Patent Application
20170174764
The invention relates to peptides derived from T-cell receptor (TCR) CDR3 segment related to self-immunity, and to antibodies to these peptide sequences. The invention also relates to methods of use of specific peptides for prevention, suppression and treatment of autoimmune diseases and allograft rejection. Also provided are antibodies specific to several CDR3 derived peptides for tumor immunotherapy and against pathogens.
The potential diversity of TCR molecules synthesized during the maturation of T cells in the thymus is estimated to be >10′5 for the mouse TCRαβ repertoire and >1010 for the TCRβ segment of the TCR. In contrast, the number of unique TCR types appearing in the peripheral lymphoid organs of an individual mouse (˜106) is many orders of magnitude less than this potential diversity. This excess of potential thymic TCR diversity leads to the expectation that different individuals would hardly ever share the same TCR recombination. Nevertheless, several reports have demonstrated identical TCR sequences occurring in the T-cell responses to defined antigens in different MHC-matched humans (V. P. Argaet et al., J Exp Med 180, 2335, 1994; P. A. Moss et al., Proc Natl Acad Sci USA 88, 8987, 1991), macaques (V. Venturi, et al., J Immunol 181, 2597, 2008) and mice (V. Venturi, et al., Nat Rev Immunol 8, 231, 2008). There have also been studies reporting substantial overlap in the naive TCR repertoire between two mice, of about 18-27%. Shared TCR molecules can be referred to as public; private T-cell responses involve little TCR sharing. It has been suggested that an adequate sampling of individual TCR repertoires would demonstrate the true prevalence of public TCR sequences (V. Venturi et al., Proc Natl Acad Sci USA 103, 18691, 2006).
T cell activation plays an important role in specific responses against pathogens, in tumor immunity and in autoimmune and inflammatory disorders. Therefore, methods of modulating the immune response and the T cell response in particular, are widely used in a plethora of medical conditions.
Tumor cells, for example, express many antigens that differ from those of healthy cells and against which the healthy immune system is posed to respond. Despite existing immunity to tumor-associated antigens, tumors can evade immune rejection by activating immune suppressor T cells of various types including CD4+ regulatory T cells (Tregs); growing tumors attract these immune suppressor cells, which down-regulate effector T cells and other immune cells that could otherwise reject the tumor. The tumor, in other words, hijacks immune regulation mechanisms that normally serve to prevent or down-regulate potential autoimmune effector reactions that might otherwise cause an autoimmune disease. The successful tumor masquerades as a normal cell population, not attacked by the immune system, despite the fact that it expresses tumor-associated antigens—body molecules that are abnormal in their structure, tissue site, or developmental timing. This new understanding of the tumor-immune relationship has led to the development of new therapies aimed at depriving the tumor of its protective immune suppression. A proof-of-concept has been demonstrated by the use of anti-PD1 and anti CTLA-4 antibodies in tumor immunotherapy (Curran M A, et al., PNAS, 107(9):4275-80, 2010); these antibodies target and disarm immune regulatory mechanisms, and thereby unleash quiescent or suppressed tumor-associated autoimmunity to attack the tumor with a destructive, autoimmune-like reaction. Initial clinical trials have been quite encouraging and major pharmaceutical companies are racing ahead to complete the development of anti-PD1 immunotherapy (Wolchok J D et al., N Engl J Med. 369(2):122-33, 2013). The disadvantage of anti-PD1 and anti-CTLA-4 treatment is that it lacks specificity; for example, the PD1 molecule is expressed on all T cells, B cells and macrophages. Specific treatment should target suppressor T cells that are specifically associated with the tumor, to reduce side effects and increase efficacy.
TCR diversity has been an obstacle for treatments such as T-cell vaccination based on specific TCR sequences. This might be alleviated if public TCRs can be used as effective T-cell vaccines. There is an unmet need to provide effective compositions for prevention, suppression and treatment of autoimmune diseases and allograft rejection and new, effective and specific therapies for cancer and against pathogens.
The present invention is based in part on high throughput study of the TCR repertoire and provides new therapeutic peptides for prevention and treatment of autoimmunity and allo-immunity, and neutralizing antibodies to promote immunity against pathogens and for cancer immunotherapy.
The proposed peptides and antibodies of the present invention emerged from the discovery of a set of T cells expressing public TCR molecules featuring CDR3 segments that are highly shared among individual mice, monkeys and humans. These public T cells represent some 5-10% of the T cell repertoire. Functionally, the public set of T cells is enriched for T cells associated with autoimmunity, with allograft immunity, and, with tumor-infiltrating T cells and T cells responsive to tumor-associated antigens such as MDM2 and HSP60. Indeed, CDR3 segments associated with tumor-related T cells are shared by humans and mice. It is thus plausible that experimental results obtained in mice are relevant to humans. Experiments in mice shown herein indicate that an antibody raised against a CDR3 peptide expressed by a relatively public T cell clonotype can activate a latent autoimmune T cell effector response in a Diabetes Type I mouse model and conversely inhibit tumor progression in a lung carcinoma mouse tumor model.
According to an aspect of some embodiments of the present invention there is provided an isolated agent capable of at least one of:
(i) binding a TCR presented on a T cell;
(ii) competing with binding of a TCR presented on a T cell to a target of the T cell;
(iii) eliciting a specific immune-response of a T cell; and
(iv) eliciting a specific immune-response against a T cell;
wherein the T cell is expressing a TCR-CDR3 sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs of Table 8, wherein when the agent is a peptide it is selected from the group consisting of SEQ ID NOs of Table 7.
According to an aspect of some embodiments of the present invention there is provided an isolated peptide of no more than 20 amino acids comprising an amino acid sequence having a CDR3 sequence of a TCR on a T cell, the CDR3 sequence being selected from the group consisting of SEQ ID NOs of Table 7.
According to an aspect of some embodiments of the present invention there is provided a use of:
(i) the isolated agent;
(ii) the isolated peptide; or
(iii) an isolated peptide of no more than 20 amino acids comprising an amino acid sequence having a CDR3 sequence of a TCR on a T cell, the CDR3 sequence being selected from the group consisting of SEQ ID NOs of Table 8,
in the manufacture of a medicament identified for treating a disease associated with the T cell.
According to an aspect of some embodiments of the present invention there is provided a method of treating a disease associated with the T cell, the method comprising administering to a subject in need thereof an effective amount of:
(i) the isolated agent;
(ii) the isolated peptide; or
(iii) an isolated peptide of no more than 20 amino acids comprising an amino acid sequence having a CDR3 sequence of a TCR on a T cell, the CDR3 sequence being selected from the group consisting of SEQ ID NOs of Table 8,
thereby treating the disease associated with the T cell.
According to some embodiments of the invention, the agent is selected from the group consisting of antibody, T cell, peptide and polynucleotide. According to an aspect of some embodiments of the present invention there is provided an isolated antibody comprising an antigen recognition domain capable of specifically binding SEQ ID NO: 1 of a TCR presented on a T cell.
According to some embodiments of the invention, there is provided a use of the isolated antibody in the manufacture of a medicament identified for treating a disease associated with the T cell.
According to some embodiments of the invention, there is provided a method of treating a disease associated with a T cell expressing a TCR-CDR3 segment comprising an amino acid sequence of SEQ ID NO: 1 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the isolated antibody, thereby treating the disease associated with a T cell expressing the TCR-CDR3 segment comprising an amino acid sequence of SEQ ID NO: 1 in the subject.
According to some embodiments of the invention, the T cell is a regulatory T cell.
According to some embodiments of the invention, the T cell is an effector T cell.
The present invention provides, according to a further aspect an isolated peptide of 8-20 amino acids, or an analog thereof, comprising a sequence of at least 6 contiguous amino acids derived from a TCR-CDR3 segment, wherein the peptide does not comprise a sequence selected from the group consisting of: ASSLGGNQD (SEQ ID NO: 2033); ASRLGNQD (SEQ ID NO: 2034); ASSLGLGANQD (SEQ ID NO: 2035); and ASSLGANQD (SEQ ID NO: 2036).
According to some embodiments, the CDR3 segment is from beta TCR.
According to some embodiments, the isolated peptide comprises an amino acid sequence which was found to be associated with immunity selected from the group consisting of: autoimmunity, pathogenic immunity, tumor immunity and, graft rejection, and was further identified in at least 75% of tested mammalian individuals.
According to other embodiments, the isolated peptide comprises an amino acid sequence which was found to be associated with immunity selected from the group consisting of: autoimmunity, pathogenic immunity, tumor immunity and, graft rejection, and was further identified in human individuals.
According to other embodiments, the isolated peptide comprises an amino acid sequence which was found to be associated with immunity selected from the group consisting of: autoimmunity, pathogenic immunity, tumor immunity and, graft rejection, was further identified in at least 75% of tested mammalian individuals, and was identified also in human individuals.
According to other embodiments, the isolated peptide comprises an amino acid sequence that was identified in at least 75% of tested mammalian individuals, and was identified also in human individuals.
According to some embodiments, the peptide or peptide analog consists of 10-16 amino acids.
According to some embodiments, the isolated peptide or analog thereof comprises 8-20 (e.g., 8-14) contiguous amino acids derived from a TCR-CDR3 segment.
According to some embodiments, the TCR-CDR3 segment is from mouse.
According to some embodiments, the TCR-CDR3 segment is from human.
According to some embodiments, the TCR-CDR3 segment is shared by human and mouse.
According to a specific embodiment, the CDR3 sequence is selected from any of the tables provided hereinbelow.
According to some embodiments of the invention, the CDR3 sequence is selected from the group consisting of the sequences in Table 2.
According to some embodiments of the invention, the peptide amino acid sequence is selected from the group consisting of SEQ ID NOs of Table 2.
Each possibility represents a separate embodiment of the present invention.
According to some embodiments of the invention, the CDR3 sequence is selected from the group consisting of the sequences in Table 3.
According to some embodiments of the invention, the peptide amino acid sequence is selected from the group consisting of SEQ ID NOs of Table 3.
Each possibility represents a separate embodiment of the present invention.
According to some embodiments of the invention, the CDR3 sequence is selected from the group consisting of the sequences in Table 4.
According to some embodiments of the invention, the peptide amino acid sequence is selected from the group consisting of SEQ ID NOs of Table 4.
Each possibility represents a separate embodiment of the present invention.
According to some embodiments of the present invention, the CDR3 sequence is selected from the group consisting of the sequences in Table 5.
According to some embodiments of the present invention, the peptide amino acid sequence is selected from the group consisting of SEQ ID NOs of Table 5.
Each possibility represents a separate embodiment of the present invention.
According to some embodiments of the present invention, the CDR3 sequence is selected from the group consisting of the sequences in Table 6.
According to some embodiments of the present invention, the peptide amino acid sequence is selected from the group consisting of SEQ ID NOs of Table 6.
Each possibility represents a separate embodiment of the present invention.
According to some embodiments of the present invention, the CDR3 sequence is selected from the group consisting of the sequences in Table 7.
According to some embodiments of the present invention, the peptide amino acid sequence is selected from the group consisting of SEQ ID NOs of Table 7.
Each possibility represents a separate embodiment of the present invention.
According to some embodiments of the present invention, the CDR3 sequence is selected from the group consisting of the sequences in Table 8.
According to some embodiments of the present invention, the peptide amino acid sequence is selected from the group consisting of SEQ ID NOs of Table 8.
Each possibility represents a separate embodiment of the present invention.
According to some embodiments of the present invention, the CDR3 sequence is selected from the group consisting of the sequences in Table 9.
According to some embodiments of the present invention, the peptide amino acid sequence is selected from the group consisting of SEQ ID NOs of Table 9.
