75 80 Lys Gln Pro Leu Pro Thr Thr Arg Gly Asn Pro Thr Asn Pro Lys Glu 85 90 95 Ser Lys Lys Glu Val Ala Ser Lys Thr Glu Thr Asn Gln Cys Asp 100 105 110 38114PRTArtificial SequenceTat derivative polypeptide 38Met Gln Gln Pro Glu Gln Glu Gln His Thr Gln Gln Lys Gln His Leu 1 5 10 15 Asp Gln Leu Glu Glu Ile Tyr Lys Glu Ala Ile Thr Asp Pro Arg Lys 20 25 30 Thr Ala Cys Thr Thr Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gln 35 40 45 Val Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys 50 55 60 Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr His Gln Ala 65 70 75 80 Ser Leu Ser Lys Gln Pro Ala Ser Gln Ser Arg Gly Asp Pro Thr Gly 85 90 95 Pro Thr Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu Thr Asp Pro 100 105 110 Phe

Asp 39126PRTArtificial SequenceTat derivative polypeptide 39Met Gln Gln Pro Glu Gln Glu Gln His Thr Gln Gln Lys Gln His Leu 1 5 10 15 Asp Gln Leu Glu Glu Ile Tyr Lys Glu Ala Ile Thr Asp Pro Arg Thr 20 25 30 Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Tyr Ala 35 40 45 Cys Phe Leu Arg Lys Gly Leu Gly Ile Thr Tyr His Ala Phe Arg Thr 50 55 60 Arg Arg Lys Lys Ile Ala Ser Ala Asp Arg Ile Pro Val Pro Gln Gln 65 70 75 80 Ser Ile Ser Ile Arg Gly Arg Asp Ser Gln Thr Thr Gln Glu Ser Gln 85 90 95 Lys Lys Val Glu Glu Gln Ala Lys Ala Asn Leu Arg Ile Ser Arg Lys 100 105 110 Asn Leu Gly Asp Glu Thr Arg Gly Pro Val Gly Ala Gly Asn 115 120 125 40120PRTArtificial SequenceTat derivative polypeptide 40Met Gln Gln Pro Glu Gln Glu Gln His Thr Gln Gln Lys Gln His Leu 1 5 10 15 Asp Gln Leu Glu Glu Ile Tyr Lys Glu Ala Ile Thr Asp Pro Leu Glu 20 25 30 Thr Cys Asn Asn Thr Cys Tyr Cys Lys Glu Cys Cys Tyr His Cys Gln 35 40 45 Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile Trp Tyr Asp Arg Lys Gly 50 55 60 Arg Arg Arg Arg Ser Pro Lys Lys Ile Lys Ala His Ser Ser Ser Ala 65 70 75 80 Ser Asp Lys Ser Ile Ser Thr Arg Thr Arg Asn Ser Gln Pro Glu Glu 85 90 95 Lys Gln Lys Lys Thr Leu Glu Thr Thr Leu Gly Thr Asp Cys Gly Pro 100 105 110 Gly Arg Ser His Ile Tyr Ile Ser 115 120 41113PRTArtificial SequenceTat derivative polypeptide 41Met Gln Gln Pro Glu Gln Glu Gln His Thr Gln Gln Lys Gln His Leu 1 5 10 15 Asp Gln Leu Glu Glu Ile Tyr Lys Glu Ala Ile Thr Asp Pro Thr Thr 20 25 30 Ala Cys Ser Lys Cys Tyr Cys Lys Met Cys Cys Trp His Cys Gln Leu 35 40 45 Cys Phe Leu Asn Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg 50 55 60 Lys Arg Arg Arg Gly Thr Pro Gln Ser Arg Gln Asp His Gln Asn Pro 65 70 75 80 Val Pro Lys Gln Pro Leu Pro Thr Thr Arg Gly Asn Pro Thr Asn Pro 85 90 95 Lys Glu Ser Lys Lys Glu Val Ala Ser Lys Thr Glu Thr Asn Gln Cys 100 105 110 Asp 42130PRTArtificial SequenceTat derivative polypeptide 42Met Glu Thr Pro Leu Lys Glu Gln Glu Ser Ser Leu Arg Ser Ser Ser 1 5 10 15 Glu Pro Ser Ser Cys Thr Ser Glu Ala Val Ala Ala Thr Pro Gly Leu 20 25 30 Ala Asn Gln Glu Glu Glu Ile Leu Trp Gln Leu Tyr Arg Pro Arg Lys 35 40 45 Thr Ala Cys Thr Thr Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gln 50 55 60 Val Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys 65 70 75 80 Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr His Gln Ala 85 90 95 Ser Leu Ser Lys Gln Pro Ala Ser Gln Ser Arg Gly Asp Pro Thr Gly 100 105 110 Pro Thr Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu Thr Asp Pro 115 120 125 Phe Asp 130 43142PRTArtificial SequenceTat derivative polypeptide 43Met Glu Thr Pro Leu Lys Glu Gln Glu Ser Ser Leu Arg Ser Ser Ser 1 5 10 15 Glu Pro Ser Ser Cys Thr Ser Glu Ala Val Ala Ala Thr Pro Gly Leu 20 25 30 Ala Asn Gln Glu Glu Glu Ile Leu Trp Gln Leu Tyr Arg Pro Arg Thr 35 40 45 Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Tyr Ala 50 55 60 Cys Phe Leu Arg Lys Gly Leu Gly Ile Thr Tyr His Ala Phe Arg Thr 65 70 75 80 Arg Arg Lys Lys Ile Ala Ser Ala Asp Arg Ile Pro Val Pro Gln Gln 85 90 95 Ser Ile Ser Ile Arg Gly Arg Asp Ser Gln Thr Thr Gln Glu Ser Gln 100 105 110 Lys Lys Val Glu Glu Gln Ala Lys Ala Asn Leu Arg Ile Ser Arg Lys 115 120 125 Asn Leu Gly Asp Glu Thr Arg Gly Pro Val Gly Ala Gly Asn 130 135 140 44136PRTArtificial SequenceTat derivative polypeptide 44Met Glu Thr Pro Leu Lys Glu Gln Glu Ser Ser Leu Arg Ser Ser Ser 1 5 10 15 Glu Pro Ser Ser Cys Thr Ser Glu Ala Val Ala Ala Thr Pro Gly Leu 20 25 30 Ala Asn Gln Glu Glu Glu Ile Leu Trp Gln Leu Tyr Arg Pro Leu Glu 35 40 45 Thr Cys Asn Asn Thr Cys Tyr Cys Lys Glu Cys Cys Tyr His Cys Gln 50 55 60 Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile Trp Tyr Asp Arg Lys Gly 65 70 75 80 Arg Arg Arg Arg Ser Pro Lys Lys Ile Lys Ala His Ser Ser Ser Ala 85 90 95 Ser Asp Lys Ser Ile Ser Thr Arg Thr Arg Asn Ser Gln Pro Glu Glu 100 105 110 Lys Gln Lys Lys Thr Leu Glu Thr Thr Leu Gly Thr Asp Cys Gly Pro 115 120 125 Gly Arg Ser His Ile Tyr Ile Ser 130 135 45129PRTArtificial SequenceTat derivative polypeptide 45Met Glu Thr Pro Leu Lys Glu Gln Glu Ser Ser Leu Arg Ser Ser Ser 1 5 10 15 Glu Pro Ser Ser Cys Thr Ser Glu Ala Val Ala Ala Thr Pro Gly Leu 20 25 30 Ala Asn Gln Glu Glu Glu Ile Leu Trp Gln Leu Tyr Arg Pro Thr Thr 35 40 45 Ala Cys Ser Lys Cys Tyr Cys Lys Met Cys Cys Trp His Cys Gln Leu 50 55 60 Cys Phe Leu Asn Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg 65 70 75 80 Lys Arg Arg Arg Gly Thr Pro Gln Ser Arg Gln Asp His Gln Asn Pro 85 90 95 Val Pro Lys Gln Pro Leu Pro Thr Thr Arg Gly Asn Pro Thr Asn Pro 100 105 110 Lys Glu Ser Lys Lys Glu Val Ala Ser Lys Thr Glu Thr Asn Gln Cys 115 120 125 Asp 46106PRTArtificial SequenceTat derivative polypeptide 46Met Asp Lys Gly Glu Glu Glu Arg Thr Val Leu His Gln Asp Leu Ile 1 5 10 15 Arg Gln Tyr Lys Lys Pro Arg Lys Thr Ala Cys Thr Thr Cys Tyr Cys 20 25 30 Lys Lys Cys Cys Phe His Cys Gln Val Cys Phe Thr Arg Lys Gly Leu 35 40 45 Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro 50 55 60 Gln Asp Ser Gln Thr His Gln Ala Ser Leu Ser Lys Gln Pro Ala Ser 65 70 75 80 Gln Ser Arg Gly Asp Pro Thr Gly Pro Thr Glu Ser Lys Lys Lys Val 85 90 95 Glu Arg Glu Thr Glu Thr Asp Pro Phe Asp 100 105 47118PRTArtificial SequenceTat derivative polypeptide 47Met Asp Lys Gly