Methods for reversing a suppressed immune state in a subject to increase survival - Patents.com

Abstract

The present disclosure provides a method for using peptides of oncogenic proteins to reverse immune suppression, induce stronger immune responses, reduce cancer recurrence rates, and prolong disease-free survival. The suppressed immune state may be due to prior treatments, including but not limited to surgery, chemotherapy, antibodies targeting the oncogenic protein, high levels of expression of the oncogenic protein, or combinations thereof. For example, in a control group of clinical studies, it was shown that when GM-CSF was administered to patients without peptides derived from the oncogenic protein, the immune response to GM-CSF alone was lower when compared with patients who were not treated with antibodies against the oncogenic protein and did not have high levels of expression of the oncogenic protein, and the immune state of these patients was suppressed, with higher recurrence rates and lower disease-free survival rates, all of which were reversed by the addition of the peptides.

Description

[Technical field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 63 / 317,601, filed March 8, 2022, and U.S. Provisional Application No. 63 / 329,409, filed April 9, 2022, each of which is incorporated by reference in its entirety herein.

[0002] (Sequence Listing) This application contains a Sequence Listing that has been submitted electronically via EFS-Web to the United States Patent and Trademark Office as an ASCII text file, which Sequence Listing is named "0629.000003WO01.xml", has a size of 2 kilobytes, and was created on March 8, 2023. The information contained in that Sequence Listing is incorporated herein by reference. [Background technology]

[0003] (background) HER2 / neu (human epidermal growth factor receptor 2 (also referred to herein as HER2)) protein is a cell surface receptor protein expressed in a variety of common cancers, including 75% of breast cancers. In breast cancer, this protein can be expressed at low (1+), moderate (2+), and high (3+ or overexpressor or positive) levels. Removal of the tumor by surgery is beneficial in treatment, and further treatment is typically recommended. There are many therapies approved for HER2 / neu expressing breast cancer. For example, subjects expressing HER2 / neu may receive trastuzumab (Herceptin®) or a trastuzumab-based derivative before surgery or as a follow-up treatment within one year after surgery to reduce the chance of recurrence. However, some patients do not respond to the trastuzumab-based treatment and recur within five years of the surgery or thereafter. Summary of the Invention [Means for solving the problem]

[0004] (Summary of the present application) GP2 is a nine amino acid transmembrane peptide of the HER2 / neu protein and is a natural cancer antigen. GP2, when combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) (these combined are called GLSI-100), is a candidate treatment for reducing cancer recurrence and therefore increasing disease-free survival in breast cancer patients with tumors that express any amount of HER2 / neu. We found that GLSI-100 safely elicited a strong immune response, evidenced by injection site reactions (ISR), which led to a reduced recurrence rate of 0% and a corresponding increased disease-free survival rate of 100% during a 5-year follow-up in HER2 3+ or positive patients. These patients received a standard course of trastuzumab after surgery and were then treated with GLSI-100. The injection site reactions of HER2 3+ patients treated with GLSI-100 were significantly greater than those of the GM-CSF alone control group, providing further evidence of the specific GP2 immune response and the boost in immune response that occurred specifically by adding GP2 alone. The lower immune response of the GM-CSF alone HER2 positive patients suggests a reduced immune status compared to the HER2 low and intermediate expressers (HER2 1+ and HER2 2+) treated with either GLSI-100 or GM-CSF alone (these patients had similar ISRs to the HER2 positive patients treated with GLSI-100). The HER2 low and intermediate expressers were not treated with trastuzumab. Thus, the addition of GP2 alleviates the reduced immune status in HER2 positive patients, which may be caused by HER2 positivity and / or trastuzumab prior treatment. Trastuzumab treatment may also include derivatives of trastuzumab (e.g., antibody drug conjugates or other antibodies that target the extracellular portion of the HER2 / neu protein).The ISR resulting from GLSI-100 administration is associated with a delayed-type hypersensitivity (DTH) immune response to GP2, and the ISR resulting from GLSI-100 administration may be used to complement or replace the delayed-type hypersensitivity (DTH) immune response to GP2.Thus, as described herein, an immune response (such as, but not limited to, a positive DTH immune response or ISR to the peptide, or a combination thereof) can be an indicator of the immune status of a subject, including whether the subject is experiencing an immune response and therefore less likely to have a recurrence of cancer and more likely to experience a longer disease-free survival period. The peptide can be a peptide that is a peptide of a protein associated with cancer (e.g., a peptide of an oncogenic protein). In one embodiment, the peptide is a part of Her2 / neu (e.g., GP2).

