PERSONALISED IMMUNOGENIC PEPTIDE IDENTIFICATION PLATFORM
The disclosure relates to methods of identifying fragments of a polypeptide that are immunogenic for a specific human subject, methods of preparing personalised pharmaceutical compositions comprising such polypeptide fragments, human subject-specific pharmaceutical compositions comprising such polypeptide fragments, and methods of treatment using such compositions. The methods comprise identifying a fragment of the polypeptide that binds to multiple HLA of the subject.
This application claims the benefit of priority to European Application No. 17159242.1, filed on Mar. 3, 2017, European Application No. 17159243.9, filed on Mar. 3, 2017, and Great Britain Application No. 1703809.2, filed on Mar. 9, 2017, each of which is incorporated herein by reference in their entireties.
SEQUENCE LISTINGThe instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created May 30, 2018, is named “52895703201_SL.txt” and is 25,675 bytes in size.
FIELDThe disclosure relates to methods of predicting whether a polypeptide is immunogenic for a specific human subject, methods of identifying fragments of a polypeptide that are immunogenic for a specific human subject, methods of preparing personalised or precision pharmaceutical compositions or kits comprising such polypeptide fragments, human subject-specific pharmaceutical compositions comprising such polypeptide fragments, and methods of treatment using such compositions.
BACKGROUNDFor decades, scientists have assumed that chronic diseases were beyond the reach of a person's natural defences. Recently, however, significant tumor regressions observed in individuals treated with antibodies that block immune inhibitory molecules have accelerated the field of cancer immunotherapy. These clinical findings demonstrate that re-activation of existing T cell responses results in meaningful clinical benefit for individuals. These advances have renewed enthusiasm for developing cancer vaccines that induce tumor specific T cell responses.
Despite the promise, current immunotherapy is effective only in a fraction of individuals. In addition, most cancer vaccine trials have failed to demonstrate statistically significant efficacy because of a low rate of tumor regression and antitumor T cell responses in individuals. Similar failures were reported with therapeutic and preventive vaccines that sought to include T cell responses in the fields of HIV and allergy. There is a need to overcome the clinical failures of immunotherapies and vaccines.
SUMMARYIn antigen presenting cells (APC) protein antigens are processed into peptides. These peptides bind to human leukocyte antigen molecules (HLAs) and are presented on the cell surface as peptide-HLA complexes to T cells. Different individuals express different HLA molecules and different HLA molecules present different peptides. Therefore, according to the state of the art, a peptide, or a fragment of a larger polypeptide, is identified as immunogenic for a specific human subject if it is presented by a HLA molecule that is expressed by the subject. In other words, the state of the art describes immunogenic peptides as HLA-restricted epitopes. However, HLA restricted epitopes induce T cell responses in only a fraction of individuals who express the HLA molecule. Peptides that activate a T cell response in one individual are inactive in others despite HLA allele matching. Therefore, it was unknown how an individual's HLA molecules present the antigen-derived epitopes that positively activate T cell responses.
As provided herein multiple HLA expressed by an individual need to present the same peptide in order to trigger a T cell response. Therefore the fragments of a polypeptide antigen that are immunogenic for a specific individual are those that can bind to multiple class I (activate cytotoxic T cells) or class II (activate helper T cells) HLAs expressed by that individual.
Accordingly, in a first aspect the disclosure provides methods of predicting whether a polypeptide or a fragment of a polypeptide is immunogenic for a specific human subject, the methods comprising the steps of
-
- (i) determining whether the polypeptide comprises:
- (a) an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or
- (b) an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class II molecules of the subject; and
- (ii) predicting
- A. that the polypeptide is immunogenic for the subject if the polypeptide comprises at least one sequence that meets the requirements of step (i); or
- B. that the polypeptide is not immunogenic for the subject if the polypeptide does not comprise at least one sequence that meets the requirements of step (i).
- (i) determining whether the polypeptide comprises:
The disclosure also provides methods of identifying a fragment of a polypeptide as immunogenic for a specific human subject, the methods comprising the steps of
-
- (i) determining that the polypeptide comprises:
- (a) an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or
- (b) an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class II molecules of the subject; and
- (ii) identifying said sequence as a fragment of the polypeptide that is immunogenic for the subject.
- (i) determining that the polypeptide comprises:
In some embodiments the methods of the disclosure comprise the step of determining or obtaining the HLA class I genotype and/or the HLA class II genotype of the specific human subject.
A specific polypeptide antigen may comprise more than one fragment that is a T cell epitope capable of binding to multiple HLA of a specific individual. The combined group of all such fragments characterize the individual's antigen specific T cell response set, wherein the amino acid sequence of each fragment characterizes the specificity of each activated T cell clone.
Accordingly in some cases the method is repeated until all of the fragments of the polypeptide that are a T cell epitope capable of binding to at least two HLA class I and/or at least two HLA class II of the subject have been identified. This method characterises the immune response of the subject to the polypeptide.
The disclosure further provides methods of treatment of a human subject in need thereof, the method comprising administering to the subject a polypeptide, pharmaceutical composition or kit of the polypeptides of a panel of polypeptides that has been identified or selected by any of the methods above or comprising a fragment of a polypeptide that has been identified or selected by any of the methods above; their use in a method of treatment of a relevant human subject; and their use in the manufacture of a medicament for treating a relevant subject.
The fragments of polypeptide that are determined to be immunogenic for a specific human subject in accordance with the methods above can be used to prepare human subject-specific immunogenic compositions.
Accordingly in a further aspect, the disclosure provides methods of designing or preparing a human subject-specific pharmaceutical composition or kit or panel of polypeptides for use in a method of treatment of a specific human subject, the methods comprising:
-
- (i) selecting a fragment of a polypeptide, which fragment has been identified as immunogenic for the subject by the method above;
- (ii) if the fragment selected in step (i) is an HLA class I-binding epitope, optionally selecting a longer fragment of the polypeptide, which longer fragment
- a. comprises the fragment selected in step (i); and
- b. is a T cell epitope capable of binding at least three or to the most possible HLA class II molecules of the subject;
- (iii) selecting a first sequence of up to 50 consecutive amino acids of the polypeptide, which consecutive amino acids comprise the amino acid sequence of the fragment selected in step (i) or the longer fragment selected in step (ii);
- (iv) repeating steps (i) to (iii) to select a second amino acid sequence of up to 50 consecutive amino acids of the same or a different polypeptide to the first amino acid sequence;
- (v) optionally further repeating steps (i) to (iii) to select one or more additional amino acid sequences of up to 50 consecutive amino acids of the same or different polypeptides to the first and second amino acid sequences; and
- (vi) designing or preparing a subject-specific pharmaceutical composition, kit or panel of polypeptides having as active ingredients one or more polypeptides that together have all of the amino acid sequences selected in the preceding steps, optionally wherein one or more or each sequence is flanked at the N and/or C terminus by additional amino acids that are not part of the sequence of the polypeptides.
In some cases each peptide either consists of one of the selected amino acid sequences, or consists of two or more of the amino acid sequences arranged end to end or overlapping in a single peptide.
The disclosure further provides a human subject-specific pharmaceutical composition, kit or panel of polypeptides for use in a method of treatment of a specific human subject in need thereof, the composition, kit or panel comprising as active ingredients a first and a second peptide and optionally one of more additional peptides, wherein each peptide comprises an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules and/or at least two HLA class II molecules of the subject, wherein the amino acid sequence of the T cell epitope of the first, second and optionally any additional peptides are different from each other, and wherein the pharmaceutical composition or kit optionally comprises at least one pharmaceutically acceptable diluent, carrier, or preservative.
The disclosure further provides a human subject-specific pharmaceutical composition, kit or panel of polypeptides for use in a method of treatment of a specific human subject in need thereof, the composition or kit comprising as an active ingredient a polypeptide comprising a first region and a second region and optionally one of more additional regions, wherein each region comprises an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules and/or at least two HLA class II molecules of the subject, wherein the amino acid sequence of the T cell epitope of the first, second and optionally any additional regions are different from each other, and wherein the pharmaceutical composition or kit optionally comprises at least one pharmaceutically acceptable diluent, carrier, or preservative.
The disclosure further provides a method of designing or preparing a polypeptide for inducing an immune response in a specific human subject the method comprising selecting an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules or at least three HLA class II molecules of the subject, and designing or preparing a polypeptide comprising the selected amino acid sequence.
In further aspects, the disclosure provides
-
- a method of inducing an immune response or a method of treatment comprising administering to a human subject in need thereof a human subject-specific pharmaceutical composition, or the polypeptides a kit or panel as described above, wherein the composition, kit or panel of polypeptides is specific for the subject;
- a human subject-specific immunogenic composition, kit or panel as described above for use in a method of inducing an immune response or a method of treatment of the specific human subject; and
- use of a human subject-specific pharmaceutical composition or the polypeptides of a kit or panel as described above in the manufacture of a medicament, wherein the medicament is for inducing an immune response in or treating the specific subject.
In a further aspect the disclosure provides a system comprising
(a) a storage module configured to store data comprising the class I and/or class II HLA genotype of a subject and the amino acid sequence of one or more test polypeptides; and
(b) a computation module configured to identify and/or quantify amino acid sequences in the one or more test polypeptides that are capable of binding to multiple HLA class I molecules of the subject and/or that are capable of binding to multiple HLA class II molecules of the subject.
The disclosure provides a method of treatment of a human subject in need thereof, the method comprising administering to the subject a polypeptide, a panel of polypeptides, a pharmaceutical composition or the active ingredient polypeptides of a kit described above, wherein the subject has been determined to express at least three HLA class I molecules and/or at least three HLA class II molecules capable of binding to the polypeptide or to one or more of the active ingredient polypeptides of the pharmaceutical composition or kit.
Disclosed herein in certain embodiments are human subject-specific pharmaceutical compositions for treatment of a disease or disorder in a specific human subject, comprising (a) at least two different polypeptides, each of the at least two different polypeptides being 10-50 amino acids in length and comprising a T cell epitope that binds at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject, and wherein the T cell epitope of each of the at least two polypeptides are different from each other; and (b) a pharmaceutically-acceptable adjuvant. In some embodiments, the composition comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides. In some embodiments, the composition comprises 3-40 different polypeptides. In some embodiments, the T cell epitope that binds at least three HLA class I molecules of the subject comprises 7 to 11 amino acids, and/or the T cell epitope that binds at least three HLA class II molecules comprises 13 to 17 amino acids. In some embodiments, the epitopes of the at least two different polypeptides are from a single antigen. In some embodiments, the epitopes of the at least two different polypeptides are from two or more different antigens. In some embodiments, the antigen is an antigen expressed by a cancer cell, a neoantigen expressed by a cancer cell, a cancer-associated antigen, a tumor-associated antigen, or an antigen expressed by a target pathogenic organism, an antigen expressed by a virus, an antigen expressed by a bacterium, an antigen expressed by a fungus, an antigen associated with an autoimmune disorder, or is an allergen. In some embodiments, the cancer cell is from the subject. In some embodiments, the antigen is selected from the antigens listed in Tables 2 to 7. In some embodiments, the at least two different polypeptides further comprise up to 10 amino acids flanking the T cell epitope that are part of a consecutive sequence flanking the epitope in a corresponding antigen. In some embodiments, the at least two different polypeptides further comprise up to 10 amino acids flanking the T cell epitope that are not part of a consecutive sequence flanking the epitope in a corresponding antigen. In some embodiments, two of the at least two polypeptides are arranged end to end or overlapping in a joined polypeptide. In some embodiments, the composition comprises two or more different joined polypeptides, wherein the two or more different joined polypeptides comprise different epitopes from each other. In some embodiments, the joined polypeptides have been screened to eliminate substantially all neoepitopes that span a junction between the two polypeptides and that (i) corresponds to a fragment of a human polypeptide expressed in healthy cells of the subject; (ii) is a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or (iii) meets both requirements (i) and (ii). In some embodiments, the at least two polypeptides do not comprise any amino acid sequences that (i) correspond to a fragment of a human polypeptide expressed in healthy cells; or (ii) correspond to a fragment of a human polypeptide expressed in healthy cells and is a T cell epitope capable of binding to at least two HLA class I molecules of the subject. In some embodiments, the composition further comprises a pharmaceutically acceptable diluent, carrier, preservative, or combination thereof. In some embodiments, the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, bacillus Calmette-Guerin (BCG), corynbacterium parvum, levamisole, azimezone, isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins (KLH), Freunds adjuvant (complete), Freunds adjuvant (incomplete), mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), and combinations thereof.
Disclosed herein in certain embodiments are kits comprising, one or more separate containers each container comprising: (i) one or more polypeptides being 10-50 amino acids in length comprising an amino acid sequence that is a T cell epitope that binds at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject; and (ii) a pharmaceutically acceptable adjuvant, diluent, carrier, preservative, or combination thereof. In some embodiments, the kit comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides, wherein the amino acid sequence of the T cell epitope of each of the different polypeptides are different from each other. In some embodiments, the kit further comprises a package insert.
Disclosed herein in certain embodiments are human subject-specific pharmaceutical compositions comprising: a nucleic acid molecule expressing two or more polypeptides, each polypeptide being 10-50 amino acids in length comprising a T cell epitope that binds at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject, wherein each of the two or more polypeptides comprises a different T cell epitope, wherein the polypeptides do not comprise amino acid sequences that are adjacent to each other in a corresponding antigen. In some embodiments, the nucleic acid molecule expresses at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides, each being 10-50 amino acids in length comprising an amino acid sequence that is a T cell epitope that binds at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject, wherein the amino acid sequence of the T cell epitope of each of the different polypeptides are different from each other.
Disclosed herein in certain embodiments are human subject-specific pharmaceutical compositions for treatment of a disease or disorder in a specific human subject, comprising at least one different polypeptides, each of the at least one different polypeptides comprising at least a first region and a second region, (i) the first region of 10-50 amino acids in length comprising an amino acid sequence that is a T cell epitope that binds at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject, (ii) the second region of 10-50 amino acids in length comprising an amino acid sequence that is a T cell epitope that binds at least three HLA class I molecules of the subject and/or at least two HLA class II molecules of the subject, wherein the amino acid sequence of the T cell epitope of each of the first and second regions of each of the at least three different polypeptides comprise different sequences. In some embodiments, the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides. In some embodiments, the composition comprises 2-40 different polypeptides. In some embodiments, the T cell epitope that binds at least three HLA class I molecules of the subject comprises 7 to 11 amino acids, and/or the T cell epitope that binds at least three HLA class II molecules comprises 13 to 17 amino acids. In some embodiments, the epitopes of the first and second regions are from a single antigen. In some embodiments, the epitopes of the first and second regions are from two or more different antigens. In some embodiments, the antigen is an antigen expressed by a cancer cell, a neoantigen expressed by a cancer cell, a cancer-associated antigen, a tumor-associated antigen, or an antigen expressed by a target pathogenic organism, an antigen expressed by a virus, an antigen expressed by a bacterium, an antigen expressed by a fungus, an antigen associated with an autoimmune disorder, or is an allergen. In some embodiments, the cancer cell is from the subject. In some embodiments, the antigen is selected from the antigens listed in Tables 2 to 7. In some embodiments, the polypeptides have been screened to eliminate substantially all neoepitopes that span a junction between the two regions and that (i) corresponds to a fragment of a human polypeptide expressed in healthy cells of the subject; (ii) is a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or (iii) meets both requirements (i) and (ii). In some embodiments, the at least one polypeptides do not comprise any amino acid sequences that (i) correspond to a fragment of a human polypeptide expressed in healthy cells; or (ii) correspond to a fragment of a human polypeptide expressed in healthy cells and is a T cell epitope capable of binding to at least two HLA class I molecules of the subject. In some embodiments, the composition further comprises a pharmaceutically acceptable adjuvant, diluent, carrier, preservative, or combination thereof. In some embodiments, the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, bacillus Calmette-Guerin (BCG), corynbacterium parvum, levamisole, azimezone, isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins (KLH), Freunds adjuvant (complete), Freunds adjuvant (incomplete), mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), and combinations thereof.
Disclosed herein in certain embodiments are methods of preparing a human subject-specific pharmaceutical composition for use in a method of treatment of a specific human subject, the method comprising:
-
- (i) selecting a fragment of a polypeptide, which fragment has been identified as immunogenic for the subject by
- a) determining whether the fragment comprises:
- 1) an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; or
- 2) an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class II molecules of the subject; or
- 3) or meets both requirements (1) and (2); and
- b) identifying the sequence as a fragment of the polypeptide that is immunogenic for the subject;
- a) determining whether the fragment comprises:
- (ii) selecting a first sequence of up to 50 consecutive amino acids of the polypeptide, which consecutive amino acids comprise the amino acid sequence of the fragment selected in step (i); and
- (iii) preparing a subject-specific pharmaceutical composition having as active ingredients one or more polypeptides that together have all of the amino acid sequences selected in the preceding steps.
In some embodiments, the method further comprises prior to the preparing step repeating steps (i) to (ii) to select a second amino acid sequence of up to 50 consecutive amino acids of the same or a different polypeptide to the first amino acid sequence. In some embodiments, the method further comprises, further repeating prior to the preparing step, steps (i) to (ii) one or more times to select one or more additional amino acid sequences of up to 50 consecutive amino acids of the same or different polypeptides to the first and second amino acid sequences. In some embodiments, the method further comprises prior to the preparing step selecting a longer fragment of the polypeptide if the fragment selected in step (i) is an HLA class I-binding epitope, which longer fragment comprises the fragment selected in step (i); and is a T cell epitope capable of binding at least three HLA class II molecules of the subject. In some embodiments, each polypeptide either consists of one of the selected amino acid sequences, or comprises or consists of two or more of the selected amino acid sequences arranged end to end or overlapping in a single joined polypeptide. In some embodiments, any neoepitopes formed at the junction between any two of the selected amino acid sequences arranged end to end in a single joined polypeptide have been screened to eliminate substantially all polypeptides comprising a neoepitope amino acid sequence that (i) corresponds to a fragment of a human polypeptide expressed in healthy cells; (ii) is a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or (iii) meets both requirements (i) and (ii). In some embodiments, the one or more polypeptides have been screened to eliminate polypeptides comprising an amino acid sequence that (i) corresponds to a fragment of a human polypeptide expressed in healthy cells; or (ii) corresponds to a fragment of a human polypeptide expressed in healthy cells and is a T cell epitope capable of binding to at least two HLA class I molecules of the subject. In some embodiments, the method further comprises determining HLA class I genotype and HLA class II genotype from a biological sample of the subject prior to step (i). In some embodiments, the biological sample is obtained from the subject. In some embodiments, the determining HLA class I genotype and HLA class II genotype is performed by sequence based typing (SBT) methods. In some embodiments, the determining HLA class I genotype and HLA class II genotype is performed by sequencing, next generation sequencing, sequence specific primer (SSP) methods, or sequence specific oligonucleotide (SSO) methods. In some embodiments, the biological sample is blood, serum, plasma, saliva, buccal swab, urine, expiration, cell, or tissue. In some embodiments, the biological sample is saliva or a buccal swab.
- (i) selecting a fragment of a polypeptide, which fragment has been identified as immunogenic for the subject by
Disclosed herein in certain embodiments are methods of treating a cancer in a specific human subject in need thereof comprising, administering to a specific human subject a pharmaceutical composition comprising at least one polypeptide, each of the at least one polypeptide being 10-50 amino acids in length comprising a first amino acid sequence that is a T cell epitope that binds at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject, wherein the T cell epitope of each of the at least one polypeptide is from an antigen that is specific for the cancer. In some embodiments, the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides, wherein the amino acid sequence of the T cell epitope of each of the different polypeptides are different from each other, and are from one or more antigens that are expressed by a cancer cell from the subject. In some embodiments, the composition comprises 2-40 different polypeptides. In some embodiments, the T cell epitope that binds at least three HLA class I molecules of the subject comprises 7 to 11 amino acids, and/or the T cell epitope that binds at least three HLA class II molecules comprises 13 to 17 amino acids. In some embodiments, the composition comprises at least two different polypeptides and the epitopes of the amino acid sequences of the at least two different polypeptides are from a single antigen. In some embodiments, the composition comprises at least two different polypeptides and the epitopes of the at least two different polypeptides are from two or more different antigens. In some embodiments, the one or more antigen is a neoantigen expressed by a cancer cell, a cancer-associated antigen, or a tumor-associated antigen. In some embodiments, the one or more antigen is selected from the antigens listed in Table 2. In some embodiments, the at least one different polypeptides further comprise up to 10 amino acids flanking the T cell epitope that are part of a consecutive sequence flanking the epitope in a corresponding antigen. In some embodiments, the at least one different polypeptides further comprise up to 10 amino acids flanking the T cell epitope that are not part of a consecutive sequence flanking the epitope in a corresponding antigen. In some embodiments, the composition comprises at least two different polypeptides and two of the polypeptides are arranged end to end or overlapping in a joined polypeptide. In some embodiments, the composition comprises two or more different joined polypeptides, wherein the two or more different joined polypeptides comprise different epitopes from each other. In some embodiments, the joined polypeptides have been screened to eliminate substantially all neoepitopes that span a junction between the two polypeptides and that (i) corresponds to a fragment of a human polypeptide expressed in healthy cells of the subject; (ii) is a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or (iii) meets both requirements (i) and (ii). In some embodiments, the at least one polypeptide does not comprise any amino acid sequences that (i) correspond to a fragment of a human polypeptide expressed in healthy cells; or (ii) correspond to a fragment of a human polypeptide expressed in healthy cells and is a T cell epitope capable of binding to at least two HLA class I molecules of the subject. In some embodiments, the composition further comprises a pharmaceutically acceptable adjuvant, diluent, carrier, preservative, or combination thereof. In some embodiments, the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, bacillus Calmette-Guerin (BCG), corynbacterium parvum, levamisole, azimezone, isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins (KLH), Freunds adjuvant (complete), Freunds adjuvant (incomplete), mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), and combinations thereof. In some embodiments, the method further comprises administering a chemotherapeutic agent, a targeted therapy, radiation therapy, a checkpoint inhibitor, another immunotherapy, or combination thereof.