Glu Glu Glu Arg Thr Val Leu His Gln Asp Leu Ile 1 5 10 15 Arg Gln Tyr Lys Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys 20 25 30 Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu Gly 35 40 45 Ile Thr Tyr His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala 50 55 60 Asp Arg Ile Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp 65 70 75 80 Ser Gln Thr Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys 85 90 95 Ala Asn Leu Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly 100 105 110 Pro Val Gly Ala Gly Asn 115 48112PRTArtificial SequenceTat derivative polypeptide 48Met Asp Lys Gly Glu Glu Glu Arg Thr Val Leu His Gln Asp Leu Ile 1 5 10 15 Arg Gln Tyr Lys Lys Pro Leu Glu Thr Cys Asn Asn Thr Cys Tyr Cys 20 25 30 Lys Glu Cys Cys Tyr His Cys Gln Leu Cys Phe Leu Asn Lys Gly Leu 35 40 45 Gly Ile Trp Tyr Asp Arg Lys Gly Arg Arg Arg Arg Ser Pro Lys Lys 50 55 60 Ile Lys Ala His Ser Ser Ser Ala Ser Asp Lys Ser Ile Ser Thr Arg 65 70 75 80 Thr Arg Asn Ser Gln Pro Glu Glu Lys Gln Lys Lys Thr Leu Glu Thr 85 90 95 Thr Leu Gly Thr Asp Cys Gly Pro Gly Arg Ser His Ile Tyr Ile Ser 100 105 110 49105PRTArtificial SequenceTat derivative polypeptide 49Met Asp Lys Gly Glu Glu Glu Arg Thr Val Leu His Gln Asp Leu Ile 1 5 10 15 Arg Gln Tyr Lys Lys Pro Thr Thr Ala Cys Ser Lys Cys Tyr Cys Lys 20 25 30 Met Cys Cys Trp His Cys Gln Leu Cys Phe Leu Asn Lys Gly Leu Gly 35 40 45 Ile Ser Tyr Gly Arg Lys Lys Arg Lys Arg Arg Arg Gly Thr Pro Gln 50 55 60 Ser Arg Gln Asp His Gln Asn Pro Val Pro Lys Gln Pro Leu Pro Thr 65 70 75 80 Thr Arg Gly Asn Pro Thr Asn Pro Lys Glu Ser Lys Lys Glu Val Ala 85 90 95 Ser Lys Thr Glu Thr Asn Gln Cys Asp 100 105 50105PRTArtificial SequenceTat derivative polypeptide 50Met Gln Pro Leu Gln Asn Arg Pro Asp Leu Gly Glu Glu Ile Leu Ser 1 5 10 15 Gln Leu Tyr Arg Pro Arg Lys Thr Ala Cys Thr Thr Cys Tyr Cys Lys 20 25 30 Lys Cys Cys Phe His Cys Gln Val Cys Phe Thr Arg Lys Gly Leu Gly 35 40 45 Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln 50 55 60 Asp Ser Gln Thr His Gln Ala Ser Leu Ser Lys Gln Pro Ala Ser Gln 65 70 75 80 Ser Arg Gly Asp Pro Thr Gly Pro Thr Glu Ser Lys Lys Lys Val Glu 85 90 95 Arg Glu Thr Glu Thr Asp Pro Phe Asp 100 105 51117PRTArtificial SequenceTat derivative polypeptide 51Met Gln Pro Leu Gln Asn Arg Pro Asp Leu Gly Glu Glu Ile Leu Ser 1 5 10 15 Gln Leu Tyr Arg Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys 20 25 30 Cys Cys Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu Gly Ile 35 40 45 Thr Tyr His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala Asp 50 55 60 Arg Ile Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp Ser 65 70 75 80 Gln Thr Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys Ala 85 90 95 Asn Leu Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly Pro 100 105 110 Val Gly Ala Gly Asn 115 52111PRTArtificial SequenceTat derivative polypeptide 52Met Gln Pro Leu Gln Asn Arg Pro Asp Leu Gly Glu Glu Ile Leu Ser 1 5 10 15 Gln Leu Tyr Arg Pro Leu Glu Thr Cys Asn Asn Thr Cys Tyr Cys Lys 20 25 30 Glu Cys Cys Tyr His Cys Gln Leu Cys Phe Leu Asn Lys Gly Leu Gly 35 40 45 Ile Trp Tyr Asp Arg Lys Gly Arg Arg Arg Arg Ser Pro Lys Lys Ile 50 55 60 Lys Ala His Ser Ser Ser Ala Ser Asp Lys Ser Ile Ser Thr Arg Thr 65 70 75 80 Arg Asn Ser Gln Pro Glu Glu Lys Gln Lys Lys Thr Leu Glu Thr Thr 85 90 95 Leu Gly Thr Asp Cys Gly Pro Gly Arg Ser His Ile Tyr Ile Ser 100 105 110 53104PRTArtificial SequenceTat derivative polypeptide 53Met Gln Pro Leu Gln Asn Arg Pro Asp Leu Gly Glu Glu Ile Leu Ser 1 5 10 15 Gln Leu Tyr Arg Pro Thr Thr Ala Cys Ser Lys Cys Tyr Cys Lys Met 20 25 30 Cys Cys Trp His Cys Gln Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile 35 40 45 Ser Tyr Gly Arg Lys Lys Arg Lys Arg Arg Arg Gly Thr Pro Gln Ser 50 55 60 Arg Gln Asp His Gln Asn Pro Val Pro Lys Gln Pro Leu Pro Thr Thr 65 70 75 80 Arg Gly Asn Pro Thr Asn Pro Lys Glu Ser Lys Lys Glu Val Ala Ser 85 90 95 Lys Thr Glu Thr Asn Gln Cys Asp 100 54130PRTArtificial SequenceTat derivative polypeptide 54Met Glu Thr Pro Leu Lys Glu Gln Glu Ser Ser Leu Glu Ser Ser Arg 1 5 10 15 Glu His Ser Ser Ser Ile Ser Glu Val Asp Ala Asp Thr Pro Glu Ser 20 25 30 Ala Ser Leu Glu Glu Glu Ile Leu Ser Gln Leu Tyr Arg Pro Arg Lys 35 40 45 Thr Ala Cys Thr Thr Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gln 50 55 60 Val Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys 65 70 75 80 Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr His Gln Ala 85 90 95 Ser Leu Ser Lys Gln Pro Ala Ser Gln Ser Arg Gly Asp Pro Thr Gly 100 105 110 Pro Thr Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu Thr Asp Pro 115 120 125 Phe Asp 130 55142PRTArtificial SequenceTat derivative polypeptide 55Met Glu Thr Pro Leu Lys Glu Gln Glu Ser Ser Leu Glu Ser Ser Arg 1 5 10 15 Glu His Ser Ser Ser Ile Ser Glu Val Asp Ala Asp Thr Pro Glu Ser 20 25 30 Ala Ser Leu Glu Glu Glu Ile Leu Ser Gln Leu Tyr Arg Pro Arg Thr 35 40 45 Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Tyr Ala 50 55 60 Cys Phe Leu Arg Lys Gly Leu Gly Ile Thr Tyr His Ala Phe Arg Thr 65 70 75 80 Arg Arg Lys Lys Ile Ala Ser Ala Asp Arg Ile Pro Val Pro Gln Gln 85 90 95 Ser Ile Ser Ile Arg Gly Arg Asp Ser Gln Thr Thr Gln Glu Ser Gln 100 105 110 Lys Lys Val Glu Glu Gln Ala Lys Ala Asn Leu Arg Ile Ser Arg Lys 115 120 125 Asn Leu Gly Asp Glu Thr Arg Gly Pro Val Gly Ala Gly Asn 130 135 140 56129PRTArtificial SequenceTat derivative polypeptide 56Met Glu Thr Pro Leu Lys Glu Gln Glu Ser Ser Leu Glu Ser Ser Arg 1 5 10 15 Glu His Ser Ser Ser Ile Ser Glu Val Asp Ala Asp Thr Pro Glu Ser 20 25 30 Ala Ser Leu Glu Glu Glu Ile Leu Ser Gln Leu Tyr Arg Pro Thr Thr 35 40 45 Ala Cys Ser Lys Cys Tyr Cys Lys Met Cys Cys Trp His Cys Gln Leu 50 55 60 Cys Phe Leu Asn Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg 65 70 75 80 Lys Arg Arg Arg Gly Thr Pro Gln Ser Arg Gln Asp His Gln Asn Pro 85 90 95 Val Pro Lys Gln Pro Leu Pro Thr Thr Arg Gly Asn Pro Thr Asn Pro 100 105 110 Lys Glu Ser Lys Lys Glu Val Ala Ser Lys Thr Glu Thr Asn Gln