[0005] Terms used in this specification are understood to have their ordinary meaning in the relevant art unless otherwise specified. Some terms used in this specification and their meanings are set forth below.

[0006] "Relapse" and "recurrence" are used interchangeably herein and refer to a diagnosis of the return of breast cancer after a period of improvement or remission, or a diagnosis of signs and symptoms of the return of breast cancer. Cancer recurrence can be diagnosed using standard of care methods (e.g., patient follow-up visits, blood tests, mammograms, and imaging).

[0007] As used herein, "oncoprotein" and "oncogenic protein" are used interchangeably and refer to proteins associated with cancer in humans. Oncoproteins are encoded by oncogenes. Oncogenes are mutated forms of normal cellular genes (proto-oncogenes). The protein products of proto-oncogenes stimulate cell division and / or inhibit cell death. Proto-oncogenes can be likened to the gas pedal of a car. Normally, internal and external signals tightly regulate the activity of the proto-oncogene, but oncogenes are flawed and stay "on" even when the oncogene does not receive the appropriate signal. Oncogenes also help cells ignore negative signals that prevent healthy cells from dividing. Oncogenes can cause cells to divide continuously even in the absence of any pro-growth signals. The following list describes the various cellular roles of a few of the many known oncogenes. HER-2 / neu: HER-2 / neu encodes a cell surface receptor that can stimulate cell division, and the HER-2 / neu gene is amplified in up to 30% of human breast cancers; RAS: Ras gene products are involved in kinase signaling pathways that ultimately control gene transcription to regulate cell growth and differentiation, and overexpression and amplification of RAS can result in continued cell proliferation; MYC: Myc protein is a transcription factor that controls the expression of several genes, Myc is thought to be involved in evading cell death mechanisms, and the MYC oncogene can be activated by gene rearrangement or amplification; SRC: SRC was the first oncogene to be discovered.Src proteins are tyrosine kinases that regulate cell activity; hTERT: hTERT codes for an enzyme (telomerase) that maintains chromosome ends, in most normal cells, telomerase is only present during fetal development, and activation of hTERT in mature cells gives them the ability to divide indefinitely; BCL-2: Bcl-2 proteins function to prevent cell death (apoptosis), and overexpression of BCL-2 allows mutated cells to continue dividing. Oncogenic proteins can be proteins that are expressed by non-cancerous cells but at higher levels in cancer cells, or proteins that are not expressed by non-cancerous cells but are expressed in cancer cells. Examples of oncogenic proteins are shown in Table 1 (and also on the World Wide Web at cancerquest.org / cancer-biology / cancer-genes). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0008] As used herein, "peptide" refers to a portion of a protein (e.g., a protein associated with cancer, e.g., an oncoprotein) that is of sufficient size to generate an immune response. A peptide can be at least 6 amino acids, at least 9 amino acids, or at least 12 amino acids in length.

[0009] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more.

[0010] As used in this specification and the appended claims, the term "or" is generally used in its sense, including "and / or," unless the context clearly indicates otherwise. The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements. The use of "and / or" in some instances does not mean that the use of "or" in other instances may not mean "and / or."

[0011] The words "preferred" and "preferably" refer to embodiments of the present disclosure that may provide certain advantages, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Moreover, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and the recitation of one or more preferred embodiments is not intended to exclude other embodiments from the scope of the present disclosure.

[0012] As used herein, the words "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising," and the like are used in their non-limiting, inclusive sense and generally mean "including, but not limited to," "includes, but not limited to," or "including, but not limited to."