Each possibility represents a separate embodiment of the present invention.
Accordingly, the peptide may have a sequence which encompasses the entire CDR3 sequences as presented in any one of the tables above.
It will be appreciated that peptide fragments are also encompassed according to the present teachings, as long as the peptide maintains its function e.g., capable of competing with binding of a TCR having the respective CDR3 sequence presented on a T cell to a target of the T cell. According to specific embodiments, the peptide may be 6-20, 8-20, 10-20, 15-20, 6-16, 8-16, 10-16, 12-16, 6-14, 8-14, 10-14 amino acids long.
According to some embodiments, the isolated peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 2-6, 8, 10, 12, 15-17, 19-25, 27, 29-32, 34, 37, 38, 40-42, 44-53, 55-58, 60-62, 65, 67-73, 77-79, 81, 82, 84-88, 90, 91, 93-100, 102-104, 106, 108-110, 1923 and 1924.
Each possibility represents a separate embodiment of the present invention.
It should be noted that any known peptide, such as the peptide ASSLGGNQDTQY (denoted C-9, SEQ ID NO: 1), is excluded from the scope of isolated peptides per se of the present invention.
According to some specific embodiments, the peptide is selected from the group consisting of SEQ ID NOs: 2-6, 8, 10, 12, 15-17, 19-25, 27, 29-32, 34, 1923 and 1924.
Each possibility represents a separate embodiment of the present invention. According to yet other embodiments, the peptide is selected from the group consisting of SEQ ID NOs: 3, 6, 8, 10, 12, 15-17, 19, 27 and 1923.
Each possibility represents a separate embodiment of the present invention.
According to yet other embodiments, the peptide is selected from the group consisting of SEQ ID NOs: 5, 6, 10, 12, 20-23, 25, 27, 30-32, 34 and 1924.
Each possibility represents a separate embodiment of the present invention.
Analogs and derivatives of the peptides are also within the scope of the present invention; as long as they maintain the peptide function e.g. compete with binding of a TCR presented on a T cell to a target of the T cell. These include but are not limited to conservative and non-conservative substitutions of amino acids, modification of the peptide's terminal (e.g. acylation of N-terminus, amidation of C-terminus etc.), insertion and deletion of amino acids within the sequence, cyclization, modification of a peptide bond, and combination of two or more such modification. Such modification and the resultant peptide analog or derivative are within the scope of the present invention as long as they confer, or even improve the immunogenicity or activity of the peptide.
Specifically, according to some embodiments, an isolated peptide analog comprising one conservative amino acid substitution, deletion or addition to the specific peptides listed above is provided.
According to some embodiments, the peptide analog, having one amino acid substitution is selected from the group consisting of SEQ ID NOs: 3, 5, 6, 8, 10, 12, 15-17, 19, 415, 1923, 2018-2032.
Each possibility represents a separate embodiment of the present invention.
The present invention further provides peptide multimers, peptide conjugates, and fusion proteins comprising peptides, analogs and derivatives according to the invention.
According to some embodiments, a fusion protein according to the invention comprises an immunogenic protein carrier, such as an immunoglobulin molecule or a T cell.
According to some embodiments, a peptide multimer comprising a plurality of identical or different peptides defined above is provided.
According to some embodiments of the invention, there is provided a multimer of the isolated peptide.
According to some embodiments of the invention, the at least two isolated peptides are identical.
According to some embodiments of the invention, the at least two isolated peptides are different.
According to some embodiments, the at least two peptides or peptide analogs are covalently linked, directly or through a spacer or a linker.
According to some embodiments, the peptide multimer comprises a linker. According to particular embodiments, the linker comprises plurality of Lysine residues. Each possibility represents a separate embodiment of the present invention.
A peptide conjugate according to the present invention comprises any peptide, peptide analog or peptide multimer defined above, conjugated or fused (e.g., covalent bond e.g., translational fusion or non-translational fusion) to a carrier protein or moiety which improves the peptide's solubility, stability or permeability (e.g., collectively termed bioavailability) or antigenicity.
According to some embodiments of the invention, the peptide is attached to a non-proteinaceous moiety.
According to some embodiments of the invention, the non-proteinaceous moiety comprises polyethylene glycol (PEG).
The peptide of present invention may be produced by any method known in the art, including recombinant and synthetic methods. According to some embodiments a synthetic peptide, peptide multimer or peptide conjugate is provided. According to other embodiments a recombinantly produced peptide, peptide multimer, peptide fusion protein or peptide conjugate with a carrier protein is provided.
Isolated polynucleotide sequences comprising at least one sequence encoding a peptide, peptide analog, conjugate or fusion protein are also included in the scope of the present invention.
According to some embodiments of the invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding the peptide (and as mentioned the modification thereof e.g., multimers, fusions as long as it is a translational product).
According to some embodiments of the invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding the agent.
According to some embodiments of the invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding the antibody.
According to some embodiments, a polynucleotide sequence encoding a peptide or peptide analog is translationally linked to another polynucleotide sequence such as an RNA or DNA molecule and is recombinantly expressed within target cells.
According to specific embodiments, said polynucleotide sequence is part of a nucleic acid construct also referred to herein as a vector such as a recombinant viral or bacterial vector. Vectors comprising the above polynucleotide sequences, as well as host cells, including hybridoma cells, comprising said vectors, are also within the scope of the present invention.
In another aspect the present invention is related to a pharmaceutical composition useful for preventing, attenuating or treating a disease or disorder associated T cell expressing a TCR with a specific CDR3 sequence, such as cancer, autoimmunity or allo-immunity.
According to some embodiments of the invention, there is provided a pharmaceutical composition comprising as an active ingredient the isolated agent, peptide or antibody and a pharmaceutically acceptable carrier or diluent.
According to some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a peptide, peptide analog, peptide multimer, fusion protein or conjugate or encoding nucleic acid sequence or viral or bacterial vector comprising them; and a pharmaceutically acceptable carrier or diluent.
According to some embodiments the pharmaceutical composition is formulated as a vaccine.
According to some embodiments of the invention, the pharmaceutical composition further comprises an adjuvant or a delivery system.
According to other embodiments, the formulation does not comprise an adjuvant or delivery system.
Pharmaceutically acceptable adjuvants include, but are not limited to water in oil emulsions, lipid emulsions, and liposomes.
In some embodiments the pharmaceutical composition is formulated for intramuscular, intranasal, oral, intraperitoneal, subcutaneous, topical, intradermal and transdermal delivery. In some embodiments the pharmaceutical composition is formulated for intramuscular administration. In yet other embodiments the pharmaceutical composition is formulated for intranasal administration.
The present invention further provides methods and uses of the peptides, peptide multimers and peptide conjugates for production of specific antibodies. According to some embodiments the antibodies are polyclonal antibodies. According to other embodiments, the antibodies are monoclonal antibodies. Any method known in the art for production of monoclonal or polyclonal antibodies may be used.
According to an aspect of some embodiments of the present invention there is provided a method of obtaining an antibody of interest, the method comprising using an isolated peptide of no more than 20 amino acids comprising an amino acid sequence having a CDR3 sequence of a TCR on a T cell selected from the group consisting of SEQ ID NO: 1 and SEQ ID NOs of Table 8 for producing or selecting an antibody specifically recognizing said peptide, thereby producing the antibody of interest.
According to some embodiments of the invention, the contacting is effected via immunization.
According to some embodiments of the invention, the selecting is from an antibody display platform.
According to some embodiments of the invention, the antibody display platform is selected from the group consisting of phage display, ribosome and mRNA display and microbial cell display.
The isolated agents and peptides of the present invention may be used for treating a disease associated with a T cell expressing the respective TCR-CDR3.
According to yet another aspect, the present invention provides a method of treating or alleviating an autoimmune or allograft disease or disorder comprising administering to a patient in need thereof, effective amount of a TCR CDR3 derived peptide, peptide analog, peptide multimer or peptide conjugate as defined above.
According to some embodiments of the invention the disease is an autoimmune disease.
According to some embodiments, the autoimmune disease is selected from the group consisting of: rheumatoid arthritis, multiple sclerosis, type-1 diabetes, chronic obstructive pulmonary disease (COPD), Crohn's disease, ulcerative colitis, and psoriasis.
According to some embodiments of the invention, the disease is a graft rejection disease.
According to some embodiments of the invention, the graft rejection disease is host vs. graft disease.
According to some embodiments of the invention, the disease is cancer.
According to some embodiments of the invention, the disease is pathogenic disease.
According to some embodiments of the invention, the pathogenic disease is human immunodeficiency virus or tuberculosis infection.
According to some particular embodiments, the peptide is selected from the group consisting of SEQ ID NOs: 5, 6, 10, 12, 20-23, 25, 27, 30-32, 34 and 1924.
Each possibility represents a separate embodiment of the present invention.
According to another aspect, the present invention provides antibody against a peptide derived from TCR-CDR3 segment, or an antibody fragment thereof comprising at least an antigen-binding portion.
Each possibility represents a separate embodiment of the present invention.
Each possibility represents a separate embodiment of the present invention.
According to yet other embodiments, the antibody comprises an antigen binding domain which specifically binds a sequence selected from the group consisting of SEQ ID NOs: 3, 6, 8, 10, 12, 15-17, 19, 27 and 1923.
Each possibility represents a separate embodiment of the present invention.
According to yet other embodiments, the antibody comprises an antigen recognition domain which specifically binds a sequence selected from the group consisting of SEQ ID NOs: 2-4, 8, 15-17, 19, 24, 29 and 1923.
Each possibility represents a separate embodiment of the present invention.
According to one embodiment of the present invention, the antibody is a monoclonal antibody (mAb). According to a specific embodiment, the mAb is selected from the group consisting of: mammalian antibody, humanized antibody, human antibody, chimeric antibody, and an antibody fragment comprising at least the antigen-binding portion of an antibody. According to a specific embodiment, the antibody fragment is selected from the group consisting of: Fab, Fab′, F(ab′)2, Fd, Fd′, Fv, dAb, isolated CDR region, single chain antibody, “diabodies”, and “linear antibodies”.
Within the scope of the present invention are also nucleic acid molecules encoding an antibody or antibody fragment or monoclonal or bispecific antibody, according to the invention, having affinity and specificity for a TCR CDR3 sequence.
An antibody or antibody fragment according to the invention may be translationally linked to another protein as part of a polynucleotide molecule such as RNA or DNA.
In another aspect the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antibody fragment comprising at least an antigen-binding portion, which specifically binds to a peptide according to the invention; and a pharmaceutically acceptable carrier or diluent.
According to some embodiments, a pharmaceutical composition comprising an antibody defined above, useful for preventing, attenuating or treating a malignancy is provided wherein the antibody recognizes a TCR CDR3 sequence and is specific for the T cells that down-regulate tumor-associated autoimmunity.
According to some embodiment, the pharmaceutical composition comprises a therapeutically effective amount of an antibody which comprises an antigen recognition domain which specifically binds a sequence selected from the group consisting of SEQ ID NOs: 2-4, 8, 15-17, 19, 24, 29 and 1923.
Each possibility represents a separate embodiment of the present invention.
In yet another aspect the present invention is related to a method of attenuating or treating a malignancy comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of an antibody which recognizes a TCR CDR3 sequence specific for the T cells that down-regulate tumor-associated autoimmunity. According to this aspect, the compositions and methods are used to up-regulate effector immunity or to deprive tumor of its ability to down-regulate T cell immune intervention.
According to some embodiments, the method comprises a combined treatment regimen of an antibody according to the invention and a peptide, analog, peptide conjugate, or fusion protein according to the invention. Such administration may be performed in a combined composition or in separate compositions administered together or at separate times.