Disclosed herein in some embodiments are human subject-specific pharmaceutical compositions for treatment of a disease or disorder in a specific human subject, comprising (a) a polypeptide of 10-50 amino acids in length and comprising a T cell epitope that binds at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject; and (b) a pharmaceutically-acceptable adjuvant. In some embodiments, the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides, each of the different polypeptides being 10-50 amino acids in length comprising a T cell epitope that binds at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject, wherein the amino acid sequence of the T cell epitope of each of the different polypeptides are different from each other. In some embodiments, the composition comprises 2-40 different polypeptides. In some embodiments, the T cell epitope that binds at least three HLA class I molecules of the subject comprises 7 to 11 amino acids, and/or the T cell epitope that binds at least three HLA class II molecules comprises 13 to 17 amino acids. In some embodiments, the composition comprises at least two different polypeptides, wherein the epitopes of the at least two different polypeptides are from a single antigen. In some embodiments, the composition comprises at least two different polypeptides, wherein the epitopes of the at least two different polypeptides are from two or more different antigens. In some embodiments, the antigen is an antigen expressed by a cancer cell, a neoantigen expressed by a cancer cell, a cancer-associated antigen, a tumor-associated antigen, or an antigen expressed by a target pathogenic organism, an antigen expressed by a virus, an antigen expressed by a bacterium, an antigen expressed by a fungus, an antigen associated with an autoimmune disorder, or is an allergen. In some embodiments, the cancer cell is from the subject. In some embodiments, the antigen is selected from the antigens listed in Tables 2 to 7. In some embodiments, the composition comprises at least two different polypeptides, wherein two of the polypeptides are arranged end to end or overlapping in a joined polypeptide. In some embodiments, the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, bacillus Calmette-Guerin (BCG), corynbacterium parvum, levamisole, azimezone, isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins (KLH), Freunds adjuvant (complete), Freunds adjuvant (incomplete), mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), and combinations thereof. In some embodiments, the composition comprises at least two different polypeptides, wherein two of the at least two polypeptides are arranged end to end or overlapping in a joined polypeptide. In some embodiments, the composition comprises two or more different joined polypeptides, wherein the two or more different joined polypeptides comprise different epitopes from each other. In some embodiments, the joined polypeptides have been screened to eliminate substantially all neoepitopes that span a junction between the two polypeptides and that (i) corresponds to a fragment of a human polypeptide expressed in healthy cells of the subject; (ii) is a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or (iii) meets both requirements (i) and (ii). In some embodiments, the at least two polypeptides do not comprise any amino acid sequences that (i) correspond to a fragment of a human polypeptide expressed in healthy cells; or (ii) correspond to a fragment of a human polypeptide expressed in healthy cells and is a T cell epitope capable of binding to at least two HLA class I molecules of the subject.
Disclosed herein in certain embodiments are kits comprising: a first human subject-specific pharmaceutical composition comprising (i) a first polypeptide of 10-50 amino acids in length and comprising a T cell epitope that binds at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject; and (ii) a pharmaceutically-acceptable adjuvant; and a second human subject-specific pharmaceutical composition comprising (i) a second polypeptide of 10-50 amino acids in length and comprising a T cell epitope that binds at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject; and (ii) a pharmaceutically-acceptable adjuvant, wherein the first and second polypeptides comprise different T cell epitopes. In some embodiments, the first composition and/or the second composition comprise one or more additional polypeptides, wherein each additional polypeptide being of 10-50 amino acids in length comprising an amino acid sequence that is a T cell epitope that binds at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject, wherein the amino acid sequences comprise different T cell epitopes.
The disclosure will now be described in more detail, by way of example and not limitation, and by reference to the accompanying drawings. Many equivalent modifications and variations will be apparent, to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the disclosure set forth are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the scope of the disclosure. All documents cited herein, whether supra or infra, are expressly incorporated by reference in their entirety.
The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or is stated to be expressly avoided. As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a peptide” includes two or more such peptides.
Section headings are used herein for convenience only and are not to be construed as limiting in any way.
SEQ ID NOs: 1 to 13 set forth the additional peptide sequences described in Table 17.
SEQ ID NOs: 14-26 set forth personalised vaccine peptides designed for patient XYZ described in Table 26.
SEQ ID NOs: 27-38 set forth personalised vaccine peptides designed for patient ABC described in Table 29.
SEQ ID NOs: 39-86 set forth further 9 mer T cell epitopes described in Table 33.
DETAILED DESCRIPTION HLA GenotypesHLAs are encoded by the most polymorphic genes of the human genome. Each person has a maternal and a paternal allele for the three HLA class I molecules (HLA-A*, HLA-B*, HLA-C*) and four HLA class II molecules (HLA-DP*, HLA-DQ*, HLA-DRB1*, HLA-DRB3*/4*/5*). Practically, each person expresses a different combination of 6 HLA class I and 8 HLA class II molecules that present different epitopes from the same protein antigen. The function of HLA molecules is to regulate T cell responses. However up to date it was unknown how the HLAs of a person regulate T cell activation.
The nomenclature used to designate the amino acid sequence of the HLA molecule is as follows: gene name*allele:protein number, which, for instance, can look like: HLA-A*02:25. In this example, “02” refers to the allele. In most instances, alleles are defined by serotypes—meaning that the proteins of a given allele will not react with each other in serological assays. Protein numbers (“25” in the example above) are assigned consecutively as the protein is discovered. A new protein number is assigned for any protein with a different amino acid sequence (e.g. even a one amino acid change in sequence is considered a different protein number). Further information on the nucleic acid sequence of a given locus may be appended to the HLA nomenclature, but such information is not required for the methods described herein.
The HLA class I genotype or HLA class II genotype of an individual may refer to the actual amino acid sequence of each class I or class II HLA of an individual, or may refer to the nomenclature, as described above, that designates, minimally, the allele and protein number of each HLA gene. In some embodiments, the HLA genotype of an individual is obtained or determined by assaying a biological sample from the individual. The biological sample typically contains subject DNA. The biological sample may be, for example, a blood, serum, plasma, saliva, urine, expiration, cell or tissue sample. In some embodiments the biological sample is a saliva sample. In some embodiments the biological sample is a buccal swab sample. An HLA genotype may be obtained or determined using any suitable method. For example, the sequence may be determined via sequencing the HLA gene loci using methods and protocols known in the art. In some embodiments, the HLA genotype is determined using sequence specific primer (SSP) technologies. In some embodiments, the HLA genotype is determined using sequence specific oligonucleotide (SSO) technologies. In some embodiments, the HLA genotype is determined using sequence based typing (SBT) technologies. In some embodiments, the HLA genotype is determined using next generation sequencing. Alternatively, the HLA set of an individual may be stored in a database and accessed using methods known in the art.
HLA-Epitope BindingA given HLA of a subject will only present to T cells a limited number of different peptides produced by the processing of protein antigens in an APC. As used herein, “display” or “present”, when used in relation to HLA, references the binding between a peptide (epitope) and an HLA. In this regard, to “display” or “present” a peptide is synonymous with “binding” a peptide.
As used herein, the term “epitope” or “T cell epitope” refers to a sequence of contiguous amino acids contained within a protein antigen that possess a binding affinity for (is capable of binding to) one or more HLAs. An epitope is HLA- and antigen-specific (HLA-epitope pairs, predicted with known methods), but not subject specific. An epitope, a T cell epitope, a polypeptide, a fragment of a polypeptide or a composition comprising a polypeptide or a fragment thereof is “immunogenic” for a specific human subject if it is capable of inducing a T cell response (a cytotoxic T cell response or a helper T cell response) in that subject. In some cases the helper T cell response is a Th1-type helper T cell response. In some cases an epitope, a T cell epitope, a polypeptide, a fragment of a polypeptide or a composition comprising a polypeptide or a fragment thereof is “immunogenic” for a specific human subject if it is more likely to induce a T cell response or immune response in the subject than a different T cell epitope (or in some cases two different T cell epitopes each) capable of binding to just one HLA molecule of the subject.
The terms “T cell response” and “immune response” are used herein interchangeably, and refer to the activation of T cells and/or the induction of one or more effector functions following recognition of one or more HLA-epitope binding pairs. In some cases an “immune response” includes an antibody response, because HLA class II molecules stimulate helper responses that are involved in inducing both long lasting CTL responses and antibody responses. Effector functions include cytotoxicity, cytokine production and proliferation. According to the present disclosure, an epitope, a T cell epitope, or a fragment of a polypeptide is immunogenic for a specific subject if it is capable of binding to at least two, or in some cases at least three, class I or at least two, or in some cases at least three or at least four class II HLAs of the subject.
For the purposes of this disclosure we have coined the term “personal epitope”, or “PEPI” to distinguish subject specific epitopes from HLA specific epitopes. A “PEPI” is a fragment of a polypeptide consisting of a sequence of contiguous amino acids of the polypeptide that is a T cell epitope capable of binding to one or more HLA class I molecules of a specific human subject. In other cases a “PEPI” is a fragment of a polypeptide consisting of a sequence of contiguous amino acids of the polypeptide that is a T cell epitope capable of binding to one or more HLA class II molecules of a specific human subject. In other words a “PEPI” is a T cell epitope that is recognised by the HLA set of a specific individual. In contrast to an “epitope”, PEPIs are specific to an individual because different individuals have different HLA molecules which each bind to different T cell epitopes.
“PEPI1” as used herein refers to a peptide, or a fragment of a polypeptide, that can bind to one HLA class I molecule (or, in specific contexts, HLA class II molecule) of an individual. “PEPI1+” refers to a peptide, or a fragment of a polypeptide, that can bind to one or more HLA class I molecule of an individual.
“PEPI2” refers to a peptide, or a fragment of a polypeptide, that can bind to two HLA class I (or II) molecules of an individual. “PEPI2+” refers to a peptide, or a fragment of a polypeptide, that can bind to two or more HLA class I (or II) molecules of an individual, i.e. a fragment identified according to a method disclosed herein.
“PEPI3” refers to a peptide, or a fragment of a polypeptide, that can bind to three HLA class I (or II) molecules of an individual. “PEPI3+” refers to a peptide, or a fragment of a polypeptide, that can bind to three or more HLA class I (or II) molecules of an individual.
“PEPI4” refers to a peptide, or a fragment of a polypeptide, that can bind to four HLA class I (or II) molecules of an individual. “PEPI4+” refers to a peptide, or a fragment of a polypeptide, that can bind to four or more HLA class I (or II) molecules of an individual.
“PEPI5” refers to a peptide, or a fragment of a polypeptide, that can bind to five HLA class I (or II) molecules of an individual. “PEPI5+” refers to a peptide, or a fragment of a polypeptide, that can bind to five or more HLA class I (or II) molecules of an individual.
“PEPI6” refers to a peptide, or a fragment of a polypeptide, that can bind to all six HLA class I (or six HLA class II) molecules of an individual.
Generally speaking, epitopes presented by HLA class I molecules are about nine amino acids long and epitopes presented by HLA class II molecules are about fifteen amino acids long. For the purposes of this disclosure, however, an epitope may be more or less than nine (for HLA Class I) or more or less than fifteen (for HLA Class II) amino acids long, as long as the epitope is capable of binding HLA. For example, an epitope that is capable of binding to class I HLA may be between 7, or 8 or 9 and 9 or 10 or 11 amino acids long. An epitope that is capable of binding to a class II HLA may be between 13, or 14 or 15 and 15 or 16 or 17 amino acids long.
Therefore the disclosure herein includes, for example, a method of predicting whether a polypeptide is immunogenic for a specific human subject or identifying a fragment of a polypeptide as immunogenic for a specific human subject, the method comprising the steps of
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- (i) determining whether the polypeptide comprises:
- a. a sequence of 7 to 11 consecutive amino acids that is capable of binding to at least two HLA class I of the subject; or
- b. a sequence of 13 to 17 consecutive amino acids that is capable of binding to at least two HLA class II of the subject; and
- (ii) predicting that the polypeptide is immunogenic for the subject if the polypeptide comprises at least one sequence that meets the requirements of step (i); or predicting that the polypeptide is not immunogenic for the subject if the polypeptide does not comprise at least one sequence that meets the requirements of step (i); or identifying said consecutive sequence of amino acids as the sequence of a fragment of the polypeptide that is immunogenic for the subject.
- (i) determining whether the polypeptide comprises:
Using techniques known in the art, it is possible to determine the epitopes that will bind to a known HLA. Any suitable method may be used, provided that the same method is used to determine multiple HLA-epitope binding pairs that are directly compared. For example, biochemical analysis may be used. It is also possible to use lists of epitopes known to be bound by a given HLA. It is also possible to use predictive or modelling software to determine which epitopes may be bound by a given HLA. Examples are provided in Table 1. In some cases a T cell epitope is capable of binding to a given HLA if it has an IC50 or predicted IC50 of less than 5000 nM, less than 2000 nM, less than 1000 nM, or less than 500 nM.
As provided herein T cell epitope presentation by multiple HLAs of an individual is generally needed to trigger a T cell response. Accordingly, the methods of the invention comprise determining whether a polypeptide has a sequence that is a T cell epitope capable of binding to at least two HLA class I molecules or at least two HLA class II (PEPI2+) molecules of a specific human subject.
The best predictor of a cytotoxic T cell response to a given polypeptide is the presence of at least one T cell epitope that is presented by three or more HLA class I molecules of an individual (≥1 PEPI3+). Accordingly, in some cases the method comprises determining whether a polypeptide has a sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of a specific human subject. In some cases the method comprises determining whether a polypeptide has a sequence that is a T cell epitope capable of binding to just three HLA class I of a specific human subject. A helper T cell response may be predicted by the presence of at least one T cell epitope that is presented by three or more (≥1 PEPI3+) or 4 or more (≥1 PEPI4+) HLA class II of an individual. Therefore in some cases, the method comprises determining whether a polypeptide has a sequence that is a T cell epitope capable of binding to at least three HLA class II of a specific human subject. In other cases, the method comprises determining whether a polypeptide has a sequence that is a T cell epitope capable of binding to at least four HLA class II of a specific human subject. In other cases, the method comprises determining whether a polypeptide has a sequence that is a T cell epitope capable of binding to at just three and/or just four HLA class II of a specific human subject.
In some cases, the disclosure may be used to predict whether a polypeptide/fragment will induce both a cytotoxic T cell response and a helper T cell response in a specific human subject. The polypeptide/fragment comprises both an amino acid sequence that is a T cell epitope capable of binding to multiple HLA class I molecules of the subject and an amino acid sequence that is a T cell epitope capable of binding to multiple HLA class II molecules of the subject. The HLA class I-binding and HLA class II-binding epitopes may fully or partially overlap. In some cases such fragments of a polypeptide may be identified by selecting an amino acid sequence that is a T cell epitope capable of binding to at multiple (e.g. at least two or at least three) HLA class I molecules of the subject, and then screening one or more longer fragments of the polypeptide that are extended at the N- and/or C-terminus for binding to one or more HLA class II molecules of the subject.
Some subjects may have two HLA alleles that encode the same HLA molecule (for example, two copies for HLA-A*02:25 in case of homozygosity). The HLA molecules encoded by these alleles bind all of the same T cell epitopes. For the purposes of this disclosure “binding to at least two HLA molecules of the subject” as used herein includes binding to the HLA molecules encoded by two identical HLA alleles in a single subject. In other words, “binding to at least two HLA molecules of the subject” and the like could otherwise be expressed as “binding to the HLA molecules encoded by at least two HLA alleles of the subject”.
Polypeptide AntigensDescribed herein are methods of predicting whether a polypeptide is immunogenic for a specific human subject and of identifying a fragment of a polypeptide as immunogenic for a specific human subject. As used herein, the term “polypeptide” refers to a full-length protein, a portion of a protein, or a peptide characterized as a string of amino acids. As used herein, the term “peptide” refers to a short polypeptide comprising between 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15 and 10, or 11, or 12, or 13, or 14, or 15, or 20, or 25, or 30, or 35, or 40, or 45, or 50 amino acids.
The terms “fragment” or “fragment of a polypeptide” as used herein refer to a string of amino acids or an amino acid sequence typically of reduced length relative to the or a reference polypeptide and comprising, over the common portion, an amino acid sequence identical to the reference polypeptide. Such a fragment according to the disclosure may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some cases the fragment may comprise the full length of the polypeptide, for example where the whole polypeptide, such as a 9 amino acid peptide, is a single T cell epitope.
In some cases the polypeptide is, or the polypeptide consists of all or part of an antigen that is, expressed by a pathogenic organism (for example, a bacteria or a parasite), a virus, or a cancer cell, that is associated with an autoimmune disorder or response or a disease-associated cell, or that is an allergen, or an ingredient of a medicine or pharmaceutical composition such as a vaccine or immunotherapy composition. In some cases the method of the disclosure comprises an initial step of identifying or selecting a suitable polypeptide, for example a polypeptide as further described below.
The polypeptide or antigen may be expressed in the cells or specifically in diseased cells of the subject (e.g. a tumor-associated antigen, a polypeptide expressed by a virus, intracellular bacteria or parasite, or the in vivo product of a vaccine or immunotherapy composition) or acquired from the environment (e.g. a food, an allergen or a drug). The polypeptide or antigen may be present in a sample taken from the specific human subject. Both polypeptide antigens and HLAs can be exactly defined by amino acid or nucleotide sequences and sequenced using methods known in the art.
The polypeptide or antigen may be a cancer- or tumor-associated antigen (TAA). TAAs are proteins expressed in cancer or tumor cells. The cancer or tumour cell may be present in a sample obtained from the subject. Examples of TAAs include new antigens (neoantigens) expressed during tumorigenesis, products of oncogenes and tumor suppressor genes, overexpressed or aberrantly expressed cellular proteins (e.g. HER2, MUC1), antigens produced by oncogenic viruses (e.g. EBV, HPV, HCV, HBV, HTLV), cancer testis antigens (CTA)(e.g. MAGE family, NY-ESO) and cell-type-specific differentiation antigens (e.g. MART-1). TAA sequences may be found experimentally, or in published scientific papers, or through publicly available databases, such as the database of the Ludwig Institute for Cancer Research (www.cta.lncc.br/), Cancer Immunity database (cancerimmunity.org/peptide/) and the TANTIGEN Tumor T cell antigen database (cvc.dfci.harvard.edu/tadb/).
In some cases the polypeptide or antigen is not expressed or is minimally expressed in normal healthy cells or tissues, but is expressed (in those cells or tissues) in a high proportion of (with a high frequency in) subjects having a particular disease or condition, such as a type of cancer or a cancer derived from a particular cell type or tissue, for example breast cancer, ovarian cancer or melanoma. A further example is colorectal cancer. Other non-limiting cancer examples include non-melanoma skin, lung, prostate, kidney, bladder, stomach, liver, cervix uteri, oesophagus, non-Hodgkin lymphoma, leukemia, pancreas, corpus uteri, lip, oral cavity, thyroid, brain, nervous system, gallbladder, larynx, pharynx, myeloma, nasopharynx, Hodgkin lymphoma, testis and Kaposi sarcoma. Alternatively, the polypeptide may be expressed at low levels in normal healthy cells, but at high levels (overexpressed) in diseased (e.g. cancer) cells or in subjects having the disease or condition. In some cases the polypeptide is expressed in, or expressed at a high level relative to normal healthy cells or subjects in, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such individuals, or of a subject-matched human subpopulation. For example the subpopulation may be matched to the subject by ethnicity, geographical location, gender, age, disease, disease type or stage, genotype, or expression of one or more biomarkers.
In some cases the expression frequencies can be determined from published figures and scientific publications. In some cases the method of the disclosure comprises a step of identifying or selecting such a polypeptide.