[0013] Whenever an embodiment is described herein using the words "have," "has," "having," "include," "includes," "including," "comprise," "comprises," or "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also presented. The term "consisting of" means to include and be limited to whatever precedes the phrase "consisting of." That is, "consisting of" indicates that the recited elements are required or mandatory, and that no other elements may be present. The term "consisting essentially of" indicates that any elements recited before the phrase are included, and that elements other than the recited elements may be included, provided that they do not interfere with or contribute to the activity or action identified in this disclosure for those recited elements.

[0014] References throughout this specification to "one embodiment," "an embodiment," "particular embodiment," or "some embodiments," etc., mean that the particular feature, configuration, composition, or characteristic described with respect to that embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification do not necessarily refer to the same embodiment of the disclosure. Moreover, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0015] Throughout this disclosure, various aspects of the disclosure may be described in range format. It should be understood that the description in range format is merely for convenience and brevity, and the description in range format should not be interpreted as an indefinite limitation on the scope of the disclosure. Thus, the description of a range should be considered to have all possible subranges specifically disclosed, as well as individual values ​​within that range. For example, the description of a range (e.g., 1-6) should be considered to have specifically disclosed subranges (e.g., 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc.), as well as individual values ​​within that range (e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6). This is true regardless of the breadth of the range.

[0016] In the description herein, specific embodiments may be described separately for clarity. A particular embodiment may include any combination of compatible features described herein in connection with one or more embodiments, unless a feature of a particular embodiment is specifically and explicitly specified as incompatible with a feature of another embodiment.

[0017] For any method disclosed herein that includes distinct steps, those steps may be carried out in any order practicable. Where appropriate, any combination of two or more steps may be carried out simultaneously.

[0018] The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The following description illustrates exemplary embodiments in more detail. In several places throughout this application, guidance is provided through the listing of examples, which examples can be used in various combinations. In each instance, the listed list serves only as a representative group and should not be interpreted as an exclusive list.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description of the exemplary embodiments of the present disclosure can be best understood when read in conjunction with the following drawings. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 shows HER2 3+ subjects who completed the primary immunization series after treatment with trastuzumab. 100.0% refers to disease-free survival for subjects treated with GLSI-100, and 89.4% refers to disease-free survival for placebo subjects treated with GM-CSF. The inset depicts the median orthogonal mean of injection site reaction (ISR) over time for the HER2 3+ subjects treated with GLSI-100 (upper trace) or HER2 3+ placebo subjects treated with GM-CSF alone (lower trace). The median orthogonal mean of the inset (Y-axis) is the median orthogonal mean of injection site reaction (ISR). HER2+ and HER2 positive are equivalent to HER2 3+; HER2- and HER2 negative are equivalent to HER2 1+ and HER2 2+. The numbers in the two rows under the x-axis, labeled "GP2" and "Placebo," refer to the number of patients in each group at each time point tested. Trastuzumab-treated HER2 3+ patients with unsuppressed immune status who received GLSI-100 (upper trace in inset) were those with 100% disease-free survival during 5 years of follow-up (upper trace).

[0021] [Diagram 2] Figure 2 shows the correlation between DTH and ISR at 6 months at the sixth injection in GLSI-100 treated subjects. One outlier was excluded from this analysis due to the effect of this point.

[0022] [Diagram 3] FIG. 3 shows the correlation between baseline DTH and ISR at the first injection in GLSI-100 treated subjects.

[0023] [Figure 4]Figure 4 shows injection site reactions (ISR) by HER2 status in treated (GLSI-100) versus placebo (GM-CSF alone) subgroups. The solid traces labeled "HER2 positive treated with GLSI-100" and "HER2 positive treated with GM-CSF alone" are the traces shown in solid lines in the Figure 1 inset. Figure 4 also includes two additional traces for "HER2 low and HER2 moderate expressing treated with GLSI-100" and "HER2 low and HER2 moderate expressing treated with GM-CSF alone." HER2+ and HER2 positive are equivalent to HER2 3+; HER2- and HER2 negative are equivalent to HER2 1+ and HER2 2+. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] (Detailed Description) The inventors have determined that GLSI-100 safely elicited a strong immune response, as shown by ISR, which correlates with and serves as a complement to immune response data (e.g., DTH). The lower immune response of the HER2-positive control population treated with trastuzumab (not shown in low HER2 expressers and moderate HER2 expressers who did not receive trastuzumab) suggests a reduced immune state that may be related to prior exposure to trastuzumab and / or HER2 positivity, and this reduced immune state is reversed with the addition of GLSI-100. In HER2-positive (3+) patients, GP2 reverses the suppressed immune state, increases immune response, reduces cancer recurrence rate, and prolongs disease-free survival.