According to some embodiments, the malignancy is a metastatic cancer.
According to other embodiments, the malignancy is a solid cancer.
According to yet another aspect, the present invention provides a method of preventing or treatment tumor metastasis comprising administering to a subject in need thereof a pharmaceutical composition comprising at least one peptide, peptide analog, peptide multimer, peptide conjugate, fusion protein, antibody, or antibody fragment disclosed above.
According to some embodiments the metastasis is decreased. According to other embodiments, the metastasis is prevented. According to yet other embodiments, the spread of tumors to the lungs of said subject is inhibited.
The pharmaceutical composition according to the present invention may be administered together with an anti-neoplastic composition. According to a specific embodiment, the anti-neoplastic composition comprises at least one chemotherapeutic agent. The chemotherapeutic agent, which could be administered separately or together with the antibody according to the present invention, may comprise any such agent known in the art exhibiting anti-cancer activity, including but not limited to: mitoxantrone, topoisomerase inhibitors, spindle poison vincas: vinblastine, vincristine, vinorelbine (taxol), paclitaxel, docetaxel; alkylating agents: mechlorethamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide; methotrexate; 6-mercaptopurine; 5-fluorouracil, cytarabine, gemcitabin; podophyllotoxins: etoposide, irinotecan, topotecan, dacarbazin; antibiotics: doxorubicin (adriamycin), bleomycin, mitomycin; nitrosoureas: carmustine (BCNU), lomustine, epirubicin, idarubicin, daunorubicin; inorganic ions: cisplatin, carboplatin; interferon, asparaginase; hormones: tamoxifen, leuprolide, flutamide, and megestrol acetate. According to a specific embodiment, the chemotherapeutic agent is selected from the group consisting of alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodopyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitor, interferons, platinum coordination complexes, anthracenedione substituted urea, methyl hydrazine derivatives, adrenocortical suppressant, adrenocorticosteroides, progestins, estrogens, antiestrogen, androgens, antiandrogen, and gonadotropin-releasing hormone analog. According to another embodiment, the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil (5-FU), leucovorin (LV), irenotecan, oxaliplatin, capecitabine, paclitaxel and doxetaxel. Two or more chemotherapeutic agents can be used in a cocktail to be administered in combination with administration of the antibody or fragment thereof.
According to a specific embodiment, the invention provides a method of treating cancer in a subject, comprising administering to the subject effective amounts of an antibody or antibody fragment according to the invention.
In another aspect, the present invention provides a method for increasing the duration of survival of a subject having cancer, comprising administering to the subject a composition comprising effective amounts of an antibody or antibody fragment defined above, and optionally an anti-neoplastic composition whereby the administration of the antibody effectively increases the duration of survival.
In yet another aspect, the present invention provides a method for increasing the progression free survival of a subject having cancer, comprising administering to the subject a composition comprising effective amounts of an antibody, or antibody fragment defined above, and optionally an anti-neoplastic composition, whereby administration of the antibody or antibody fragment effectively increases the duration of progression free survival.
Furthermore, the present invention provides a method for treating a subject having cancer, comprising administering to the subject effective amounts of an antibody or antibody fragment defined above, and optionally anti-neoplastic composition whereby administration of the antibody or antibody fragment effectively increases the response incidence in the group of subjects.
In yet another aspect, the present invention provides a method for increasing the duration of response of a subject having cancer, comprising administering to the subject a composition comprising effective amounts of an antibody or antibody fragment defined above, and optionally an anti-neoplastic composition, wherein said anti-neoplastic composition comprises at least one chemotherapeutic agent, whereby administration of the antibody or antibody fragment effectively increases the duration of response.
In another aspect, the invention provides a method of preventing or inhibiting development of metastasis in a patient having cancer, comprising administering to the subject a composition comprising effective amounts of an antibody or antibody fragment defined above and optionally an anti-neoplastic composition, whereby administration of the antibody or antibody fragment effectively increases the duration of response.
Another aspect of the present invention relates to the use of an antibody defined above or an antibody fragment thereof, for the manufacture of a therapeutic composition for the treatment of a cancer.
According to another aspect, the present invention provides a method of preventing tumor recurrence comprising administering to a subject in need thereof an antibody or antibody fragment defined above, in conjugation with surgery, radio- or chemotherapy.
A pharmaceutical composition according to the invention, comprising an antibody or fragment thereof may be administered to a subject in need thereof, by any administration route, including but not limited to: intramuscular, intravenous, oral, intraperitoneal, subcutaneous, topical, intradermal or transdermal delivery.
According to some embodiments, the composition is administered by a route selected from the group consisting of: subcutaneous injection (SC), intra-peritoneal (IP) injection, intra-muscular (IM) injection and intra-venous (IV) injection.
According to some embodiments, the compositions and treatments comprising antibody or antibody fragment are specific for the T cells that down-regulate tumor-associated autoimmunity and can unleash the otherwise suppressed effector immunity without affecting unrelated T cell responses.
According to yet another aspect, the invention provides method for up-regulating effector immunity against pathogens, comprising administering to a patient in need thereof a pharmaceutical composition comprising an antibody, or a fragment thereof, which recognizes a TCR CDR3 peptide.
According to some embodiments, the pathogen is selected from the group consisting of: human immunodeficiency virus or tuberculosis.
The present invention provides, according to another aspect a method of selecting a TCR CDR3 peptide relevant to human immunity, comprising the steps of:
According to some embodiments, the immune function is selected from the group consisting of autoimmunity, pathogenic immunity, tumor immunity and graft rejection. Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter.
The CDR30 segment of the T-cell receptor (TCR), which recognizes antigen epitopes, is formed by random recombination of V-D-J gene segments, junctional nucleotide deletions and non-templated nucleotide insertions, which renders unlikely any sharing of CDR3 sequences among individuals. Nevertheless, reports of shared TCR sequences are accumulating. To gain a repertoire-wide view of TCR sharing, CDR3 β-chain sequences were studied using high-throughput TCR-sequencing in CD4+ splenic T cells of 28 healthy C57BL/6 mice. A few hundreds of relatively public and public sequences shared by most mice were uncovered. These highly shared sequences differed from more private ones: they are two orders of magnitude more abundant on average, feature a restricted V/J segment usage, and exhibit much higher convergent recombination—tens of different nucleotide (nt) recombinations encode the same CDR3 amino-acid (aa) sequence. Public sequences were found to be enriched for previously defined, MHC-diverse CDR3 sequences that were functionally associated with autoimmune, allograft and tumor-related reactions, but less with anti-pathogen-related CDR3 sequences. Thus, public/private CDR3 discrimination marks functionally different T-cell response categories. These results suggest an ongoing positive selection of a restricted subset of self-associated, public T-cell clones and invite reexamination of the basic mechanisms of T-cell repertoire formation.
To investigate TCR publicness in a repertoire-wide manner, high-resolution maps of TCRβ repertoires of splenic CD4+ T cells in 28 individual C57BL/6 mice, were generated based on massive parallel sequencing (TCR-seq) of T-cell mRNA (W. Ndifon et al., Proc Natl Acad Sci USA 109, 15865, 2012). The mice included 12 untreated, 7 immunized with complete Freund's adjuvant (CFA) and 9 immunized with CFA+ovalbumin (OVA). About 2.4×106 TCRβ CDR3 nt sequence reads were obtained, which corresponded to about 3.5×105 unique (non-redundant) TCR aa sequences. A summary of the samples is presented in Table 1 (in the “Examples” section below). The analysis is focused mainly on the as sequences of the TCRβ complementarity determining region 3 (CDR3), which is the most diverse region of the TCR molecule and is associated with antigen epitope recognition. Due to the degeneracy of the genetic code, the same functional CDR3 aa sequence could result from different nt recombinations—a phenomenon termed convergent recombination (V. Venturi, et al., 2006, 2008 ibid).
It was unexpectedly found that on average, any two mice in the dataset share 10.5±1.8% of their expressed CDR3 as sequences. There was no significant difference in pairwise sharing between the naïve and immunized groups of mice; hence, all 28 mice were combined for further analysis. Unique CDR3 as sequences were next binned according to the number of mice in which they occurred (
The frequency of each CDR3 as sequence was next analyzed as a function of its degree of sharing. CDR3 sequence frequency reflects two factors: the number of T cells bearing that aa sequence (herein termed the CDR3-type) and the amount of relevant mRNA produced by a cell. Thus, the frequency of a sequence reflects the numbers and the activity state of the T cells that express the specific receptor sequence. A gradual increase in median frequency as a function of sharing was observed; CDR3 sequences with higher levels of sharing tended to be more abundant. Interestingly, very prominent CDR3 as sequences (relative frequency>5×10−4) appear both among private or relatively private sequences as well as among more public sequences; the frequency curve seems to dip for intermediate levels of sharing, suggesting the distinctness of the most highly public subset of sequences. Since increased frequencies of TCR sequences probably result from antigen-specific T-cell clonal expansion, it is likely that the most public CDR3-types, as well as a fraction of the private CDR3-types, reflect T-cell expansion following antigen-activation.
Previous studies reported that public TCRs manifest a higher level of convergent recombination (H. Li et al., J Immunol 189, 2404, 2012; M. F. Quigley et al., Proc Natl Acad Sci USA 107, 19414, 2010; V. Venturi et al., J Immunol 186, 4285, 2011). The analysis demonstrated in the present invention, of a large number of individuals revealed a continuous trend; increased sharing was associated with a gradual increase in the degree of convergent recombination (
The pattern of convergent recombination of nt sequences for 4 CDR3 as sequences were studied. The two more public sequences, found in 28 and 27 of the mice, show high convergent recombination (encoded by 105 and 53 nt sequences, respectively). There is no dominating nt sequence in any mouse, nor a dominant nt sequence across mice. In contrast, two relatively private CDR3 as sequences, present in 7 and 3 mice, manifest a limited number of nt sequences. Thus, private and public CDR3 segments differ markedly in their detectable degree of convergent recombination.
Further analysis of the public CDR3 sequences revealed other distinct characteristics.
The marked differences between public and private CDR3 sequences suggest that each class might be driven by different classes of antigens. Interestingly, a sequence (C9: CASSLGGNQDTQYF, SEQ ID NO: 1), which was previously found to be public in NOD mice that spontaneously develop autoimmune type 1 diabetes (Y. Tikochinski et al., Int Immunol 11, 951, 1999), is relatively public in the dataset of healthy C57BL/6 mice (shared by 27 mice). The C9 CDR3 sequence was found to recognize a peptide epitope (p277) in the mouse/human HSP60 molecule; administration of peptide p277 to NOD mice activates anti-C9 and other regulatory T cells (D. Elias, et al., Int Immunol 11, 957, 1999), and arrests the destruction of pancreatic beta cells both in NOD mice (D. Elias, I. R. Cohen, Lancet 343, 704, 1994) and in humans with recent-onset type 1 diabetes (I. Raz et al., Lancet 358, 1749, 2001). Despite the fact that the NOD and the C57BL/6 mouse strains differ in their MHC haplotypes (H2g7 and H2b, respectively), it was now found that the same CDR30 as sequence is public in both.