In some cases the polypeptide is associated with or highly (over-) expressed in cancer cells, or in solid tumors. Exemplary cancers include carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, or blastomas. The cancer may or may not be a hormone related or dependent cancer (e.g., an estrogen or androgen related cancer). The tumor may be malignant or benign. The cancer may or may not be metastatic.
In some cases the polypeptide is a cancer testis antigens (CTA). CTA are not typically expressed beyond embryonic development in healthy cells. In healthy adults, CTA expression is limited to male germ cells that do not express HLAs and cannot present antigens to T cells. Therefore, CTAs are considered expressional neoantigens when expressed in cancer cells. CTA expression is (i) specific for tumor cells, (ii) more frequent in metastases than in primary tumors and (iii) conserved among metastases of the same patient (Gajewski ed. Targeted Therapeutics in Melanoma. Springer New York. 2012).
The polypeptide may be a mutational neoantigen, which is expressed by a cell, for example a cancer cell, of the individual, but altered from the analogous protein in a normal or healthy cell. In some cases the methods of the disclosure comprise the step of identifying a polypeptide that is a mutational neoantigen, or that is a mutational neoantigen in the specific human subject, or of identifying a neoepitope. For example the neoantigen may be present in a sample obtained from the subject. Mutational neoantigens or neoepitopes can be used to target disease-associated cells, such as cancer cells, that express the neoantigen or a neoantigen comprising the neoepitope. Mutations in a polypeptide expressed by a cell, for example a cell in a sample taken from a subject, can be detected by, for example, sequencing, but the majority do not induce an immune response against the neoantigen-expressing cells. Currently, the identification of mutational neoantigens that do induce an immune response is based on prediction of mutational HLA restricted epitopes and further in vitro testing of the immunogenicity of predicted epitopes in individual's blood specimen. This process is inaccurate, long and expensive.
As provided herein the identification of mutational epitopes (neoepitopes) that bind to multiple HLA molecules reproducibly define the immunogenicity of mutational neoantigens. Therefore, in some cases in accordance with the disclosure, the polypeptide is a mutational neoantigen, and the immunogenic fragment of the polypeptide comprises a neoantigen specific mutation (or consists of a neoepitope).
The polypeptide may be a viral protein that is expressed intracellularly. Examples include HPV16 E6, E7; HIV Tat, Rev, Gag, Pol, Env; HTLV-Tax, Rex, Gag, Env, Human herpes virus proteins, Dengue virus proteins. The polypeptide may be a parasite protein that is expressed intracellularly, for example malaria proteins.
The polypeptide may be an active ingredient of a pharmaceutical composition, such as a vaccine or immunotherapy composition, optionally a candidate active ingredient for a new pharmaceutical composition. The term “active ingredient” as used herein refers to a polypeptide that is intended to induce an immune response and may include a polypeptide product of a vaccine or immunotherapy composition that is produced in vivo after administration to a subject. For a DNA or RNA immunotherapy composition, the polypeptide may be produced in vivo by the cells of a subject to whom the composition is administered. For a cell-based composition, the polypeptide may be processed and/or presented by cells of the composition, for example autologous dendritic cells or antigen presenting cells pulsed with the polypeptide or comprising an expression construct encoding the polypeptide. The pharmaceutical composition may comprise a polynucleotide or cell encoding one or more active ingredient polypeptides.
In other cases the polypeptide may be a target polypeptide antigen of a pharmaceutical, vaccine or immunotherapy composition. A polypeptide is a target polypeptide antigen if the composition is intended or designed to induce an immune response (e.g. a cytotoxic T cell response) that targets or is directed at the polypeptide. A target polypeptide antigen is typically a polypeptide that is expressed by a pathogenic organism, a virus or a diseased cell such as a cancer cell. A target polypeptide antigens may be a TAA or a CTA.
Presently, >200 clinical trials are investigating cancer vaccines with tumor antigens.
The polypeptide may be an allergen that enters the body of an individual through, for example, the skin, lung or oral routes.
Non-limiting examples of suitable polypeptides include those listed in one or more of Tables 2 to 7.
Genetic sequences may be obtained from the sequencing of biological materials. Sequencing can be done by any suitable method that determines DNA and/or RNA and/or amino acid sequences. The disclosure utilizes both the HLA genotypes and amino acid sequences. However, methods to identify HLA genotype from genetic sequences of an individual and methods of obtaining amino acid sequences derived from DNA or RNA sequence data are not the subject of the disclosure.
Specific polypeptide antigens induce immune responses in only a fraction of human subjects. Currently, there is no diagnostic test that can predict whether a polypeptide antigen would likely induce an immune response in an individual. In particular, there is a need for a test that can predict whether a person is an immune responder to a vaccine or immunotherapy composition.
According to the present disclosure, the polypeptide antigen-specific T cell response of an individual is defined by the presence within the polypeptide of one or more fragments that may be presented by multiple HLA class I or multiple HLA class II molecules of the individual.
In some cases the disclosure provides a method of predicting whether a subject will have an immune response to administration of a polypeptide, wherein an immune response is predicted if the polypeptide is immunogenic according to any method described herein. A cytotoxic T cell response is predicted if the polypeptide comprises at least one amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules of the subject. A helper T cell response is predicted if the polypeptide comprises at least one amino acid sequence that is a T cell epitope capable of binding to at least two HLA class II molecules of the subject. No cytotoxic T cell response is predicted if the polypeptide does not comprise any amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules of the subject. No helper T cell response is predicted if the polypeptide does not comprise any amino acid sequence that is a T cell epitope capable of binding to at least two HLA class II molecules of the subject.
In some cases the polypeptide is an active component of a pharmaceutical composition, and the method comprises predicting the development or production of anti-drug antibodies (ADA) to the polypeptide. The pharmaceutical composition may be a drug selected from those listed in Table 8. According to the present disclosure, ADA development will occur if, or to the extent that, an active component polypeptide is recognised by multiple HLA class II molecules of the subject, resulting in a helper T cell response to support an antibody response to the active component. The presence of such epitopes (PEPIs) may predict the development of ADA in the subject. The method may further comprise selecting or recommending for treatment of the specific human subject administration to the subject of a pharmaceutical composition that is predicted to induce low or no ADA, and optionally further administering the composition to the subject. In other cases the method predicts that the pharmaceutical composition will induce unacceptable ADA and the method further comprises selecting or recommending or treating the subject with a different treatment or therapy. The polypeptide may be a checkpoint inhibitor. The method may comprise predicting whether the subject will respond to treatment with the checkpoint inhibitor.
There is also currently no test that can predict the likelihood that a person will have a clinical response to, or derive clinical benefit from, a vaccine or immunotherapy composition. This is important because currently T cell responses measured in a cohort of individuals participating in vaccine or immunotherapy clinical trials poorly correlate with clinical responses. That is, the clinical responder subpopulation is substantially smaller than the immune responder subpopulation. Therefore, to enable the personalization of vaccines and immunotherapies it is important to predict not only the likelihood of an immune response in a specific subject, but also whether the immune response induced by the drug will be clinically effective (e.g. can kill cancer cells or pathogen infected cells or pathogens).
The inventors have discovered that the presence in a vaccine or immunotherapy composition of at least two polypeptide fragments (epitopes) that can bind to at least three HLA class I of an individual (≥2 PEPI3+) is predictive for a clinical response. In other words, if ≥2 PEPI3+ can be identified within the active ingredient polypeptide(s) of a vaccine or immunotherapy composition, then an individual is a likely clinical responder. A “clinical response” or “clinical benefit” as used herein may be the prevention of or a delay in the onset of a disease or condition, the amelioration of one or more symptoms, the induction or prolonging of remission, or the delay of a relapse or recurrence or deterioration, or any other improvement or stabilisation in the disease status of a subject. Where appropriate, a “clinical response” may correlate to “disease control” or an “objective response” as defined by the Response Evaluation Criteria In Solid Tumors (RECIST) guidelines.
Therefore, in some cases the disclosure provides a method of predicting whether the subject will have a clinical response to administration of a pharmaceutical composition such as a vaccine or immunotherapy composition comprising one or more polypeptides as active ingredients. The method may comprise determining whether the one or more polypeptides together comprise at least two different sequences each of which is a T cell epitope capable of binding to at least two, or in some cases at least three HLA class I molecules of the subject; and predicting that the subject will have a clinical response to administration of the pharmaceutical composition if the one or more polypeptides together comprise at least two different sequences each of which is a T cell epitope capable of binding to at least two, or in some cases at least three HLA class I molecules of the subject; or that the subject will not have a clinical response to administration of the pharmaceutical composition if the one or more polypeptides together comprise no more that one sequence that is a T cell epitope capable of binding to at least two, or in some cases at least three HLA class I molecules of the subject.
For the purposes of this method two T cell epitopes are “different” from each other if they have different sequences, and in some cases also if they have the same sequence that is repeated in a target polypeptide antigen. In some cases the different T cell epitopes in a target polypeptide antigen do not overlap with one another.
In some cases all of the fragments of one or more polypeptides or active ingredient polypeptides that are immunogenic for a specific human subject are identified using the methods described herein. The identification of at least one fragment of the polypeptide(s) that is a T cell epitope capable of binding to at least two, or at least three HLA class I molecules of the subject predicts that the polypeptide(s) will elicit or is likely to elicit a cytotoxic T cell response in the subject. The identification of at least one fragment of the polypeptide(s) that is a T cell epitope capable of binding to at least two, or at least three, or at least four HLA class II molecules of the subject predicts that the polypeptide(s) will elicit or is likely to elicit a helper T cell response in the subject. The identification of no fragments of the polypeptide(s) that are T cell epitopes capable of binding to at least two, or at least three HLA class I molecules of the subject predicts that the polypeptide(s) will not elicit or is not likely to elicit a cytotoxic T cell response in the subject. The identification of no fragments of the polypeptide(s) that are T cell epitopes capable of binding to at least two, or at least three, or at least four HLA class II molecules of the subject predicts that the polypeptide(s) will not elicit or is not likely to elicit a helper T cell response in the subject. The identification of at least two fragments of one or more active ingredient polypeptides of a vaccine or immunotherapy composition, wherein each fragment is a T cell epitope capable of binding to at least two, or at least three HLA class I molecules of the subject predicts that the subject is more likely to have, or will have a clinical response to the composition. The identification of less than two fragments of the one or more polypeptides that are T cell epitopes capable of binding to at least two, or at least three HLA class I molecules of the subject predicts that the subject is less likely to have, or will not have, a clinical response to the composition.
Without wishing to be bound by theory, one reason for the increased likelihood of deriving clinical benefit from a vaccine/immunotherapy comprising at least two multiple-HLA binding PEPIs, is that diseased cell populations, such as cancer or tumor cells or cells infected by viruses or pathogens such as HIV, are often heterogenous both within and between effected subjects. A specific cancer patient, for example, may or may not express or overexpress a particular cancer associated target polypeptide antigen of a vaccine, or their cancer may comprise heterogeneous cell populations, some of which (over-)express the antigen and some of which do not. In addition, the likelihood of developing resistance is decreased when more multiple HLA-binding PEPIs are included or targeted by a vaccine/immunotherapy because a patient is less likely to develop resistance to the composition through mutation of the target PEPI(s).
The likelihood that a subject will respond to treatment is therefore increased by (i) the presence of more multiple HLA-binding PEPIs in the active ingredient polypeptides; (ii) the presence of PEPIs in more target polypeptide antigens; and (iii) (over-)expression of the target polypeptide antigens in the subject or in diseased cells of the subject. In some cases expression of the target polypeptide antigens in the subject may be known, for example if target polypeptide antigens are in a sample obtained from the subject. In other cases, the probability that a specific subject, or diseased cells of a specific subject, (over-)express a specific or any combination of target polypeptide antigens may be determined using population expression frequency data. The population expression frequency data may relate to a subject- and/or disease-matched population or the intent-to-treat population. For example, the frequency or probability of expression of a particular cancer-associated antigen in a particular cancer or subject having a particular cancer, for example breast cancer, can be determined by detecting the antigen in tumor, e.g. breast cancer tumor samples. In some cases such expression frequencies may be determined from published figures and scientific publications. In some cases a method of the invention comprises a step of determining the expression frequency of a relevant target polypeptide antigen in a relevant population.
Disclosed is a range of pharmacodynamic biomarkers to predict the activity/effect of vaccines in individual human subjects as well as in populations of human subjects. The biomarkers have been developed specifically for cancer vaccines, but similar biomarkers could be used for other vaccines or immunotherapy compositions. These biomarkers expedite more effective vaccine development and also decrease the development cost and may be used to assess and compare different compositions. Exemplary biomarkers are as follows.
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- AG95—potency of a vaccine: The number of antigens in a cancer vaccine that a specific tumor type expresses with 95% probability. AG95 is an indicator of the vaccine's potency, and is independent of the immunogenicity of the vaccine antigens. AG95 is calculated from the tumor antigen expression rate data. Such data may e obtained from experiments published in peer reviewed scientific journals. Technically, AG95 is determined from the binomial distribution of antigens in the vaccine, and takes into account all possible variations and expression rates.
- PEPI3+ count—immunogenicity of a vaccine in a subject: Vaccine-derived PEPI3+ are personal epitopes that bind to et least 3 HLAs of a subject and induce T cell responses. PEPI3+ can be determined using the PEPI3+ Test in subjects who's complete 4-digit HLA genotype is known.
- AP count—antigenicity of a vaccine in a subject: Number of vaccine antigens with PEPI3+. Vaccines contain sequences from target polypeptide antigens expressed by diseased cells. AP count is the number of antigens in the vaccine that contain PEPI3+, and the AP count represents the number of antigens in the vaccine that can induce T cell responses in a subject. AP count characterizes the vaccine-antigen specific T cell responses of the subject since it depends only on the HLA genotype of the subject and is independent of the subject's disease, age, and medication. The correct value is between 0 (no PEPI presented by the antigen) and maximum number of antigens (all antigens present PEPIs).
- AP50—antigenicity of a vaccine in a population: The mean number of vaccine antigens with a PEPI in a population. The AP50 is suitable for the characterization of vaccine-antigen specific T cell responses in a given population since it depends on the HLA genotype of subjects in a population.
- AGP count—effectiveness of a vaccine in a subject: Number of vaccine antigens expressed in the tumor with PEPI. The AGP count indicates the number of tumor antigens that vaccine recognizes and induces a T cell response against (hit the target). The AGP count depends on the vaccine-antigen expression rate in the subject's tumor and the HLA genotype of the subject. The correct value is between 0 (no PEPI presented by expressed antigen) and maximum number of antigens (all antigens are expressed and present a PEPI).
- AGP50—effectiveness of a cancer vaccine in a population: The mean number of vaccine antigens expressed in the indicated tumor with PEPI (i.e., AGP) in a population. The AGP50 indicates the mean number of tumor antigens that the T cell responses induced by the vaccine can recognize. AGP50 is dependent on the expression rate of the antigens in the indicated tumor type and the immunogenicity of the antigens in the target population. AGP50 can estimate a vaccine's effectiveness in different populations and can be used to compare different vaccines in the same population. The computation of AGP50 is similar to that used for AG50, except the expression is weighted by the occurrence of the PEPI3+ in the subject on the expressed vaccine antigens. In a theoretical population, where each subject has a PEPI from each vaccine antigen, the AGP50 will be equal to AG50. In another theoretical population, where no subject has a PEPI from any vaccine antigen, the AGP50 will be 0. In general, the following statement is valid: 0≤AGP50≤AG50.
- mAGP—a candidate biomarker for the selection of likely responders: Likelihood that a cancer vaccine induces T cell responses against multiple antigens expressed in the indicated tumor. mAGP is calculated from the expression rates of vaccine-antigens in e.g. the tumor and the presence of vaccine derived PEPIs in the subject. Technically, based on the AGP distribution, the mAGP is the sum of probabilities of the multiple AGP (≥2 AGPs).
The results of a prediction as set out above may be used to inform a physician's decisions concerning treatment of the subject. Accordingly, in some cases the polypeptide is an active ingredient, for example of a vaccine or immunotherapy composition, the method of the disclosure predicts that the subject will have, is likely to have, or has above a threshold minimum likelihood of having an immune response and/or a clinical response to a treatment comprising administering the active ingredient polypeptide to the subject, and the method further comprises selecting the treatment for or selecting the vaccine or immunotherapy composition for treatment of the specific human subject. Also provided is a method of treatment with a subject-specific pharmaceutical composition, kit or panel of polypeptides comprising one or more polypeptides as active ingredients, wherein the pharmaceutical composition, kit or panel of polypeptides has been determined to have a threshold minimum likelihood of inducing a clinical response in the subject, wherein the likelihood of response has been determined using a method described herein. In some cases the minimum threshold is defined by one or more of the pharmacodynamic biomarkers described herein, for example a minimum PEPI3+ count (for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more PEPI3+), a minimum AGP count (for example AGP=at least 2 or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more) and/or a minimum mAGP (for example AGP=at least 2 or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more). For example, in some cases a subject is selected for treatment if their likelihood of a response targeted at a predefined number of target polypeptide antigens, optionally wherein the target polypeptide antigens are (predicted to be) expressed, is above a predetermined threshold (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more). Alternatively, the method may predict that the one or more polypeptide(s) of the composition will not elicit a T cell response and/or a clinical response in the subject and further comprise selecting a different treatment for the specific human subject.
Predicting an Autoimmune or Toxic Immune Response to a Polypeptide AntigenThe differences among HLAs may influence the probability of developing an autoimmune disease, condition or response. In some cases the method of the disclosure may be used to identify a polypeptide or a fragment of a polypeptide that is immunogenic and/or associated with an auto-immune disorder or response. In some cases, the method comprises determining whether a polypeptide comprises an amino acid sequence that is a T cell epitope capable of binding to at least three, or at least four, or at least five HLA class I of a subject; or in other cases a sequence that is a T cell epitope capable of binding to at least four, or at least five, or at least six HLA class II of a subject; and identifying the polypeptide or said sequence as immunogenic or as being related to or associated with an auto-immune disorder or an auto-immune response in the subject.
The differences among HLAs may also influence the probability that a subject will experience immune-toxicity from a drug or polypeptide administered to the subject. There may be a toxic immune response if a polypeptide administered to the subject comprises a fragment that corresponds to a fragment of an antigen expressed in normal healthy cells of the subject and that comprises an amino acid that is a T cell epitope capable of binding to multiple HLA class I molecules of the subject. Therefore, in some cases in accordance with the disclosure, the method is used to identify a toxic immunogenic region or fragment of a polypeptide or to identify subjects who are likely to experience immune-toxicity in response to administration of one or more polypeptides or a fragments thereof. The polypeptide may be an active ingredient of a vaccine or immunotherapy composition.
The method may comprise determining whether the polypeptide(s) comprises a sequence that is a T cell epitope capable of binding to at least two, or in other cases to at least three HLA class I molecules of the subject. In some cases the method comprises determining that the polypeptide comprises a sequence that is a T cell epitope capable of binding to at least four, or at least five HLA class I molecules of the subject; or an amino acid sequence that is a T cell epitope capable of binding to at least four, or at least five, or at least six or at least seven HLA class II of the subject. The method may further comprise identifying said sequence as toxic immunogenic for the subject or predicting a toxic immune response in the subject. In other cases no such amino acid sequence is identified and the method further comprises predicting no toxic immune response in the subject. The method may further comprise selecting or recommending for treatment of the subject administration of one or more polypeptides or a pharmaceutical composition that is predicted to induce no or low immune-toxicity, and optionally further treating the subject by administering the polypeptide. The disclosure also provides a method of treating a subject in need thereof by administrating to the subject such a polypeptide or composition.
In some cases a method described herein further comprises mutating a polypeptide that is predicted to be immunogenic for a specific human subject, or that is predicted to be immunogenic in a proportion of subjects in a human population. Also provided is a method of reducing the immunogenicity of a polypeptide that has been identified as immunogenic in a specific human subject or in a proportion of a human population using any one of the methods described herein. The polypeptide may be mutated to reduce the number of PEPIs in the polypeptide or to reduce the number of HLA class I or class II molecules of the subject or of said population that bind to the fragment of the polypeptide that is identified as immunogenic in the subject or in a proportion of said population. In some cases the mutation may reduce or prevent a toxic immune response or may increase the efficacy by preventing the ADA development in the subject or in a proportion of said population. The mutated polypeptide may be further selected or recommended for treatment of the subject or of a subject of said population. The subject may further be treated by administration of the mutated polypeptide. The disclosure also provides a method of treating a subject in need thereof by administrating to the subject such a mutated polypeptide.
