The content of the ASCII text file of the sequence listing named Seq_listing_ST25, which is 17 kb in size was created on 02/26/2020 and electronically submitted via EFS-Web along with the present application is incorporated by reference in its entirety
The present disclosure relates to compositions and methods of ascertaining or predicting an immune response against an antigen, and most typically a therapeutic antigen in an antitumor vaccine.
The background description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
While numerous cancer vaccines have been developed targeting a number of molecular targets, clinical success is often unpredictable and may vary significantly from one antigen to another or among different patients where common antigens are targeted. Indeed, predicting the likelihood of a therapeutic effect (whether or not a functional immune response against the tumor is elicited) has been elusive, and therapeutic effect, and with that a determination as to continued treatment with the vaccine, is in many cases only based on quantitation of retarded or reversed tumor growth. Consequently, significant time will have lapsed between start of treatment and a first significant indication of treatment effect.
More recently, T cells from the tumor microenvironment (e.g., tumor infiltrating lymphocytes (TIL)) or adjacent lymphatic organs have been examined to determine reactivity and quantity of antigen reactive T cells. However, collecting these cells can be highly invasive and uncomfortable for the patient, and most typically such cells are obtained from tumor biopsies or otherwise surgically removed tumor sample. For example, antigen reactive tumor infiltrating lymphocytes have been used as therapeutic entities in combination with further immune modulatory agents as reported in Nature Medicine (URL: doi.org/10.1038/s41591-018-0040-8). While notably effective, the TIL were obtained from a surgical sample.
Assessment of an immune response in vitro has been described in U.S. Pat. No. 8,697,371 where an artificial immune system was proposed comprising T cells, B cells, and dendritic cells for in vitro testing of vaccines, adjuvants, immunotherapy candidates, biologics, etc. Unfortunately, such system will not provide an indication of a likely immune response in vivo where a subject was already exposed to an antigen or anti-cancer vaccine. Likewise, due to high inter-subject variability results from such in vitro tests will not necessarily equally translate to a large number of different subjects.
Thus, even though various systems and methods of identifying an immune response are known in the art, all or almost all of them suffer from various disadvantages. Consequently, there is a need to provide improved compositions and methods that provide improved systems and methods that help identify or even predict an immune response in an individual in a safe, simple, and expeditious manner.
The inventor has now discovered that an immune response can be ascertained or predicted in a subject, and preferably in a subject selected to receive or having received an antitumor vaccine, typically using whole blood of the subject and recombinant antigen as a starting material. Thus, contemplated compositions and methods are conceptually simple, significantly reduce risk and discomfort to the patient otherwise necessitated by a biopsy or surgery, and can provide results within a fraction of time (e.g., several days) otherwise required by in vivo observation (e.g., several months).
In one aspect, a method is disclosed of ascertaining an immune response against an antigen in a subject previously exposed to the antigen that includes a step of generating dendritic cells from peripheral blood of the subject, and exposing the dendritic cells to an antigen-containing composition to generate antigen presenting dendritic cells. In another step, T cells are isolated from peripheral blood of the subject, and the isolated T cells are then contacted with the antigen presenting dendritic cells. To expand antigen-reactive T cells, the isolated T cells and the antigen presenting dendritic cells are exposed to a cytokine-containing composition, and in still another step, the expanded antigen-reactive T cells are detected, whereby expanded antigen-reactive T cells ascertain an immune response against the antigen.
In some embodiments, the subject was previously exposed to a vaccine containing the antigen or nucleic acid encoding the antigen. For example, the vaccine containing the antigen may be a recombinant viral vaccine, a recombinant yeast vaccine, and/or a recombinant bacterial vaccine, and it is generally preferred (but not necessary) that the antigen is a patient and tumor specific neoantigen. Most typically, the dendritic cells are generated from monocytes in the peripheral blood.
In further embodiments, the antigen in the antigen-containing composition is a patient and tumor specific neoantigen, and/or the antigen in the antigen-containing composition is a full-length protein that contains a neoantigen. For example, the antigen-containing composition may be a recombinant antigen-containing composition. Where desired, the antigen-containing composition may comprise a polytope containing a plurality of distinct antigens or an antigen pool derived from a full-length protein. Most typically, the cytokine-containing composition may comprise IL7, IL15, and IL21, or the cytokine-containing composition may comprise an IL7/N803/IL21 TxM.