The literature was then searched for additional annotated TCRβ sequences in various models in different strains of mice bearing varying MHC haplotypes. 252 TCRβ sequences that were previously annotated to be associated with defined immune functions were collected from the literature, and compared with the CDR3 dataset. The annotated sequences were associated with four categories of immune reactions: a) Immunity to foreign pathogens; b) Allograft reactions; c) Tumor-related T cells; and d) Autoimmune conditions. Of the 252 annotated CDR3 sequences, 124 sequences were identified that were also present in one or more of our 28 healthy C57BL/6 mice (see Table 11 hereinbelow). The 124 annotated sequences associated functionally with autoimmunity, allograft rejection and cancer (self or modified self) were relatively enriched with shared, relatively public and public sequences compared with the sequences associated with anti-virus or anti-malarial immunity. This is evident from
As noted above, the annotated sequences were derived from various mouse strains that differed in their MHC haplotypes. To further explore the MHC restrictions of the public sequences, TCR-sequencing was used to map the repertoires of T cells interacting with different MHC molecules: C57BL/6 CD8+ T cells (which are restricted by MHC class-I H2b); C3H.SW CD4+ T cells (which have the H2b MHC allele, but different genetic background than C57BL/6); and C3H.HeSnJ CD4+ T cells (which are congenic with the C3H.SW strain but bear the H2k allele). These repertories were compared with those of the MHC-II H2b restricted CD4+ T cells of the C57BL/6 mice. It was found that >82% of the 289 public CDR3 as sequences were also present in the other T cell repertories. Interestingly, most of these public CDR3 sequences were associated with several different TCRβ V region gene segments. Moreover, the V gene segments associated with each shared CDR3 as sequence tended to differ between the different MHC-restricted T cell groups. A global analysis of the degree of similarity in V-segment usage of the public CDR3 sequences between C57BL/6 (H2b) CD4+ T cells and the other T cell groups shows that differences in MHC restriction are associated with more diverse V gene usage (
Without wishing to be bound to any theory or mechanism of action, it is suggested that the high level of convergent recombination of public sequences together with their greater abundance relative to the more private sequences could result mainly from two mechanisms: a) biases in the recombination process that favor the generation of certain sequences, which renders them more abundant and more public, and b) different degrees of positive selection by particular antigen epitopes, such that the more public CDR3 aa types would enjoy a selective advantage, particularly in the process of tonic stimulation needed to preserve TCR repertoires in the periphery (K. Hochweller et al., Proc Natl Acad Sci USA 107, 5931, 2010), which is where they were sampled. The two mechanisms can function together: recombination biases ensure the initial presence of certain public clones in different individuals, and selection that leads to clonal expansion differentially prevents their subsequent loss. The restricted pattern of V/J segment usage by public clones, the high level of convergent recombination and the finding of the same CDR3 as sequences among T cells interacting with different types of MHC molecules, are all in favor of positive selection as a dominant mechanism in the generation of public CDR3-types. According to this hypothesis, public CDR3-types are those stimulated to proliferate by frequent contact with high amounts of their cognate antigens. In contrast, private CDR3-types would be those that meet their cognate antigens only rarely or sporadically, and so would proliferate less often without accumulation of convergent recombined sequences over time. In other words, private and public CDR3-types might express the degree and dynamics of their contact with cognate antigens subsequent to genetic recombination in the thymus.
The finding of annotated CDR3 sequences (see Table 11 hereinbelow) in the dataset of healthy mice, presented herein for the first time, highlights a functional difference between the more private TCR sequences, which was found to be associated with all classes of antigens, and the more public sequences, which appear to be associated mainly with autoimmune conditions, allograft reactions and tumor infiltration (
The high frequency of public CDR3 TCR as sequences associated with autoimmunity is used, according to the present invention, as a source of therapeutic peptides against autoimmune disorders and graft rejection and neutralizing antibodies for cancer therapy; Modulating public CDR3-types identified herein might provide a new therapeutic approach to modulating autoimmune disease. TCR diversity has been an obstacle for treatments such as T-cell vaccination based on specific TCR sequences (I. R. Cohen, Vaccine 20, 706, 2001), which might be alleviated if public TCRs can be used as effective T-cell vaccines.
Based on the present teachings, the inventors were able to show that an antibody raised against the C9 relatively public peptide was able to unleash an autoimmune response in NOD mice, a model for type 1 Diabetes (Example 2 and
Currently, there are no specific drugs or clinically-used antibodies that target CDR3 peptides for cancer immunotherapy. This is mainly due to 2 reasons: a) The enormous size of the TCR repertoire, which precluded effective identification of potential targets; b) The fact that most CDR3 sequences are private or exist only in a small number of individuals, thus making them highly individualized targets that cannot be hit by simple reagents.
The discoveries represented herein for the first time open a possibility to overcome both limitations. First, using high-throughput methodologies, millions of CDR3 sequences were scanned. Using these new methodologies, CDR3 sequences that are highly shared among mice (also across MHC barriers) were identified; some of these are shared also in humans, and are related to sequences annotated in various cancer models. Thus, a set of specific candidate sequences that can serve as potential targets was identified. Second, the high level of sharing of these CDR3 sequences among individuals can provide highly specific targets that are still found in a large fraction of patients, indicating the universality of these novel therapeutic agents.
Thus, the present inventors have identified CDR3 sequences which are shared between mice strains and even by human and mice which may be used per se or as targets for immunotherapy using dedicated agents.
Thus, according to a specific embodiment, there is provided an isolated agent capable of at least one of:
According to a specific embodiment, the CDR3 sequence is selected from the group consisting of the sequences in Table 2.
According to a specific embodiment, the CDR3 sequence is selected from the group consisting of the sequences in Table 9.
According to a specific embodiment, the CDR3 sequence is selected from the group consisting of the sequences in Table 3.
According to another specific embodiment, the CDR3 sequence is selected from the group consisting of the sequences in Table 4.
According to another specific embodiment, the CDR3 sequence is selected from the group consisting of the sequences in Table 5.
According to yet another specific embodiment, the CDR3 sequence is selected from the group consisting of the sequences in Table 6.
According to another specific embodiment, the CDR3 sequence is selected from the group consisting of the sequences in Table 7.
Additional CDR3 sequences which can be used according to the present teachings can be identified according to a method comprising the steps of:
According to specific embodiments, the immune function is selected from the group consisting of: autoimmunity, pathogenic immunity, tumor immunity and graft rejection.
According to a specific embodiment, the CDR3 of the invention are selected from the group of private, public, relatively private and relatively public.
As used herein the term “private” refers to a CDR3 sequence present in 1 of the mammalian individuals tested in a dataset.
As used herein, the term “relatively private” refers to a CDR3 sequence present in at least 2 and not more than 25% of the mammalian individuals tested in the dataset.
As used herein, the term “relatively public” refers to a CDR3 sequence present in 75%-98% of the mammalian individuals tested in the dataset.
As used herein, the term “public” refers to a CDR3 sequence present in 98%-100% of the mammalian individuals tested in the dataset.
As used herein the term “T cell” refers to a differentiated lymphocyte with a CD3+, TCR+ having either CD4+ or CD8+ phenotype. The T cell may be either an effector or a regulatory T cells.
As used herein, the term “effector T cells” refers to a T cell that activates and direct other immune cells e.g. by producing cytokines or has a cytotoxic activity e.g., CD4+, Th1/Th2, CD8+ cytotoxic T lymphocyte.
As used herein, the term “regulatory T cell” or “Treg” refers to a T cell that negatively regulates the activation of other T cells, including effector T cells, as well as innate immune system cells. Treg cells are characterized by sustained suppression of effector T cell responses. According to a specific embodiment, the Treg is a CD4+CD25+Foxp3+ T cell.
As used herein the term “T cell receptor” or “TCR” refers to an antigen-recognition molecule present on the surface of T cells and may comprise the TCRα chain, the TCRβ chain, the TCRγ chain or the TCRδ chain.
According to a specific embodiment, TCR refers to the TCRβ chain.
As used herein, the terms “complementarity-determining region” or “CDR” are used interchangeably to refer to the hypervariable regions found within the variable region of an antibody or a TCR chain. Generally, each of a heavy chain of an antibody, a light chain of an antibody, a TCRα chain and TCRβ chain comprise three CDRs, CDR1, CDR2 and CDR3. Typically, CDR3 in TCR is the main CDR responsible for recognizing processed antigen.
The identity of the amino acid residues in a particular TCR that make up a variable region or a CDR can be determined using methods well known in the art and include methods such as sequence variability as defined by Kabat et al. (See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), location of the structural loop regions as defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877-883, 1989.), a compromise between Kabat and Chothia using Oxford Molecular's AbM antibody modeling software (now Accelrys®, see, Martin et al., 1989, Proc. Natl Acad Sci USA. 86:9268; and world wide web site www.bioinf-org.uk/abs), available complex crystal structures as defined by the contact definition (see MacCallum et al., J. Mol. Biol. 262:732-745, 1996), the “conformational definition” (see, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008) and IMGT [Lefranc M P, et al. (2003) IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol 27: 55-77]. According to a specific embodiment, the CDR3 region is defined as starting from the last conserved cysteine of Vβ and ending at the first position of the conserved amino acid motif [F|H][A|G]XG of Jβ, where X denotes any amino acid.
As used herein the term “agent” refers to a substance capable of at least one of:
The agent may be capable of at least one or two of the above-provided properties i.e.: (i); (ii); (iii); (iv), (i)+(iii); (ii)+(iii); (i)+(iv) and (ii)+(iv).
Thus, on-limiting examples of an agent include antibody, T cell, peptide and polynucleotide.
According to specific embodiments the agent is a peptide.
Thus, according to an aspect of the present invention there is provided an isolated peptide of no more than 20 amino acids comprising an amino acid sequence having a CDR3 sequence of a TCR on a T cell, said CDR3 sequence being selected from the group consisting of SEQ ID NOs of Table 7.
According to a specific embodiment, the peptide amino acid sequence is selected from the group consisting of SEQ ID NOs of Table 2.
According to a specific embodiment, the peptide amino acid sequence is selected from the group consisting of SEQ ID NOs of Table 9.
According to a specific embodiment, the peptide amino acid sequence is selected from the group consisting of SEQ ID NOs of Table 3.
According to a specific embodiment the peptide amino acid sequence is selected from the group consisting of SEQ ID NOs of Table 4.
According to a specific embodiment, the peptide amino acid sequence is selected from the group consisting of SEQ ID NOs of Table 5.
According to a specific embodiment, the peptide amino acid sequence is selected from the group consisting of SEQ ID NOs of Table 6.
According to a specific embodiment, the peptide amino acid sequence is selected from the group consisting of SEQ ID NOs of Table 7.
According to yet other embodiments, the peptide is selected from the group consisting of SEQ ID NOs: 5, 6, 10, 12, 20-23, 25, 27, 30-32, 34 and 1924.
According to an embodiment, the peptides are selected non-immunogenic in a subject.
The term “peptide” as used herein encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder. Although peptide mimetics, analogs and derivatives are contemplated, it is still very important to maintain the function of the peptides either in vivo (ex-vivo) or in-vitro such as for generating antibodies to TCR-CDR3.
Peptide bonds (—CO—NH—) within the peptide may be substituted, for example, by N-methylated amide bonds (—N(CH3)-CO—), ester bonds (—C(═O)—O—), ketomethylene bonds (—CO—CH2-), sulfinmylmethylene bonds (—S(═O)—CH2-), α-aza bonds (—NH—N(R)—CO—), wherein R is any alkyl (e.g., methyl), amine bonds (—CH2-NH—), sulfide bonds (—CH2-S—), ethylene bonds (—CH2-CH2-), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds (—CS—NH—), olefinic double bonds (—CH═CH—), fluorinated olefinic double bonds (—CF═CH—), retro amide bonds (—NH—CO—), peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” side chain, naturally present on the carbon atom.
These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) bonds at the same time.
Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted by non-natural aromatic amino acids such as 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine, ring-methylated derivatives of Phe, halogenated derivatives of Phe or O-methyl-Tyr.
The peptides of some embodiments of the invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
The term “amino acid” or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term “amino acid” includes both D- and L-amino acids.