Predicting the Response of an Individual to Treatment with a Checkpoint Inhibitor
Typically some or all of the tumor specific T cell clones that are induced by a tumor are inactive or poorly functional in metastatic cancer patients. Inactive tumor specific T cells cannot kill the tumor cells. A fraction of these inactive T cells may be re-activated by checkpoint inhibitors (such as Ipilimumab), for example monoclonal antibodies that recognize checkpoint molecules (e.g. CTLA-4, PD-1, Lag-3, Tim-3, TIGIT, BTLA). According to the present disclosure, treating a subject with a checkpoint inhibitor will only be effective if or to the extent to which expressed cancer-antigens can be adequately recognised by the HLA of the individual, i.e. if there are epitopes in cancer- or disease-associated antigens that are recognised by multiple, preferably at least three, HLA class I molecules of the subject. Therefore, in some cases, the methods of the disclosure may be used to identify one or more or the subset of T cell clones that may be reactivated by a checkpoint inhibitor or to predict likely responders to checkpoint inhibitor (immuno)therapies.
Accordingly in some cases the disclosure provides a method of predicting whether a subject will respond to of cancer with a checkpoint inhibitor. In some cases the method comprises the step of identifying or selecting one or more polypeptides or polypeptide fragments that are associated with the disease or condition that is to be treated or that is associated with achieving an immune or clinical response to treatment with a checkpoint inhibitor. In some cases the polypeptide is a tumor-associated and/or mutational antigen. The polypeptide may be present in a sample obtained from the subject. The polypeptide may be one that is frequently (over-) expressed in a subject- and/or disease-matched population. The polypeptide may consist of or comprise a PEPI (or PEPI3+) identified in a subject that is known to have positively responded to a, or the, checkpoint inhibitor. The polypeptide may comprise or consist of an amino acid sequence that is stored or recorded in or retrieved from a database.
In some cases the method comprises determining whether the polypeptide(s) comprise a sequence that is a T cell epitope capable of binding to multiple HLA class I molecules of the subject. In some cases the presence of at least two, or at least three, or four or five or six or seven or eight different such amino acid sequences is determined, and/or the presence of such an amino acid sequence in at least two, or at least three, or four or five different target polypeptide antigens. In some cases the method comprises determining whether the polypeptide(s) comprise a sequence that is a T cell epitope capable of binding to at least two, or in some cases at least three or at least four HLA class II molecules of the subject. A response to treatment with the or a checkpoint inhibitor may be predicted if the above requirement(s) is met. No response or no clinical response may be predicted if the above requirement(s) is not met.
The disclosure also provides a method of identifying a fragment of a polypeptide or a T cell epitope in a polypeptide that may be targeted by the subject's immune response following treatment with a checkpoint inhibitor, or that will be targeted by T cells that are re-activated by treatment with a checkpoint inhibitor.
The method may further comprise selecting, recommending and/or administering a checkpoint inhibitor to a subject who is predicted to respond, or selecting, recommending and/or administering a different treatment to a subject that is predicted not to respond to a checkpoint inhibitor. In other cases the disclosure provides a method of treatment of a human subject in need thereof, the method comprising administering to the subject a checkpoint inhibitor, wherein the subject has been predicted to respond to administration of a checkpoint inhibitor by the method described herein.
Checkpoint inhibitors include, but are not limited to, PD-1 inhibitors, PD-L1 inhibitors, Lag-3 inhibitors, Tim-3 inhibitors, TIGIT inhibitors, BTLA inhibitors and CTLA-4 inhibitors, for example. Co-stimulatory antibodies deliver positive signals through immune-regulatory receptors including but not limited to ICOS, CD137, CD27 OX-40 and GITR. In one embodiment the checkpoint inhibitor is a CTLA-4 inhibitor.
Design and Preparation of Pharmaceutical Compositions for an Individual Human SubjectIn some aspects the disclosure provides a method of designing or preparing a polypeptide, or a polynucleic acid that encodes a polypeptide, for inducing an immune response, a cytotoxic T cell response or a helper T cell response in a specific human subject. The disclosure also provides a human subject-specific drug, immunogenic composition, or pharmaceutical composition, kit or panel of peptides, methods of designing or preparing the same, compositions that may be obtained by those methods, and their use in a method of inducing an immune response, a cytotoxic T cell response, or a helper T cell response in the subject, or a method of treating, vaccinating or providing immunotherapy to the subject. The pharmaceutical composition, kit or panel of peptides has as active ingredients one or more polypeptides that together comprising two or more T cell epitopes (PEPIs) capable of binding to multiple HLA class I or multiple HLA class II molecules of the subject that are immunogenic for the subject as described herein or that have been identified as immunogenic for the subject by a method described herein.
The composition/kit may optionally further comprise at least one pharmaceutically acceptable diluent, carrier, or preservative and/or additional polypeptides that do not comprise any PEPIs. The polypeptides may be engineered or non-naturally occurring. The kit may comprise one or more separate containers each containing one or more of the active ingredient peptides. The composition/kit may be a personalised medicine to prevent, diagnose, alleviate, treat, or cure a disease of an individual, such as a cancer.
Typically each PEPI is a fragment of a target polypeptide antigen and polypeptides that comprise one or more of the PEPIs are the target polypeptide antigens for the treatment, vaccination or immunotherapy. The method may comprise the step of identifying one or more suitable target polypeptide antigens. Typically each target polypeptide antigen will be associated with the same disease or condition, pathogenic organism or group of pathogenic organisms or virus, or type of cancer.
The composition, kit or panel may comprise, or the method may comprise selecting, for each PEPI a sequence of up to 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or 9 consecutive amino acids of the target polypeptide antigen, such as a polypeptide described herein, which consecutive amino acids comprise the amino acid sequence of the PEPI.
In some cases the amino acid sequence is flanked at the N and/or C terminus by additional amino acids that are not part of the consecutive sequence of the target polypeptide antigen. In some cases the sequence is flanked by up to 41 or 35 or 30 or 25 or 20 or 15 or 10, or 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 additional amino acid at the N and/or C terminus or between target polypeptide fragments. In other cases each polypeptide either consists of a fragment of a target polypeptide antigen, or consists of two or more such fragments arranged end to end (arranged sequentially in the peptide end to end) or overlapping in a single peptide (where two or more of the fragments comprise partially overlapping sequences, for example where two PEPIs in the same polypeptide are within 50 amino acids of each other).
When fragments of different polypeptides or from different regions of the same polypeptide are joined together in an engineered peptide there is the potential for neoepitopes to be generated around the join or junction. Such neoepitopes encompass at least one amino acid from each fragment on either side of the join or junction, and may be referred to herein as junctional amino acid sequences. The neoepitopes may induce undesired T cell responses against healthy cells (autoimmunity). The peptides may be designed, or the peptides may be screened, to avoid or eliminate neoepitopes that correspond to a fragment of a protein expressed in normal healthy human cells and/or neoepitopes that are capable of binding to at least two, or in some cases at least three, or at least four HLA class I molecules of the subject, or in some cases at least two, or at least three or four or five HLA class II molecules of the subject. The methods of the disclosure may be used to identify or screen for such neoepitopes as described herein. Alignment may be determined using known methods such as BLAST algorithms. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
The at least two multiple HLA-binding PEPIs of the composition polypeptides may both target a single antigen (e.g a polypeptide vaccine comprising two multiple HLA-binding PEPIs derived from a single antigen, for example a tumor associated antigen, targeted by the vaccine/immunotherapy) or may target different antigens (e.g. a polypeptide vaccine comprising one multiple HLA-binding PEPI derived from one antigen, e.g. a tumor associated antigen, and a second multiple HLA-binding PEPI derived from a different antigen, e.g. a different tumor associated antigen, both targeted by the vaccine/immunotherapy).
In some cases the active ingredient polypeptide(s) together comprise, or the method comprises selecting, a total of or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more different PEPIs. The PEPIs may be fragments of one or more different target polypeptide antigens. By identifying the specific fragments of each target polypeptide antigen that are immunogenic for a specific subject it is possible to incorporate multiple such fragments, optionally from multiple different target polypeptide antigens, in a single active ingredient polypeptide or multiple active ingredient polypeptides intended for use in combination or to maximise the number of T cell clones that can be activated by one or more polypeptides of a certain length.
Currently most vaccines and immunotherapy compositions target only a single polypeptide antigen. However according to the present disclosure it is in some cases beneficial to provide a pharmaceutical composition or an active ingredient polypeptide that targets two or more different polypeptide antigens. For example, most cancers or tumors are heterogeneous, meaning that different cancer or tumor cells of a subject (over-)express different antigens. The tumour cells of different cancer patients also express different combinations of tumour-associated antigens. The anti-cancer immunogenic compositions that are most likely to be effective are those that target multiple antigens expressed by the tumor, and therefore more cancer or tumor cells, in an individual human subject or in a population.
The beneficial effect of combining multiple PEPIs in a single treatment (administration of one or more pharmaceutical compositions that together comprise multiple PEPIs), can be illustrated by the personalised vaccine polypeptides described in Examples 17 and 18 below. Exemplary CTA expression probabilities in ovarian cancer are as follows: BAGE: 30%; MAGE A9: 37%; MAGE A4: 34%; MAGE A10: 52%. If patient XYZ were treated with a vaccine comprising PEPIs in only BAGE and MAGE A9, then the probability of having a mAGP (multiple expressed antigens with PEPI) would be 11%. If patent XYZ were treated with a vaccine comprising only PEPIs for the MAGE A4 and MAGE A10 CTAs, then the probability of having a multiAGP would be 19%. However if a vaccine contained all 4 of these CTAs (BAGE, MAGE A9, MAGE A4 and MAGE A10), then the probability of having a mAGP would be 50%. In other words the effect would be greater than the combined probabilities of mAGP for both two-PEPI treatments (probability mAGP for BAGE/MAGE+ probability mAGP for MAGE A4 and MAGE A10). Patient XYZ's PIT vaccine described in Example 17 contains a further 9 PEPIs, and thus, the probability of having a mAGP is over 99.95%.
Likewise exemplary CTA expression probabilities in breast cancer are as follows: MAGE C2: 21%; MAGE A1: 37%; SPC1: 38%; MAGE A9: 44%. Treatment of patient ABC with a vaccine comprising PEPIs in only MAGE C2: 21% and MAGE A1 has a mAGP probability of 7%. Treatment of patient ABC with a vaccine comprising PEPIs in only SPC1: 38%; MAGE A9 has a mAGP probability of 11%. Treatment of patient ABC with a vaccine comprising PEPIs in MAGE C2: 21%; MAGE A1: 37%; SPC1: 38%; MAGE A9 has a mAGP probability of 44% (44>7+11). Patient ABC's PIT vaccine described in Example 18 contains a further 8 PEPIs, and thus, the probability of having a mAGP is over 99.93%.
Accordingly in some cases the PEPIs of the active ingredient polypeptides are from two or more different target polypeptide antigens, for example different antigens associated with a specific disease or condition, for example different cancer- or tumor-associated antigens or antigens expressed by a target pathogen. In some cases the PEPIs are from a total of or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more different target polypeptide antigens. The different target polypeptide antigens may be any different polypeptides that it is useful to target or that can be selectively targeted with different PEPI3+s. In some cases different target polypeptide antigens are non-homologues or non-paralogues or have less than 95%, or 90%, or 85% or 80% or 75% or 70% or 60% or 50% sequence identity across the full length of each polypeptide. In some cases different polypeptides are those that do not share any PEPI3+s Alternatively, in some cases the PEPI3+s are from different target polypeptide antigens when they are not shared with other polypeptide antigens targeted by the active ingredient polypeptides.
In some cases one or more or each of the immunogenic polypeptide fragments is from a polypeptide that is present in a sample taken from the specific human subject. This indicates that the polypeptide is expressed in the subject, for example a cancer- or tumor-associated antigen or a cancer testis antigen expressed by cancer cells of the subject. In some cases one or more or each of the polypeptides is a mutational neoantigen, or an expressional neoantigen of the subject. One or more or each fragment may comprise a neoantigen specific mutation. Since mutational neoantigens are subject specific, a composition that targets one or more neoantigen specific mutations is personalised with regard to both their specific disease and their specific HLA set.
In other cases one or more or each of the immunogenic polypeptide fragments is from a target polypeptide antigen that is not generally expressed or is minimally expressed in normal healthy cells or tissue, but is expressed in a high proportion of (with a high frequency in) subjects or in the diseased cells of a subject having a particular disease or condition, as described above. The method my comprise identifying or selecting such a target polypeptide antigen. In some cases two or more or each of the immunogenic polypeptide fragments/PEPIs are from different cancer- or tumor-associated antigens that are each (over-)expressed with a high frequency in subjects having a type of cancer or a cancer derived from a particular cell type or tissue. In some cases the immunogenic polypeptide fragments are from a total of or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 different cancer- or tumor-associated polypeptides. In some cases one or more or each or at least one, at least two, at least three, at least four, at least five or at least six or at least seven of the polypeptides are selected from the antigens listed in any one of Tables 2 to 7.
In some cases one or more or each of the target polypeptide antigens is a cancer testis antigen (CTA). In some cases the immunogenic polypeptide fragments/PEPIs are from at least 1, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 CTAs, or from a total of 3 or more different target polypeptide antigens, optionally wherein 1, 2, or all three or at least three are CTAs, or from 4 or more different polypeptide antigens, optionally wherein 1, 2, 3 or all four or at least 1, 2, 3 or 4 are CTAs, or from 5 or more different polypeptide antigens, optionally wherein 1, 2, 3, 4 or all five or at least 1, 2, 3, 4, or 5 are CTAs, or from 6 or more different polypeptide antigens, optionally wherein 1, 2, 3, 4, 5 or all six or at least 1, 2, 3, 4, 5, or 6 are CTAs, or from 7 or more different polypeptide antigens, optionally wherein 1, 2, 3, 4, 5, 6 or all 7 or at least 1, 2, 3, 4, 5, 6 or 7 are CTAs, or from 8 or more different polypeptide antigens, optionally wherein 1, 2, 3, 4, 5, 6, 7 or all 8 or at least 1, 2, 3, 4, 5, 6, 7 or 8 are CTAs. In some cases one or more or each of the target polypeptide antigens is expressed by a bacteria, a virus, or a parasite.
In some cases one or more of the polypeptide fragments comprises an amino acid sequence that is a T cell epitope capable of binding to at least two, or at least three HLA class I of the subject and one or more of the polypeptide fragments comprises an amino acid sequence that is a T cell epitope capable of binding to at least two, or at least three, or at least four HLA class II of the subject, wherein the HLA class I and HLA class II binding fragments may optionally overlap. A composition prepared by such a method may elicit both a cytotoxic T cell response and a helper T cell response in the specific human subject.
Immunogenic and Pharmaceutical Compositions, Methods of Treatment and Modes of AdministrationIn some aspects the disclosure relates to a pharmaceutical composition, kit, or panels of polypeptides as described above having one or more polypeptides as active ingredient(s). These may be for use in a method of inducing an immune response, treating, vaccinating or providing immunotherapy to a subject, and the pharmaceutical composition may be a vaccine or immunotherapy composition. Such a treatment comprises administering one or more polypeptides or pharmaceutical compositions that together comprise all of the active ingredient polypeptides of the treatment to the subject. Multiple polypeptides or pharmaceutical compositions may be administered together or sequentially, for example all of the pharmaceutical compositions or polypeptides may be administered to the subject within a period of 1 year, or 6 months, or 3 months, or 60 or 50 or 40 or 30 days.
The immunogenic or pharmaceutical compositions or kits described herein may comprise, in addition to one or more immunogenic peptides, a pharmaceutically acceptable excipient, carrier, diluent, buffer, stabiliser, preservative, adjuvant or other materials well known to those skilled in the art. Such materials are preferably non-toxic and preferably do not interfere with the pharmaceutical activity of the active ingredient(s). The pharmaceutical carrier or diluent may be, for example, water containing solutions. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intradermal, and intraperitoneal routes.
The pharmaceutical compositions of the disclosure may comprise one or more “pharmaceutically acceptable carriers”. These are typically large, slowly metabolized macromolecules such as proteins, saccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose (Paoletti et al., 2001, Vaccine, 19:2118), trehalose (WO 00/56365), lactose and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. The pharmaceutical compositions may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. Sterile pyrogen-free, phosphate buffered physiologic saline is a typical carrier (Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20th edition, ISBN:0683306472).
The pharmaceutical compositions of the disclosure may be lyophilized or in aqueous form, i.e. solutions or suspensions. Liquid formulations of this type allow the compositions to be administered direct from their packaged form, without the need for reconstitution in an aqueous medium, and are thus ideal for injection. The pharmaceutical compositions may be presented in vials, or they may be presented in ready filled syringes. The syringes may be supplied with or without needles. A syringe will include a single dose, whereas a vial may include a single dose or multiple doses.
Liquid formulations of the disclosure are also suitable for reconstituting other medicaments from a lyophilized form. Where a pharmaceutical composition is to be used for such extemporaneous reconstitution, the disclosure provides a kit, which may comprise two vials, or may comprise one ready-filled syringe and one vial, with the contents of the syringe being used to reconstitute the contents of the vial prior to injection.
The pharmaceutical compositions of the disclosure may include an antimicrobial, particularly when packaged in a multiple dose format. Antimicrobials may be used, such as 2-phenoxyethanol or parabens (methyl, ethyl, propyl parabens). Any preservative is preferably present at low levels. Preservative may be added exogenously and/or may be a component of the bulk antigens which are mixed to form the composition (e.g. present as a preservative in pertussis antigens).
The pharmaceutical compositions of the disclosure may comprise detergent e.g. Tween (polysorbate), DMSO (dimethyl sulfoxide), DMF (dimethylformamide). Detergents are generally present at low levels, e.g. <0.01%, but may also be used at higher levels, e.g. 0.01-50%.
The pharmaceutical compositions of the disclosure may include sodium salts (e.g. sodium chloride) and free phosphate ions in solution (e.g. by the use of a phosphate buffer).
In certain embodiments, the pharmaceutical composition may be encapsulated in a suitable vehicle either to deliver the peptides into antigen presenting cells or to increase the stability. As will be appreciated by a skilled artisan, a variety of vehicles are suitable for delivering a pharmaceutical composition of the disclosure. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers and other phospholipid-containing systems. Methods of incorporating pharmaceutical compositions into delivery vehicles are known in the art.
In order to increase the immunogenicity of the composition, the pharmacological compositions may comprise one or more adjuvants and/or cytokines.
Suitable adjuvants include an aluminum salt such as aluminum hydroxide or aluminum phosphate, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, or may be cationically or anionically derivatised saccharides, polyphosphazenes, biodegradable microspheres, monophosphoryl lipid A (MPL), lipid A derivatives (e.g. of reduced toxicity), 3-O-deacylated MPL [3D-MPL], quil A, Saponin, QS21, Freund's Incomplete Adjuvant (Difco Laboratories, Detroit, Mich.), Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.), AS-2 (Smith-Kline Beecham, Philadelphia, Pa.), CpG oligonucleotides, bio adhesives and mucoadhesives, microparticles, liposomes, polyoxyethylene ether formulations, polyoxyethylene ester formulations, muramyl peptides or imidazoquinolone compounds (e.g. imiquamod and its homologues). Human immunomodulators suitable for use as adjuvants in the disclosure include cytokines such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc), macrophage colony stimulating factor (M-CSF), tumour necrosis factor (TNF), granulocyte, macrophage colony stimulating factor (GM-CSF) may also be used as adjuvants.
In some embodiments, the compositions comprise an adjuvant selected from the group consisting of Montanide ISA-51 (Seppic, Inc., Fairfield, N.J., United States of America), QS-21 (Aquila Biopharmaceuticals, Inc., Lexington, Mass., United States of America), GM-CSF, cyclophosamide, bacillus Calmette-Guerin (BCG), corynbacterium parvum, levamisole, azimezone, isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins (KLH), Freunds adjuvant (complete and incomplete), mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, diphtheria toxin (DT).
By way of example, the cytokine may be selected from the group consisting of a transforming growth factor (TGF) such as but not limited to TGF-α and TGF-β; insulin-like growth factor-I and/or insulin-like growth factor-II; erythropoietin (EPO); an osteoinductive factor; an interferon such as but not limited to interferon-.α, -β, and -γ; a colony stimulating factor (CSF) such as but not limited to macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF). In some embodiments, the cytokine is selected from the group consisting of nerve growth factors such as NGF-β; platelet-growth factor; a transforming growth factor (TGF) such as but not limited to TGF-α. and TGF-β; insulin-like growth factor-I and insulin-like growth factor-II; erythropoietin (EPO); an osteoinductive factor; an interferon (IFN) such as but not limited to IFN-α, IFN-β, and IFN-γ; a colony stimulating factor (CSF) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); an interleukin (I1) such as but not limited to IL-1, IL-1.alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18; LIF; kit-ligand or FLT-3; angiostatin; thrombospondin; endostatin; a tumor necrosis factor (TNF); and LT.
It is expected that an adjuvant or cytokine can be added in an amount of about 0.01 mg to about 10 mg per dose, preferably in an amount of about 0.2 mg to about 5 mg per dose. Alternatively, the adjuvant or cytokine may be at a concentration of about 0.01 to 50%, preferably at a concentration of about 2% to 30%.
In certain aspects, the pharmaceutical compositions of the disclosure are prepared by physically mixing the adjuvant and/or cytokine with the PEPIs under appropriate sterile conditions in accordance with known techniques to produce the final product.