Cys 115 120 125 Asp 57111PRTArtificial SequenceTat derivative polypeptide 57Met Asp Ala Gly Lys Ala Val Ser Asp Lys Lys Glu Gly Asp Val Thr 1 5 10 15 Pro Tyr Asp Pro Phe Arg Asp Arg Thr Thr Pro Arg Lys Thr Ala Cys 20 25 30 Thr Thr Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gln Val Cys Phe 35 40 45 Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln 50 55 60 Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr His Gln Ala Ser Leu Ser 65 70 75 80 Lys Gln Pro Ala Ser Gln Ser Arg Gly Asp Pro Thr Gly Pro Thr Glu 85 90 95 Ser Lys Lys Lys Val Glu Arg Glu Thr Glu Thr Asp Pro Phe Asp 100 105 110 58123PRTArtificial SequenceTat derivative polypeptide 58Met Asp Ala Gly Lys Ala Val Ser Asp Lys Lys Glu Gly Asp Val Thr 1 5 10 15 Pro Tyr Asp Pro Phe Arg Asp Arg Thr Thr Pro Arg Thr Ala Cys Asn 20 25 30 Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Leu 35 40 45 Arg Lys Gly Leu Gly Ile Thr Tyr His Ala Phe Arg Thr Arg Arg Lys 50 55 60 Lys Ile Ala Ser Ala Asp Arg Ile Pro Val Pro Gln Gln Ser Ile Ser 65 70 75 80 Ile Arg Gly Arg Asp Ser Gln Thr Thr Gln Glu Ser Gln Lys Lys Val 85 90 95 Glu Glu Gln Ala Lys Ala Asn Leu Arg Ile Ser Arg Lys Asn Leu Gly 100 105 110 Asp Glu Thr Arg Gly Pro Val Gly Ala Gly Asn 115 120 59110PRTArtificial SequenceTat derivative polypeptide 59Met Asp Ala Gly Lys Ala Val Ser Asp Lys Lys Glu Gly Asp Val Thr 1 5 10 15 Pro Tyr Asp Pro Phe Arg Asp Arg Thr Thr Pro Thr Thr Ala Cys Ser 20 25 30 Lys Cys Tyr Cys Lys Met Cys Cys Trp His Cys Gln Leu Cys Phe Leu 35 40 45 Asn Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Lys Arg Arg 50 55 60 Arg Gly Thr Pro Gln Ser Arg Gln Asp His Gln Asn Pro Val Pro Lys 65 70 75 80 Gln Pro Leu Pro Thr Thr Arg Gly Asn Pro Thr Asn Pro Lys Glu Ser 85 90 95 Lys Lys Glu Val Ala Ser Lys Thr Glu Thr Asn Gln Cys Asp 100 105 110 60117PRTArtificial SequenceTat derivative polypeptide 60Met Glu Pro Ser Gly Lys Glu Asp His Asn Cys Pro Pro Gln Asp Ser 1 5 10 15 Gly Gln Glu Glu Ile Asp Tyr Lys Gln Leu Leu Glu Glu Tyr Tyr Gln 20 25 30 Pro Arg Lys Thr Ala Cys Thr Thr Cys Tyr Cys Lys Lys Cys Cys Phe 35 40 45 His Cys Gln Val Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly 50 55 60 Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr 65 70 75 80 His Gln Ala Ser Leu Ser Lys Gln Pro Ala Ser Gln Ser Arg Gly Asp 85 90 95 Pro Thr Gly Pro Thr Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu 100 105 110 Thr Asp Pro Phe Asp 115 61129PRTArtificial SequenceTat derivative polypeptide 61Met Glu Pro Ser Gly Lys Glu Asp His Asn Cys Pro Pro Gln Asp Ser 1 5 10 15 Gly Gln Glu Glu Ile Asp Tyr Lys Gln Leu Leu Glu Glu Tyr Tyr Gln 20 25 30 Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe His 35 40 45 Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu Gly Ile Thr Tyr His Ala 50 55 60 Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala Asp Arg Ile Pro Val 65 70 75 80 Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp Ser Gln Thr Thr Gln 85 90 95 Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys Ala Asn Leu Arg Ile 100 105 110 Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly Pro Val Gly Ala Gly 115 120 125 Asn 62123PRTArtificial SequenceTat derivative polypeptide 62Met Glu Pro Ser Gly Lys Glu Asp His Asn Cys Pro Pro Gln Asp Ser 1 5 10 15 Gly Gln Glu Glu Ile Asp Tyr Lys Gln Leu Leu Glu Glu Tyr Tyr Gln 20 25 30 Pro Leu Glu Thr Cys Asn Asn Thr Cys Tyr Cys Lys Glu Cys Cys Tyr 35 40 45 His Cys Gln Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile Trp Tyr Asp 50 55 60 Arg Lys Gly Arg Arg Arg Arg Ser Pro Lys Lys Ile Lys Ala His Ser 65 70 75 80 Ser Ser Ala Ser Asp Lys Ser Ile Ser Thr Arg Thr Arg Asn Ser Gln 85 90 95 Pro Glu Glu Lys Gln Lys Lys Thr Leu Glu Thr Thr Leu Gly Thr Asp 100 105 110 Cys Gly Pro Gly Arg Ser His Ile Tyr Ile Ser 115 120 63116PRTArtificial SequenceTat derivative polypeptide 63Met Glu Pro Ser Gly Lys Glu Asp His Asn Cys Pro Pro Gln Asp Ser 1 5 10 15 Gly Gln Glu Glu Ile Asp Tyr Lys Gln Leu Leu Glu Glu Tyr Tyr Gln 20 25 30 Pro Thr Thr Ala Cys Ser Lys Cys Tyr Cys Lys Met Cys Cys Trp His 35 40 45 Cys Gln Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile Ser Tyr Gly Arg 50 55 60 Lys Lys Arg Lys Arg Arg Arg Gly Thr Pro Gln Ser Arg Gln Asp His 65 70 75 80 Gln Asn Pro Val Pro Lys Gln Pro Leu Pro Thr Thr Arg Gly Asn Pro 85 90 95 Thr Asn Pro Lys Glu Ser Lys Lys Glu Val Ala Ser Lys Thr Glu Thr 100 105 110 Asn Gln Cys Asp 115 64111PRTArtificial SequenceTat derivative polypeptide 64Met Asp Val Gly Glu Val Ala Ser Asp Lys Lys Glu Glu Asp Ile Thr 1 5 10 15 His Phe Asp Pro Phe Arg Ala Arg Thr Thr Pro Arg Lys Thr Ala Cys 20 25 30 Thr Thr Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gln Val Cys Phe 35 40 45 Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln 50 55 60 Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr His Gln Ala Ser Leu Ser 65 70 75 80 Lys Gln Pro Ala Ser Gln Ser Arg Gly Asp Pro Thr Gly Pro Thr Glu 85 90 95 Ser Lys Lys Lys Val Glu Arg Glu Thr Glu Thr Asp Pro Phe Asp 100 105 110 65123PRTArtificial SequenceTat derivative polypeptide 65Met Asp Val Gly Glu Val Ala Ser Asp Lys Lys Glu Glu Asp Ile Thr 1 5 10 15 His Phe Asp Pro Phe Arg Ala Arg Thr Thr Pro Arg Thr Ala Cys Asn 20 25 30 Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Leu 35 40 45 Arg Lys Gly Leu Gly Ile Thr Tyr His Ala Phe Arg Thr Arg Arg Lys 50 55 60 Lys Ile Ala Ser Ala Asp Arg Ile Pro Val Pro Gln Gln Ser Ile Ser 65 70 75 80 Ile Arg Gly Arg Asp Ser Gln Thr Thr Gln Glu Ser Gln Lys Lys Val 85 90 95 Glu Glu Gln Ala Lys Ala Asn Leu Arg Ile Ser Arg Lys Asn Leu Gly 100 105 110 Asp Glu Thr Arg Gly Pro Val Gly Ala Gly Asn 115 120 66117PRTArtificial SequenceTat derivative polypeptide 66Met Asp Val Gly Glu Val Ala Ser Asp Lys Lys Glu Glu Asp Ile Thr 1 5 10 15 His Phe Asp Pro Phe Arg Ala Arg Thr Thr Pro Leu Glu Thr Cys Asn 20 25 30 Asn Thr Cys Tyr Cys Lys Glu Cys Cys Tyr His Cys Gln Leu Cys Phe 35 40 45 Leu Asn Lys Gly Leu Gly Ile Trp Tyr Asp Arg Lys Gly Arg Arg Arg 50 55 60 Arg Ser Pro Lys Lys Ile Lys Ala His Ser Ser Ser Ala Ser Asp Lys 65 70 75 80 Ser Ile Ser Thr Arg Thr Arg Asn Ser Gln Pro Glu Glu Lys Gln Lys 85 90 95 Lys Thr Leu Glu Thr Thr Leu Gly Thr Asp Cys Gly Pro Gly Arg Ser 100 105 110 His Ile Tyr Ile Ser 115 67110PRTArtificial SequenceTat derivative polypeptide 67Met Asp Val Gly Glu Val Ala Ser Asp Lys Lys Glu Glu Asp Ile Thr 1 5 10 15 His Phe Asp Pro Phe Arg Ala Arg Thr Thr Pro Thr Thr Ala Cys Ser 20 25 30 Lys Cys Tyr Cys Lys Met Cys Cys Trp His Cys Gln Leu Cys Phe Leu 35 40 45 Asn Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Lys Arg Arg 50 55 60 Arg Gly Thr Pro Gln Ser Arg Gln Asp His Gln Asn Pro Val Pro Lys 65 70 75 80 Gln Pro Leu Pro Thr Thr Arg Gly Asn Pro Thr Asn Pro Lys Glu Ser 85 90 95 Lys Lys Glu Val Ala Ser Lys Thr Glu Thr Asn Gln Cys Asp 100 105 110 68103PRTArtificial SequenceTat derivative polypeptide 68Met Asp Ala Arg Lys Val Asp Leu Asp Gln Gln Asp Ala Gly Thr His 1 5 10 15 Phe Glu Pro Arg Lys Thr Ala Cys Thr Thr Cys Tyr Cys Lys Lys Cys 20 25 30 Cys Phe His Cys Gln Val Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser 35 40 45 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser 50 55 60 Gln Thr His Gln Ala Ser Leu Ser Lys Gln Pro Ala Ser Gln Ser Arg 65 70 75 80 Gly Asp Pro Thr Gly Pro Thr Glu Ser Lys Lys Lys Val Glu Arg Glu 85 90 95 Thr Glu Thr Asp Pro Phe Asp 100 69115PRTArtificial SequenceTat derivative polypeptide 69Met Asp Ala Arg Lys Val Asp Leu Asp Gln Gln Asp Ala Gly Thr His 1 5 10 15 Phe Glu Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys 20 25 30 Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu Gly Ile Thr Tyr 35 40 45 His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala Asp Arg Ile 50 55 60 Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp Ser Gln Thr 65 70 75 80 Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys Ala Asn Leu 85 90 95 Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly Pro Val Gly 100 105 110 Ala Gly Asn 115 70109PRTArtificial SequenceTat derivative polypeptide 70Met Asp Ala Arg Lys Val Asp Leu Asp Gln Gln Asp Ala Gly Thr His 1 5 10 15 Phe Glu Pro Leu Glu Thr Cys Asn Asn Thr Cys Tyr Cys Lys Glu Cys 20 25 30 Cys Tyr His Cys Gln Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile Trp 35 40 45 Tyr Asp Arg Lys Gly Arg Arg Arg Arg Ser Pro Lys Lys Ile Lys Ala 50 55 60 His Ser Ser Ser Ala Ser Asp Lys Ser Ile Ser Thr Arg Thr Arg Asn 65 70 75 80 Ser Gln Pro Glu Glu Lys Gln Lys Lys Thr Leu Glu Thr Thr Leu Gly 85 90 95 Thr Asp Cys Gly Pro Gly Arg Ser His Ile Tyr Ile Ser 100 105 71102PRTArtificial SequenceTat derivative polypeptide 71Met Asp Ala Arg Lys Val Asp Leu Asp Gln Gln Asp Ala Gly Thr His 1 5 10 15 Phe Glu Pro Thr Thr Ala Cys Ser Lys Cys Tyr Cys Lys Met Cys Cys 20 25 30 Trp His Cys Gln Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile Ser Tyr 35 40 45 Gly Arg Lys Lys Arg Lys Arg Arg Arg Gly Thr Pro Gln Ser Arg Gln 50 55 60 Asp His Gln Asn Pro Val Pro Lys Gln Pro Leu Pro Thr Thr Arg Gly 65 70 75 80 Asn Pro Thr Asn Pro Lys Glu Ser Lys Lys Glu Val Ala Ser Lys Thr 85 90 95 Glu Thr Asn Gln Cys Asp 100 72107PRTArtificial SequenceTat derivative polypeptide 72Met Ser Ser Lys Glu Glu Leu Arg Thr Thr Pro Ile Ser Asp Pro Phe 1 5 10 15 Gln Glu Glu Gly Arg Gly Pro Arg Lys Thr Ala Cys Thr Thr Cys Tyr 20 25 30 Cys Lys Lys Cys Cys Phe His Cys Gln Val Cys Phe Thr Arg Lys Gly 35 40 45 Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala 50 55 60 Pro Gln Asp Ser Gln Thr His Gln Ala Ser Leu Ser Lys Gln Pro Ala 65 70 75 80 Ser Gln Ser Arg Gly Asp Pro Thr Gly Pro Thr Glu Ser Lys Lys Lys 85 90 95 Val Glu Arg Glu Thr Glu Thr Asp Pro Phe Asp 100 105 73119PRTArtificial SequenceTat derivative polypeptide 73Met Ser Ser Lys Glu Glu Leu Arg Thr Thr Pro Ile Ser Asp Pro Phe 1 5 10 15 Gln Glu Glu Gly Arg Gly Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys 20 25 30 Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu 35 40 45 Gly Ile Thr Tyr His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser 50 55 60 Ala Asp Arg Ile Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg 65 70 75 80 Asp Ser Gln Thr Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala 85 90 95 Lys Ala Asn Leu Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg 100 105 110 Gly Pro Val Gly Ala Gly Asn 115 74113PRTArtificial SequenceTat derivative polypeptide 74Met Ser Ser Lys Glu Glu Leu Arg Thr Thr Pro Ile Ser Asp Pro Phe 1 5 10 15 Gln Glu Glu Gly Arg Gly Pro Leu Glu Thr Cys Asn Asn Thr Cys Tyr 20 25 30 Cys Lys Glu Cys Cys Tyr His Cys Gln Leu Cys Phe Leu Asn Lys Gly 35 40 45 Leu Gly Ile Trp Tyr Asp Arg Lys Gly Arg Arg Arg Arg Ser Pro Lys 50 55 60 Lys Ile Lys Ala His Ser Ser Ser Ala Ser Asp Lys Ser Ile Ser Thr 65 70 75 80 Arg Thr Arg Asn Ser Gln Pro Glu Glu Lys Gln Lys Lys Thr Leu Glu 85 90 95 Thr Thr Leu Gly Thr Asp Cys Gly Pro Gly Arg Ser His Ile Tyr Ile 100 105 110 Ser 75106PRTArtificial SequenceTat derivative polypeptide 75Met Ser Ser Lys Glu Glu Leu Arg Thr Thr Pro Ile Ser Asp Pro Phe 1 5 10 15 Gln Glu Glu Gly Arg Gly Pro Thr Thr Ala Cys Ser Lys Cys Tyr Cys 20 25 30 Lys Met Cys Cys Trp His Cys Gln Leu Cys Phe Leu Asn Lys Gly Leu 35 40 45 Gly Ile Ser Tyr Gly Arg Lys Lys Arg Lys Arg Arg Arg Gly Thr Pro 50 55 60 Gln Ser Arg Gln Asp His Gln Asn Pro Val Pro Lys Gln Pro Leu Pro 65 70 75 80 Thr Thr Arg Gly Asn Pro Thr Asn Pro Lys Glu Ser Lys Lys Glu Val 85