Tables 12 and 13 below list naturally occurring amino acids (Table 12), and non-conventional or modified amino acids (e.g., synthetic, Table 13) which can be used with some embodiments of the invention.
The amino acids of the peptides of the present invention may be substituted either conservatively or non-conservatively.
The term “conservative substitution” as used herein, refers to the replacement of an amino acid present in the native sequence in the peptide with a naturally or non-naturally occurring amino or a peptidomimetics having similar steric properties. Where the side-chain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).
As naturally occurring amino acids are typically grouped according to their properties, conservative substitutions by naturally occurring amino acids can be easily determined bearing in mind the fact that in accordance with the invention replacement of charged amino acids by sterically similar non-charged amino acids are considered as conservative substitutions.
For producing conservative substitutions by non-naturally occurring amino acids it is also possible to use amino acid analogs (synthetic amino acids) well known in the art. A peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled practitioner.
When affecting conservative substitutions the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.
The following six groups each contain amino acids that are conservative substitutions for one another:
2) Aspartic acid (D), Glutamic acid (E);
“Derivatives” of the peptides of the invention as used herein covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the activity of the peptide, do not confer toxic properties on compositions containing it and do not adversely affect the antigenic properties thereof.
These derivatives may, for example, include aliphatic esters of the carboxyl groups, amides of the carboxyl groups produced by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed by reaction with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group (for example that of seryl or threonyl residues) formed by reaction with acyl moieties.
The term “analog” further indicates a molecule which has the amino acid sequence according to the invention except for one or more amino acid changes. Analogs according to the present invention may comprise also peptidomimetics. “Peptidomimetic” means that a peptide according to the invention is modified in such a way that it includes at least one non-coded residue or non-peptidic bond. Such modifications include, e.g., alkylation and more specific methylation of one or more residues, insertion of or replacement of natural amino acid by non-natural amino acids, replacement of an amide bond with other covalent bond. A peptidomimetic according to the present invention may optionally comprises at least one bond which is an amide-replacement bond such as urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond. The design of appropriate “analogs” may be computer assisted.
According to a specific embodiment, the peptide analogs are as said forth in SEQ ID NOs: 3, 5, 6, 8, 10, 12, 15-17, 19, 415, 1923 and 2018-2032.
“Salts” of the peptides of the invention contemplated by the invention are physiologically acceptable organic and inorganic salts.
The peptides of some embodiments of the invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.
According to specific embodiments the isolated peptide comprises a multimer. The multimer comprises at least two isolated peptide (e.g., 3 or 4), which may be identical or different peptides.
According to a specific embodiment the at least two isolated peptides are identical.
The term identical in this case refers to the chemical composition of the peptide per se.
According to another specific embodiment, the at least two isolated peptides are different. The term different as used in this case, refers to peptides having a different chemical composition. Thus the peptides may have different biological properties e.g., bind different targets or the same target in a different manner (e.g., difference in affinities).
According to specific embodiments, there is provided a fusion protein comprising at least one of the isolated peptide.
According to specific embodiment the agent is an isolated antibody.
According to specific embodiments the antibody comprises an antigen recognition domain capable of specifically binding an epitope on CDR3 of a TCR presented on a T cell. As used herein, the term “epitope” refers to any antigenic determinant on an antigen to which the paratope of an antibody or a TCR binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. According to a specific embodiment, the isolated antibody comprises an antigen recognition domain capable of specifically binding SEQ ID NO: 1 of a TCR presented on a T cell.
Antibodies, or immunoglobulins, comprise two heavy (H) chains linked together by disulfide bonds and two light (L) chains, each L chain being linked to a respective H chain by disulfide bonds in a “Y” shaped configuration. Proteolytic digestion of an antibody yields Fv (Fragment variable) and Fc (fragment crystalline) domains. The antigen binding domains, Fab's, include regions where the polypeptide sequence varies. The term F(ab′)2 represents two Fab′ arms linked together by disulfide bonds. The central axis of the antibody is termed the Fc fragment. Each H chain has at its N-terminal end a variable (V) domain (VH) followed by a number of constant (C) domains (CH). Each L chain has a V domain (VL) at one end and a C domain (CL) at its other end, the VL domain being aligned with the VH domain and the CL domain being aligned with the first CH domain (CH1). The V domains of each pair of L and H chains form the antigen-binding site. The domains on the L and H chains have the same general structure, and each domain comprises four framework regions (FRs), whose sequences are relatively conserved, joined by three hypervariable domains known as complementarity determining regions (CDR1-3). These domains contribute specificity and affinity of the antigen-binding site. The isotype of the H chain (gamma-γ, alpha-α, delta-δ, epsilon-ε or mu-μ) determines immunoglobulin class (IgG, IgA, IgD, IgE or IgM, respectively). There are several subtypes of IgG (IgG1, IgG2, IgG3, and IgG4). The L chain is either of two isotypes (kappa, κ or lambda, λ) found in all antibody classes.
The term “antibody” is used in the broadest sense and includes mAbs (including full-length or intact mAbs), polyclonal antibodies, multivalent antibodies, multi-specific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.
The antibody according to the present invention is a molecule comprising at least the antigen-binding portion of an antibody. Antibody or antibodies according to the invention include intact antibodies, such as polyclonal antibodies or mAbs, as well as proteolytic fragments thereof such as Fab or F(ab′)2 fragments. Further included within the scope of the invention are chimeric antibodies; human and humanized antibodies; recombinant and engineered antibodies, bi-specific antibodies, and fragments thereof. Furthermore, the DNA encoding the V region of the antibody can be inserted into the DNA encoding the C regions of other antibodies to produce chimeric antibodies. Single chain antibodies also fall within the scope of the present invention.
“Antibody fragments” comprise only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CH1 domains; (ii) the Fab′ fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 domain; (iii) the Fd fragment having VH and CH1 domains; (iv) the Fd′ fragment having VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., Nature 1989, 341, 544-546) which consists of a VH domain; (vii) isolated CDR regions; (viii) F(ab′)2 fragments, a bivalent fragment including two Fab′ fragments linked by a disulfide bridge at the hinge region; (ix) single chain antibody molecules (e.g. single chain Fv; scFv) (Bird et al., Science 1988, 242, 423-426; and Huston et al., PNAS (USA) 1988, 85, 5879-5883); (x) “diabodies” with two antigen binding sites, comprising a VH domain connected to a VL domain in the same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448); (xi) “linear antibodies” comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary L chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng., 1995, 8, 1057-1062; and U.S. Pat. No. 5,641,870).
Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab′-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab′)2 fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another approach, F(ab′)2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv).
Single-chain antibodies can be single-chain composite polypeptides having antigen binding capabilities and comprising amino acid sequences homologous or analogous to VH and VL, i.e., linked VH-VL or single-chain Fv (scFv).
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. MAbs are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method. mAbs may be obtained by methods known to those skilled in the art. For example, the mAbs to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 1975, 256, 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 1991, 352, 624-628 or Marks et al., J. Mol. Biol., 1991, 222:581-597, for example. The mAbs may be isolated from a library from human lymphocytes and selected according to their specificity.
The mAbs of the present invention may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof. A hybridoma producing a mAb may be cultivated in vitro or in vivo. High titers of mAbs can be obtained by production in recombinant mammalian cells that contain the nucleic acids encoding the H and L chains of the mAb under the control of a cell-specific promoter. Such recombinant expresser cells are cultivated in large volumes in bioreactors. mAbs of any isotype are purified from culture supernatants, using filtration and column chromatography methods well known to those of skill in the art.
The mAbs herein specifically include “chimeric” antibodies in which a portion of the H and/or L chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). In addition, CDR grafting may be performed to alter certain properties of the antibody molecule including affinity or specificity. A non-limiting example of CDR grafting is disclosed in U.S. Pat. No. 5,225,539.
Chimeric antibodies are molecules, the different portions of which are derived from different animal species, such as those having a variable region derived from a murine mAb and a human C region. Antibodies which have V region FR residues substantially from human antibody (termed an acceptor antibody) and CDRs substantially from a mouse antibody (termed a donor antibody) are also referred to as humanized antibodies. Chimeric antibodies are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher immunogenicity in humans (HAMA, which is human anti-mouse antibody response), such that human/murine chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (for example PCT patent applications WO 86/01533, WO 97/02671, WO 90/07861, WO 92/22653 and U.S. Pat. Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101 and 5,225,539).
“Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from CDRs of the recipient are replaced by residues from CDRs of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in either the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance, specificity, affinity and reduced immunogenicity. In general, the humanized antibody will comprise substantially all of at least one, and typically two, V domains, in which all or substantially all of the CDR loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin C region (Fc), typically that of a human immunoglobulin in order to provide for a full mAb and appropriate effector functions as desired. For further details, see Jones et al., Nature 1986, 321, 522-525; Riechmann et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol., 1992 2, 593-596.
A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or encoded by the human genome and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human CDR residues. Human antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et al. Nature Biotechnology 1996 14,309-314; Sheets et al. PNAS (USA), 1998, 95, 6157-6162); Hoogenboom and Winter, J. Mol. Biol., 1991, 227, 381; Marks et al., J. Mol. Biol., 1991, 222, 581). Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al, Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995). Alternatively, the human antibody may be prepared via immortalization of human B lymphocytes producing an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or may have been immunized in vitro) followed by screening with the antigen of interest for a specific antibody.
By the term “single-chain variable fragment (scFv)” is meant a fusion of the VH and VL regions, linked together with a short (usually serine, glycine) linker. Single-chain antibodies can be single chain composite polypeptides having antigen binding capabilities and comprising amino acid sequences homologous or analogous to VH and VL VL (linked VH-VL or single chain Fv (scFv)). Both VH and VL may copy natural mAb sequences or one or both of the chains may comprise a CDR-FR construct of the type described in U.S. Pat. No. 5,091,513, the entire contents of which are incorporated herein by reference. The separate polypeptides analogous to the VH and VL regions are held together by a polypeptide linker. Methods of production of such single chain antibodies, particularly where the DNA encoding the polypeptide structures of the VH and VL chains are known, may be accomplished in accordance with the methods described, for example, in U.S. Pat. Nos. 4,946,778, 5,091,513 and 5,096,815, the entire contents of each of which are incorporated herein by reference.
A “molecule having the antigen-binding portion of an antibody” as used herein is intended to include not only intact immunoglobulin molecules of any isotype and generated by any animal cell line or microorganism, but also the antigen-binding reactive fraction thereof, including, but not limited to, the Fab fragment, the Fab′ fragment, the F(ab′)2 fragment, the variable portion of the heavy and/or light chains thereof, Fab mini-antibodies (see WO 93/15210, U.S. patent application Ser. No. 08/256,790, WO 96/13583, U.S. patent application Ser. No. 08/817,788, WO 96/37621, U.S. patent application Ser. No. 08/999,554, the entire contents of which are incorporated herein by reference), dimeric bispecific mini-antibodies (see Muller et al., 1998) and chimeric or single-chain antibodies incorporating such reactive fraction, as well as any other type of molecule or cell in which such antibody reactive fraction has been physically inserted, such as a chimeric T-cell receptor or a T-cell having such a receptor, or molecules developed to deliver therapeutic moieties by means of a portion of the molecule containing such a reactive fraction. Such molecules may be provided by any known technique, including, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques.
Antibodies according to the invention can be obtained by administering a peptide, peptide analog, or cells expressing these, to an animal, preferably a nonhuman, using routine protocols. According to specific embodiment, the antibody of interest is obtained by a method comprising using the CDR3 peptides as described herein for producing or selecting an antibody specifically recognizing said peptide, thereby producing the antibody of interest. For preparation of Abs, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc. (1985). According to specific embodiments, the antibodies are obtained by immunization of an animal.