Examples of suitable compositions of polypeptide fragments and methods of administration are provided in Esseku and Adeyeye (2011) and Van den Mooter G. (2006). Vaccine and immunotherapy composition preparation is generally described in Vaccine Design (“The subunit and adjuvant approach” (eds Powell M. F. & Newman M. J. (1995) Plenum Press New York). Encapsulation within liposomes, which is also envisaged, is described by Fullerton, U.S. Pat. No. 4,235,877.
In some embodiments, the compositions disclosed herein are prepared as a nucleic acid vaccine. In some embodiments, the nucleic acid vaccine is a DNA vaccine. In some embodiments, DNA vaccines, or gene vaccines, comprise a plasmid with a promoter and appropriate transcription and translation control elements and a nucleic acid sequence encoding one or more polypeptides of the disclosure. In some embodiments, the plasmids also include sequences to enhance, for example, expression levels, intracellular targeting, or proteasomal processing. In some embodiments, DNA vaccines comprise a viral vector containing a nucleic acid sequence encoding one or more polypeptides of the disclosure. In additional aspects, the compositions disclosed herein comprise one or more nucleic acids encoding peptides determined to have immunoreactivity with a biological sample. For example, in some embodiments, the compositions comprise one or more nucleotide sequences encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more peptides comprising a fragment that is a T cell epitope capable of binding to at least three HLA class I molecules and/or at least three HLA class II molecules of a patient. In some embodiments, the peptides are derived from an antigen that is expressed in cancer. In some embodiments the DNA or gene vaccine also encodes immunomodulatory molecules to manipulate the resulting immune responses, such as enhancing the potency of the vaccine, stimulating the immune system or reducing immunosuppression. Strategies for enhancing the immunogenicity of DNA or gene vaccines include encoding of xenogeneic versions of antigens, fusion of antigens to molecules that activate T cells or trigger associative recognition, priming with DNA vectors followed by boosting with viral vector, and utilization of immunomodulatory molecules. In some embodiments, the DNA vaccine is introduced by a needle, a gene gun, an aerosol injector, with patches, via microneedles, by abrasion, among other forms. In some forms the DNA vaccine is incorporated into liposomes or other forms of nanobodies. In some embodiments, the DNA vaccine includes a delivery system selected from the group consisting of a transfection agent; protamine; a protamine liposome; a polysaccharide particle; a cationic nanoemulsion; a cationic polymer; a cationic polymer liposome; a cationic nanoparticle; a cationic lipid and cholesterol nanoparticle; a cationic lipid, cholesterol, and PEG nanoparticle; a dendrimer nanoparticle. In some embodiments, the DNA vaccines is administered by inhalation or ingestion. In some embodiments, the DNA vaccine is introduced into the blood, the thymus, the pancreas, the skin, the muscle, a tumor, or other sites.
In some embodiments, the compositions disclosed herein are prepared as an RNA vaccine. In some embodiments, the RNA is non-replicating mRNA or virally derived, self-amplifying RNA. In some embodiments, the non-replicating mRNA encodes the peptides disclosed herein and contains 5′ and 3′ untranslated regions (UTRs). In some embodiments, the virally derived, self-amplifying RNA encodes not only the peptides disclosed herein but also the viral replication machinery that enables intracellular RNA amplification and abundant protein expression. In some embodiments, the RNA is directly introduced into the individual. In some embodiments, the RNA is chemically synthesized or transcribed in vitro. In some embodiments, the mRNA is produced from a linear DNA template using a T7, a T3, or an Sp6 phage RNA polymerase, and the resulting product contains an open reading frame that encodes the peptides disclosed herein, flanking UTRs, a 5′ cap, and a poly(A) tail. In some embodiments, various versions of 5′ caps are added during or after the transcription reaction using a vaccinia virus capping enzyme or by incorporating synthetic cap or anti-reverse cap analogues. In some embodiments, an optimal length of the poly(A) tail is added to mRNA either directly from the encoding DNA template or by using poly(A) polymerase. The RNA encodes one or more peptides comprising a fragment that is a T cell epitope capable of binding to at least three HLA class I and/or at least three HLA class II molecules of a patient. In some embodiments, the fragments are derived from an antigen that is expressed in cancer. In some embodiments, the RNA includes signals to enhance stability and translation. In some embodiments, the RNA also includes unnatural nucleotides to increase the half-life or modified nucleosides to change the immunostimulatory profile. In some embodiments, the RNAs is introduced by a needle, a gene gun, an aerosol injector, with patches, via microneedles, by abrasion, among other forms. In some forms the RNA vaccine is incorporated into liposomes or other forms of nanobodies that facilitate cellular uptake of RNA and protect it from degradation. In some embodiments, the RNA vaccine includes a delivery system selected from the group consisting of a transfection agent; protamine; a protamine liposome; a polysaccharide particle; a cationic nanoemulsion; a cationic polymer; a cationic polymer liposome; a cationic nanoparticle; a cationic lipid and cholesterol nanoparticle; a cationic lipid, cholesterol, and PEG nanoparticle; a dendrimer nanoparticle; and/or naked mRNA; naked mRNA with in vivo electroporation; protamine-complexed mRNA; mRNA associated with a positively charged oil-in-water cationic nanoemulsion; mRNA associated with a chemically modified dendrimer and complexed with polyethylene glycol (PEG)-lipid; protamine-complexed mRNA. in a PEG-lipid nanoparticle; mRNA associated with a cationic polymer such as polyethylenimine (PEI); mRNA associated with a cationic polymer such as PEI and a lipid component; mRNA associated with a polysaccharide (for example, chitosan) particle or gel; mRNA in a cationic lipid nanoparticle (for example, 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP) or dioleoylphosphatidylethanolamine (DOPE) lipids); mRNA complexed with cationic lipids and cholesterol; or mRNA complexed with cationic lipids, cholesterol and PEG-lipid. In some embodiments, the RNA vaccine is administered by inhalation or ingestion. In some embodiments, the RNA is introduced into the blood, the thymus, the pancreas, the skin, the muscle, a tumor, or other sites, and/or by an intradermal, intramuscular, subcutaneous, intranasal, intranodal, intravenous, intrasplenic, intratumoral or other delivery route.
Polynucleotide or oligonucleotide components may be naked nucleotide sequences, or be in combination with cationic lipids, polymers or targeting systems. They may be delivered by any available technique. For example, the polynucleotide or oligonucleotide may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the polynucleotide or oligonucleotide may be delivered directly across the skin using a delivery device such as particle-mediated gene delivery. The polynucleotide or oligonucleotide may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, or intrarectal administration.
Uptake of polynucleotide or oligonucleotide constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents. Examples of these agents include cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectam and transfectam. The dosage of the polynucleotide or oligonucleotide to be administered can be altered.
Administration is typically in a “prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to result in a clinical response or to show clinical benefit to the individual, e.g. an effective amount to prevent or delay onset of the disease or condition, to ameliorate one or more symptoms, to induce or prolong remission, or to delay relapse or recurrence.
The dose may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the individual to be treated; the route of administration; and the required regimen. The amount of antigen in each dose is selected as an amount which induces an immune response. A physician will be able to determine the required route of administration and dosage for any particular individual. The dose may be provided as a single dose or may be provided as multiple doses, for example taken at regular intervals, for example 2, 3 or 4 doses administered hourly. Typically peptides, polynucleotides or oligonucleotides are typically administered in the range of 1 pg to 1 mg, more typically 1 pg to 10 μg for particle mediated delivery and 1 μg to 1 mg, more typically 1-100 μg, more typically 5-50 μg for other routes. Generally, it is expected that each dose will comprise 0.01-3 mg of antigen. An optimal amount for a particular vaccine can be ascertained by studies involving observation of immune responses in subjects.
Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.
In some cases in accordance with the disclosure, more than one peptide or composition of peptides is administered. Two or more pharmaceutical compositions may be administered together/simultaneously and/or at different times or sequentially. Thus, the disclosure includes sets of pharmaceutical compositions and uses thereof. The use of combination of different peptides, optionally targeting different antigens, is important to overcome the challenges of genetic heterogeneity of tumors and HLA heterogeneity of individuals. Multiple pharmaceutical compositions of PEPIs, manufactured for use in one regimen, may define a drug product.
Routes of administration include but are not limited to intranasal, oral, subcutaneous, intradermal, and intramuscular. The subcutaneous administration is particularly preferred. Subcutaneous administration may for example be by injection into the abdomen, lateral and anterior aspects of upper arm or thigh, scapular area of back, or upper ventrodorsal gluteal area.
The skilled artisan will recognize that compositions of the disclosure may also be administered in one, or more doses, as well as, by other routes of administration. For example, such other routes include, intracutaneously, intravenously, intravascularly, intraarterially, intraperitnoeally, intrathecally, intratracheally, intracardially, intralobally, intramedullarly, intrapulmonarily, and intravaginally. Depending on the desired duration of the treatment, the compositions according to the disclosure may be administered once or several times, also intermittently, for instance on a monthly basis for several months or years and in different dosages.
Solid dosage forms for oral administration include capsules, tablets, caplets, pills, powders, pellets, and granules. In such solid dosage forms, the active ingredient is ordinarily combined with one or more pharmaceutically acceptable excipients, examples of which are detailed above. Oral preparations may also be administered as aqueous suspensions, elixirs, or syrups. For these, the active ingredient may be combined with various sweetening or flavoring agents, coloring agents, and, if so desired, emulsifying and/or suspending agents, as well as diluents such as water, ethanol, glycerin, and combinations thereof.
One or more compositions of the disclosure may be administered, or the methods and uses for treatment according to the disclosure may be performed, alone or in combination with other pharmacological compositions or treatments, for example chemotherapy and/or immunotherapy and/or vaccine. The other therapeutic compositions or treatments may for example be one or more of those discussed herein, and may be administered either simultaneously or sequentially with (before or after) the composition or treatment of the disclosure.
In some cases the treatment may be administered in combination with checkpoint blockade therapy/checkpopint inhibitors, co-stimulatory antibodies, cytotoxic or non-cytotoxic chemotherapy and/or radiotherapy, targeted therapy or monoclonal antibody therapy. It has been demonstrated that chemotherapy sensitizes tumors to be killed by tumor specific cytotoxic T cells induced by vaccination (Ramakrishnan et al. J Clin Invest. 2010; 120(4):1111-1124). Examples of chemotherapy agents include alkylating agents including nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; anthracyclines; epothilones; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin); triazenes such as decarbazine (DTIC; dimethyltriazenoimidazole-carboxamide; ethylenimines/methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulfonates such as busulfan; Antimetabolites including folic acid analogues such as methotrexate (amethopterin); alkylating agents, antimetabolites, pyrimidine analogs such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); purine analogues and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and pentostatin (2′-deoxycoformycin); epipodophylotoxins; enzymes such as L-asparaginase; biological response modifiers such as IFNα, IL-2, G-CSF and GM-CSF; platinum coordination complexes such as cisplatin (cis-DDP), oxaliplatin and carboplatin; anthracenediones such as mitoxantrone and anthracycline; substituted urea such as hydroxyurea; methylhydrazine derivatives including procarbazine (N-methylhydrazine, MIH) and procarbazine; adrenocortical suppressants such as mitotane (o,p′-DDD) and aminoglutethimide; taxol and analogues/derivatives; hormones/hormonal therapy and agonists/antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide, progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate, estrogen such as diethylstilbestrol and ethinyl estradiol equivalents, antiestrogen such as tamoxifen, androgens including testosterone propionate and fluoxymesterone/equivalents, antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide and non-steroidal antiandrogens such as flutamide; natural products including vinca alkaloids such as vinblastine (VLB) and vincristine, epipodophyllotoxins such as etoposide and teniposide, antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C), enzymes such as L-asparaginase, and biological response modifiers such as interferon alphenomes.
In some cases the method of treatment is a method of vaccination or a method of providing immunotherapy. As used herein, “immunotherapy” is the treatment of a disease or condition by inducing or enhancing an immune response in an individual. In certain embodiments, immunotherapy refers to a therapy that comprises the administration of one or more drugs to an individual to elicit T cell responses. In a specific embodiment, immunotherapy refers to a therapy that comprises the administration or expression of polypeptides that contain one or more PEPIs to an individual to elicit a T cell response to recognize and kill cells that display the one or more PEPIs on their cell surface in conjunction with a class I HLA. In another specific embodiment, immunotherapy comprises the administration of one or more PEPIs to an individual to elicit a cytotoxic T cell response against cells that display tumor associated antigens (TAAs) or cancer testis antigens (CTAs) comprising the one or more PEPIs on their cell surface. In another embodiment, immunotherapy refers to a therapy that comprises the administration or expression of polypeptides that contain one or more PEPIs presented by class II HLAs to an individual to elicit a T helper response to provide co-stimulation to cytotoxic T cells that recognize and kill diseased cells that display the one or more PEPIs on their cell surface in conjunction with a class I HLAs. In still another specific embodiment, immunotherapy refers to a therapy that comprises administration of one or more drugs to an individual that re-activate existing T cells to kill target cells. The theory is that the cytotoxic T cell response will eliminate the cells displaying the one or more PEPIs, thereby improving the clinical condition of the individual. In some instances, immunotherapy may be used to treat tumors. In other instances, immunotherapy may be used to treat intracellular pathogen-based diseases or disorders.
In some cases the disclosure relates to the treatment of cancer or the treatment of solid tumors. The treatment may be of cancers or malignant or benign tumors of any cell, tissue, or organ type. The cancer may or may not be metastatic. Exemplary cancers include carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, or blastomas. The cancer may or may not be a hormone related or dependent cancer (e.g., an estrogen or androgen related cancer).
In other cases the disclosure relates to the treatment of a viral, bacterial, fungal or parasitic infection, or any other disease or condition that may be treated by immunotherapy.
SystemsThe disclosure provides a system comprising a storage module configured to store data comprising the class I and/or class II HLA genotype of a subject and the amino acid sequence of one or more test polypeptides; and a computation module configured to identify and/or quantify amino acid sequences in the one or more test polypeptides that are capable of binding to multiple HLA of the subject. The system may be for obtaining data from at least one sample from at least one subject. The system may comprise a an HLA genotyping module for determining the class I and/or class II HLA genotype of a subject. The storage module may be configured to store the data output from the genotyping module. The HLA genotyping module may receive a biological sample obtained from the subject and determines the subject's class I and/or class II HLA genotype. The sample typically contains subject DNA. The sample may be, for example, a blood, serum, plasma, saliva, urine, expiration, cell or tissue sample. The system may further comprise an output module configured to display the sequence of one or more fragments of the one or more polypeptides that are predicted to be immunogenic for the subject, or any output prediction or treatment selection or recommendation described herein or the value of any pharmodynamic biomarker described herein.
Further Embodiments of the Disclosure1. A method of predicting whether a polypeptide or a fragment of a polypeptide is immunogenic for a specific human subject, the method comprising the steps of
-
- (i) determining whether the polypeptide comprises:
- (a) an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or
- (b) an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class II molecules of the subject; and
- (ii) predicting
- A. that the polypeptide is immunogenic for the subject if the polypeptide comprises at least one sequence that meets the requirements of step (i); or
- B. that the polypeptide is not immunogenic for the subject if the polypeptide does not comprise at least one sequence that meets the requirements of step (i)
- (i) determining whether the polypeptide comprises:
2. A method of identifying a fragment of a polypeptide as immunogenic for a specific human subject, the method comprising the steps of
-
- (i) determining that the polypeptide comprises:
- (a) an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or
- (b) an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class II molecules of the subject; and
- (ii) identifying said sequence as a fragment of the polypeptide that is immunogenic for the subject.
- (i) determining that the polypeptide comprises:
3. The method of item 1 or item 2, wherein the T cell epitope is capable of binding to at least two HLA class I molecules of the subject and consists of 9 consecutive amino acids of the polypeptide, or wherein the T cell epitope is capable of binding to at least two HLA class II molecules of the subject and consists of 15 consecutive amino acids of the polypeptide.
4. The method of any one of the preceding items, wherein step (i) comprises determining that the polypeptide comprises an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules of the subject.
5. The method of any one of the preceding items, wherein step (i) comprises determining that the polypeptide comprises an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject.
6. The method of any one of items 1 to 3, wherein step (i) comprises determining that the polypeptide comprises an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class II molecules of the subject.
7. The method of item 4 or item 5 further comprising identifying a fragment of the polypeptide that is a T cell epitope capable of binding to at least one HLA class II molecule of the subject, wherein the HLA class II-binding epitope comprises the amino acid sequence of the HLA class I-binding T cell epitope.
8. The method of any one of the preceding items, wherein the polypeptide is expressed by a pathogenic organism, a virus or a cancer cell, is associated with an autoimmune disorder, or is an allergen or an ingredient of a pharmaceutical composition.
9. The method of any one of the preceding items, wherein the polypeptide is selected from the antigens listed in Tables 2 to 6.
10. The method of any one of the preceding items, wherein the polypeptide is an antigen or neoantigen expressed by a cancer cell, optionally wherein the cancer cell, the antigen or the neoantigen is in a sample taken from the subject.
11. The method of any one of the preceding items, wherein the polypeptide is a mutational neoantigen, optionally wherein
-
- (a) the neoantigen is present in a sample obtained from the subject; and/or
- (b) the immunogenic fragment comprises a neoantigen specific mutation.
12. The method of any one of items 1 to 11, wherein all of the fragments of the polypeptide that are a T cell epitope capable of binding to at least two HLA class I molecules and/or all of the fragments of the polypeptide that are a T cell epitope capable of binding to at least two HLA class II molecules of the subject are identified, optionally wherein the method is repeated for each polypeptide that is an active ingredient of a specific pharmaceutical composition.
13. The method of any one of the preceding items, further comprising predicting whether the subject will have a cytotoxic T cell response or a helper T cell response to administration of one or more polypeptide or a pharmaceutical composition or kit comprising one or more polypeptides as active ingredients, wherein
-
- A. a cytotoxic T cell response is predicted if the polypeptide(s) comprises at least one amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject;
- B. a helper T cell response is predicted if the polypeptide(s) comprises at least one amino acid sequence that is a T cell epitope capable of binding to at least three HLA class II molecules of the subject;
- C. no cytotoxic T cell response is predicted if the polypeptide(s) does not comprise any amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; or
- D. no helper T cell response is predicted if the polypeptide(s) does not comprise any amino acid sequence that is a T cell epitope capable of binding to at least three HLA class II molecules of the subject.
14. The method of item 13, wherein the subject is predicted to have a cytotoxic T cell and/or a helper T cell response, and the method further comprises determine the likelihood that the subject will have a cytotoxic T cell response and/or a helper T cell response that targets a polypeptide antigen that is expressed in the subject, the method comprising
-
- (i) identifying one or more polypeptide antigens that comprises an amino acid sequence that is
- (a) a T cell epitope capable of binding to at least three HLA class I or at least three HLA class II molecules of the subject; and
- (b) comprised in the amino acid sequence of the polypeptide(s)
- (ii) using population expression frequency data for the one or more polypeptide antigens identified in step (i) to determine the likelihood that the subject will have a cytotoxic T cell response and/or a helper T cell response that targets a polypeptide antigen that is expressed in the subject.
- (i) identifying one or more polypeptide antigens that comprises an amino acid sequence that is
15. The method of item 13 wherein the polypeptide is a component of a pharmaceutical composition and the method comprises determining the likelihood that the subject will develop anti-drug antibodies (ADA) following administration of the polypeptide, wherein a predicted T helper cell response corresponds to a higher likelihood of ADA and no predicted T helper cell response corresponds to a lower likelihood of ADA.
16. The method of item 15, wherein the polypeptide is a checkpoint inhibitor.
17. The method of any one of items 1 to 14 further comprising predicting whether the subject will have a clinical response to administration of a pharmaceutical composition, kit or panel of polypeptides comprising one or more polypeptides as active ingredients, the method comprising determining whether the one or more active ingredient polypeptides together comprise at least two different amino acid sequences each of which is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; and predicting
-
- A. that the subject will have a clinical response to administration of the pharmaceutical composition, kit or panel of polypeptides if the one or more active ingredient polypeptides together comprise at least two different sequences each of which is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; or
- B. that the subject will not have a clinical response to administration of the pharmaceutical composition, kit or panel of polypeptides if the one or more active ingredient polypeptides together comprise no more than one sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject.
18. The method of item 17, wherein the at least two different amino acid sequences are comprised in the amino acid sequence of two different polypeptide antigens targeted by the active ingredient polypeptide(s).