90 95 Ala Ser Lys Thr Glu Thr Asn Gln Cys Asp 100 105 76105PRTArtificial SequenceTat derivative polypeptide 76Met Asp Pro Ser Val Glu Glu Leu Pro Lys Glu Gln Arg Pro Gly Ala 1 5 10 15 Ala Pro Ala Thr Pro Arg Lys Thr Ala Cys Thr Thr Cys Tyr Cys Lys 20 25 30 Lys Cys Cys Phe His Cys Gln Val Cys Phe Thr Arg Lys Gly Leu Gly 35 40 45 Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln 50 55 60 Asp Ser Gln Thr His Gln Ala Ser Leu Ser Lys Gln Pro Ala Ser Gln 65 70 75 80 Ser Arg Gly Asp Pro Thr Gly Pro Thr Glu Ser Lys Lys Lys Val Glu 85 90 95 Arg Glu Thr Glu Thr Asp Pro Phe Asp 100 105 77117PRTArtificial SequenceTat derivative polypeptide 77Met Asp Pro Ser Val Glu Glu Leu Pro Lys Glu Gln Arg Pro Gly Ala 1 5 10 15 Ala Pro Ala Thr Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys 20 25 30 Cys Cys Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu Gly Ile 35 40 45 Thr Tyr His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala Asp 50 55 60 Arg Ile Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp Ser 65 70 75 80 Gln Thr Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys Ala 85 90 95 Asn Leu Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly Pro 100 105 110 Val Gly Ala Gly Asn 115 78111PRTArtificial SequenceTat derivative polypeptide 78Met Asp Pro Ser Val Glu Glu Leu Pro Lys Glu Gln Arg Pro Gly Ala 1 5 10 15 Ala Pro Ala Thr Pro Leu Glu Thr Cys Asn Asn Thr Cys Tyr Cys Lys 20 25 30 Glu Cys Cys Tyr His Cys Gln Leu Cys Phe Leu Asn Lys Gly Leu Gly 35 40 45 Ile Trp Tyr Asp Arg Lys Gly Arg Arg Arg Arg Ser Pro Lys Lys Ile 50 55 60 Lys Ala His Ser Ser Ser Ala Ser Asp Lys Ser Ile Ser Thr Arg Thr 65 70 75 80 Arg Asn Ser Gln Pro Glu Glu Lys Gln Lys Lys Thr Leu Glu Thr Thr 85 90 95 Leu Gly Thr Asp Cys Gly Pro Gly Arg Ser His Ile Tyr Ile Ser 100 105 110 79104PRTArtificial SequenceTat derivative polypeptide 79Met Asp Pro Ser Val Glu Glu Leu Pro Lys Glu Gln Arg Pro Gly Ala 1 5 10 15 Ala Pro Ala Thr Pro Thr Thr Ala Cys Ser Lys Cys Tyr Cys Lys Met 20 25 30 Cys Cys Trp His Cys Gln Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile 35 40 45 Ser Tyr Gly Arg Lys Lys Arg Lys Arg Arg Arg Gly Thr Pro Gln Ser 50 55 60 Arg Gln Asp His Gln Asn Pro Val Pro Lys Gln Pro Leu Pro Thr Thr 65 70 75 80 Arg Gly Asn Pro Thr Asn Pro Lys Glu Ser Lys Lys Glu Val Ala Ser 85 90 95 Lys Thr Glu Thr Asn Gln Cys Asp 100 80103PRTArtificial SequenceTat derivative polypeptide 80Met Glu Glu Glu Met Asp Leu Phe Gln Gly Arg Gly Arg Gly Glu Ala 1 5 10 15 Asn His Pro Arg Lys Thr Ala Cys Thr Thr Cys Tyr Cys Lys Lys Cys 20 25 30 Cys Phe His Cys Gln Val Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser 35 40 45 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser 50 55 60 Gln Thr His Gln Ala Ser Leu Ser Lys Gln Pro Ala Ser Gln Ser Arg 65 70 75 80 Gly Asp Pro Thr Gly Pro Thr Glu Ser Lys Lys Lys Val Glu Arg Glu 85 90 95 Thr Glu Thr Asp Pro Phe Asp 100 81115PRTArtificial SequenceTat derivative polypeptide 81Met Glu Glu Glu Met Asp Leu Phe Gln Gly Arg Gly Arg Gly Glu Ala 1 5 10 15 Asn His Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys 20 25 30 Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu Gly Ile Thr Tyr 35 40 45 His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala Asp Arg Ile 50 55 60 Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp Ser Gln Thr 65 70 75 80 Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys Ala Asn Leu 85 90 95 Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly Pro Val Gly 100 105 110 Ala Gly Asn 115 82109PRTArtificial SequenceTat derivative polypeptide 82Met Glu Glu Glu Met Asp Leu Phe Gln Gly Arg Gly Arg Gly Glu Ala 1 5 10 15 Asn His Pro Leu Glu Thr Cys Asn Asn Thr Cys Tyr Cys Lys Glu Cys 20 25 30 Cys Tyr His Cys Gln Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile Trp 35 40 45 Tyr Asp Arg Lys Gly Arg Arg Arg Arg Ser Pro Lys Lys Ile Lys Ala 50 55 60 His Ser Ser Ser Ala Ser Asp Lys Ser Ile Ser Thr Arg Thr Arg Asn 65 70 75 80 Ser Gln Pro Glu Glu Lys Gln Lys Lys Thr Leu Glu Thr Thr Leu Gly 85 90 95 Thr Asp Cys Gly Pro Gly Arg Ser His Ile Tyr Ile Ser 100 105 83102PRTArtificial SequenceTat derivative polypeptide 83Met Glu Glu Glu Met Asp Leu Phe Gln Gly Arg Gly Arg Gly Glu Ala 1 5 10 15 Asn His Pro Thr Thr Ala Cys Ser Lys Cys Tyr Cys Lys Met Cys Cys 20 25 30 Trp His Cys Gln Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile Ser Tyr 35 40 45 Gly Arg Lys Lys Arg Lys Arg Arg Arg Gly Thr Pro Gln Ser Arg Gln 50 55 60 Asp His Gln Asn Pro Val Pro Lys Gln Pro Leu Pro Thr Thr Arg Gly 65 70 75 80 Asn Pro Thr Asn Pro Lys Glu Ser Lys Lys Glu Val Ala Ser Lys Thr 85 90 95 Glu Thr Asn Gln Cys Asp 100 8499PRTArtificial SequenceTat derivative polypeptide 84Met Asn Ala Asp Ser Ile Asp Pro Phe Ala Gly Asn Lys Thr Pro Arg 1 5 10 15 Lys Thr Ala Cys Thr Thr Cys Tyr Cys Lys Lys Cys Cys Phe His Cys 20 25 30 Gln Val Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys 35 40 45 Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr His Gln 50 55 60 Ala Ser Leu Ser Lys Gln Pro Ala Ser Gln Ser Arg Gly Asp Pro Thr 65 70 75 80 Gly Pro Thr Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu Thr Asp 85 90 95 Pro Phe Asp 85111PRTArtificial SequenceTat derivative polypeptide 85Met Asn Ala Asp Ser Ile Asp Pro Phe Ala Gly Asn Lys Thr Pro Arg 1 5 10 15 Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Tyr 20 25 30 Ala Cys Phe Leu Arg Lys Gly Leu Gly Ile Thr Tyr His Ala Phe Arg 35 40 45 Thr Arg Arg Lys Lys Ile Ala Ser Ala Asp Arg Ile Pro Val Pro Gln 50 55 60 Gln Ser Ile Ser Ile Arg Gly Arg Asp Ser Gln Thr Thr Gln Glu Ser 65 70 75 80 Gln Lys Lys Val Glu Glu Gln Ala Lys Ala Asn Leu Arg Ile Ser Arg 85 90 95 Lys Asn Leu Gly Asp Glu Thr Arg Gly Pro Val Gly Ala Gly Asn 100 105 110 86105PRTArtificial SequenceTat derivative polypeptide 86Met Asn Ala Asp Ser Ile Asp Pro Phe Ala Gly Asn Lys Thr Pro Leu 1 5 10 15 Glu Thr Cys Asn Asn Thr Cys Tyr Cys Lys Glu Cys Cys Tyr His Cys 20 25 30 Gln Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile Trp Tyr Asp Arg Lys 35 40 45 Gly Arg Arg Arg Arg Ser Pro Lys Lys Ile Lys Ala His Ser Ser Ser 50 55 60 Ala Ser Asp Lys Ser Ile Ser Thr Arg Thr Arg Asn Ser Gln Pro Glu 65 70 75 80 Glu Lys Gln Lys Lys Thr Leu Glu Thr Thr Leu Gly Thr Asp Cys Gly 85 90 95 Pro Gly Arg Ser His Ile Tyr Ile Ser 100 105 8798PRTArtificial SequenceTat derivative polypeptide 87Met Asn Ala Asp Ser Ile Asp Pro Phe Ala Gly Asn Lys Thr Pro Thr 1 5 10 15 Thr Ala Cys Ser Lys Cys Tyr Cys Lys Met Cys Cys Trp His Cys Gln 20 25 30 Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys 35 40 45 Arg Lys Arg Arg Arg Gly Thr Pro Gln Ser Arg Gln Asp His Gln Asn 50 55 60 Pro Val Pro Lys Gln Pro Leu Pro Thr Thr Arg Gly Asn Pro Thr Asn 65 70 75 80 Pro Lys Glu Ser Lys Lys Glu Val Ala Ser Lys Thr Glu Thr Asn Gln 85 90 95 Cys Asp 88104PRTArtificial SequenceTat derivative polypeptide 88Met Asp Pro Lys Gly Glu Glu Asp Gln Asp Val Ser His Gln Asp Leu 1 5 10 15 Ile Lys Gln Tyr Arg Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys 20 25 30 Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Leu Arg Lys Gly Leu 35 40 45 Phe Leu Gln Lys Gly Leu Gly Ile Ser Tyr Arg Ser Tyr Ser Lys Lys 50 55 60 Thr Lys Pro Asp Thr Thr Thr Ala Ala Ser Arg Asx Leu Gly Arg Val 65 70 75 80 Thr Leu Ser Leu Tyr Leu Ser Arg Thr Thr Ser Thr Thr Trp Lys Arg 85 90 95 Asp Ser Lys Thr Ala Lys Lys Glu 100 89130PRTArtificial SequenceTat derivative polypeptide 89Met Asp Pro Lys Gly Glu Glu Asp Gln Asp Val Ser His Gln Asp Leu 1 5 10 15 Ile Lys Gln Tyr Arg Lys Pro Ala Cys Tyr Cys Arg Ile Pro Ala Cys 20 25 30 Ile Ala Gly Glu Arg Arg Tyr Gly Thr Cys Ile Tyr Gln Gly Arg Leu 35 40 45 Trp Ala Phe Cys Cys Phe Leu Arg Lys Gly Leu Gly Ile Thr Tyr His 50 55 60 Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala Asp Arg Ile Pro 65 70 75 80 Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp Ser Gln Thr Thr 85 90 95 Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys Ala Asn Leu Arg 100 105 110 Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly Pro Val Gly Ala 115 120 125 Gly Asn 130 90132PRTArtificial SequenceTat derivative polypeptide 90Met Asp Pro Lys Gly Glu Glu Asp Gln Asp Val Ser His Gln Asp Leu 1 5 10 15 Ile Lys Gln Tyr Arg Lys Pro Thr Cys Leu Lys Ser Gly Ala Ile Cys 20 25 30 His Pro Val Phe Cys Pro Arg Arg Tyr Lys Gln Ile Gly Thr Cys Gly 35 40 45 Leu Pro Gly Thr Lys Cys Cys Phe Leu Arg Lys Gly Leu Gly Ile Thr 50 55 60 Tyr His Ala Phe Arg Thr Arg Arg Lys Lys Ile Ala Ser Ala Asp Arg 65 70 75 80 Ile Pro Val Pro Gln Gln Ser Ile Ser Ile Arg Gly Arg Asp Ser Gln 85 90 95 Thr Thr Gln Glu Ser Gln Lys Lys Val Glu Glu Gln Ala Lys Ala Asn 100 105 110 Leu Arg Ile Ser Arg Lys Asn Leu Gly Asp Glu Thr Arg Gly Pro Val 115 120 125 Gly Ala Gly Asn 130 9187PRTArtificial SequenceTat derivative polypeptide 91Met Asp Pro Lys Gly Glu Glu Asp Gln Asp Val Ser His Gln Asp Leu 1 5 10 15 Ile Lys Gln Tyr Arg Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys 20 25 30 Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Thr Lys Lys Gly Leu 35 40 45 Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Arg Pro Ala Arg Thr Ala 50 55 60 Asp Lys Asp Gln Asp Asn Gln Asp Pro Val Ser Lys Gln Ser Leu Ala 65 70 75 80 Gly Thr Arg Ser Gln Gln Glu 85 92141PRTArtificial SequenceTat derivative polypeptide 92Met Asp Pro Lys Gly Glu Glu Asp Gln Asp Val Ser His Gln Asp Leu 1 5 10 15 Ile Lys Gln Tyr Arg Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys 20 25 30 Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Thr Lys Lys Ala Leu 35 40 45 Gly Ile Ser Tyr Gly Arg Lys Arg Arg Gly Arg Lys Ser Ala Gly Asp 50 55 60 Asn Lys Thr His Gln Asp Pro Val Arg Gln Gln Ser Leu Pro Lys Arg 65 70 75 80 Ser Arg Ile Gln Ser Ser Gln Glu Glu Ser Gln Lys Glu Val Glu Thr 85 90 95 Glu Ala Gly Ser Gly Gly Arg Pro Arg Pro Glu Asp Ser Ser Ala Ser 100 105 110 Ser Gly Arg Thr Ser Gly Thr Ser Ser Ser Gly Ser Thr Arg Pro Val 115 120 125 Ser Thr Ser Ser Gly Cys Trp Gly Pro Tyr Ser Lys Pro 130 135 140 93117PRTArtificial SequenceTat derivative polypeptide 93Met Asp Pro Lys Gly Glu Glu Asp Gln Asp Val Ser His Gln Asp Leu 1 5 10 15 Ile Lys Gln Tyr Arg Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys 20 25 30 Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Leu Thr Lys Gly Leu 35 40 45 Gly Ile Ser Tyr Gly Arg Lys Arg Lys Arg Arg Arg Ala Thr Ser Pro 50 55 60 Val Pro Gly Leu Ser Ser Ser Lys Asn Pro Ala Arg Lys Gln Gly Arg 65 70 75 80 Asp Thr Leu Phe Phe Leu Leu Arg Ser Leu Ser His Pro Thr Arg Asp 85 90 95 Ser Gln Arg Pro Thr Glu Gln Ala Gln Ala Val Ala Thr Ala Ala Thr 100 105 110 Pro Asp Arg Gln His 115 94136PRTArtificial SequenceTat derivative polypeptide 94Met Glu Thr Pro Leu Arg Glu Gln Glu Asn Ser Leu Lys Ser Ser Asn 1 5 10 15 Gly Arg Ser Ser Cys Thr Ser Glu Ala Ala Ala Pro Thr Leu Glu Ser 20 25 30 Ala Asn Leu Glu Glu Glu Ile Leu Ser Gln Leu Tyr Arg Pro Leu Glu 35 40 45 Thr Cys Asn Asn Thr Cys Tyr Cys Lys Glu Cys Cys Tyr His Cys Gln 50 55 60 Leu Cys Phe Leu Asn Lys Gly Leu Gly Ile Trp Tyr Asp Arg Lys Gly 65 70 75 80 Arg Arg Arg Arg Ser Pro Lys Lys Ile Lys Ala His Ser Ser Ser Ala 85 90 95 Ser Asp Lys Ser Ile Ser Thr Arg Thr Arg Asn Ser Gln Pro Glu Glu 100 105 110 Lys Gln Lys Lys Thr Leu Glu Thr Thr Leu Gly Thr Asp Cys Gly Pro 115 120 125 Gly Arg Ser His Ile Tyr Ile Ser 130 135 95108PRTArtificial SequenceTat derivative polypeptide 95Met Asp Pro Lys Gly Glu Glu Asp Gln Asp Val Ser His Gln Asp

