Patent Grant
10159707
The present invention relates to the field of immune-based therapeutic agents for cancer.
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.
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.
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.
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.
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.
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.
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.
Also disclosed herein is a pharmaceutical composition comprising a Tat derivative polypeptide disclosed herein.
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 α-defensin or a β-defensin. In yet another embodiment, the Tat derivative polypeptide further comprises an arginine-rich domain from HIV-1 or HIV-2 Tat.
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.
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.
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.
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.
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.
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.
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.
In one embodiment of the methods, the Tat derivative polypeptide has greater than 85% sequence identity to one of SEQ ID NOs 5-95.
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.
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, Sézary 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, Waldenström macroglobulinemia, or Wilms tumor.
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.
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.
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.
The HIV-Tat protein can repetitively 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.
Tat Derivative Polypeptides
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 α-interferon (α-interferon is a well established immunosuppressive cytokine).
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 α-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.
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).
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.
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.
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.
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.
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 α-defensin or β-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.
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.
The TF domain, cysteine-rich domain, and C-terminal domain sequences are arranged in the Tat derivative polypeptide in that order.
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.
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.
MEPVDANLEAWKHAGSQP
RKTACTTCYCKK
CCFHCQVCFTRKGLGISYGRKKRRQRRRAP
MDPKGEEDQDVSHQDLIKQYRKP
RTACNNC
YCKKCCFHCYACFLRKGLGITYHAFRTRRK
METPLKEQENSLESCREHSSSISEVDVPTP
VSCLRKGGRCWNRCIGNTRQIGSCGVPFLK
CCKRKPFTRKGLGISYGRKKRRQRRRAPQD
METPLKEQESSLESSREHSSSISEVDADTP
ESASLEEEILSQLYRP
LETCNNTCYCKECC
YHCQLCFLNKGLGIWYDRKGRRRRSPKKIK
MDAGKAVSDKKEGDVTPYDPFRDRTTP
LET
CNNTCYCKECCYHCQLCFLNKGLGIWYDRK
MDVQGVGLEHPEEVILYDP
RTACNNCYCKK
CCFHCYACFLQKGLGINYASRARRRRSKEE
MDKGEEERTVLHQDLIRQYKKP
RTACNNCY
CKKCCFHCYACFLRKGLGITYHAFRTRRKK
MDPKGEEDQDVSHQDLIKQYRKP
RTACNNC
YCKKCCFHCYACFHCYACFLQKGLGVTYHA
MDQEQEARPQVWEELQEELHRP
RTACNNCY
CKKCCFHCYACFLRKGLGITYHAFRTRRKK
MDPKGEEDQDVSHQDLIKQYRKP
RTACNNC
YCKKCCFHCYACFHKKALGIRYYVPRPRRA
MESEGDGMAESLLQDLHRP
RTACNNCYCKK
CCFHCYACFLRKGLGITYHAFRTRRKKIAS
MDPKGEEDQDVSHQDLIKQYRKP
RTACNNC
YCKKCCFHCYACFHCYACFLQKGLGITYHV
MDKGEAEQIVSHQDLSEDYQKP
RTACNNCY
CKKCCFHCYACFLRKGLGITYHAFRTRRKK
MDPKGEEDQDVSHQDLIKQYRKP
RTACNNC
YCKKCCFHCYACFLQKGLGVTYHAPRTRRK
MDKEEEPHPLLQDLHRPLQP
RTACNNCYCK
KCCFHCYACFLRKGLGITYHAFRTRRKKIA
MDPKGEEDQDVSHQDLIKQYRKP
RTACNNC
YCKKCCFHCYACFLQKGLGVRYHVSRKRRK
MAQEEGLQVWEELQEELQRP
RTACNNCYCK
KCCFHCYACFLRKGLGITYHAFRTRRKKIA
MDPKGEEDQDVSHQDLIKQYRKP
RTACNNC
YCKKCCFHCYACFTQKGLGIAYYVPRTRRT
MDKGEDEQGAYHQDLIEQLKAP
RTACNNCY
CKKCCFHCYACFLRKGLGITYHAFRTRRKK
MDPKGEEDQDVSHQDLIKQYRKP
RTACNNC
YCKKCCFHCYACFFLQKGLGVTYHAPRIRR
MDVRAVGSERIEEETLYNP
RKTACTTCYCK
KCCFHCQVCFTRKGLGISYGRKKRRQRRRA
MDVRAVGSERIEEETLYNP
RTACNNCYCKK
CCFHCYACFLRKGLGITYHAFRTRRKKIAS
MDVRAVGSERIEEETLYNP
LETCNNTCYCK
ECCYHCQLCFLNKGLGIWYDRKGRRRRSPK
MDVRAVGSERIEEETLYNP
TTACSKCYCKM
CCWHCQLCFLNKGLGISYGRKKRKRRRGTP
MSSTDQICQTQRVPPSFLEGTFLEKGPPTP
RKTACTTCYCKKCCFHCQVCFTRKGLGISY
MSSTDQICQTQRVPPSFLEGTFLEKGPPTP
RTACNNCYCKKCCFHCYACFLRKGLGITYH
MSSTDQICQTQRVPPSFLEGTFLEKGPPTP
LETCNNTCYCKECCYHCQLCFLNKGLGIWY
MSSTDQICQTQRVPPSFLEGTFLEKGPPTP
TTACSKCYCKMCCWHCQLCFLNKGLGISYG
MDGQEAGLERQEEETLYNPFQSVETP
RKTA
CTTCYCKKCCFHCQVCFTRKGLGISYGRKK
MDGQEAGLERQEEETLYNPFQSVETP
RTAC
NNCYCKKCCFHCYACFLRKGLGITYHAFRT
MDGQEAGLERQEEETLYNPFQSVETP
LETC
NNTCYCKECCYHCQLCFLNKGLGIWYDRKG
MDGQEAGLERQEEETLYNPFQSVETP
TTAC