19. The method of any one of items 1 to 14, 17 and 18, further comprising determining the likelihood that the specific human subject will have a clinical response to administration of a pharmaceutical composition, kit or panel of polypeptides comprising one or more polypeptides as active ingredients, wherein one or more of the following factors corresponds to a higher likelihood of a clinical response:
-
- (a) presence in the active ingredient polypeptide(s) of a higher number of amino acid sequences and/or different amino acid sequences that are each a T cell epitope capable of binding to at least three HLA class I of the subject;
- (b) a higher number of target polypeptide antigens, comprising at least one amino acid sequence that is both
- A. comprised in an active ingredient polypeptide; and
- B. a T cell epitope capable of binding to at least three HLA class I of the subject; optionally wherein the target polypeptide antigens are expressed in the subject, further optionally wherein the target polypeptides antigens are in one or more samples obtained from the subject;
- (c) a higher probability that the subject expresses target polypeptide antigens, optionally a threshold number of the target polypeptide antigens and/or optionally target polypeptide antigens that have been determined to comprise at least one amino acid sequence that is both
- A. comprised in an active ingredient polypeptide; and
- B. a T cell epitope capable of binding to at least three HLA class I of the subject; and/or
- (d) a higher number of target polypeptide antigens that the subject is predicted to express, optionally a higher number of target polypeptide antigens that the subject expresses with a threshold probability, and/or optionally the target polypeptide antigens that have been determined to comprise at least one amino acid sequence that is both
- A. comprised in an active ingredient polypeptide; and
- B. a T cell epitope capable of binding to at least three HLA class I of the subject.
20. The method of any one of items 1 to 14, and 17 to 19, comprising determining the likelihood that the specific human subject will have a clinical response to administration of a pharmaceutical composition, kit or panel of polypeptides comprising one or more polypeptides as active ingredients, wherein the method comprises
-
- (i) identifying which polypeptide antigens targeted by the active ingredient polypeptide(s) comprise an amino acid sequence that is both
- A. comprised in an active ingredient polypeptide; and
- B. a T cell epitope capable of binding to at least three HLA class I of the subject;
- (ii) using population expression data for each antigen identified in step (i) to determine the probability that the subject expresses one or more of the antigens identified in step (i) that together comprise at least two different amino acid sequences of step (i); and
- (iii) determining the likelihood that the subject will have a clinical response to administration of the pharmaceutical composition, kit or panel of polypeptides, wherein a higher probability determined in step (ii) corresponds to a more likely clinical response.
- (i) identifying which polypeptide antigens targeted by the active ingredient polypeptide(s) comprise an amino acid sequence that is both
21. The method of item 20, wherein step (ii) comprises using population expression data for each antigen identified in step (i) to determine the probability that the subject expresses two or more of the antigens identified in step (i) that together comprise at least two different amino acid sequences of step (i).
22. The method of item 21, wherein the at least two different amino acid sequences are comprised in the amino acid sequence of two different polypeptide antigens targeted by the active ingredient polypeptide(s).
23. The method of any one of items 19 to 22, wherein one or more of the following factors
further correspond to a higher likelihood of a clinical response:
-
- (a) presence in the active ingredient polypeptide(s) of a higher number of amino acid sequences and/or different amino acid sequences that are each a T cell epitope capable of binding to at least three HLA class II of the subject;
- (b) a higher number of target polypeptide antigens comprising at least one amino acid sequence that is both
- A. comprised in an active ingredient polypeptide; and
- B. a T cell epitope-capable of binding to at least three HLA class II of the subject, optionally wherein the target polypeptide antigens are expressed in the subject, optionally wherein the target polypeptides antigens are in one or more samples obtained from the subject;
- (c) a higher number of target polypeptide antigens comprising
- i. at least one amino acid sequence that is both
- A. comprised in an active ingredient polypeptide; and
- B. a T cell epitope capable of binding to at least three HLA class I of the subject; and
- ii. at least one amino acid sequence that is both
- A. comprised in an active ingredient polypeptide; and
- B. a T cell epitope capable of binding to at least three HLA class II of the subject;
- i. at least one amino acid sequence that is both
- (d) a higher probability that the subject expresses target polypeptide antigens, optionally a threshold number of the target polypeptide antigens, that have been determined to comprise at least one amino acid sequence that is both
- A. comprised in an active ingredient polypeptide; and
- B. a T cell epitope capable of binding to at least three HLA class II of the subject
- (e) a higher probability that the subject expresses target polypeptide antigens, optionally a threshold number of the target polypeptide antigens, that have been determined to comprise
- i. at least one amino acid sequence that is both
- A. comprised in an active ingredient polypeptide; and
- B. a T cell epitope capable of binding to at least three HLA class I of the subject; and
- ii. at least one amino acid sequence that is both
- A. comprised in an active ingredient polypeptide; and
- B. a T cell epitope capable of binding to at least three HLA class II of the subject;
- i. at least one amino acid sequence that is both
- (f) a higher number of target polypeptide antigens that the subject is predicted to express, optionally a higher number of target polypeptide antigens that the subject expresses with a threshold probability, and that have been determined to comprise at least one amino acid sequence that is both
- A. comprised in an active ingredient polypeptide; and
- B. a T cell epitope capable of binding to at least three HLA class II of the subject;
- and/or
- (g) a higher number of target polypeptide antigens that the subject is predicted to express, optionally a higher number of target polypeptide antigens that the subject expresses with a threshold probability, and that have been determined to comprise
- i. at least one amino acid sequence that is both
- A. comprised in an active ingredient polypeptide; and
- B. a T cell epitope capable of binding to at least three HLA class I of the subject; and
- ii. at least one amino acid sequence that is both
- A. comprised in an active ingredient polypeptide; and
- B. a T cell epitope capable of binding to at least three HLA class II of the subject.
- i. at least one amino acid sequence that is both
24. The method of any one of items 19 to 23, further comprising repeating the method for one or more further pharmaceutical compositions, kits or panels of polypeptides and ranking the compositions, kits or panels of polypeptides by their likelihood to induce a clinical response in the subject.
25. The method of any one of items 1 to 24, further comprising predicting whether administration of the polypeptide, pharmaceutical composition, kit or panel of polypeptides will induce a toxic immune response in the subject, wherein
-
- (a) the polypeptide(s) comprises at least one amino acid sequence that
- i. is capable of binding to at least three HLA class I of the subject; and
- ii. corresponds to a fragment of a human polypeptide expressed in healthy cells;
- and a toxic immune response is predicted; or
- (b) the polypeptide(s) do not comprise any amino acid sequence that
- A. is capable of binding to at least three HLA class I of the subject; and
- B. corresponds to a fragment of a human polypeptide expressed in healthy cells;
- and no toxic immune response is predicted.
- (a) the polypeptide(s) comprises at least one amino acid sequence that
26. The method of any one of the preceding items further comprising selecting or recommending for treatment of the specific human subject administration to the subject of a polypeptide that comprises a polypeptide fragment that is identified as immunogenic for the subject, or of a polypeptide that is predicted to be immunogenic, or to induce a cytotoxic T cell or helper T cell response, or of a pharmaceutical composition, kit or panel of polypeptides that is predicted to induce a clinical response, or of a polypeptide or pharmaceutical composition that is predicted not to induce a toxic immune response or not to induce ADA in the subject.
27. The method of item 26, further comprising administering one or more of the selected or recommended polypeptides or pharmaceutical compositions or the polypeptides of one or more kits or panels of polypeptides to the subject.
28. A method of treatment of a human subject in need thereof, the method comprising administering to the subject a polypeptide that comprises a polypeptide fragment that has been identified as immunogenic, or a polypeptide that has been predicted to be immunogenic, or a polypeptide or pharmaceutical composition that has been predicted to induce a cytotoxic T cell or helper T cell response, or a pharmaceutical composition, kit or panel of polypeptides that has been predicted to induce a clinical response, or a pharmaceutical composition, kit or panel of polypeptides that has been determined to have a threshold minimum likelihood of inducing a clinical response, or a polypeptide or pharmaceutical composition that is predicted not to induce a toxic immune response or ADA development in the subject using a method according to any one of items 1 to 23, or one or more polypeptides or pharmaceutical compositions that have been selected or recommended for treatment of the subject using a method according to item 26.
29. The method of any one of items 1 to 11, wherein the polypeptide is associated with or suspected of being associated with an autoimmune disorder or an autoimmune response in the subject and determining that the polypeptide comprises an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject identifies the polypeptide and/or the fragment as immunogenic or associated with the autoimmune disorder or autoimmune response in the subject.
30. The method of any one of items 1 to 12 further comprising predicting whether the subject will have a clinical response to administration of a checkpoint inhibitor to treat cancer, the method comprising determining whether one or more cancer associated antigens together comprise at least two different amino acid sequences each of which is a T cell epitope capable of binding to at least three HLA class I of the subject and predicting
-
- that the subject will have a clinical response to administration of a checkpoint inhibitor if the one or more cancer associated antigens together comprise at least two different sequences each of which is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; or
- B. that the subject will not have a clinical response to administration of a checkpoint inhibitor if the one or more cancer associated antigens together comprise no more than one sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject.
31. The method of any one of items 1 to 12, further comprising determining the likelihood that the subject will have a clinical response to administration of a checkpoint inhibitor to treat cancer, the method comprising
-
- (i) selecting a plurality of polypeptide antigens that are associated with the cancer type of the subject;
- (ii) identifying which of said cancer associated antigens comprise an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; and
- (iii) using population expression data for each cancer associated antigen identified in step to determine the likelihood that the subject will have a clinical response to administration of a checkpoint inhibitor to treat cancer, wherein a higher probability that the subject expresses one or more of the cancer associated antigens identified in step (ii) that together comprise at least two amino acid sequences each of which is a T cell epitope capable of binding to at least three HLA class I molecules of the subject corresponds to a more likely clinical response.
32. The method of item 30 or item 31 further comprising selecting or recommending administration of a checkpoint inhibitor for treatment of the subject.
33. The method of item 32 further comprising administering a checkpoint inhibitor to the subject.
34. A method of treatment of a human subject in need thereof, the method comprising administering to the subject a checkpoint inhibitor, wherein the subject has been predicted to respond, or to be likely to respond, to administration of a checkpoint inhibitor by a method according to item 30 or item 31.
35. The method of any one of items 13, 15 to 18 and 30, wherein the subject has been predicted to have a toxic immune response or ADA development, or not to have a cytotoxic T cell or helper T cell or clinical response, or not to respond to treatment with a checkpoint inhibitor and the method further comprises selecting or recommending a different treatment for the subject.
36. A method of designing or preparing a human subject-specific pharmaceutical composition or kit or panel of polypeptides for use in a method of treatment of a specific human subject, the method comprising:
-
- (i) selecting a fragment of a polypeptide, which fragment has been identified as immunogenic for the subject by the method of any one of items 2 to 11;
- (ii) if the fragment selected in step (i) is an HLA class I-binding epitope, optionally selecting a longer fragment of the polypeptide, which longer fragment
- a. comprises the fragment selected in step (i); and
- b. is a T cell epitope capable of binding at least three or to the most possible HLA class II molecules of the subject;
- (iii) selecting a first sequence of up to 50 consecutive amino acids of the polypeptide, which consecutive amino acids comprise the amino acid sequence of the fragment selected in step (i) or the longer fragment selected in step (ii);
- (iv) repeating steps (i) to (iii) to select a second amino acid sequence of up to 50 consecutive amino acids of the same or a different polypeptide to the first amino acid sequence;
- (v) optionally further repeating steps (i) to (iii) to select one or more additional amino acid sequences of up to 50 consecutive amino acids of the same or different polypeptides to the first and second amino acid sequences; and
- (vi) designing or preparing a subject-specific pharmaceutical composition, kit or panel of polypeptides having as active ingredients one or more polypeptides that together have all of the amino acid sequences selected in the preceding steps, optionally wherein one or more or each sequence is flanked at the N and/or C terminus by additional amino acids that are not part of the sequence of the polypeptides.
37. The method of item 36, wherein each polypeptide either consists of one of the selected amino acid sequences, or comprises or consists of two or more of the selected amino acid sequences arranged end to end or overlapping in a single peptide.
38. The method of item 37, wherein all of the neoepitopes formed at the join between any two of the selected amino acid sequences arranged end to end in a single polypeptide have been screened to eliminate polypeptides comprising a neoepitope amino acid sequence that
-
- (i) corresponds to a fragment of a human polypeptide expressed in healthy cells;
- (ii) is a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or
- (iii) meets both requirements (i) and (ii).
39. The method of any of items 36 to 38, wherein the one or more polypeptides have been screened to eliminate polypeptides comprising an amino acid sequence that
-
- (i) corresponds to a fragment of a human polypeptide expressed in healthy cells; or
- (ii) corresponds to a fragment of a human polypeptide expressed in healthy cells and is a T cell epitope capable of binding to at least two HLA class I molecules of the subject.
40. A human subject-specific pharmaceutical composition, kit or panel of polypeptides for use in a method of inducing an immune response in a specific human subject, and designed or prepared for the subject according to the method of any one of items 36 to 39, wherein the composition or kit optionally comprises at least one pharmaceutically acceptable diluent, carrier, or preservative.
41. A human subject-specific pharmaceutical composition, kit or panel of polypeptides for use in a method of treatment of a specific human subject in need thereof, the composition, kit or panel comprising as active ingredients a first and a second peptide and optionally one of more additional peptides, wherein each peptide comprises an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules and/or at least two HLA class II molecules of the subject, wherein the amino acid sequence of the T cell epitope of the first, second and optionally any additional peptides are different from each other, and wherein the pharmaceutical composition or kit optionally comprises at least one pharmaceutically acceptable diluent, carrier, or preservative.
42. A human subject-specific pharmaceutical composition, kit or panel of polypeptides for use in a method of treatment of a specific human subject in need thereof, the composition or kit comprising as an active ingredient a polypeptide comprising a first region and a second region and optionally one of more additional regions, wherein each region comprises an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules and/or at least two HLA class II molecules of the subject, wherein the amino acid sequence of the T cell epitope of the first, second and optionally any additional regions are different from each other, and wherein the pharmaceutical composition or kit optionally comprises at least one pharmaceutically acceptable diluent, carrier, or preservative.
43. The human subject-specific pharmaceutical composition, kit or panel of item 41 or item 42, wherein one or more or each of the peptides or regions comprises an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules of the subject.
44. The human subject-specific pharmaceutical composition, kit or panel of any one of items 41 to 43, wherein one or more or each of the peptides or regions comprises an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject.
45. The human subject-specific pharmaceutical composition, kit or panel of any one of items 41 to 44, wherein one or more or each of the peptides or regions comprises an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class II molecules of the subject.
46. The human subject-specific pharmaceutical composition, kit or panel of item 44 or item 45 wherein one or more or each of the peptides or regions comprises an amino acid sequence that is a T cell epitope capable of binding at least one HLA class II molecule of the subject, wherein the HLA class II-binding T cell epitope comprises an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules of the subject.
47. The human subject-specific pharmaceutical composition, kit or panel of any one of items 41 to 46, wherein one or more or each of the peptides or regions comprises a sequence of up to 50 consecutive amino acids of a polypeptide that is expressed by a pathogenic organism, a virus or a cancer cell, is associated with an autoimmune disorder, or is an allergen, wherein the sequence comprises the T cell epitope of the peptide or region that is capable of binding to at least two HLA class I or class II molecules of the subject, optionally wherein one or more or each of the polypeptide sequences is flanked at the N and/or C terminus by additional amino acids that are not part of the amino acid sequence of the polypeptide(s).
48. The human subject-specific pharmaceutical composition, kit or panel of any one of items 41 to 47, wherein one or more of the polypeptide(s) are selected from the antigens listed in Tables 2 to 6.
49. The human subject-specific pharmaceutical composition, kit or panel of any one of items 41 to 48, wherein the polypeptide(s) are antigens or neoantigens expressed by a cancer cell, optionally wherein the cancer cell is in a sample taken from the subject.
50. The human subject-specific pharmaceutical composition, kit or panel of any one of items 41 to 49, wherein the polypeptide(s) are mutational neoantigen(s), optionally wherein the neoantigen(s) are present in a sample obtained from the subject; and/or the T cell epitope(s) each comprise a neoantigen specific mutation.
51. The human subject-specific pharmaceutical composition, kit or panel of any one of items 47 to 50 wherein two or more or each of the polypeptide sequences of up to 50 consecutive amino acids are from different polypeptides.
52. The human subject-specific pharmaceutical composition, kit or panel of any one of items 47 to 51, wherein one or more or each of the sequences of up to 50 consecutive amino acids comprises an amino acid sequence that
-
- (a) comprises an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; and
- (b) is a T cell epitope capable of binding to at least three HLA class II molecules of the subject or to the most possible HLA class II molecules of the subject for a sequence comprising the HLA class I-binding epitope of (a).
53. The human subject-specific pharmaceutical composition, kit or panel of item 47 to 52 wherein one or more or each polypeptide either
-
- (a) consists of one of said sequence of up to 50 consecutive amino acids from a polypeptide that is expressed by a pathogenic organism, a virus or a cancer cell, is associated with an autoimmune disorder or is an allergen; or
- (b) comprises or consist of two or more of said sequences of up to 50 consecutive amino acids arranged end to end or overlapping in a single peptide.
54. The human subject-specific pharmaceutical composition, kit or panel of item 53 wherein the one or more peptides do not comprise any neoepitopes that span a join between any two of said amino acid sequences that are arranged end to end in a single peptide and that
-
- (i) corresponds to a fragment of a human polypeptide expressed in healthy cells;
- (ii) is a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or
- (iii) meets both requirements (i) and (ii).
55. The human subject-specific pharmaceutical composition, kit or panel of any of items 41 to 54 wherein the one or more polypeptides do not comprise any amino acid sequences that
-
- (i) corresponds to a fragment of a human polypeptide expressed in healthy cells; or
- (ii) corresponds to a fragment of a human polypeptide expressed in healthy cells and is a T cell epitope capable of binding to at least two HLA class I molecules of the subject.
56. A method of treatment comprising administering to a human subject in need thereof a human subject-specific pharmaceutical composition or the polypeptides of a kit or panel of polypeptides according to any one of items 41 to 55, wherein the pharmaceutical composition, kit or panel is specific for the subject, optionally wherein the method is for the treatment of cancer.
57. The method of treatment according to any one of items 28, 34 and 56 wherein the treatment is administered in combination with chemotherapy, targeted therapy or a checkpoint inhibitor.
58. A method of designing or preparing a polypeptide for inducing an immune response in a specific human subject the method comprising selecting an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules or at least three HLA class II molecules of the subject, and designing or preparing a polypeptide comprising the selected amino acid sequence.
59. The method of item 58, which is
-
- (a) a method of designing or preparing a polypeptide for inducing a cytotoxic T cell response in a specific human subject, the method comprising selecting an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject, and designing or preparing a polypeptide comprising the selected amino acid sequence; or
- (b) a method of designing or preparing a polypeptide for inducing a helper T cell response, the method comprising selecting an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class II molecules of the subject, and designing or preparing a polypeptide comprising the selected amino acid sequence.
60. The method of item 58 or claim 59 further comprising administering the polypeptide to the subject.
61. A method of inducing an immune response in a subject, the method comprising administering to the subject a polypeptide designed according to the method of item 58 or item 59.
62. A method of inducing an immune response in a specific human subject, the method comprising designing or preparing a peptide according to the method of item 58 or item 59, and administering the peptide to the subject.
63. A system comprising
-
- (c) a method of designing or preparing a polypeptide for inducing a cytotoxic T cell response in a specific human subject, the method comprising selecting an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject, and designing or preparing a polypeptide comprising the selected amino acid sequence; or
- (d) a method of designing or preparing a polypeptide for inducing a helper T cell response, the method comprising selecting an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class II molecules of the subject, and designing or preparing a polypeptide comprising the selected amino acid sequence.
64. The storage system of item 63 further comprising
-
- (c) an output module configured to display
- (i) a prediction of whether the one or more polypeptides is immunogenic for the subject; or the sequence of one or more fragments of the one or more polypeptides that are predicted to be immunogenic for the subject;
- (ii) a prediction of whether the individual will have an immune response to administration of the one or more polypeptides or one or more pharmaceutical compositions comprising the one or more polypeptides as active ingredients;
- (iii) a prediction of whether the subject will have a clinical response to a method of treatment comprising administering to the subject one or more pharmaceutical compositions comprising the one or more polypeptides as active ingredients;
- (iv) the likelihood that the subject will have a clinical response to administration of one or more pharmaceutical compositions comprising the one or more polypeptides as active ingredients;
- (v) a prediction of whether administration of the one or more polypeptides or one or more pharmaceutical compositions comprising the one or more polypeptides will induce a toxic immune response in the subject;
- (vi) a prediction that the one or more polypeptides is associated with an autoimmune disorder in the subject;
- (vii) a prediction of whether the subject will have a clinical response to administration of a checkpoint inhibitor;
- (viii) a recommendation of whether or not the subject should be treated by administration of the one or more polypeptides and/or one or more pharmaceutical compositions.
- (c) an output module configured to display
Predicted binding between particular HLA and epitopes (9 mer peptides) was based on the Immune Epitope Database tool for epitope prediction (www.iedb.org).