Leu 1 5 10 15 Ile Lys Gln Tyr Arg Lys Pro Arg Thr Ala Cys Asn Asn Cys Tyr Cys 20 25 30 Lys Lys Cys Cys Phe His Cys Tyr Ala Cys Phe Phe Met Lys Lys Gly 35 40 45 Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Gly Ala 50 55 60 Ser Lys Ser Asn Gln Asn His Gln Asp Ser Ile Pro Glu Gln Pro Phe 65 70 75 80 Ser Gln Ser Arg Gly Asp Gln Ser Ser Pro Glu Lys Gln Glu Lys Lys 85 90 95 Val Glu Ser Lys Thr Thr Ser Asp Pro Phe Gly Cys 100 105 9618PRTHuman immunodeficiency virus type 1 96Met Glu Pro Val Asp Ala Asn Leu Glu Ala Trp Lys His Ala Gly Ser 1 5 10 15 Gln Pro 9723PRTSimian immunodeficiency virus 97Met Asp Pro Lys Gly Glu Glu Asp Gln Asp Val Ser His Gln Asp Leu 1 5 10 15 Ile Lys Gln Tyr Arg Lys Pro 20 9828PRTSimian immunodeficiency virus 98Met Glu Thr Pro Leu Lys Glu Gln Glu Asn Ser Leu Glu Ser Cys Arg 1 5 10 15 Glu His Ser Ser Ser Ile Ser Glu Val Asp Val Pro 20 25 9946PRTSimian immunodeficiency virus 99Met Glu Thr Pro Leu Lys Glu Gln Glu Ser Ser Leu Glu Ser Ser Arg 1 5 10 15 Glu His Ser Ser Ser Ile Ser Glu Val Asp Ala Asp Thr Pro Glu Ser 20 25 30 Ala Ser Leu Glu Glu Glu Ile Leu Ser Gln Leu Tyr Arg Pro 35 40 45 10027PRTSimian immunodeficiency virus 100Met Asp Ala Gly Lys Ala Val Ser Asp Lys Lys Glu Gly Asp Val Thr 1 5 10 15 Pro Tyr Asp Pro Phe Arg Asp Arg Thr Thr Pro 20 25 10119PRTSimian immunodeficiency virus 101Met Asp Val Gln Gly Val Gly Leu Glu His Pro Glu Glu Val Ile Leu 1 5 10 15 Tyr Asp Pro 10222PRTSimian immunodeficiency virus 102Met Asp Lys Gly Glu Glu Glu Arg Thr Val Leu His Gln Asp Leu Ile 1 5 10 15 Arg Gln Tyr Lys Lys Pro 20 10322PRTSimian immunodeficiency virus 103Met Asp Gln Glu Gln Glu Ala Arg Pro Gln Val Trp Glu Glu Leu Gln 1 5 10 15 Glu Glu Leu His Arg Pro 20 10419PRTSimian immunodeficiency virus 104Met Glu Ser Glu Gly Asp Gly Met Ala Glu Ser Leu Leu Gln Asp Leu 1 5 10 15 His Arg Pro 10522PRTSimian immunodeficiency virus 105Met Asp Lys Gly Glu Ala Glu Gln Ile Val Ser His Gln Asp Leu Ser 1 5 10 15 Glu Asp Tyr Gln Lys Pro 20 10620PRTSimian immunodeficiency virus 106Met Asp Lys Glu Glu Glu Pro His Pro Leu Leu Gln Asp Leu His Arg 1 5 10 15 Pro Leu Gln Pro 20 10720PRTSimian immunodeficiency virus 107Met Ala Gln Glu Glu Gly Leu Gln Val Trp Glu Glu Leu Gln Glu Glu 1 5 10 15 Leu Gln Arg Pro 20 10822PRTSimian immunodeficiency virus 108Met Asp Lys Gly Glu Asp Glu Gln Gly Ala Tyr His Gln Asp Leu Ile 1 5 10 15 Glu Gln Leu Lys Ala Pro 20 10919PRTSimian immunodeficiency virus 109Met Asp Val Arg Ala Val Gly Ser Glu Arg Ile Glu Glu Glu Thr Leu 1 5 10 15 Tyr Asn Pro 11030PRTSimian immunodeficiency virus 110Met Ser Ser Thr Asp Gln Ile Cys Gln Thr Gln Arg Val Pro Pro Ser 1 5 10 15 Phe Leu Glu Gly Thr Phe Leu Glu Lys Gly Pro Pro Thr Pro 20 25 30 11126PRTSimian immunodeficiency virus 111Met Asp Gly Gln Glu Ala Gly Leu Glu Arg Gln Glu Glu Glu Thr Leu 1 5 10 15 Tyr Asn Pro Phe Gln Ser Val Glu Thr Pro 20 25 11228PRTSimian immunodeficiency virus 112Met Ser Thr Gln Gly His Gln Gln Asp Gln Asp Gln Gly Lys Gly Thr 1 5 10 15 Leu Glu Glu Ala Tyr Lys Thr Asn Leu Glu Ala Pro 20 25 11330PRTSimian immunodeficiency virus 113Met Gln Gln Pro Glu Gln Glu Gln His Thr Gln Gln Lys Gln His Leu 1 5 10 15 Asp Gln Leu Glu Glu Ile Tyr Lys Glu Ala Ile Thr Asp Pro 20 25 30 11446PRTSimian immunodeficiency virus 114Met Glu Thr Pro Leu Lys Glu Gln Glu Ser Ser Leu Arg Ser Ser Ser 1 5 10 15 Glu Pro Ser Ser Cys Thr Ser Glu Ala Val Ala Ala Thr Pro Gly Leu 20 25 30 Ala Asn Gln Glu Glu Glu Ile Leu Trp Gln Leu Tyr Arg Pro 35 40 45 11521PRTSimian immunodeficiency virus 115Met Gln Pro Leu Gln Asn Arg Pro Asp Leu Gly Glu Glu Ile Leu Ser 1 5 10 15 Gln Leu Tyr Arg Pro 20 11633PRTSimian immunodeficiency virus 116Met Glu Pro Ser Gly Lys Glu Asp His Asn Cys Pro Pro Gln Asp Ser 1 5 10 15 Gly Gln Glu Glu Ile Asp Tyr Lys Gln Leu Leu Glu Glu Tyr Tyr Gln 20 25 30 Pro 11727PRTSimian immunodeficiency virus 117Met Asp Val Gly Glu Val Ala Ser Asp Lys Lys Glu Glu Asp Ile Thr 1 5 10 15 His Phe Asp Pro Phe Arg Ala Arg Thr Thr Pro 20 25 11819PRTSimian immunodeficiency virus 118Met Asp Ala Arg Lys Val Asp Leu Asp Gln Gln Asp Ala Gly Thr His 1 5 10 15 Phe Glu Pro 11923PRTSimian immunodeficiency virus 119Met Ser Ser Lys Glu Glu Leu Arg Thr Thr Pro Ile Ser Asp Pro Phe 1 5 10 15 Gln Glu Glu Gly Arg Gly Pro 20 12021PRTSimian immunodeficiency virus 120Met Asp Pro Ser Val Glu Glu Leu Pro Lys Glu Gln Arg Pro Gly Ala 1 5 10 15 Ala Pro Ala Thr Pro 20 12119PRTSimian immunodeficiency virus 121Met Glu Glu Glu Met Asp Leu Phe Gln Gly Arg Gly Arg Gly Glu Ala 1 5 10 15 Asn His Pro 12215PRTSimian immunodeficiency virus 122Met Asn Ala Asp Ser Ile Asp Pro Phe Ala Gly Asn Lys Thr Pro 1 5 10 15 12346PRTSimian immunodeficiency virus 123Met Glu Thr Pro Leu Arg Glu Gln Glu Asn Ser Leu Lys Ser Ser Asn 1 5 10 15 Gly Arg Ser Ser Cys Thr Ser Glu Ala Ala Ala Pro Thr Leu Glu Ser 20 25 30 Ala Asn Leu Glu Glu Glu Ile Leu Ser Gln Leu Tyr Arg Pro 35 40 45 12421PRTHuman immunodeficiency virus type 1 124Arg Lys Thr Ala Cys Thr Thr Cys Tyr Cys Lys Lys Cys Cys Phe His 1 5 10 15 Cys Gln Val Cys Phe 20 12520PRTHuman immunodeficiency virus type 1 125Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys 1 5 10 15 Tyr Ala Cys Phe 20 12617PRTHuman immunodeficiency virus type 1 126Thr Pro Val Ser Cys Leu Arg Lys Gly Gly Arg Cys Trp Asn Arg Cys 1 5 10 15 Ile 12721PRTHuman immunodeficiency virus type 2 127Leu Glu Thr Cys Asn Asn Thr Cys Tyr Cys Lys Glu Cys Cys Tyr His 1 5 10 15 Cys Gln Leu Cys Phe 20 12826PRTHuman immunodeficiency virus type 1 128Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys 1 5 10 15 Tyr Ala Cys Phe His Cys Tyr Ala Cys Phe 20 25 12920PRTHuman immunodeficiency virus type 1 129Thr Thr Ala Cys Ser Lys Cys Tyr Cys Lys Met Cys Cys Trp His Cys 1 5 10 15 Gln Leu Cys Phe 20 13031PRTHomo sapiens 