Besides the conventional method of raising antibodies in vivo, antibodies can be generated in vitro using antibody display platforms such as, but not limited to, phage display, ribosome and mRNA display and microbial cell display technologies. Such a production of recombinant antibodies is much faster compared to conventional antibody production and they can be generated against an enormous number of antigens. Furthermore, when using the conventional method, many antigens prove to be non-immunogenic or extremely toxic, and therefore cannot be used to generate antibodies in animals. Moreover, affinity maturation (i.e., increasing the affinity and specificity) of recombinant antibodies is very simple and relatively fast. Finally, large numbers of different antibodies against a specific antigen can be generated in one selection procedure. To generate recombinant mAbs one can use various methods all based on display libraries to generate a large pool of antibodies with different antigen recognition sites. Such a library can be made in several ways: One can generate a synthetic repertoire by cloning synthetic CDR regions in a pool of H chain germline genes and thus generating a large antibody repertoire, from which recombinant antibody fragments with various specificities can be selected. One can use the lymphocyte pool of humans as starting material for the construction of an antibody library. It is possible to construct naive repertoires of human IgM antibodies and thus create a human library of large diversity. This method has been widely used successfully to select a large number of antibodies against different antigens. Protocols for bacteriophage library construction and selection of recombinant antibodies are provided in the well-known reference text Current Protocols in Immunology, Colligan et al (Eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1.
Non-human antibodies may be humanized by any methods known in the art. In one method, the non-human CDRs are inserted into a human antibody or consensus antibody FR sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.
For example, U.S. Pat. No. 5,585,089 of Queen et al. discloses a humanized immunoglobulin and methods of preparing same, wherein the humanized immunoglobulin comprises CDRs from a donor immunoglobulin and VH and VL region FRs from human acceptor immunoglobulin H and L chains, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin FR outside the Kabat and Chothia CDRs, wherein the donor amino acids replace corresponding amino acids in the acceptor immunoglobulin H or L chain frameworks.
U.S. Pat. No. 5,225,539, of Winter, also discloses an altered antibody or antigen-binding fragment thereof and methods of preparing same, wherein a V domain of the antibody or antigen-binding fragment has the FRs of a first immunoglobulin H or L chain V domain and the CDRs of a second immunoglobulin VH or VL domain, wherein said second immunoglobulin VH or VL domain is different from said first immunoglobulin VH or VL domain in antigen binding specificity, antigen binding affinity, stability, species, class or subclass.
The above-described antibodies can be employed to isolate or to identify clones expressing the polypeptides to purify the polypeptides by, for example, affinity chromatography.
Both neutralizing and activating antibodies are encompassed by the present invention.
According to a specific embodiment, the antibody is a neutralizing antibody.
A “neutralizing antibody” as used herein refers to an antibody capable of preventing, reducing, inhibiting or interfering the activity or signaling through a TCR, as determined by in vivo or in vitro assays, as per the specification, thereby suppressing activity of the T cell it binds to.
According to another specific embodiment, the antibody is an activating antibody.
An “activating antibody” as used herein refers to an antibody capable of eliciting activity or signaling through a TCR, as determined by in vivo or in vitro assays, as per the specification, thereby activating the T cell it binds to.
According to specific embodiments, the proteinaceous agents e.g., peptide of the present invention may be attached to a proteinaceous moiety which is heterologous to the CDR3 sequence (the heterologous sequence is not contiguously found in nature along with the CD3 sequence). Such a moiety may be an immunoglobulin fragment such as an Fc which is known to increase the bioavailability of protein based agents (e.g., peptides).
According to specific embodiments, the agent of the present invention may be attached to a non-proteinaceous moiety. It will be appreciated that the attachment of heterologous moieties, proteinaceous or non-proteinaceous, is contemplated herein for any agent used according to the present teachings. The elaboration of such a modification with respect to peptides should not be interpreted as limiting.
According to a specific embodiment the non-proteinaceous or proteinaceous moiety is a non-toxic moiety.
The phrase “non-proteinaceous moiety” as used herein refers to a molecule not including peptide bonded amino acids that is attached to the above-described peptide. Exemplary non-proteinaceous moieties which may be used according to the present teachings include, but are not limited to, polyethylene glycol (PEG), Polyvinyl pyrrolidone (PVP), poly(styrene comaleic anhydride) (SMA), and divinyl ether and maleic anhydride copolymer (DIVEMA). According to a specific embodiment, the non-proteinaceous moiety comprises polyethylene glycol (PEG).
Such a molecule is highly stable (resistant to in-vivo proteolytic activity probably due to steric hindrance conferred by the non-proteinaceous moiety) and may be produced using common solid phase synthesis methods which are inexpensive and highly efficient, as further described hereinbelow. However, it will be appreciated that recombinant techniques may still be used, whereby the recombinant peptide product is subjected to in-vitro modification (e.g., PEGylation as further described hereinbelow).
Bioconjugation of the agent e.g., peptide amino acid sequence, with PEG (i.e., PEGylation) can be effected using PEG derivatives such as N-hydroxysuccinimide (NHS) esters of PEG carboxylic acids, monomethoxyPEG2-NHS, succinimidyl ester of carboxymethylated PEG (SCM-PEG), benzotriazole carbonate derivatives of PEG, glycidyl ethers of PEG, PEG p-nitrophenyl carbonates (PEG-NPC, such as methoxy PEG-NPC), PEG aldehydes, PEG-orthopyridyl-disulfide, carbonyldimidazol-activated PEGs, PEG-thiol, PEG-maleimide. Such PEG derivatives are commercially available at various molecular weights [See, e.g., Catalog, Polyethylene Glycol and Derivatives, 2000 (Shearwater Polymers, Inc., Huntsvlle, Ala.)]. If desired, many of the above derivatives are available in a monofunctional monomethoxyPEG (mPEG) form. In general, the PEG added to the peptide of the present invention should range from a molecular weight (MW) of several hundred Daltons to about 100 kDa (e.g., between 3-30 kDa). Larger MW PEG may be used, but may result in some loss of yield of PEGylated peptides. The purity of larger PEG molecules should be also watched, as it may be difficult to obtain larger MW PEG of purity as high as that obtainable for lower MW PEG. It is preferable to use PEG of at least 85% purity, and more preferably of at least 90% purity, 95% purity, or higher. PEGylation of molecules is further discussed in, e.g., Hermanson, Bioconjugate Techniques, Academic Press San Diego, Calif. (1996), at Chapter 15 and in Zalipsky et al., “Succinimidyl Carbonates of Polyethylene Glycol,” in Dunn and Ottenbrite, eds., Polymeric Drugs and Drug Delivery Systems, American Chemical Society, Washington, D.C. (1991).
Conveniently, PEG can be attached to a chosen position in the peptide by site-specific mutagenesis as long as the activity of the conjugate is retained. A target for PEGylation could be any Cysteine residue at the N-terminus or the C-terminus of the peptide sequence. Additionally or alternatively, other Cysteine residues can be added to the peptide amino acid sequence (e.g., at the N-terminus or the C-terminus) to thereby serve as a target for PEGylation. Computational analysis may be effected to select a preferred position for mutagenesis without compromising the activity.
Various conjugation chemistries of activated PEG such as PEG-maleimide, PEG-vinylsulfone (VS), PEG-acrylate (AC), PEG-orthopyridyl disulfide can be employed. Methods of preparing activated PEG molecules are known in the arts. For example, PEG-VS can be prepared under argon by reacting a dichloromethane (DCM) solution of the PEG-OH with NaH and then with di-vinylsulfone (molar ratios: OH 1:NaH 5:divinyl sulfone 50, at 0.2 gram PEG/mL DCM). PEG-AC is made under argon by reacting a DCM solution of the PEG-OH with acryloyl chloride and triethylamine (molar ratios: OH 1: acryloyl chloride 1.5: triethylamine 2, at 0.2 gram PEG/mL DCM). Such chemical groups can be attached to linearized, 2-arm, 4-arm, or 8-arm PEG molecules.
Resultant conjugated molecules (e.g., PEGylated or PVP-conjugated peptide) are separated, purified and qualified using e.g., high-performance liquid chromatography (HPLC) as well as biological assays.
The agents e.g., peptides of some embodiments of the invention may be synthesized by any techniques that are known to those skilled in the art of peptide synthesis. For solid phase peptide synthesis, a summary of the many techniques may be found in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, W. H. Freeman Co. (San Francisco), 1963 and J. Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (New York), 1973. For classical solution synthesis see G. Schroder and K. Lupke, The Peptides, vol. 1, Academic Press (New York), 1965.
In general, these methods comprise the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then either be attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions suitable for forming the amide linkage. The protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support) are removed sequentially or concurrently, to afford the final peptide compound. By simple modification of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under conditions which do not racemize chiral centers) a protected tripeptide with a properly protected dipeptide to form, after deprotection, a pentapeptide and so forth. Further description of peptide synthesis is disclosed in U.S. Pat. No. 6,472,505.
A preferred method of preparing the peptide compounds of some embodiments of the invention involves solid phase peptide synthesis.
Large scale peptide synthesis is described by Andersson Biopolymers 2000; 55(3):227-50.
Any of the proteinaceous agents described herein can be encoded from a polynucleotide. These polynucleotides can be used as therapeutics per se or in the recombinant production of the agent.
Thus, according to specific embodiments there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding the agent, the peptide, or the antibody of the present invention.
The phrase “an isolated polynucleotide” refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
The isolated polynucleotide may be ligated into an expression construct which can be used as a shuttle vector (for the mere replication of the polynucleotide) or an expression vector whereby the isolated polynucleotide is typically ligated in a cis-acting manner to a cis acting element such as a promoter.
Such an expression vectors can be typically classified as viral vectors and bacterial vectors. The term “viral vector” or “bacterial vector” refers to a virus or bacteria, respectively, which can be administered to a human host without causing any disease or pathology and which encodes a protein or peptide or epitope not present in the native virus of bacteria. Such viral and bacterial vectors can be readily produced by recombinant methods well known in the art. Non-limiting examples include poxviruses, adenoviruses, alphaviruses, lentiviruses, Listeria monocytogenes, Salmonella typhi, Fibrio cholerae, Shigella sonnei, Mycobacterium bovis, and Bacillus anthracis.
The term “nucleic acid” in the context of vaccine refers to the injection of DNA to the host, whereby DNA is taken up by cells, transcribed and translated to protein or peptide that is presented to the immune system and thus elicit antibody- and cell-based immune responses specific to the peptide of interest. Non-limiting examples of such nucleic acid vaccines are purified nucleic acid administered alone, DNA-liposome complexes, DNA-coated polymers, and metal-coated DNA.
The agents, the peptides, the antibodies and polynucleotides of the present invention can be used to treat a disease associated with a T cell expressing a specific CDR3-TCR.
Thus, according to specific embodiments, (i) the isolated agent; (ii) the isolated peptide; or (iii) an isolated peptide of no more than 20 amino acids comprising an amino acid sequence having a CDR3 sequence of a TCR on a T cell, said CDR3 sequence being selected from the group consisting of SEQ ID NOs of Table 8, is used in the manufacture of a medicament identified for treating a disease associated with the T cell.
According to specific embodiments, there is provided a method of treating a disease associated with the T cell, the method comprising administering to a subject in need thereof an effective amount of: (i) the isolated agent; (ii) the isolated peptide; or (iii) an isolated peptide of no more than 20 amino acids comprising an amino acid sequence having a CDR3 sequence of a TCR on a T cell, said CDR3 sequence being selected from the group consisting of SEQ ID NOs of Table 8, thereby treating the disease associated with the T cell.