SKCYCKMCCWHCQLCFLNKGLGISYGRKKR
MSTQGHQQDQDQGKGTLEEAYKTNLEAP
RK
TACTTCYCKKCCFHCQVCFTRKGLGISYGR
MSTQGHQQDQDQGKGTLEEAYKTNLEAP
RT
ACNNCYCKKCCFHCYACFLRKGLGITYHAF
MSTQGHQQDQDQGKGTLEEAYKTNLEAP
LE
TCNNTCYCKECCYHCQLCFLNKGLGIWYDR
MSTQGHQQDQDQGKGTLEEAYKTNLEAP
TT
ACSKCYCKMCCWHCQLCFLNKGLGISYGRK
MQQPEQEQHTQQKQHLDQLEEIYKEAITDP
RKTACTTCYCKKCCFHCQVCFTRKGLGISY
MQQPEQEQHTQQKQHLDQLEEIYKEAITDP
RTACNNCYCKKCCFHCYACFLRKGLGITYH
MQQPEQEQHTQQKQHLDQLEEIYKEAITDP
LETCNNTCYCKECCYHCQLCFLNKGLGIWY
MQQPEQEQHTQQKQHLDQLEEIYKEAITDP
TTACSKCYCKMCCWHCQLCFLNKGLGISYG
METPLKEQESSLRSSSEPSSCTSEAVAATP
GLANQEEEILWQLYRP
RKTACTTCYCKKCC
FHCQVCFTRKGLGISYGRKKRRQRRRAPQD
METPLKEQESSLRSSSEPSSCTSEAVAATP
GLANQEEEILWQLYRP
RTACNNCYCKKCCF
HCYACFLRKGLGITYHAFRTRRKKIASADR
METPLKEQESSLRSSSEPSSCTSEAVAATP
GLANQEEEILWQLYRP
LETCNNTCYCKECC
YHCQLCFLNKGLGIWYDRKGRRRRSPKKIK
METPLKEQESSLRSSSEPSSCTSEAVAATP
GLANQEEEILWQLYRP
TTACSKCYCKMCCW
HCQLCFLNKGLGISYGRKKRKRRRGTPQSR
MDKGEEERTVLHQDLIRQYKKP
RKTACTTC
YCKKCCFHCQVCFTRKGLGISYGRKKRRQR
MDKGEEERTVLHQDLIRQYKKP
RTACNNCY
CKKCCFHCYACFLRKGLGITYHAFRTRRKK
MDKGEEERTVLHQDLIRQYKKP
LETCNNTC
YCKECCYHCQLCFLNKGLGIWYDRKGRRRR
MDKGEEERTVLHQDLIRQYKKP
TTACSKCY
CKMCCWHCQLCFLNKGLGISYGRKKRKRRR
MQPLQNRPDLGEEILSQLYRP
RKTACTTCY
CKKCCFHCQVCFTRKGLGISYGRKKRRQRR
MQPLQNRPDLGEEILSQLYRP
RTACNNCYC
KKCCFHCYACFLRKGLGITYHAFRTRRKKI
MQPLQNRPDLGEEILSQLYRP
LETCNNTCY
CKECCYHCQLCFLNKGLGIWYDRKGRRRRS
MQPLQNRPDLGEEILSQLYRP
TTACSKCYC
KMCCWHCQLCFLNKGLGISYGRKKRKRRRG
METPLKEQESSLESSREHSSSISEVDADTP
ESASLEEEILSQLYRP
RKTACTTCYCKKCC
FHCQVCFTRKGLGISYGRKKRRQRRRAPQD
METPLKEQESSLESSREHSSSISEVDADTP
ESASLEEEILSQLYRP
RTACNNCYCKKCCF
HCYACFLRKGLGITYHAFRTRRKKIASADR
METPLKEQESSLESSREHSSSISEVDADTP
ESASLEEEILSQLYRP
TTACSKCYCKMCCW
HCQLCFLNKGLGISYGRKKRKRRRGTPQSR
MDAGKAVSDKKEGDVTPYDPFRDRTTP
RKT
ACTTCYCKKCCFHCQVCFTRKGLGISYGRK
MDAGKAVSDKKEGDVTPYDPFRDRTTP
RTA
CNNCYCKKCCFHCYACFLRKGLGITYHAFR
MDAGKAVSDKKEGDVTPYDPFRDRTTP
TTA
CSKCYCKMCCWHCQLCFLNKGLGISYGRKK
MEPSGKEDHNCPPQDSGQEEIDYKQLLEEY
YQP
RKTACTTCYCKKCCFHCQVCFTRKGLG
MEPSGKEDHNCPPQDSGQEEIDYKQLLEEY
YQP
RTACNNCYCKKCCFHCYACFLRKGLGI
MEPSGKEDHNCPPQDSGQEEIDYKQLLEEY
YQP
LETCNNTCYCKECCYHCQLCFLNKGLG
MEPSGKEDHNCPPQDSGQEEIDYKQLLEEY
YQP
TTACSKCYCKMCCWHCQLCFLNKGLGI
MDVGEVASDKKEEDITHFDPFRARTTP
RKT
ACTTCYCKKCCFHCQVCFTRKGLGISYGRK
MDVGEVASDKKEEDITHFDPFRARTTP
RTA
CNNCYCKKCCFHCYACFLRKGLGITYHAFR
MDVGEVASDKKEEDITHFDPFRARTTP
LET
CNNTCYCKECCYHCQLCFLNKGLGIWYDRK
MDVGEVASDKKEEDITHFDPFRARTTP
TTA
CSKCYCKMCCWHCQLCFLNKGLGISYGRKK
MDARKVDLDQQDAGTHFEP
RKTACTTCYCK