[0025] The present inventors have also determined that peptides (e.g., peptides of oncogenic proteins) reverse the suppressed immune state in subjects. The suppressed immune state may be due to, for example, prior treatment. Prior treatment may include, but is not limited to, surgery, chemotherapy, and antibody therapy targeting oncogenic proteins, or combinations thereof. The administration of the peptides (e.g., peptides of oncogenic proteins) reverses immune suppression, induces stronger immune responses, reduces recurrence rates, and / or prolongs disease-free survival. For example, in a control group of clinical studies, it was shown that when GM-CSF was administered to these patients without the peptide derived from the oncogenic protein, the immune response to GM-CSF was suppressed when compared with patients who were not treated with antibodies against the oncogenic protein and did not have high levels of expression of the oncogenic protein.

[0026] Examples of cancers that can be treated to reverse suppressed immune states using the methods described herein include Her2 / neu positive cancers, such as breast cancer, ovarian cancer, stomach cancer, prostate cancer, lung cancer, colon and rectum cancer, urinary bladder and urinary corpus cancer, pancreatic cancer, liver cancer, esophageal cancer, ovary cancer, and many other cancers, which can include many different types of proteins (e.g., oncoproteins) or combinations thereof. Other examples of cancers that can be analyzed using the methods described herein include, but are not limited to, the cancers listed in Table 1. Oncoproteins associated with various cancers are encoded by the oncogenes listed in Table 1. The peptides can be any set of consecutive amino acids derived from a protein (e.g., a protein associated with cancer, such as an oncoprotein). In one embodiment, the peptide is a peptide that has therapeutic activity by activating the immune system, for example, the peptide can be used to treat the subject to increase the immune response against cancer, thus reducing one aspect of cancer and improving the health of the subject.For example, the peptide can be a peptide that reduces the recurrence of cancer in the subject (including recurrence caused by metastatic cancer) by training the immune system to attack the cancer.The therapeutic activity can result from the peptide or from the combination of the peptide with one or more compounds.An example of a peptide that has therapeutic activity is GP2, which is typically administered with GMCSF.

[0027] The methods may include determining the subject's immune response to a peptide, such as a protein associated with cancer (e.g., a peptide of an oncogenic protein). The methods include a subject that is a human or an animal that is typically used as a model system to evaluate cancer-related treatments. In one embodiment, the subject is a human. In one embodiment, the subject may be a human at risk of cancer, and the peptide used in the method is derived from an oncogenic protein associated with the cancer. A human at risk of cancer includes, but is not limited to, a human who has been treated for cancer and is in complete or partial remission. A human at risk of cancer also includes, but is not limited to, a human who has one or more biomarkers or a human who has a genetic risk for cancer. A biomarker is a molecule (e.g., a gene or protein) that is present in a subject and is indicative of the progression of cancer. Examples of biomarkers include BRCA1, BRCA2, and prostate-specific antigen (PSA). A human at genetic risk is a human who has a history of cancer in a family member (e.g., a first-degree relative). In one embodiment, the subject may be a subject who has not been previously diagnosed with cancer. Such a person may be a person at risk for cancer, for example, the subject may be a subject who has not been diagnosed with cancer but has one or more biomarkers and / or has a genetic risk for cancer.