130Ala Cys Tyr Cys Arg Ile Pro Ala Cys Ile Ala Gly Glu Arg Arg Tyr 1 5 10 15 Gly Thr Cys Ile Tyr Gln Gly Arg Leu Trp Ala Phe Cys Cys Phe 20 25 30 13133PRTHomo sapiens 131Thr Cys Leu Lys Ser Gly Ala Ile Cys His Pro Val Phe Cys Pro Arg 1 5 10 15 Arg Tyr Lys Gln Ile Gly Thr Cys Gly Leu Pro Gly Thr Lys Cys Cys 20 25 30 Phe 13214PRTHomo sapiens 132Arg Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe 1 5 10 13363PRTHuman immunodeficiency virus type 1 133Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln 1 5 10 15 Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr His Gln Ala Ser Leu Ser 20 25 30 Lys Gln Pro Ala Ser Gln Ser Arg Gly Asp Pro Thr Gly Pro Thr Glu 35 40 45 Ser Lys Lys Lys Val Glu Arg Glu Thr Glu Thr Asp Pro Phe Asp 50 55 60 13476PRTSimian immunodeficiency virus 134Leu Arg Lys Gly Leu Gly Ile Thr Tyr His Ala Phe Arg Thr Arg Arg 1 5 10 15 Lys Lys Ile Ala Ser Ala Asp Arg Ile Pro Val Pro Gln Gln Ser Ile 20 25 30 Ser Ile Arg Gly Arg Asp Ser Gln Thr Thr Gln Glu Ser Gln Lys Lys 35 40 45 Val Glu Glu Gln Ala Lys Ala Asn Leu Arg Ile Ser Arg Lys Asn Leu 50 55 60 Gly Asp Glu Thr Arg Gly Pro Val Gly Ala Gly Asn 65 70 75 13585PRTHuman immunodeficiency virus type 1 135Gly Asn Thr Arg Gln Ile Gly Ser Cys Gly Val Pro Phe Leu Lys Cys 1 5 10 15 Cys Lys Arg Lys Pro Phe Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly 20 25 30 Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr 35 40 45 His Gln Ala Ser Leu Ser Lys Gln Pro Ala Ser Gln Ser Arg Gly Asp 50 55 60 Pro Thr Gly Pro Thr Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu 65 70 75 80 Thr Asp Pro Phe Asp 85 13669PRTHuman immunodeficiency virus type 2 136Leu Asn Lys Gly Leu Gly Ile Trp Tyr Asp Arg Lys Gly Arg Arg Arg 1 5 10 15 Arg Ser Pro Lys Lys Ile Lys Ala His Ser Ser Ser Ala Ser Asp Lys 20 25 30 Ser Ile Ser Thr Arg Thr Arg Asn Ser Gln Pro Glu Glu Lys Gln Lys 35 40 45 Lys Thr Leu Glu Thr Thr Leu Gly Thr Asp Cys Gly Pro Gly Arg Ser 50 55 60 His Ile Tyr Ile Ser 65 13763PRTSimian immunodeficiency virus 137Leu Gln Lys Gly Leu Gly Ile Asn Tyr Ala Ser Arg Ala Arg Arg Arg 1 5 10 15 Arg Ser Lys Glu Glu Asn Lys Ala Asp Lys Phe Pro Val Pro Asn His 20 25 30 Arg Ser Ile Ser Thr Thr Arg Gly Asn Arg Lys Leu Gln Glu Lys Lys 35 40 45 Glu Lys Thr Val Glu Lys Lys Val Ala Thr Ser Thr Thr Ile Gly 50 55 60 13876PRTSimian immunodeficiency virus 138Leu Gln Lys Gly Leu Gly Val Thr Tyr His Ala Pro Arg Thr Arg Arg 1 5 10 15 Lys Lys Ser Val Gln Pro Asn Arg Leu Ser Gln Gln Asp Gln Ser Ile 20 25 30 Ser Thr Arg Gly Arg Asp Gly Gln Ala Thr Gln Glu Ser Gln Lys Lys 35 40 45 Val Glu Arg Glu Thr Thr Thr Ala Gln Ile Leu Gly Arg Lys Asp Leu 50 55 60 Glu Arg Asp Lys Arg Glu Ala Val Gly Ala Asn Ala 65 70 75 13930PRTSimian immunodeficiency virus 139His Lys Lys Ala Leu Gly Ile Arg Tyr Tyr Val Pro Arg Pro Arg Arg 1 5 10 15 Ala Ser Lys Lys Ile Ser His Asn Gln Val Ser Leu His Asn 20 25 30 14067PRTSimian immunodeficiency virus 140His Cys Tyr Ala Cys Phe Leu Gln Lys Gly Leu Gly Ile Thr Tyr His 1 5 10 15 Val Ser Arg Ile Arg Arg Pro Lys Lys Asn His Ser Asn His Gln Asn 20 25 30 Leu Val Ser Gln Gln Ser Ile Ser Ala Trp Gly Gly Asn Ser Gln Thr 35 40 45 Thr Gln Glu Glu Lys Thr Lys Ile Pro Ala Ala Ala Glu Thr Ser Arg 50 55 60 Arg Pro Gln 65 14156PRTSimian immunodeficiency virus 141Gln Lys Gly Leu Gly Val Thr Tyr His Ala Pro Arg Thr Arg Arg Lys 1 5 10 15 Lys Ile Arg Ser Leu Asn Leu Ala Pro Leu Gln His Gln Ser Ile Ser 20 25 30 Thr Lys Trp Gly Arg Asp Gly Gln Thr Thr Pro Thr Ser Gln Glu Lys 35 40 45 Val Glu Thr Thr Ala Gly Ser Asn 50 55 14256PRTSimian immunodeficiency virus 142Leu Gln Lys Gly Leu Gly Val Arg Tyr His Val Ser Arg Lys Arg Arg 1 5 10 15 Lys Thr Ser Thr Gln Asp Asn Gln Asp Pro Ile Arg Gln Gln Ser Ile 20 25 30 Ser Thr Val Gln Arg Asn Gly Gln Thr Thr Glu Glu Gly Lys Thr Glu 35 40 45 Val Glu Lys Ala Ala Ala Ala Asn 50 55 14365PRTSimian immunodeficiency virus 143Thr Gln Lys Gly Leu Gly Ile Ala Tyr Tyr Val Pro Arg Thr Arg Arg 1 5 10 15 Thr Val Lys Lys Ile Gln Asn Asn Gln Val Pro Ile His Asn Gln Ser 20 25 30 Ile Ser Thr Trp Thr Arg Asn Ser Gln Ala Glu Lys Lys Ser Gln Thr 35 40 45 Lys Val Gly Gln Ala Ala Thr Ala Asp His Thr Pro Gly Arg Lys Asn 50 55 60 Ser 65 14477PRTSimian immunodeficiency virus 144Phe Leu Gln Lys Gly Leu Gly Val Thr Tyr His Ala Pro Arg Ile Arg 1 5 10 15 Arg Lys Lys Ile Ala Pro Leu Asp Arg Phe Pro Glu Gln Lys Gln Ser 20 25 30 Ile Ser Thr Arg Gly Arg Asp Ser Gln Thr Thr Gln Lys Gly Gln Glu 35 40 45 Lys Val Glu Thr Ser Ala Arg Thr Ala Pro Ser Leu Gly Arg Lys Asn 50 55 60 Leu Ala Gln Gln Ser Gly Arg Ala Thr Gly Ala Ser Asp 65 70 75 14563PRTHuman immunodeficiency virus type 1 145Leu Asn Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Lys Arg 1 5 10 15 Arg Arg Gly Thr Pro Gln Ser Arg Gln Asp His Gln Asn Pro Val Pro 20 25 30 Lys Gln Pro Leu Pro Thr Thr Arg Gly Asn Pro Thr Asn Pro Lys Glu 35 40 45 Ser Lys Lys Glu Val Ala Ser Lys Thr Glu Thr Asn Gln Cys Asp 50 55 60 14661PRTSimian immunodeficiency virus 146Leu Arg Lys Gly Leu Phe Leu Gln Lys Gly Leu Gly Ile Ser Tyr Arg 1 5 10 15 Ser Tyr Ser Lys Lys Thr Lys Pro Asp Thr Thr Thr Ala Ala Ser Arg 20 25 30 Asx Leu Gly Arg Val Thr Leu Ser Leu Tyr Leu Ser Arg Thr Thr Ser 35 40 45 Thr Thr Trp Lys Arg Asp Ser Lys Thr Ala Lys Lys Glu 50 55 60 14744PRTSimian immunodeficiency virus 147Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Arg 1 5 10 15 Pro Ala Arg Thr Ala Asp Lys Asp Gln Asp Asn Gln Asp Pro Val Ser 20 25 30 Lys Gln Ser Leu Ala Gly Thr Arg Ser Gln Gln Glu 35 40 14898PRTSimian immunodeficiency virus 148Thr Lys Lys Ala Leu Gly Ile Ser Tyr Gly Arg Lys Arg Arg Gly Arg 1 5 10 15 Lys Ser Ala Gly Asp Asn Lys Thr His Gln Asp Pro Val Arg Gln Gln 20 25 30 Ser Leu Pro Lys Arg Ser Arg Ile Gln Ser Ser Gln Glu Glu Ser Gln 35 40 45 Lys