Among other suitable choices, the expanded antigen-reactive T cells may be detected using an ELISPOT assay or a FACS assay. As will also be readily appreciated, the expanded antigen-reactive T cells may be administered to the subject.
In another aspect, a method is disclosed of predicting a likely immune response against an antigen in a subject that is selected to receive a vaccine containing the antigen. Such method may include a step of generating dendritic cells from peripheral blood of the subject, and exposing the dendritic cells to an antigen-containing composition to generate antigen presenting dendritic cells, and a further step of isolating T cells from peripheral blood of the subject, and contacting the isolated T cells with the antigen presenting dendritic cells. The isolated T cells and the antigen presenting dendritic cells are then exposed to a cytokine-containing composition to expand antigen-reactive T cells, expanded antigen-reactive T cells are quantified, and in a still further step the subject is identified as a likely immune responder when the quantified expanded antigen-reactive T cells exceed a predetermined threshold quantity.
For example, the vaccine will typically be a recombinant viral vaccine, a recombinant yeast vaccine, and/or a recombinant bacterial vaccine, and the antigen will be a patient and tumor specific neoantigen. As noted above, the dendritic cells may be generated from monocytes in the peripheral blood. It is further contemplated that the antigen in the antigen-containing composition will be a patient and tumor specific neoantigen. Most typically, the antigen in the antigen-containing composition will be included in the vaccine. In still further contemplated embodiments, the vaccine may comprise a plurality of antigens and the antigen-containing composition comprises a plurality of antigens as an antigen pool or as a polytope, and the plurality of antigens in the vaccine is encoded or present as a polytope.
Preferably, but not necessarily, the cytokine-containing composition comprises IL7, IL15, and IL21, or the cytokine-containing composition comprises an IL7/N803/IL21 TxM. Likewise, it is preferred that the step of quantifying the expanded antigen-reactive T cells uses an ELISPOT assay or a FACS assay. Most typically, the predetermined threshold quantity is presence of the expanded antigen-reactive T cells at an abundance of at least 1.0% within an expansion culture.
Various objects, features, aspects, and advantages will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing in which like numerals represent like components.
The inventor has now discovered that successful generation of antigen-reactive T cells in a subject in response to immune therapy can be detected from whole blood in a conceptually simple and effective manner. More particularly, and based on the previously known antigens in the vaccine, an in vitro assay can be performed that uses monocyte derived dendritic cells of the subject that are exposed to the antigen or antigens (e.g., as an antigen pool or polytope), and the so generated antigen presenting dendritic cells are then contacted with T cells of the same subject to generate antigen-reactive T cells that are subsequently expanded using a specific cytokine-containing composition to obtain expanded antigen-reactive T cells for detection and/or quantification. As will be readily appreciated, presence of antigen-reactive T cells will be indicative of an immune response, and especially a therapeutically effective immune response where the quantity of expanded antigen-reactive T cells exceeds a predetermined threshold. Advantageously, detection and/or quantification can be performed using routine methods and equipment. Therefore, it should be recognized that the successful generation of antigen-reactive T cells in a subject in response to immune therapy can be verified (or even predicted) within only days from the subject receiving immune therapy. Consequently, and viewed from a different perspective, contemplated compositions and methods will significantly reduce the time spent between administration of a cancer vaccine and determination of its efficacy in a specific patient.
As will be readily appreciated, the nature of the immune therapy may vary considerably and will generally include direct or indirect administration of one or more disease related antigens. In preferred embodiments, the antigen is a cancer associated (e.g., MUC-1, CEA, etc.) or a cancer specific (e.g., PSA, PSMA, BRCA1, etc.) antigen, and most preferably a patient and tumor specific neoantigen. Thus, identification of suitable antigens may include a literature review, and more typically omics sequencing (e.g., whole genome sequencing, exon sequencing, RNA-seq, protein mass spectroscopy, etc.). In further preferred aspects, the neoantigens will be confirmed to be expressed in the tumor, and expressed neoantigens may be further filtered to those having a minimal binding affinity (e.g., equal or less than 500 nM, or equal or less than 200 nM, or equal or less than 100 nM) to the subjects HLA type. There are various manners of calculating minimal binding affinity and typical examples include NetMHC4.0, NetMHCpan, PSSNetMHCpan, MHCflurry, etc.