KCCFHCQVCFTRKGLGISYGRKKRRQRRRA
MDARKVDLDQQDAGTHFEP
RTACNNCYCKK
CCFHCYACFLRKGLGITYHAFRTRRKKIAS
MDARKVDLDQQDAGTHFEP
LETCNNTCYCK
ECCYHCQLCFLNKGLGIWYDRKGRRRRSPK
MDARKVDLDQQDAGTHFEP
TTACSKCYCKM
CCWHCQLCFLNKGLGISYGRKKRKRRRGTP
MSSKEELRTTPISDPFQEEGRGP
RKTACTT
CYCKKCCFHCQVCFTRKGLGISYGRKKRRQ
MSSKEELRTTPISDPFQEEGRGP
RTACNNC
YCKKCCFHCYACFLRKGLGITYHAFRTRRK
MSSKEELRTTPISDPFQEEGRGP
LETCNNT
CYCKECCYHCQLCFLNKGLGIWYDRKGRRR
MSSKEELRTTPISDPFQEEGRGP
TTACSKC
YCKMCCWHCQLCFLNKGLGISYGRKKRKRR
MDPSVEELPKEQRPGAAPATP
RKTACTTCY
CKKCCFHCQVCFTRKGLGISYGRKKRRQRR
MDPSVEELPKEQRPGAAPATP
RTACNNCYC
KKCCFHCYACFLRKGLGITYHAFRTRRKKI
MDPSVEELPKEQRPGAAPATP
LETCNNTCY
CKECCYHCQLCFLNKGLGIWYDRKGRRRRS
MDPSVEELPKEQRPGAAPATP
TTACSKCYC
KMCCWHCQLCFLKGLGISYGRKKRKRRRGT
MEEEMDLFQGRGRGEANHP
RKTACTTCYCK
KCCFHCQVCFTRKGLGISYGRKKRRQRRRA
MEEEMDLFQGRGRGEANHP
RTACNNCYCKK
CCFHCYACFLRKGLGITYHAFRTRRKKIAS
MEEEMDLFQGRGRGEANHP
LETCNNTCYCK
ECCYHCQLCFLNKGLGIWYDRKGRRRRSPK
MEEEMDLFQGRGRGEANHP
TTACSKCYCKM
CCWHCQLCFLNKGLGISYGRKKRKRRRGTP
MNADSIDPFAGNKTP
RKTACTTCYCKKCCF
HCQVCFTRKGLGISYGRKKRRQRRRAPQDS
MNADSIDPFAGNKTP
RTACNNCYCKKCCFH
CYACFLRKGLGITYHAFRTRRKKIASADRI
MNADSIDPFAGNKTP
LETCNNTCYCKECCY
HCQLCFLNKGLGIWYDRKGRRRRSPKKIKA
MNADSIDPFAGNKTP
TTACSKCYCKMCCWH
CQLCFLNKGLGISYGRKKRKRRRGTPQSRQ
MDPKGEEDQDVSHQDLIKQYRKP
RTACNNC
YCKKCCFHCYACFLRKGLFLQKGLGISYRS
MDPKGEEDQDVSHQDLIKQYRKP
ACYCRIP
ACIAGERRYGTCIYQGRLWAFCCFLRKGLG
MDPKGEEDQDVSHQDLIKQYRKP
TCLKSGA
ICHPVFCPRRYKQIGTCGLPGTKCCFLRKG
MDPKGEEDQDVSHQDLIKQYRKP
RTACNNC
YCKKCCFHCYACFTKKGLGISYGRKKRRRP
MDPKGEEDQDVSHQDLIKQYRKP
RTACNNC
YCKKCCFHCYACFTKKALGISYGRKRRGRK
MDPKGEEDQDVSHQDLIKQYRKP
RTACNNC
YCKKCCFHCYACFLTKGLGISYGRKRKRRR
METPLREQENSLKSSNGRSSCTSEAAAPTL
ESANLEEEILSQLYRP
LETCNNTCYCKECC
YHCQLCFLNKGLGIWYDRKGRRRRSPKKIK
MDPKGEEDQDVSHQDLIKQYRKP
RTACNNC
YCKKCCFHCYACFFMKKGLGISYGRKKRRQ
‡Cysteine-rich region is underlined
Chlorocebus pygetythrus
Chlorocebus aethiops
Chlorocebus tantalus
Chlorocebus sabaeus
Cercocebus torquatus torquatus
Cercopithecus albogularis
Cercocebus agilis
Cercopithecus solatus
Cercopithecus lhoesti
Macaca arctoides
Macaca mulatta
Cercocebus atys atys
Mandrillus sphinx
Mandrillus leucophaeus
Miopithecus talapoin
Cercopithecus cephus
Cercopithecus neglectus
Cercopithecus denti
Cercopithecus mona
Gorilla
Gorilla gorilla
Procolobus verus
Pan Troglodytes Troglodytes
Pan troglodytes troglodytes
Pan Troglodytes Schweinfurthi
Pan troglodytes schweinfurthii
Macaca nemestrina
Cercopithecus ascanius schmidti
Papio spp.