Glu Val Glu Thr Glu Ala Gly Ser Gly Gly Arg Pro Arg Pro Glu 50 55 60 Asp Ser Ser Ala Ser Ser Gly Arg Thr Ser Gly Thr Ser Ser Ser Gly 65 70 75 80 Ser Thr Arg Pro Val Ser Thr Ser Ser Gly Cys Trp Gly Pro Tyr Ser 85 90 95 Lys Pro 14974PRTSimian immunodeficiency virus 149Leu Thr Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys Arg Lys Arg Arg 1 5 10 15 Arg Ala Thr Ser Pro Val Pro Gly Leu Ser Ser Ser Lys Asn Pro Ala 20 25 30 Arg Lys Gln Gly Arg Asp Thr Leu Phe Phe Leu Leu Arg Ser Leu Ser 35 40 45 His Pro Thr Arg Asp Ser Gln Arg Pro Thr Glu Gln Ala Gln Ala Val 50 55 60 Ala Thr Ala Ala Thr Pro Asp Arg Gln His 65 70 15071PRTSimian immunodeficiency virus 150His Cys Tyr Ala Cys Phe Phe Met Lys Lys Gly Leu Gly Ile Ser Tyr 1 5 10 15 Gly Arg Lys Lys Arg Arg Gln Arg Arg Gly Ala Ser Lys Ser Asn Gln 20 25 30 Asn His Gln Asp Ser Ile Pro Glu Gln Pro Phe Ser Gln Ser Arg Gly 35 40 45 Asp Gln Ser Ser Pro Glu Lys Gln Glu Lys Lys Val Glu Ser Lys Thr 50 55 60 Thr Ser Asp Pro Phe Gly Cys 65 70