The HLA I-epitope binding prediction process was validated by comparison with HLA I-epitope pairs determined by laboratory experiments. A dataset was compiled of HLA I-epitope pairs reported in peer reviewed publications or public immunological databases.
The rate of agreement with the experimentally determined dataset (Table 9) was determined. The binding HLA I-epitope pairs of the dataset were correctly predicted with a 93% probability. Coincidentally the non-binding HLA I-epitope pairs were also correctly predicted with a 93% probability.
The accuracy of the prediction of multiple HLA binding epitopes was determined. Based on the analytical specificity and sensitivity using the 93% probability for both true positive and true negative prediction and 7% (=100%-93%) probability for false positive and false negative prediction, the probability of the existence of a multiple HLA binding epitope in a person can be calculated. The probability of multiple HLA binding to an epitope shows the relationship between the number of HLAs binding an epitope and the expected minimum number of real binding. Per PEPI definition three is the expected minimum number of HLA to bind an epitope (bold).
The validated HLA-epitope binding prediction process was used to determine all HLA-epitope binding pairs described in the Examples below.
Example 2—Epitope Presentation by Multiple HLA Predicts Cytotoxic T Lymphocyte (CTL) ResponseThe presentation of one or more epitopes of a polypeptide antigen by one or more HLA I of an individual is predictive for a CTL response was determined.
The study was carried out by retrospective analysis of six clinical trials, conducted on 71 cancer and 9 HIV-infected patients (Table 11)1-7. Patients from these studies were treated with an HPV vaccine, three different NY-ESO-1 specific cancer vaccines, one HIV-1 vaccine and a CTLA-4 specific monoclonal antibody (Ipilimumab) that was shown to reactivate CTLs against NY-ESO-1 antigen in melanoma patients. All of these clinical trials measured antigen specific CD8+ CTL responses (immunogenicity) in the study subjects after vaccination. In some cases, correlation between CTL responses and clinical responses were reported.
No patient was excluded from the retroactive study for any reason other than data availability. The 157 patient datasets (Table 11) were randomized with a standard random number generator to create two independent cohorts for training and evaluation studies. In some cases the cohorts contained multiple datasets from the same patient, resulting in a training cohort of 76 datasets from 48 patients and a test/validation cohort of 81 datasets from 51 patients.
The reported CTL responses of the training dataset were compared with the HLA I restriction profile of epitopes (9 mers) of the vaccine antigens. The antigen sequences and the HLA I genotype of each patient were obtained from publicly available protein sequence databases or peer reviewed publications and the HLA I-epitope binding prediction process was blinded to patients' clinical CTL response data. The number of epitopes from each antigen predicted to bind to at least 1 (PEPI1+), or at least 2 (PEPI2+), or at least 3 (PEPI3+), or at least 4 (PEPI4+), or at least 5 (PEPI5+), or all 6 (PEPI6) HLA class I molecules of each patient was determined and the number of HLA bound were used as classifiers for the reported CTL responses. The true positive rate (sensitivity) and true negative rate (specificity) were determined from the training dataset for each classifier (number of HLA bound) separately.
ROC analysis was performed for each classifier. In a ROC curve, the true positive rate (Sensitivity) was plotted in function of the false positive rate (1-Specificity) for different cut-off points (
The analysis unexpectedly revealed that predicted epitope presentation by multiple class I HLAs of a subject (PEPI2+, PEPI3+, PEPI4+, PEPI5+, or PEPI6), was in every case a better predictor of CTL response than epitope presentation by merely one or more HLA class I (PEPI1+, AUC=0.48, Table 12).
The CTL response of an individual was best predicted by considering the epitopes of an antigen that could be presented by at least 3 HLA class I of an individual (PEPI3+, AUC=0.65, Table 12). The threshold count of PEPI3+(number of antigen-specific epitopes presented by 3 or more HLA of an individual) that best predicted a positive CTL response was 1 (Table 13). In other words, at least one antigen-derived epitope is presented by at least 3 HLA class I of a subject (≥1 PEPI3+), then the antigen can trigger at least one CTL clone, and the subject is a likely CTL responder. Using the ≥1 PEPI3+ threshold to predict likely CTL responders (“≥1 PEPI3+ Test”) provided 76% diagnostic sensitivity (Table 13).
The test cohort of 81 datasets from 51 patients was used to validate the ≥1 PEPI3+ threshold to predict an antigen-specific CTL response. For each dataset in the test cohort it was determined whether the ≥1 PEPI3+ threshold was met (at least one antigen-derived epitope presented by at least three class I HLA of the individual). This was compared with the experimentally determined CTL responses reported from the clinical trials (Table 14).
The clinical validation demonstrated that a PEPI3+ peptide induce CTL response in an individual with 84% probability. 84% is the same value that was determined in the analytical validation of the PEPI3+ prediction, epitopes that binds to at least 3 HLAs of an individual (Table 10). These data provide strong evidences that immune responses are induced by PEPIs in individuals.
ROC analysis determined the diagnostic accuracy, using the PEPI3+ count as cut-off values (
A PEPI3+ count of at least 1 (≥1 PEPI3+) best predicted a CTL response in the test dataset (Table 15). This result confirmed the threshold determined during the training (Table 12).
The ≥1 PEPI3+ Test was compared with a previously reported method for predicting a specific human subject's CTL response to peptide antigens.
The HLA genotypes of 28 cervical cancer and VIN-3 patients that received the HPV-16 synthetic long peptide vaccine (LPV) in two different clinical trials were determined from DNA samples8 8 9 10. The LPV consists of long peptides covering the HPV-16 viral oncoproteins E6 and E7. The amino acid sequence of the LPV was obtained from these publications. The publications also report the T cell responses of each vaccinated patient to pools of overlapping peptides of the vaccine.
For each patient epitopes (9 mers) of the LPV that are presented by at least three patient class I HLA (PEPI3+s) were identified and their distribution among the peptide pools was determined. Peptides that comprised at least one PEPI3+(≥1 PEPI3+) were predicted to induce a CTL response. Peptides that comprised no PEPI3+ were predicted not to induce a CTL response.
The ≥1 PEPI3+ Test correctly predicted 489 out of 512 negative CTL responses and 8 out of 40 positive CTL responses measured after vaccination (
For each patient the distribution among the peptide pools of epitopes that are presented by at least one patient class I HLA (≥1 PEPI1+, HLA restricted epitope prediction, prior art method) was also determined. ≥1 PEPI1+ correctly predicted 116 out of 512 negative CTL responses and 37 out of 40 positive CTL responses measured after vaccination (
The 28 cervical cancer and VIN-3 patients that received the HPV-16 synthetic long peptide vaccine (LPV) in two different clinical trials (as detailed in Example 4) were investigated for CD4+T helper responses following LPV vaccination (
Using the same reported studies as Examples 4 and 5, the ≥1 PEPI3+ Test was used to predict patient CD8+ and CD4+ T cell responses to the full length E6 and E7 polypeptide antigens of the LPV vaccine. Results were compared to the experimentally determined responses were reported. The Test correctly predicted the CD8+ T cell reactivity (PEPI3+) of 11 out of 15 VIN-3 patients with positive CD8+ T cell reactivity test results (sensitivity 73%, PPV 85%) and of 2 out of 5 cervical cancer patients (sensitivity 40%, PPV 100%). The CD4+ T cell reactivities (PEPI4+) were correctly predicted 100% both of VIN-3 and cervical cancer patients (
Class I and class II HLA restricted PEPI3+ count was also observed to correlate with the reported clinical benefit to LPV vaccinated patients. Patients with higher PEPI3+ counts had either complete or partial response already after 3 months.
Example 7—Case StudypGX3001 is an HPV16 based DNA vaccine containing full length E6 and E7 antigens with a linker in between. pGX3002 is an HPV18 based DNA vaccine containing full length E6 and E7 antigens with a linker in between. A Phase II clinical trial investigated the T cell responses of 17 HPV-infected patients with cervical cancer who were vaccinated with both pGX3001 and pGX3002 (VGX-3100 vaccination)1.
Patient 12-11 had an overall PEPI1+ count of 54 for the combined vaccines (54 epitopes presented by one or more class I HLA). Patient 14-5 had a PEPI1+ count of 91. Therefore patient 14-5 has a higher PEPI1+ count than patient 12-11 with respect to the four HPV antigens. The PEPI1+s represent the distinct vaccine antigen specific HLA restricted epitope sets of patients 12-11 and 14-5. Only 27 PEPI1+s were common between these two patients.
For the PEPI3+ counts (number of epitopes presented by three or more patient class I HLA), the results for patients 12-11 and 14-5 were reversed. Patient 12-11 had a PEPI3+ count of 8, including at least one PEPI3+ in each of the four HPV16/18 antigens. Patient 14-5 had a PEPI3+ count of 0.
The reported immune responses of these two patients matched the PEPI3+ counts, not the PEPI1+ counts. Patient 12-11 developed immune responses to each of the four antigens post-vaccination as measured by ELISpot, whilst patient 14-5 did not develop immune responses to any of the four antigens of the vaccines. A similar pattern was observed when the PEPI1+ and PEPI3+ sets of all 17 patients in the trial were compared. There was no correlation between the PEPI1+ count and the experimentally determined T cell responses reported from the clinical trial. However, correlation between the T cell immunity predicted by the ≥1 PEPI3+ Test and the reported T cell immunity was observed. The ≥1 PEPI3+ Test predicted the immune responders to HPV DNA vaccine.
Moreover, the diversity of the patient's PEPI3+ set resembled the diversity of T cell responses generally found in cancer vaccine trials. Patients 12-3 and 12-6, similar to patient 14-5, did not have PEPI3+s predicting that the HPV vaccine could not trigger T cell immunity. All other patients had at least one PEPI3 predicting the likelihood that the HPV vaccine can trigger T cell immunity. 11 patients had multiple PEPI3+ predicting that the HPV vaccine likely triggers polyclonal T cell responses. Patients 15-2 and 15-3 could mount high magnitude T cell immunity to E6 of both HPV, but poor immunity to E7. Other patients 15-1 and 12-11 had the same magnitude response to E7 of HPV18 and HPV16, respectively.
Example 8—Design of a Model Population for Conducting in Silico Trials and Identifying Candidate Precision Vaccine Targets for Large PopulationAn in silico human trial cohort of 433 subjects with complete 4-digit HLA class I genotype (2×HLA-A*xx:xx; 2×HLA-B*xx:xx; 2×HLA-C*xx:xx) and demographic information. This Model Population has subjects with mixed ethnicity having a total of 152 different HLA alleles that are representative for >85% of presently known allele G-groups.
A database of a “Big Population” containing 7,189 subjects characterized with 4-digit HLA genotype and demographic information was also established. The Big Population has 328 different HLA class I alleles. The HLA allele distribution of the Model Population significantly correlated with the Big Population (Table 16) (Pearson p<0.001). Therefore the 433 patient Model Population is representative for a 16 times larger population.
The Model Population is representative for 85% of the human race as given by HLA diversity as well as HLA frequency.
The objective of this study was to determine whether a model population, such as the one described in Example 8, may be used to predict CTL reactivity rates of vaccines, i.e. used in an in silico efficacy trials.
Twelve peptide vaccines derived from cancer antigens that induced T cell responses in a subpopulation of subjects were identified from peer reviewed publications. These peptides have been investigated in clinical trials enrolling a total of 172 patients (4 ethnicities). T cell responses induced by the vaccine peptides have been determined from blood specimens and reported. The immune response rate as the percentage of study subjects with positive T cell responses measured in the clinical trials was determined (
The 12 peptides were investigated with the ≥1 PEPI3+ Test in each of the 433 subjects of the Model Population described in Example 8. The “≥1 PEPI3+ Score” for each peptide was calculated as the proportion of subjects in the Model Population having at least one vaccine derived epitope that could bind to at least three subject-specific HLA class I (≥1 PEPI3+). If the corresponding clinical trial stratified patients for HLA allele selected population, the Model Population was also filtered for subjects with the respective allele(s) (Example: WT1, HLA-A*0201).
The experimentally determined response rates reported from the trials were compared with the ≥1 PEPI3+ Scores. The Overall Percentage of Agreements (OPA) were calculated on the paired data (Table 18). We also found a linear correlation between ≥1 PEPI3+ Score and response rate (R2=0.77) (
Nineteen clinical trials with published immune response rates (IRR) conducted with peptide or DNA based vaccines were identified (Table 19). These trials involved 604 patients (9 ethnicities) and covered 38 vaccines derived from tumor and viral antigens. Vaccine antigen specific CTL responses were measured in each study patient and the response rate in the clinical study populations was calculated and reported.
Each vaccine peptide of the 19 clinical trials was investigated with the ≥1 PEPI3+ Test in each subject of the Model Population. The ≥1 PEPI3+ Score for each peptide was calculated as the proportion of subjects in the Model Population having at least one vaccine derived PEPI3+. The experimentally determined response rates reported from the trials were compared with the PEPI Scores, as in Example 9 (Table 20). A linear correlation between the response rate and ≥1 PEPI3+ Score (R2=0.70) was observed (
IMA901 is a therapeutic vaccine for renal cell cancer (RCC) comprising 9 peptides derived from tumor-associated peptides (TUMAPs) that are naturally presented in human cancer tissue. A total of 96 HLA-A*02+ subjects with advanced RCC were treated with IMA901 in two independent clinical studies (phase I and phase II). Each of the 9 peptides of IMA901 have been identified in the prior art as HLA-A2-restricted epitopes. Based on currently accepted standards, they are all strong candidate peptides to boost T cell responses against renal cancer in the trial subjects, because their presence has been detected in renal cancer patients, and because the trial patients were specifically selected to have at least one HLA molecule capable of presenting each of the peptides.
For each subject in the Model population how many of the nine peptides of the IMA901 vaccine were capable of binding to three or more HLA was determined Since each peptide in the IMA901 vaccine is a 9 mer this corresponds to the PEPI3+ count. The results were compared with the immune response rates reported for the Phase I and Phase II clinical trials (Table 21).
The phase I and phase II study results show the variability of the immune responses to the same vaccine in different trial cohorts. Overall, however, there was a good agreement between response rates predicted by the ≥2 PEPI3+ Test and the reported clinical response rates.
In a retrospective analysis, the clinical investigators of the trials discussed above found that subjects who responded to multiple peptides of the IMA901 vaccine were significantly (p=0.019) more likely to experience disease control (stable disease, partial response) than subjects who responded only to one peptide or had no response. 6 of 8 subjects (75%) who responded to multiple peptides experienced clinical benefit in the trial, in contrast to 14% and 33% of 0 and 1 peptide responders, respectively. The randomized phase II trial confirmed that immune responses to multiple TUMAPs were associated with a longer overall survival.
Since the presence of PEPIs accurately predicted responders to TUMAPs, clinical responders to IMA901 are likely patients who can present ≥2 PEPIs from TUMAPs. This subpopulation is only 27% of HLA-A*02 selected patients, and according to the clinical trial result, 75% of this subpopulation is expected to experience clinical benefit. The same clinical results suggest that 100% of patients would experience clinical benefit if patient selection is based on ≥3 PEPIs from TUMAPs, albeit this population would represent only 3% of the HLA-A*02 selected patient population. These results suggest that the disease control rate (stable disease or partial response) is between 3% and 27% in the patient population which was investigated in the IMA901 clinical trials. In the absence of complete response, only a portion of these patients can experience survival benefit.
These findings explain the absence of improved survival in the Phase III IMA901 clinical trial. These results also demonstrated that HLA-A*02 enrichment of the study population was not sufficient to reach the primary overall survival endpoint in the Phase III IMA901 trial. As the IMA901 trial investigators noted, there is a need for the development of a companion diagnostic (CDx) to select likely responders to peptide vaccines. These findings also suggest that selection of patients with ≥2 TUMAP specific PEPIs may provide sufficient enrichment to demonstrate significant clinical benefit of IMA901.
Example 12—In Silico Trial Based on the Identification of Vaccine-Derived Multiple HLA Binding Epitopes Predict Reported Experimental Clinical Response RatesA correlation between the ≥2 PEPI3+ Score of immunotherapy vaccines determined in the Model Population described in Example 8 and the reported Disease Control Rate (DCR, proportion of patients with complete responses and partial responses and stable disease) determined in clinical trials was determined.
Seventeen clinical trials, conducted with peptide- and DNA-based cancer immunotherapy vaccines that have published Disease Control Rates (DCRs) or objective response rate (ORR) were identified from peer reviewed scientific journals (Table 22). These trials involved 594 patients (5 ethnicities) and covered 29 tumor and viral antigens. DCRs were determined according to the Response Evaluation Criteria in Solid Tumors (RECIST), which is the current standard for clinical trials, in which clinical responses are based on changes in maximum cross-sectional dimensions42, 43, 44. In case there was no available DCR data, objective response rate (ORR) data was used, which is also defined according to the RECIST guidelines.
Table 23 compares the ≥2 PEPI3+ Score for each vaccine in the Model Population and the published DCR or ORR. A correlation between the predicted and measured DCR was observed providing further evidence that not only the immunogenicity but also the potency of cancer vaccines depends on the multiple HLA sequences of individuals (R2=0.76) (
Whether survival benefit of melanoma patients treated with the checkpoint inhibitor Ipilimumab can be predicted by the number of melanoma-specific PEPI3+s that are potentially expressed in the patient's tumor was determined.
Eighty melanoma associated antigens (TAAs) were identified from which a panel of PEPI3+s (IPI-PEPI panel: 627 PEPIs) that are shared by Ipilimumab treated melanoma patients with a prolonged clinical benefit and are absent in those without a prolonged benefit was selected. These PEPI3+ define the specific T cells that are re-activated by Ipilimumab to attack the patient's tumor cells. Patients with certain HLA sequences that can present more melanoma-specific PEPIs have more T cells re-activated by Ipilimumab and a higher chance to benefit from Ipilimumab immunotherapy.
The clinical benefit from Ipilimumab treatment for 160 patients from four independent clinical trial cohorts was determined. Two cohorts were from the trials CA184-007 (10 mg/kg Ipilimumab) and CA184-002 (3 mg/kg Ipilimumab) and two cohorts from published clinical trials 10 mg/kg and 3/mg/kg Ipilimumab datasets5, 38, 39.
Epitopes from 80 melanoma antigens restricted to all the 6 HLA class I of each patient were predicted and the number of melanoma-specific PEPI3+s restricted to at least 3 class I HLAs of each patient (4,668 PEPIs) was then computed. Each patient with at least one out of 627 PEPIs qualified as responder. The IPI-PEPI panel predicts the overall survival of both 10 mg/kg and 3 mg/kg Ipilimumab. Results were highly significant and consistent in the four independent cohort (
The capability of the PEPI3+ to identify neoantigens from mutations was determined. PEPI3+s of 110 melanoma patients treated with Ipilimumab was determined using published exome mutation data39. From the exome mutation data, mutations in 9,502 antigens from the 110 patients (
Mutational PEPI3+ neoepitopes from the published mutations were determined (
Results show that PEPIs define the mutational neoantigens derived from genetically altered proteins expressed in an individual. Such neoantigens are PEPI3+ peptides that capable to activate T cells in the patient's body. If a genetic alteration occurs in the tumor cell of the individual that creates a PEPI3+ then this PEPI3+ can induce T cell responses. These PEPI3+ containing peptides could be included in a drug (e.g. vaccine, T cell therapy) to induce immune response against the individual tumor.
The epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific mutation broadly expressed in glioblastoma multiforme (GBM) and other neoplasms. The mutation comprises an in-frame deletion of 801 bp from the extracellular domain of the EGFR that splits a codon and yields a novel glycine at the fusion junction.1, 2 This mutation encodes a constitutively active tyrosine kinase that increases tumor formation and tumor cell migration and enhances resistance against radiation and chemotherapy.3, 4, 5, 6, 7, 8, 9 This insertion results in a tumor-specific epitope which is not found in normal adult tissues making EGFRvIII a suitable target candidate for antitumor immunotherapy.10 Rindopepimut is a 13-amino-acid peptide vaccine (LEEKKGNYVVTDHC (SEQ ID NO: 87)) spanning the EGFRvIII mutation with an additional C-terminal cysteine residue.11
In a phase II clinical study, the peptide conjugated to keyhole limpet hemocyanin (KLH) was administered to newly diagnosed EGFRvIII-expressing GBM patients. The first three vaccinations were given biweekly, starting 4 weeks after the completion of radiation. Subsequent vaccines were given monthly until radiographic evidence of tumor progression or death. All vaccines were given intradermally in the inguinal region. Immunologic evaluation showed only 3 out of 18 patients developing cellular immune response assessed by DTH reaction test.
An in silico trial with the Model Population of 433 subjects with Rindopepimut sequence was conducted. 4 out of 433 subjects had PEPI3+, confirming the low immunogenicity found in the phase II study (Table 25).