According to other specific embodiments, the isolated antibody is used in the manufacture of a medicament identified for treating a disease associated with said T cell.
According to specific embodiments, there is provided a method of treating a disease associated with a T cell expressing a TCR-CDR3 segment comprising an amino acid sequence of SEQ ID NO: 1 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the isolated antibody of claim 7, thereby treating the disease associated with a T cell expressing said TCR-CDR3 segment comprising an amino acid sequence of SEQ ID NO: 1 in the subject.
As used herein, the term “disease associated with a T cell” refers to a pathological condition which onset or progression is associated with under activity or over activity of T cells expressing a specific CDR3. The T cell may be an effector T cell or a regulatory T cell.
According to specific embodiments, wherein the disease is associated with activity of the T cell then the immune response induced by the agent is dowregulation of the activity.
According to other specific embodiments, wherein the disease is associated with suppression of activity of the T cell then the immune response induced by the agent is upregulation of the activity.
This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include cancer e.g. benign and malignant tumors; leukemias and lymphoid malignancies; autoimmune diseases, graft rejection disease (e.g. graft vs. host disease), neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic, immunologic disorders or hyperpermeability states.
Typically, activating the public clonotype is good for autoimmune disease therapy, and inactivating the public clonotype is likely to be good for tumor immunotherapy. The experimental results demonstrated herein for the first time demonstrate that it should be possible to unleash a tumor-specific “autoimmune” response by administering antibodies to the public CDR3 TCR peptides of tumor-infiltrating T cells.
As used herein, the term “subject” includes mammals, preferably human beings at any age which suffer from or is at risk of the disease.
“Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
Immunotherapy of tumor growth was approached by the use of anti PD1 antibodies demonstrating immune regulatory mechanism to attack tumor cells. To overcome some lack of specificity, use of T cells expressing TCR molecules which share common CDR3 could induce an autoimmune reaction specific to the tumor cells.
Thus, according to specific embodiments, the disease is cancer.
The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, lung cancer (including small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome. Preferably, the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. The cancerous conditions amenable for treatment of the invention include metastatic cancers.
According to specific embodiments, the cancer is lung cancer.
According to a specific embodiment, the cancer is lung carcinoma.
According to specific embodiments, the disease is an autoimmune disease. Specific examples of autoimmune diseases which may be treated according to the teachings of the present invention include, but are not limited to, rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 July; 15 (3):791), spondylitis, ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3): 189), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998; 17 (1-2):49), sclerosis, systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol. 1999 March; 6 (2):156); Chan O T. et al., Immunol Rev 1999 June; 169:107), glandular diseases, glandular autoimmune diseases, pancreatic autoimmune diseases, diabetes, Type I diabetes (Zimmet P. Diabetes Res Clin Pract 1996 October; 34 Suppl:S125), thyroid diseases, autoimmune thyroid diseases, Graves' disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 June; 29 (2):339), thyroiditis, spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec. 15; 165 (12):7262), Hashimoto's thyroiditis (Toyoda N. et al., Nippon Rinsho 1999 August; 57 (8):1810), myxedema, idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 August; 57 (8):1759); autoimmune reproductive diseases, ovarian diseases, ovarian autoimmunity (Garza K M. et al., J Reprod Immunol 1998 February; 37 (2):87), autoimmune anti-sperm infertility (Diekman A B. et al., Am J Reprod Immunol. 2000 March; 43 (3):134), repeated fetal loss (Tincani A. et al., Lupus 1998; 7 Suppl 2:S107-9), neurodegenerative diseases, neurological diseases, neurological autoimmune diseases, multiple sclerosis (Cross A H. et al., J Neuroimmunol 2001 Jan. 1; 112 (1-2):1), Alzheimer's disease (Oron L. et al., J Neural Transm Suppl. 1997; 49:77), myasthenia gravis (Infante A J. And Kraig E, Int Rev Immunol 1999; 18 (1-2):83), motor neuropathies (Kornberg A J. J Clin Neurosci. 2000 May; 7 (3):191), Guillain-Barre syndrome, neuropathies and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 April; 319 (4):234), myasthenic diseases, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 April; 319 (4):204), paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy, non-paraneoplastic stiff man syndrome, cerebellar atrophies, progressive cerebellar atrophies, encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourette syndrome, polyendocrinopathies, autoimmune polyendocrinopathies (Antoine J C. and Honnorat J. Rev Neurol (Paris) 2000 January; 156 (1):23); neuropathies, dysimmune neuropathies (Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl 1999; 50:419); neuromyotonia, acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13; 841:482), Chronic obstructive pulmonary disease (COPD), cardiovascular diseases, cardiovascular autoimmune diseases, atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998; 7 Suppl 2:S132), thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2:S107-9), granulomatosis, Wegener's granulomatosis, arteritis, Takayasu's arteritis and Kawasaki syndrome (Praprotnik S. et al., Wien Klin Wochenschr 2000 Aug. 25; 112 (15-16):660); anti-factor VIII autoimmune disease (Lacroix-Desmazes S. et al., Semin Thromb Hemost. 2000; 26 (2):157); vasculitises, necrotizing small vessel vasculitises, microscopic polyangiitis, Churg and Strauss syndrome, glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis (Noel L H. Ann Med Interne (Paris). 2000 May; 151 (3):178); antiphospholipid syndrome (Flamholz R. et al., J Clin Apheresis 1999; 14 (4):171); heart failure, agonist-like beta-adrenoceptor antibodies in heart failure (Wallukat G. et al., Am J Cardiol. 1999 Jun. 17; 83 (12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 April-June; 14 (2):114); hemolytic anemia, autoimmune hemolytic anemia (Efremov D G. et al., Leuk Lymphoma 1998 January; 28 (3-4):285), gastrointestinal diseases, autoimmune diseases of the gastrointestinal tract, intestinal diseases, chronic inflammatory intestinal disease (Garcia Herola A. et al., Gastroenterol Hepatol. 2000 January; 23 (1):16), celiac disease (Landau Y E. and Shoenfeld Y. Harefuah 2000 Jan. 16; 138 (2):122), Crohn's disease, ulcerative colitis, psoriasis autoimmune diseases of the musculature, myositis, autoimmune myositis, Sjogren's syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 September; 123 (1):92); smooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother 1999 June; 53 (5-6):234), hepatic diseases, hepatic autoimmune diseases, autoimmune hepatitis (Manns M P. J Hepatol 2000 August; 33 (2):326) and primary biliary cirrhosis (Strassburg C P. et al., Eur J Gastroenterol Hepatol. 1999 June; 11 (6):595).
According to specific embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, type-1 diabetes, Chronic obstructive pulmonary disease (COPD), Crohn's disease, ulcerative colitis, and psoriasis.
According to other specific embodiments, the disease is a transplantation related disease i.e. graft rejection disease.
Specific examples of transplantation-related diseases which may be treated according to the teachings of the present invention include but are not limited to host vs. graft disease, chronic graft rejection, subacute graft rejection, hyperacute graft rejection, acute graft rejection, allograft rejection, xenograft rejection and graft-versus-host disease (GVHD).
According to specific embodiments the graft rejection disease is host vs. graft disease.
According to other specific embodiments, the disease is pathogenic disease. Specific examples of intracellular pathogens infections which may be treated according to the teachings of the present invention include, but are not limited to, infections by viral pathogens, intracellular mycobacterial pathogens (such as, for example, Mycobacterium tuberculosis), intracellular bacterial pathogens (such as, for example, Listeria monocytogenes), or intracellular protozoan pathogens (such as, for example, Leishmania and Trypanosoma).
Specific types of viral pathogens causing infectious diseases treatable according to the teachings of the present invention include, but are not limited to, retroviruses, circoviruses, parvoviruses, papovaviruses, adenoviruses, herpesviruses, iridoviruses, poxviruses, hepadnaviruses, picornaviruses, caliciviruses, togaviruses, flaviviruses, reoviruses, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, coronaviruses, arenaviruses, and filoviruses.
Specific examples of viral infections which may be treated according to the teachings of the present invention include, but are not limited to, human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS), influenza, rhinoviral infection, viral meningitis, Epstein-Barr virus (EBV) infection, hepatitis A, B or C virus infection, measles, papilloma virus infection/warts, cytomegalovirus (CMV) infection, Herpes simplex virus infection, yellow fever, Ebola virus infection, rabies, etc.
According to specific embodiments, the pathogenic disease is human immunodeficiency virus or tuberculosis infection.
The isolated agents, peptides antibodies and polynucleotides of the present invention can be used to treat a disease or a condition associated with a pathological T cell alone or in combination with other established or experimental therapeutic regimen for such disorders. Thus for example, antibodies can be used in combination with an anti-neoplastic composition. The term “anti-neoplastic composition” refers to a composition useful in treating cancer comprising at least one active therapeutic agent capable of inhibiting or preventing tumor growth or function or metastasis, and/or causing destruction of tumor cells. Therapeutic agents suitable in an anti-neoplastic composition for treating cancer include, but not limited to, chemotherapeutic agents, radioactive isotopes, toxins, cytokines such as interferons, and antagonistic agents targeting cytokines, cytokine receptors or antigens associated with tumor cells. For example, therapeutic agents useful in the present invention can be antibodies such as anti-HER2 antibody and anti-CD20 antibody, or small molecule tyrosine kinase inhibitors such as VEGF receptor inhibitors and EGF receptor inhibitors. Preferably the therapeutic agent is a chemotherapeutic agent.
A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew, Chem Intl. Ed. Engl. 33:183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′, 2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOLR® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISORR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf and H-Ras; ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME® ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapy DNA-based vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN®rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
Qualification of the agents, peptides, antibodies and polynucleotides of the resent invention for the treatment of a specific disease may be effected by testing them in a typical animal model.
Typically shared, relatively public or public CDR3 sequences, that have been annotated to be associated with a specific disease or are suspected to be involved in a specific disease are selected after confirming that they are also relatively public or public in human TCR repertoires. These peptides are synthesized using methods known in the art, typically automated solid-phase synthesis, purified and used to immunize rabbits in order to obtain high-titer antibodies.
Thus, for example, public CDR3 peptides reactive with known tumor-associated antigens such as MDM2 and HSP60 or expressed by tumor-infiltrating T cells and shared by mice and humans, can be synthesized based on the high-throughput screening results and the dataset of public annotated CDR3 sequences.
The respective agent is chosen e.g. isolated peptide, antibody, T cell or polynucleotide and produced by any method known in the art as further disclosed hereinabove. In the next step, the effect of administering the agent to an animal model is evaluated. Non-limiting examples of animal models that can be used include syngeneic tumor models such as the B16 melanoma in both local, subcutaneous growth and dispersed lung seeding, the 3LL tumor in local growth with spontaneous metastasis to the lung and in intravenous dispersal to the lungs and the GL261 Glioblastoma.
Thus, for example, the effect of administering CDR3 antibodies or control antibodies to mice bearing tumors is studied aiming to achieve tumor rejection or inhibition of tumor growth. The mice are followed and evaluated for tumor growth and tumor spread. In addition, the anti-tumor T cell and B cell immune responses responsible for tumor rejection are characterized by determining cytotoxic T cell reactions and serum antibody responses to the tumor cells in vitro; and adoptive transfer of T cells from antibody-treated mice to naïve mice, which is then challenged with the tumors. Specific sets of monoclonal and humanized antibodies can then be developed for unleashing controlled anti-tumor “autoimmunity” as an effective tumor immunotherapy. The optimal doses and dose schedules of the antibodies are then determined for anti-CDR3 antibodies showing positive results in the tumor models.