In still further contemplated aspects, suitable antigens in the cancer vaccine will be a plurality of antigens, typically arranged in a polytope in which neoantigens are sequentially arranged with interspersed (flexible) linker domains, typically having three to fifteen amino acids in length. For example, contemplated vaccine compositions especially include recombinant bacteria (e.g., E. coli, and especially E. coli engineered to lack LPS expression), viruses (e.g., Ad5, and especially Ad5[E1−Eb2−]), and/or yeast (e.g., Saccharomyces) that include a recombinant nucleic acid that encodes the antigen or polytope. Of course, it should be recognized that the subject may further receive additional therapeutic agents to stimulate an immune response such as immune stimulating cytokines (e.g., IL15, N803, etc.), checkpoint inhibitors (e.g., targeting CTLA4, PD-1, PD-L1, etc.), and cell based therapies such as T cells and/or NK cells (preferably genetically modified to express a chimeric antigen receptor or other tumor targeting entity).
Most typically, with respect to dendritic cells and T cells it is preferred that these cells are generated/obtained from peripheral blood. In most cases, PBMCs are obtained from the peripheral using standard methods well known in the art such as Ficoll density gradient centrifugation to obtain a buffy coat or leukapheresis. While dendritic cells may be isolated from PBMC, it is generally preferred that the dendritic cells are derived from monocytes in the PBMC (typically using anti-CD14 antibodies as is well known in the art) to so allow for relatively large quantities and relatively pure dendritic cell populations. Preparation of such monocyte derived dendritic cells is well known in the art (see e.g., J Vis Exp 2016) and will in most cases include a selected cytokine mixture including IL4 and GM-CSF. However, it should also be appreciated that the dendritic cells and/or T cells may also be from a heterologous source, and especially contemplated heterologous sources include HLA matched donors (e.g., with an HLA match to at least 4 digits or at least 6 digits for at least two HLA types (HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, HLA-DP)). In still further contemplated aspects, the dendritic cells and/or the T cells will be fresh cells, however, in some instances such cells may be previously frozen, particularly where the subject has a low count of dendritic cells and/or T cells due to chemotherapy.
It is still further contemplated that the blood draw may be performed prior to the patient receiving the immune therapy where prediction of an immune response is desired. On the other hand, where the patient has already received the anti-cancer vaccine, the blood draw may be performed between 1 and 7 days, or between 7 and 14 days, or between 14-28 days after first administration of the vaccine. Of course, it should be noted that more than a single blood draw and subsequent analysis is contemplated to allow for monitoring a dynamic immune response (e.g., where individual tests are used to monitor distinct neoantigens to identify antigen spread or to monitor strength of immune response over time to identify optimum response time and then switch to new and distinct vaccine).
Once blood is drawn and dendritic cells are generated, the dendritic cells can be contacted with the antigen or antigens in numerous manners. Among other options, the dendritic cells may be exposed to one or more individual purified antigens, to a at least partially purified polytope containing at least two antigens (typically separated by a linker peptide), or to crude extracts from cells expressing the antigen or polytope. In still further contemplated aspects, the antigen may also be prepared from an in vitro transcription/translation reaction, and so prepared antigens may be used directly in the transcription/translation mix or be further purified. Upon suitable exposure time, typically between 2-6 hours, or between 6-12 hours, or between 12-24 hours (and in some cases even longer), T cells will be added to the pulsed dendritic cells. Most typically, the T cells will be present relative to the dendritic cells at a ratio of about 10:1, or 7:1, or 5:1, or 3:1, or 1:1, or 1:3, or 1:5, or 1:7, or 1:10. Where desired, the exposure of the T cells to the primed dendritic cells may further include one or more immune stimulating cytokines.
Regardless of the specific exposure, so activated T cells are then expanded (after optional isolation using a Ficoll gradient) in an expansion medium that contains a cytokine composition to preferentially stimulate cell division of activated T cells. Most typically, the cytokine composition will comprise IL7, IL15, and IL21, or an IL7/N803/IL21 TxM. Expansion will be performed over a period of about 7-20 days, typically for less than two weeks with media change every 2-5 days (e.g., 3-4 days).