Claims

1. A polypeptide having an amino acid sequence comprising, in the following order: (i) the amino acid sequence of a transcription factor (TF) domain of a simian immunodeficiency virus (SIV) trans-activator of transcription (Tat) protein, wherein the SIV is African Green Monkey Vervet SIV (SIVagmVer), (ii) the amino acid sequence of a cysteine-rich sequence of SIV, human immunodeficiency virus (HIV), or a defensin, and (iii) the amino acid sequence of a C-terminal domain of HIV or SIV Tat protein; wherein the polypeptide is not SEQ ID NO: 3, and wherein the amino acid sequences of (i), (ii), and (iii) are not all from the same source.

2. The polypeptide of claim 1, wherein the HIV of (ii) or (iii) is independently HIV-1 or HIV-2, or wherein the SIV. of (ii) or (iii) is independently from a host selected from Table 2.

3. The polypeptide of claim 2, wherein the HIV-1 Tat is from a long-term non-progressor.

4. The polypeptide of claim 1, wherein the TF domain comprises the sequence of any one of SEQ ID NOs: 97, 102, or 108.

5. The polypeptide of claim 1, wherein the defensin is an .alpha.-defensin or a .beta.-defensin.

6. The polypeptide of claim 1, wherein the cysteine-rich domain comprises an amino acid sequence of one of SEQ ID NOs:124-132.

7. The polypeptide of claim 1, wherein the C-terminal domain comprises an amino acid sequence of one of SEQ ID NOs:133-150.

8. The polypeptide of claim 1, having greater than 85% sequence identity to one of SEQ ID NOs: 8, 9, 11, 13, 15, 17, 19-21, 46-49, 88-93, or 95.

9. A pharmaceutical composition comprising a polypeptide according to claim 1.

10. A method of treating cancer comprising: administering a therapeutically effective amount of the polypeptide of claim 1, or the pharmaceutical composition of claim 10, to a subject in need thereof; and causing cessation of growth of the cancer or regression of the cancer in the subject.

11. The method of claim 11, wherein the cancer is adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal-cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, bronchial adenomas/carcinoids, carcinoid tumor, cervical cancer, chronic myeloproliferative disorders, colon cancer, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, eye cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, glioma, gastric carcinoid, head and neck cancer, heart cancer, hepatocellular cancer, Hodgkin's lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi sarcoma, kidney cancer, leukemias, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancer, lymphomas, macroglobulinemia, medulloblastoma, melanoma, merkel cell carcinoma, mesothelioma, mouth cancer, multiple myeloma/plasma cell neoplasm, mycosis fungoides, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma, pituitary adenoma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, throat cancer, thymoma, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, or Wlms tumor.

12. The method of claim 11, wherein the cancer is breast cancer.

13. A method of inhibiting the suppression of an anti-tumor immune response in a subject with cancer, the method comprising: administering a therapeutically effective amount of the polypeptide of claim 1, or the pharmaceutical composition of claim 9, to the subject; wherein the administration results in reduction or inhibition of growth of the cancer or in regression of the cancer in the subject.

14. The method of claim 13, wherein at least one pre-treatment tumor from the subject contains at least 5% PD-L1-expressing cells.

15. The method of claim 13, wherein at least one pre-treatment tumor from the subject contains at least 15% PD-L1-expressing cells.