[0028] In another embodiment, the subject may be a human who has been previously treated for cancer, and the peptide used in the method is derived from a protein (e.g., a protein associated with cancer, e.g., an oncogenic protein). In one embodiment, the subject is a subject who has been previously treated for a cancer that was HER2 / neu positive at low, moderate, or high levels. HER2-positive cancers (e.g., breast cancer) are typically assessed for HER2 / neu expression and assigned an immunohistochemistry (IHC) score of 0 or 1+ (which may be referred to as HER2 / neu low), HER2 / neu 2+ (which may be referred to as HER2 / neu intermediate), or HER2 / neu 3+ (which may be referred to as HER2 / neu high or HER2 / neu 3+ or HER2 / neu positive). "HER2 positive" or "HER2 3+" patients are defined by the ASCO CAP guidelines (American Society of Clinical Oncology and College of American Pathologists) and also include a fluorescent in situ hybridization (FISH) score of about 2.0 or greater for HER2 / neu gene expression. Methods for assessing HER2 / neu expression and for assigning IHC or FISH scores are known in the art. In this embodiment, the cancer can be, but is not limited to, HER2 / neu positive breast cancer, HER2 / neu positive ovarian cancer, or HER2 / neu positive gastric cancer, or any other HER2 / neu positive cancer. In one embodiment, the cancer is HER2 / neu positive breast cancer.

[0029] In one embodiment, the method may be used to treat a patient with a peptide, such as a peptide derived from an oncogenic protein. In one embodiment, the peptide is derived from the Her2 / neu protein. The peptide may be derived from the cytoplasmic, extracellular, or transmembrane domain of the Her2 / neu protein. In one embodiment, the peptide is derived from the transmembrane domain, a non-limiting example of a peptide derived from the transmembrane domain is GP2 (Ile Ile Ser Ala Val Val Gly Ile Leu (SEQ ID NO: 1)). GP2, in combination with granulocyte macrophage colony stimulating factor (GM-CSF), is a therapy used to stimulate an immune response in breast cancer patients with tumors that express any HER2 / neu, thus reducing cancer recurrence. (Patel et al., Five year median follow-up data from a prospective, randomized, placebo-controlled, single-blinded, multicenter, Phase IIb study evaluating the reduction of recurrences using HER2 / neu peptide GP2+GM-CSF vs. GM-CSF alone after adjuvant trastuzumab in HER2 positive women with operable breast cancer., presented at the 2020 San Antonio Breast Cancer Symposium, December 8-11, 2020.)

[0030] The immune response to a peptide (e.g., a peptide of a protein associated with cancer (e.g., an oncogenic protein)) can be determined by any method. The immune response can be determined by (but is not limited to) delayed type hypersensitivity (DTH), injection site reaction (ISR), binding assays for T cells, functional cell-based assays for cytokine secretion (e.g., Elispot (including Elispot assays to quantify T cell viability), measurement of antibody titers, and T cell assays (including identification of specific T cells for any cancer target, including T cell binding assays for HLA-peptide reagents to quantify peptide-specific T cells). An immune response is considered a positive immune response if it is greater than that of an appropriate negative control. Thus, any increase in immune response greater than an appropriate negative control is a positive immune response. One of skill in the art can readily determine negative controls that can be used to determine whether there is an immune response to a peptide (e.g., a cancer-associated peptide (e.g., an oncogenic protein)). In one embodiment, the immune response is measured by testing the subject's DTH response to intradermal injection of a peptide (e.g., a cancer-associated peptide (e.g., a protein)) at an amount less than, similar to, or greater than the treatment dose. Methods for testing DTH responses are known and routine in the art. In one embodiment, induration (e.g., localized hardening) of the skin at the site of administration is measured. If induration is determined, the perpendicular diameter of the induration is measured. The diameter of the skin reaction is typically measured. Measurements may be by any suitable method, including caliper, ruler, or ballpoint pen techniques. The measurement may be expressed as the maximum diameter of the skin reaction, or the orthogonal mean of the diameters is determined. An induration measurement of at least 3 mm, at least 5 mm, at least 7 mm, or at least 10 mm or more may be considered a positive DTH response. A subject's DTH response may be measured 24 to 72 hours, typically 48 to 72 hours, after administration of the peptide at the selected site.

[0031] In the embodiment where the peptide is a peptide having therapeutic activity, the peptide can be used to determine immune response before the peptide is used for treatment. For example, the peptide can be used to determine immune response after the subject is diagnosed with cancer or after the subject is treated for cancer, for example by surgery or chemotherapy. In one embodiment, GP2 is used as the peptide, and it is administered to determine immune response before the GP2 is used therapeutically. In another embodiment, GP2 is used as the peptide, and it is administered to determine immune response at about the same time as the GP2 is administered therapeutically. Typically, the immune response resulting from the test administered before or at about the same time as the treatment is started (e.g., before the subject can mount an immune response to the administered peptide) is referred to herein as "baseline" response.