Upon conclusion of expansion of the antigen reactive T cells, the population of antigen-reactive T cells can then be determined using various methods well known in the art. However, it is generally preferred that the determination will use an ELISPOT assay and/or a FACS assay in which a labeled construct comprising MHC-bound neoantigen is used as a fluorescence marker as is described in more detail below. As will be readily appreciated, such methods not only provide a qualitative result, but may also be used to quantify the immune response in a subject. Most typically, a threshold value is established that is reflective or predictive of an immune response (e.g., expanded antigen-reactive T cells present at an abundance of at least 0.5%, or at least 1.0%, or at least 1.5%, or at least 3% within an expansion culture).
The following examples use CMV as a model system for a viral vaccine in human, which is a common and well characterized virus. In particular, the 65 kDa lower matrix phosphoprotein (pp65) is the main component of the enveloped subviral particle and an immunodominant antigen recognized by both CD4 and CD8 T cells as is schematically shown in
While synthetic peptides can be used for all of the single peptide, the examples below employed a recombinant pp65495-503 peptide that was produced from a construct as is shown in
More particularly, peripheral blood was drawn from two CMV-seropositive, HLA A2 0201 positive subjects using venipuncture. Monocytes were isolated using the EasySep™ Human Monocyte Isolation Kit (commercially available from Stem cell Technologies) or following other known methods of CD14-based enrichment from PBMCs. To further mature and differentiate the monocytes to dendritic cells, IL-4, GM-CSF, and TNF-α were employed. To that end, monocytes were treated with the CellXVivo Human Monocyte-derived DC Differentiation Kit (commercially available from RD Systems).
The so prepared dendritic cells were then exposed to pp65 as full-length protein (see full length sequence below), as polytope (see full length sequence below), as crude cell lysate of recombinant E. coli expressing pp65, as His-purified pp65, as pp65 peptide pool as described above, or as pp65495-503 peptide fragment (see
As can be readily seen from
Antigen reactive T cells were then detected and/or quantified using a standard ELISPOT assay as well as a FACS analysis using fluorescence labeled dextramer that was decorated with MHC to which was bound the peptide antigen (e.g., pp65495-503 peptide fragment). As can be seen from the FACS results shown in
In particular, T cell lines were generated as described for
In addition, as can be seen from the key data shown in
Sequences
The amino acid sequence of the pp65 full length protein is shown in SEQ ID NO:1.
The amino acid sequence of the pp65 Polytope (31mers with flexible linker) is shown in SEQ ID NO:2. The calculated binding affinities for sequences within the polytope are shown below:
An exemplary synthetic DNA template for creating neoepitope peptides in vitro is depicted below, and the corresponding sequences are shown in SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21 respectively. Table 3 lists a variety of other sequence constructs used in the experiment of
As shown in
As used herein, the term “administering” a pharmaceutical composition or drug refers to both direct and indirect administration of the pharmaceutical composition or drug, wherein direct administration of the pharmaceutical composition or drug is typically performed by a health care professional (e.g., physician, nurse, etc.), and wherein indirect administration includes a step of providing or making available the pharmaceutical composition or drug to the health care professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.). It should further be noted that the terms “prognosing” or “predicting” a condition, a susceptibility for development of a disease, or a response to an intended treatment is meant to cover the act of predicting or the prediction (but not treatment or diagnosis of) the condition, susceptibility and/or response, including the rate of progression, improvement, and/or duration of the condition in a subject.
The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the full scope of the present disclosure, and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the claimed invention.
It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the full scope of the concepts disclosed herein. The disclosed subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C . . . and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.
This application claims priority to our co-pending U.S. provisional patent application Ser. No. 62/992,794, filed Mar. 20, 2020, and 63/003,496, filed Apr. 1, 2020. Both of these applications are incorporated by reference herein in its entirety, including the drawings.
Filing Document | Filing Date | Country | Kind |
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PCT/IB21/51786 | 3/4/2021 | WO |
Number | Date | Country | |
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62992794 | Mar 2020 | US | |
63003496 | Apr 2020 | US |