The agent, the isolated peptide, antibody or polynucleotide can be administered to the subject per se, or in a pharmaceutical composition where each or both are mixed with suitable carriers or excipients.
According to specific embodiments, the pharmaceutical composition further comprises an adjuvant or a delivery system.
As used herein a “pharmaceutical composition” refers to a preparation of the active ingredient described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
Pharmaceutical compositions comprising peptides and analog or antibodies or fragments thereof, are disclosed in the present invention, together with novel formulations, for use in prevention, suppression or treatment of a disease associated with a T cell expressing a specific CDR3-TCR, as disclosed hereinabove.
Herein the term “active ingredient” refers to the isolated agent, peptide, antibody and/or the polynucleotide accountable for the biological effect.
Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.
Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.
Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
Apart from other considerations, the fact that some of the novel active ingredients of the invention are peptides, peptide analogs or peptidomimetics, dictates that the formulation be suitable for delivery of these type of compounds. In general, peptides are less suitable for oral administration due to susceptibility to digestion by gastric acids or intestinal enzymes, but it is now disclosed that the compositions according to the present invention are also suitable for oral administration. Other routes of administration according to the present invention are intra-articular, intravenous, intramuscular, subcutaneous, intradermal, or intrathecal.
Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing or liposome capturing processes.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants for example polyethylene glycol are generally known in the art.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the variants for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the peptide and a suitable powder base such as lactose or starch.
Pharmaceutical compositions for parenteral administration include aqueous solutions of the active ingredients in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable natural or synthetic carriers are well known in the art (Pillai et al., Curr. Opin. Chem. Biol. 5, 447, 2001). Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds, to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
The compounds of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. As used herein, the phrase “therapeutically effective amount” refers to an amount of active ingredient effective to prevent, delay, alleviate or ameliorate symptoms of a disease of the subject being treated or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
In the case of cancer, the therapeutically effective amount may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) local cancer cell growth, inhibit cancer cell infiltration into peripheral organs; inhibit tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), the response rates (RR), duration of response, and/or quality of life.
For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in-vitro, in cell cultures or experimental animals, e.g., by determining the IC50 (the concentration which provides 50% inhibition) and the LD50 (lethal dose causing death in 50% of the tested animals) for a subject compound. The data obtained from these in-vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (e.g. Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1).
Dosage amount and interval may be adjusted individually to provide levels of the active ingredient that are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
Depending on the severity and responsiveness of the condition to be treated, dosing can also be a single administration of a slow release composition, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and all other relevant factors.
In one particularly preferred embodiment according to the present invention, the active ingredients are administered orally (e.g. as a syrup, capsule, or tablet).
In certain embodiments, delivery of the active ingredient can be enhanced by the use of protective excipients. This is typically accomplished either by complexing the active ingredient with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the active ingredient in an appropriately resistant carrier such as a liposome. Attempts of protecting e.g. polypeptides for oral delivery have been published (e.g., U.S. Pat. Nos. 8,093,207, 7,666,446 and 7,316,819).
Elevated serum half-life can be maintained by the use of sustained-release protein “packaging” systems. Such sustained release systems are well known to those of skill in the art. In one preferred embodiment, the ProLease biodegradable microsphere delivery system for proteins and peptides (Tracy, 1998, Biotechnol. Prog. 14, 108; Johnson et al., 1996, Nature Med. 2, 795; Herbert et al., 1998, Pharmaceut. Res. 15, 357) a dry powder composed of biodegradable polymeric microspheres containing the protein in a polymer matrix that can be compounded as a dry formulation with or without other agents.
The foregoing formulations and administration methods are intended to be illustrative and not limiting. It will be appreciated that, using the teaching provided herein, other suitable formulations and modes of administration can be readily devised.
Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets, lozenges comprising the peptide(s) in a flavoured base, usually sucrose and acacia and tragacanth; pastilles comprising the active ingredient(s) in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouth washes comprising the active ingredient(s) in a suitable liquid carrier. Each formulation generally contains a predetermined amount of the active peptide(s); as a powder or granules; or a solution or suspension in an aqueous or non-aqueous liquid such as a syrup, an elixir, an emulsion or draught and the like.
A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active peptide(s) in a free-flowing form such as a powder or granules, optionally mixed with a binder, (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered peptide(s) moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile.
A syrup may be made by adding the active ingredient(s) to a concentrated, aqueous solution of a sugar, for example, sucrose, to which may also be added any necessary ingredients. Such accessory ingredients) may include flavourings, an agent to retard crystallisation of the sugar or an agent to increase the solubility of any other ingredients, such as a polyhydric alcohol, for example, glycerol or sorbitol.
In addition to the aforementioned ingredients, the formulations of this invention may further include one or more accessory ingredient(s) selected from diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives, (including antioxidants) and the like.
Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
The following examples are intended to illustrate how to make and use the compounds and methods of this invention and are in no way to be construed as a limitation. Although the invention will now be described in conjunction with specific embodiments thereof, it is evident that many modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such modifications and variations that fall within the spirit and broad scope of the amended claims.
Libraries were prepared and pre-processed as published (Ndifon et al. 2012, Proc Natl Acad Sci USA 109, 39, 15865-15870). Briefly, total RNA was extracted from T cells using RNeasy Mini Kit (Qiagen, Hilden, Germany). The RNA was then reverse transcribed using SuperScript II reverse transcriptase (RT) enzyme (Invitrogen, La Jolla, Calif.). The primer for the RT reaction was a TCR Cβ-specific primer linked to the 3′-end Illumina sequencing adapter. The resulting cDNA was then amplified using PCR (Phusion; Finnzymes) with a Cβ-3′adp primer and Vβ-specific 5′ primers. Each Vβ-specific primer was anchored to a restriction site sequence for the ACUI restriction enzyme. PCR products were then cleaned using QIAquick PCR purification kit (Qiagen, Hilden, Germany), followed by enzymatic digestion with ACUI enzyme (New England BioLabs, Ipswich, Mass.). Then, the 5′Illumina adaptor (dsDNA, with NN overhang) were ligated (T4 ligase; Fermentas, Vilnius, Lithuania). The adaptors also contained 3-nucleotide long tags for multiplexing of samples to the same Illumina sequencing run. A second round of PCR amplification was performed, using universal primers for the 5′ and 3′ Illumina adapters. Final PCR products were run on a 2% agarose gel, cut at the desired length (˜250 bp), and purified using Wizard SV Gel and PCR Clean-Up System (Promega, Madison, Wis.) to produce the final library. The libraries were sequenced using Genome Analyzer II or HiSeq2000 (Illumina).
Polyclonal antibodies were raised in rabbits (by EZBiolab Inc., USA) against the C9 peptide (SEQ ID NO: 1). Protein A/G agarose beads were used for antibody purification. Serum of un-immunized rabbits was used as a control (pre-immune serum, also after purification).
20 C57BL/6 mice were injected intravenous (iv) with 5×105 cells of a syngeneic mouse Lewis lung carcinoma cell line (D-122). Mice were divided into 2 groups, 10 animals in each group. On the following day, the mice of group 1 were injected inter-peritonealy (ip) with 100 μg of control pre-immune serum and the mice of group 2 were injected ip with 100 μg of polyclonal anti-C9-CDR3 antibody. 10 days later mice were boosted with another 100 μg of the control serum or the polyclonal anti-C9-CDR3 antibody, respectively. One month post tumor injection all mice were sacrificed and tumor nodules in lungs were counted.
Raw reads containing bases with Q-value≦30 were filtered out, and then the remaining reads were separated according to their barcodes. Next, the reads were aligned to each of the germline Vβ/Jβ gene segments from IMGT (Lefiranc et al. 2009, Nucleic Acids Res 37 (Database issue): D1006-1012) using the Smith-Waterman algorithm. Each read was assigned its best-aligning Vβ/Jβ if the number of matching nucleotides (alignment length) was above a threshold, 11 nt for Vβ, 9 nt for Jβ. To reduce the effect of sequencing errors, hierarchical clustering to group reads assigned the same Vβ and Jβ genes and are with an edit distance less than 2 were used. Then, the sequences were annotated by matching the D3 to the junction, identifying deleted/inserted nucleotides and elongated the read to its full CDR30 length (by IMGT convention). Finally, the nt sequences were translated into amino-acid (aa) CDR30. Only sequences that are in-frame (i.e. no stop codons), have a copy number of at least 2 and have less than 2 bp enzyme cleavage error, were used. These are referred to as annotated reads (Table 1). The copy-number was also corrected, to adjust for PCR and sub-sampling bias, as published in Ndifon 2012 ibid.
Most of the CDR3 aa sequences were found in only one mouse (˜69% of all sequences). However, hundreds of sequences were highly shared among individual mice; 1,908 sequences were shared by more than 75% (n>21) of the mice (Table 10). Notably, 289 CDR3 aa sequences were found that were shared by all 28 mice (˜0.08% of all sequences) (Table 10).
In the next step, the literature was searched for additional annotated TCRβ sequences in various models in different strains of mice bearing varying MHC haplotypes. 252 TCRβ sequences that were previously annotated to be associated with defined immune functions were collected from the literature, and compared with the CDR3 dataset. The annotated sequences were associated with four categories of immune reactions: a) Immunity to foreign pathogens; b) Allograft reactions; c) Tumor-related T cells; and d) Autoimmune conditions. Of the 252 annotated CDR3 sequences, 124 sequences were identified that were also present in one or more of the 28 healthy C57BL/6 mice (see Table 11).
The finding of annotated sequences in the dataset of healthy mice, presented herein for the first time (Table 11), highlights a functional difference between the more private TCR sequences, which was found to be associated with all classes of antigens, and the more public sequences, which appear to be associated mainly with autoimmune conditions, allograft reactions and tumor infiltration (
Schistosoma
mansoni
S. mansoni
Trypanosoma cruzi
T. cruzi antigen.
Histoplasma
capsulatum
H. capsulatum
P. berghei
P. berghei
Without being bound by theory, since healthy C57BL/6 mice harbored public CDR3 TCR clonotypes associated with self-reactivity and tumor reactivity, it was thought that these sets of T cells probably served as regulatory T cells, rather than directly as effector T cells. In the case of an autoimmune disease such as Type 1 Diabetes, the public C9 CDR3 clonotype functioned to prevent or down-regulate the disease—this has been shown to be case both in NOD mice and humans with Type 1 Diabetes: a DiaPep277 peptide works in both NOD mice and in human phase 3 clinical trials to arrest beta-cell destruction by activating C9 regulatory T cells (Schloot N C, Cohen I R., Clin Immunol. 149(3):307-16, 2013). Consequently, targeting the C9 CDR3 T cell set by a specific antibody should unleash more severe autoimmune diabetes. To this end, NOD mice were injected with antibodies against the CDR3 peptide of a relatively public TCR clone from the dataset, which was previously associated with type 1 diabetes. The graphs of
Tumor immunotherapy and autoimmune disease immunotherapy are two sides of the same coin: in autoimmune disease it is desired to activate natural regulatory T clonotypes by activating a disease-associated public CDR3 clonotype using its specific antigen. In tumor immunotherapy, in contrast, it is desired to inactivate the natural regulatory, public CDR3 clonotypes mobilized by the tumor for its own protection.
Consequently, targeting the C9 CDR3 T cell set by a specific antibody may inhibit the growth of tumors. To this end, C57BL/6 mice were injected with syngeneic mouse Lewis lung carcinoma cell line and treated with antibodies against the C9-CDR3 peptide or with un-immunized serum control. As clearly shown in
While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, rather the scope, spirit and concept of the invention will be more readily understood by reference to the claims which follow.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL15/50329 | 3/26/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61970933 | Mar 2014 | US |