Cercopithecus satanas satanas
Lophocebus aterrimus
Cercopithecus mitis
Colobus guereza
procolobus verus
Cercopithecus nictitans
Procolobus [Piliocolobus]
rufomitratus tephrosceles
Erythrocebus patas
Cercopithecus preussi
Cercopithecus erythrotis erythrotis
Piliocolobus tholloni
Cercocebus torquatus lunulatus
Cercopithecus woffi
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
“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.
Override of Immune Checkpoints
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.
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.
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.
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.
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.
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.
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.
Use of Tat Derivative Polypeptides
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, Sézary 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, Waldenström macroglobulinemia, and Wilms tumor.
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.
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.
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.
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 immunofluorescence, and surface plasmon resonance. Non immunological assays such as quantitative polymerase chain reaction (qPCR), and determination of messenger RNA can also be used.
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.
Pharmaceutical Compositions
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.
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.
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.
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.
Transdermal administration includes percutaneous absorption of the composition through the skin. Transdermal formulations include patches, iontophoresis devices, ointments, creams, gels, salves and the like.
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.
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.
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.
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.
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.
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.
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.
Effects of Tat Derivative Polypeptides in Breast Cancer
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-γ 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.
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.
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® (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.
Established herein is that the Tat derivatives activate the INF-γ arm of the anti-cancer T cell immune response (
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.
Human monocytes were cultured for 24-48 hours with a Tat derivative (Nani-P2), an immunostimulatory sequence (ISS) of a toll-like receptor (TLR) (
Materials and Methods
Animals.
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.
Cell Lines.
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 μM 2-mercaptoethanol (Sigma), and 50 mg/L gentamicin (Lanza).
Tumor Challenge and Treatment.
BALB/c mice were injected (SC) with 1×104 4 T1, 1×105 TS/A or 2×105 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.
Detection of Lung Metastases.
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.
ELISA Analysis of IFN-γ Production by Immune Spleen Cells.
Splenocyte secretion of IFN-γ was assessed by an OptEIA™ ELISA kit (BD Biosciences). Briefly, spleen cells (1×105/well) from 4T1 tumor-bearing mice were cultured with or without 5×103/well mitomycin C (50 μg/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° C. until analysis without loss of activity. IFN-γ was measured in cell-free supernatants of duplicate wells by ELISA according to the manufacturer's instructions. Tumor-specific IFN-γ 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-γ using a recombinant IFN-γ standard curve. Stimulation index (SI) was calculated as the ratio of IFN-γ in stimulated versus control cultures.
Statistical Analysis.
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).
Results
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 1×104 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.
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 (
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 (
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.
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. With Nani-P2 administration (4 doses over 21 days), 35% of treated mice were still alive at day 48 (p<0.001,
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 (
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 (
The SM1 mammary carcinoma model (
To determine whether cytotoxic T-lymphocytes play a role in tumor rejection induced by Nani-P2 therapy, an IFN-γ ELISA assay (
IFN-γ concentrations, a standard surrogate for CTL activation, were quantitated by commercial ELISA (BD Biosciences). IFN-γ 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-γ 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.
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 (
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**,
Four groups of 10 BALB/c mice were implanted with 1×104 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 mm3) 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.
The decreased tumor burden in the Nani-P2/CY group compared to the CY group is very highly statistically significant (
The survival benefit of Nani-P2 bolus treatment alternating with cyclophosphamide vs. weekly cyclophosphamide is highly statistically significant (
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.
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.
As depicted in
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.
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.
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.
As depicted in
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.
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.
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.
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.
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.
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.
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.
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.
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.
The present application is a continuation of U.S. patent application Ser. No. 14/505,977 filed Oct. 3, 2014, which claims the benefit under 35 USC § 119(e) to U.S. Provisional Patent Application 61/887,166 filed Oct. 4, 2013, the entire contents of both of which are incorporated by reference herein.
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| Alignment of cited sequence and NaniP-2. |
| Number | Date | Country | |
|---|---|---|---|
| 20170274040 A1 | Sep 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61887166 | Oct 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14505977 | Oct 2014 | US |
| Child | 15587262 | US |