An HLA map of the Rindopepimut on the HLA alleles of the subjects in the Model Population (
In a recent phase III clinical study the ineffectiveness was further demonstrated when 745 patients were enrolled and randomly assigned to Rindopepimut and temozolomide (n=371) or control and temozolomide (n=374) arms.12 The trial was terminated for ineffectiveness after the interim analysis. The analysis showed no significant difference in overall survival: median overall survival was 20.1 months (95% CI 18.5-22.1) in the Rindopepimut group versus 20.0 months (18.1-21.9) in the control group (HR 1.01, 95% CI 0.79-1.30; p=0.93).
REFERENCES FOR EXAMPLE 15
- 1 Bigner et al. Characterization of the epidermal growth factor receptor in human glioma cell lines and xenografts. Cancer Res 1990; 50: 8017-22.
- 2 Libermann et al. Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin. Nature 1985; 313: 144-7.
- 3 Chu et al. Receptor dimerization is not a factor in the signalling activity of a transforming variant epidermal growth factor receptor (EGFRvIII). Biochem J 1997; 324: 855-61.
- 4 Batra et al. Epidermal growth factor ligand-independent, unregulated, cell-transforming potential of a naturally occurring human mutant EGFRvIII gene. Cell Growth Differ 1995; 6: 1251-9.
- 5 Nishikawa et al. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. PNAS 1994; 91: 7727-31.
- 6 Lammering et al. Inhibition of the type III epidermal growth factor receptor variant mutant receptor by dominant-negative EGFR-CD533 enhances malignant glioma cell radiosensitivity. Clin Cancer Res 2004; 10: 6732-43.
- 7 Nagane et al. A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis. Cancer Res 1996; 56: 5079-86.
- 8 Lammering et al. Radiation-induced activation of a common variant of EGFR confers enhanced radioresistance. Radiother Oncol 2004; 72: 267-73.
- 9 Montgomery et al. Expression of oncogenic epidermal growth factor receptor family kinases induces paclitaxel resistance and alters β-tubulin isotype expression. J Biol Chem 2000; 275: 17358-63.
- 10 Humphrey et al. Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma. PNAS 1990; 87: 4207-11.
- 11 Sampson et al. Immunologic Escape After Prolonged Progression-Free Survival With Epidermal Growth Factor Receptor Variant III Peptide Vaccination in Patients With Newly Diagnosed Glioblastoma. J Clin Oncol 28:4722-4729.
- 12 Weller at al. Rindopepimut with temozolomide for patients with newly diagnosed, EGFRvIII-expressing glioblastoma (ACT IV): a randomised, double-blind, international phase 3 trial. Lancet Oncol 2017; 18(10): 1373-1385.
Thrombopoietin (TPO) is a highly immunogenic protein drug causing toxicity in many patients. EpiVax/Genentech used State of Art technology to identify class II HLA restricted epitopes and found that the most immunogenic region of the TPO is located in the C-terminal end of TPO (US20040209324 A1).
According to the present disclosure we defined the multiple class II HLA binding epitopes (PEPI3+s) from TPO in 400 HLA class II genotyped US subjects were determined. Most of the PEPI3+ peptides of these individuals located within the N terminal region of the TPO between 1-165 amino acids. PEPI3+ were sporadically identified in some subjects also in the C terminal region. However, these results were different from the State of Art.
The published literature confirmed the disclosed results, demonstrating experimental proof for the immunotoxic region being located at the N-terminal end of TPO40, 41. Most individuals treated with TPO drug made anti-drug antibodies (ADA) ADA against this region of the drug. These antibodies not only abolished the therapeutic effect of the drug but also caused systemic adverse events, i.e. immune-toxicity, like antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity associated with thrombocytopenia, neutropenia and anemia. These data demonstrate that the identification of multiple HLA binding peptides of individuals predicts the immune-toxicity of TPO. Therefore, the disclosure is useful to identify the toxic immunogenic region of drugs, to identify subjects who likely experience immune-toxicity from drugs, to identify regions of a polypeptide drug that may be targeted by ADAs, and to identify subjects who likely experience ADA.
Example 17 Personalised Immunotherapy Composition for Treatment of Ovarian CancerThis example describes the treatment of an ovarian cancer patient with a personalised immunotherapy composition, wherein the composition was specifically designed for the patient based on her HLA genotype based on the disclosure described herein. This Example and Example 19 below provide clinical data to support the principals regarding binding of epitopes by multiple HLA of a subject to induce a cytotoxic T cell response on which the present disclosure is based.
The HLA class I and class II genotype of metastatic ovarian adenocarcinoma cancer patient XYZ was determined from a saliva sample.
To make a personalized pharmaceutical composition for patient XYZ thirteen peptides were selected, each of which met the following two criteria: (i) derived from an antigen that is expressed in ovarian cancers, as reported in peer reviewed scientific publications; and (ii) comprises a fragment that is a T cell epitope capable of binding to at least three HLA class I of patient XYZ (Table 26). In addition, each peptide is optimized to bind the maximum number of HLA class II of the patient.
Eleven PEPI3 peptides in this immunotherapy composition can induce T cell responses in XYZ with 84% probability and the two PEPI4 peptides (POC01-P2 and POC01-P5) with 98% probability, according to the validation of the PEPI Test shown in Table 10. T cell responses target 13 antigens expressed in ovarian cancers. Expression of these cancer antigens in patient XYZ was not tested. Instead the probability of successful killing of cancer cells was determined based on the probability of antigen expression in the patient's cancer cells and the positive predictive value of the ≥1 PEPI3+ Test (AGP count). AGP count predicts the effectiveness of a vaccine in a subject: Number of vaccine antigens expressed in the patient's tumor (ovarian adenocarcinoma) with PEPI. The AGP count indicates the number of tumor antigens that vaccine recognizes and induces a T cell response against the patient's tumor (hit the target). The AGP count depends on the vaccine-antigen expression rate in the subject's tumor and the HLA genotype of the subject. The correct value must be between 0 (no PEPI presented by expressed antigen) and maximum number of antigens (all antigens are expressed and present a PEPI).
The probability that patient XYZ will express one or more of the 12 antigens is shown in
A pharmaceutical composition for patient XYZ may be comprised of at least 2 from the 13 peptides (Table 26), because the presence in a vaccine or immunotherapy composition of at least two polypeptide fragments (epitopes) that can bind to at least three HLA of an individual (≥2 PEPI3+) was determined to be predictive for a clinical response. The peptides are synthesized, solved in a pharmaceutically acceptable solvent and mixed with an adjuvant prior to injection. It is desirable for the patient to receive personalized immunotherapy with at least two peptide vaccines, but preferable more to increase the probability of killing cancer cells and decrease the chance of relapse.
For treatment of patient XYZ the 12 peptides were formulated as 4×3/4 peptide (POC01/1, POC01/2, POC01/3, POC01/4). One treatment cycle is defined as administration of all 13 peptides within 30 days.
Patient History:Diagnosis: Metastatic ovarian adenocarcinoma
Age: 51Family anamnesis: colon and ovary cancer (mother) breast cancer (grandmother)
Tumor Pathology: BRCA1-185delAG, BRAF-D594Y, MAP2K1-P293S, NOTCH1-S2450N
-
- 2011: first diagnosis of ovarian adenocarcinoma; Wertheim operation and chemotherapy; lymph node removal
- 2015: metastasis in pericardial adipose tissue, excised
- 2016: hepatic metastases
- 2017: retroperitoneal and mesenteric lymph nodes have progressed; incipient peritoneal carcinosis with small accompanying ascites
-
- 2012: Paclitaxel-carboplatin (6×)
- 2014: Caelyx-carboplatin (1×)
- 2016-2017 (9 months): Lymparza (Olaparib) 2×400 mg/day, oral
- 2017: Hycamtin inf. 5×2.5 mg (3× one seria/month)
- PIT vaccine treatment began on 21 Apr. 2017.
-
- Disease was confined primarily to liver and lymph nodes. The use of MRI limits detection of lung (pulmonary) metastasis
- May 2016-January 2017: Olaparib treatment
- Dec. 25, 2016 (before PIT vaccine treatment) There was dramatic reduction in tumor burden with confirmation of response obtained at FU2
- January-March 2017—TOPO protocol (topoisomerase)
- Apr. 6, 2017 FU3 demonstrated regrowth of existing lesions and appearance of new lesions leading to disease progression
- Apr. 21 2017 START PIT
- Jul. 21, 2017 (after the 2nd Cycle of PIT) FU4 demonstrated continued growth in lesions and general enlargement of pancreas and abnormal para pancreatic signal along with increased ascites
- Jul. 26, 2017—CBP+Gem+Avastin
- Sep. 20, 2017 (after 3 Cycles of PIT) FU5 demonstrated reversal of lesion growth and improved pancreatic/parapancreatic signal. The findings suggest pseudo progression
- Nov. 28, 2017 (after 4 Cycles of PIT) FU6 demonstrated best response with resolution of non target lesions
MRI data for patient XYZ is shown in Table 28 andFIG. 14 .
The HLA class I and class II genotype of metastatic breast cancer patient ABC was determined from a saliva sample. To make a personalized pharmaceutical composition for patient ABC twelve peptides were selected, each of which met the following two criteria: (i) derived from an antigen that is expressed in breast cancers, as reported in peer reviewed scientific publications; and (ii) comprises a fragment that is a T cell epitope capable of binding to at least three HLA class I of patient ABC (Table 29). In addition, each peptide is optimized to bind the maximum number of HLA class II of the patient. The twelve peptides target twelve breast cancer antigens. The probability that patient ABC will express one or more of the 12 antigens is shown in
Predicted efficacy: AGP95=4; 95% likelihood that the PIT Vaccine induces CTL responses against 4 CTAs expressed in the breast cancer cells of BRC09. Additional efficacy parameters: AGP50=6.3, mAGP=100%, AP=12.
Detected efficacy after the 1st vaccination with all 12 peptides: 83% reduction of tumor metabolic activity (PET CT data).
For treatment of patient ABC the 12 peptides were formulated as 4×3 peptide (PBR01/1, PBR01/2, PBR01/3, PBR01/4). One treatment cycle is defined as administration of all 12 different peptide vaccines within 30 days.
Patient HistoryDiagnosis: bilateral metastatic breast carcinoma: Right breast is ER positive, PR negative, Her2 negative; Left Breast is ER, PR and Her2 negative.
First diagnosis: 2013 (4 years before PIT vaccine treatment)
2016: extensive metastatic disease with nodal involvement both above and below the diaphragm. Multiple liver and pulmonar metastases.
2016-2017 treatment: Etrozole, Ibrance (Palbociclib) and Zoladex
Mar. 7, 2017: Prior PIT Vaccine treatment
Hepatic multi-metastatic disease with truly extrinsic compression of the origin of the choledochal duct and massive dilatation of the entire intrahepatic biliary tract. Celiac, hepatic hilar and retroperitoneal adenopathy
May 26 2017: After 1 cycle of PIT
Detected efficacy: 83% reduction of tumor metabolic activity (PET CT) liver, lung lymphnodes and other metastases. Detected safety: Skin reactions
Local inflammation at the site of the injections within 48 hours following vaccine administrations
BRC-09 was treated with 5 cycles of PIT vaccine. She was feeling very well and she refused a PET CT examination in September 2017. In November she had symptoms, PET CT scan showed progressive disease, but she refused all treatments. In addition, her oncologist found out that she did not take Palbocyclib since spring/summer. Patient ABC passed away in January 2018.
The combination of palbocyclib and the personalised vaccine was likely to have been responsible for the remarkable early response observed following administration of the vaccine. Palbocyclib has been shown to improve the activity of immunotherapies by increases CTA presentation by HLAs and decreasing the proliferation of Tregs: (Goel et al. Nature. 2017:471-475). The PIT vaccine may be used as add-on to the state-of-art therapy to obtain maximal efficacy.
Example 19—Personalised Immunotherapy Composition for Treatment of Patient with Late Stage Metastatic Breast CancerPatient BRC05 was diagnosed with inflammatory breast cancer on the right with extensive lymphangiosis carcinomatose. Inflammatory breast cancer (IBC) is a rare, but aggressive form of locally advanced breast cancer. It's called inflammatory breast cancer because its main symptoms are swelling and redness (the breast often looks inflamed). Most inflammatory breast cancers are invasive ductal carcinomas (begin in the milk ducts). This type of breast cancer is associated with the expression of oncoproteins of high risk Human Papilloma Virus. Indeed, HPV16 DNA was diagnosed in the tumor of this patient.
Patient's stage in 2011 (6 years prior to PIT vaccine treatment)
T4: Tumor of any size with direct extension to the chest wall and/or to the skin (ulceration or skin nodules)
pN3a: Metastases in ≥10 axillary lymph nodes (at least 1 tumor deposit>2.0 mm); or metastases to the infraclavicular (level III axillary lymph) nodes.
14 vaccine peptides were designed and prepared for patient BRC05 (Table 30). Peptides PBRC05-P01-P10 were made for this patient based on population expression data. The last 3 peptides in the Table 29 (SSX-2, MORC, MAGE-B1) were designed from antigens that expression was measured directly in the tumor of the patient.
T cell responses were measured cells in peripheral mononuclear cells 2 weeks after the 1st vaccination with the mix of peptides PBRC05_P1, PBRC05_P2, PBRC05_P3, PBRC05_P4, PBRC05_P5, PBRC05_P6, PBRC05_P7.
The results show that a single immunization with 7 peptides induced potent T cell responses against 3 out of the 7 peptides demonstrating potent MAGE-A11, NY-SAR-35, FSIP1 and MAGE-A9 specific T cell responses. There were weak responses against AKAP4 and NY-BR-1 and no response against SPAG9.
Example 20—Personalised Immunotherapy Composition for Treatment of Patient with Early Stage Metastatic Breast CancerHISTORY: In 2011 left breast sector excision due to neoplasm. Treatment: aromatase inhibitor and lumbar spine irradiation (osseal mets).
In 2017, before PIT vaccine treatment was administered, a metastatic lesion was observed on the ventral bow of the right 5th rib and in the right 3rd rib. In the left breast recurrent malignancy has to be ruled out. In the righ breast a malignancy with metastatic right axillary lymph node may exist.
The patient obtained 2 cycles of PIT vaccine.
Example 21—Characterization of Toxicity—immunoBLASTA method was developed for performing on any antigen to determine its potential to induce toxic immune reaction, such as autoimmunity. The method is referred to herein as immunoBLAST.
PolyPEPI1018 contains six 30-mer polypeptides. Each polypeptide consists of two 15-mer peptide fragments derived from antigens expressed in CRC. Neoepitopes might be generated in the joint region of the two 15-mer peptides and could induce undesired T cell responses against healthy cells (autoimmunity). This was assesses using the immunoBLAST methodology.
A 16-mer peptide for each of the 30-mer components of PolyPEP1018 was designed. Each 16-mer contains 8 amino acids from the end of the first 15 residues of the 30-mer and 8 amino acids from the beginning of the second 15 residues of the 30-mer—thus precisely spanning the joint region of the two 15-mers. These 16-mers are then analysed to identify cross-reactive regions of local similarity with human sequences using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), which compares protein sequences to sequence databases and calculates the statistical significance of matches. 8-mers within the 16-mers were selected as the examination length since that length represents the minimum length needed for a peptide to form an epitope, and is the distance between the anchor points during HLA binding.
As shown in
The PEPI3+ Test was used to identify neoepitopes and neoPEPI among the 9-mer epitopes in the joint region. The risk of PolyPEPI1018 inducing unwanted T cell responses was assessed in the 433 subjects in the Model Population by determining the proportion of subjects with PEPI3+ among the 9-mers in the joint region. The result of neoepitope/neoPEPI analysis is summarized in Table 33. In the 433 subjects of the Model Population, the average predicted epitope number that could be generated by intracellular processing was 40.12. Neoepitopes were frequently generated; 11.61 out of 40.12 (28.9%) epitopes are neoepitopes. Most of the peptides were able to be identified as a neoepitope, but the number of subjects that present neoepitopes varied.
Epitopes harbored by PolyPEPI1018 create an average of 5.21 PEPI3+. These PEPIs can activate T cells in a subject. The amount of potential neoPEPIs was much lower than neoepitopes (3.7%). There is a marginal possibility that these neoPEPIs compete on T cell activation with PEPIs in some subjects. Importantly, the activated neoPEPI specific T cells had no targets on healthy tissue.
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Claims
1. A human subject-specific pharmaceutical composition for treatment of a disease or disorder in a specific human subject, comprising:
- (a) at least two different polypeptides, each of the at least two different polypeptides comprising 10-50 amino acids comprising a T cell epitope that binds at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject, and wherein the T cell epitopes of each of the at least two polypeptides are different; and
- (b) a pharmaceutically-acceptable adjuvant.
2. The human subject-specific pharmaceutical composition of claim 1, comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides.
3. The human subject-specific pharmaceutical composition of claim 1, comprising 3-40 different polypeptides.
4. The human subject-specific pharmaceutical composition of claim 1, wherein the T cell epitope that binds at least three HLA class I molecules of the subject comprises 7 to 17 amino acids.
5. The human subject-specific pharmaceutical composition of claim 1, wherein the T cell epitopes of the at least two different polypeptides are from a single antigen.
6. The human subject-specific pharmaceutical composition of claim 1, wherein the T cell epitopes of the at least two different polypeptides are from two or more different antigens.
7. The human subject-specific pharmaceutical composition of claim 5, wherein the antigen is an antigen expressed by a cancer cell, a neoantigen expressed by a cancer cell, a cancer-associated antigen, a tumor-associated antigen, an antigen expressed by a target pathogenic organism, an antigen expressed by a virus, an antigen expressed by a bacterium, an antigen expressed by a fungus, an antigen associated with an autoimmune disorder, or is an allergen.
8. The human subject-specific pharmaceutical composition of claim 7, wherein the cancer cell is from the subject.
9. The human subject-specific pharmaceutical composition of claim 5, wherein the antigen is selected from Tables 2 to 7.
10. The human subject-specific pharmaceutical composition of claim 1, wherein the at least two different polypeptides further comprise up to 10 amino acids flanking the T cell epitope that are part of a consecutive sequence flanking the epitope in the corresponding antigen.
11. The human subject-specific pharmaceutical composition of claim 1, wherein the at least two different polypeptides further comprise up to 10 amino acids flanking the T cell epitope that are not part of a consecutive sequence flanking the epitope in the corresponding antigen.
12. The human subject-specific pharmaceutical composition of claim 1, wherein two of the at least two polypeptides are arranged end to end or overlapping in a joined polypeptide.
13. The human subject-specific pharmaceutical composition of claim 12, comprising two or more different joined polypeptides, wherein the two or more different joined polypeptides comprise different T cell epitopes.
14. The human subject-specific pharmaceutical composition of claim 13, wherein the joined polypeptides do not substantially comprise neoepitopes that span a junction between the two polypeptides and that
- (i) corresponds to a fragment of a human polypeptide expressed in healthy cells of the subject;
- (ii) is a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or
- (iii) meets both requirements (i) and (ii).
15. The human subject-specific pharmaceutical composition of claim 1, wherein the at least two polypeptides do not comprise any amino acid sequences that:
- (i) correspond to a fragment of a human polypeptide expressed in healthy cells; or
- (ii) correspond to a fragment of a human polypeptide expressed in healthy cells and is a T cell epitope capable of binding to at least two HLA class I molecules of the subject.
16. The human subject-specific pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable diluent, carrier, preservative, or combination thereof.
17. The human subject-specific pharmaceutical composition of claim 1, wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, bacillus Calmette-Guerin (BCG), corynbacterium parvum, levamisole, azimezone, isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins (KLH), Freunds adjuvant (complete), Freunds adjuvant (incomplete), mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), and combinations thereof.
18.-20. (canceled)
21. A human subject-specific pharmaceutical composition comprising:
- a nucleic acid molecule expressing two or more polypeptides, each polypeptide comprising 10-50 amino acids comprising a T cell epitope that binds at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject, wherein the two or more polypeptides comprise different T cell epitopes, wherein the two or more polypeptides do not comprise amino acid sequences that are adjacent to each other in a corresponding antigen.
22.-47. (canceled)
48. A method of treating a cancer in a specific human subject in need thereof comprising:
- administering to a human subject a pharmaceutical composition comprising at least one polypeptide,
- the at least one polypeptide comprising 10-50 amino acids comprising a first T cell epitope that binds at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject,
- wherein the first T cell epitope is from an antigen that is specific for the cancer.
49-54. (canceled)
55. The method of claim 48, wherein the antigen is listed in Table 2.
56.-81. (canceled)
Type: Application
Filed: Mar 2, 2018
Publication Date: Sep 20, 2018
Inventors: Julianna LISZIEWICZ (Balatonalmadi), Levente MOLNÁR (Felsopakony), Enikö R. TÖKE (Felsopakony), József TOTH (Gyor), Orsolya LORINCZ (Budapest), Zsolt CSISZOVSZKI (Budapest), Eszter SOMOGYI (Balatonalmádi), Katalin PÁNTYA (Budapest), Mónika MEGYESI (Mezokeresztes)
Application Number: 15/910,930