[0032] In embodiments in which the peptide is not a therapeutic peptide, the peptide can be used to determine the immune response at any time (e.g., before the subject is diagnosed with cancer, after the subject is diagnosed with cancer, or after the subject has been treated for cancer, e.g., by surgery or chemotherapy).

[0033] The amount of peptide administered to a subject to measure an immune response may vary and is not intended to be limiting. For example, the range may be 0.01 milligrams per milliliter (mg / ml) to 1 mg / ml. If the peptide is a Her2 / neu peptide (e.g., GP2), the amount may be 0.05 mg / ml to 0.4 mg / ml, and in one embodiment, is 0.2 mg / ml. In one embodiment, the composition of the peptide administered does not include an immunomodulatory agent (e.g., GMCSF). The volume used may vary and is not intended to be limiting. For example, the volume may be 0.8 ml to 0.2 ml, and in one embodiment, is 0.5 ml. One skilled in the art will recognize that saline injection may be used as a negative control.

[0034] After a positive immune response to the peptide, the subject can be treated with a therapy to reduce existing cancer, reduce the risk of recurrence of the cancer, or a combination thereof, which may include, but is not limited to, antibodies, antibody drug conjugates, checkpoint inhibitors, hormone therapy, tyrosine kinase inhibition, surgery, chemotherapy, or radiation therapy.

[0035] In some embodiments where the method may be used to treat a subject, the method includes administering a peptide to the subject. The peptide may be at least 6 amino acids, at least 9 amino acids, or at least 12 amino acids in length. The peptide may be a peptide of an oncogenic protein (e.g., an oncogenic protein in Table 1). In one embodiment, the oncogenic protein is HER2 / neu. In one embodiment, the peptide is GP2 or a derivative thereof. The amount of peptide administered to a subject for treatment may vary and is not intended to be limiting. For example, the range may be 0.01 milligrams per milliliter (mg / ml) to 5 mg / ml. When the peptide is a Her2 / neu peptide (e.g., GP2), the amount may be 0.2 mg / ml to 2 mg / ml, and in one embodiment is 1 mg / ml. The volume used may vary and is not intended to be limiting. For example, the volume may be 0.1 ml to 1 ml, and in one embodiment is 0.5 ml. One of skill in the art will recognize that saline injection may be used as a negative control.

[0036] In one embodiment, the treatment of the subject may include administration of an adjuvant. An example of an adjuvant is granulocyte macrophage colony stimulating factor (GM-CSF). Examples of GM-CSF include, but are not limited to, derivatized GM-CSF, non-glycosylated or partially non-glycosylated GM-CSF, or truncated GM-CSF. GM-CSF may be produced (e.g., recombinantly) from any genetically modified cell line, may be synthesized, or may be obtained from natural sources. In one embodiment, the GM-CSF is sargramostim (Leukine®). The peptide and the adjuvant may be administered together in a composition, or may be administered independently at the same time or at different times. The administration may be intradermal, subcutaneous, intramuscular, or by inhalation.

[0037] The subject to be treated may be a subject with or have had cancer. The cancer may be a cancer listed in Table 1. In one embodiment, the cancer is a HER2 / neu expressing cancer (e.g., breast cancer, ovarian cancer, gastric cancer, prostate cancer, lung cancer, colon and rectum cancer, urinary bladder and urinary corpus cancer, pancreatic cancer, liver cancer, esophageal cancer, or ovary cancer). A HER2 / neu expressing cancer is a cancer that contains HER2 / neu expressing cells. The HER2 / neu expressing cells may have expression that is HER2 / neu 3+ (also called HER2 / neu positive), HER2 / neu intermediate expression, or HER2 / neu low expression.

[0038] The subject may have undergone a prior treatment for the cancer. Examples of prior treatment include, but are not limited to, surgery, chemotherapy (e.g., therapeutic agents or radiation), antibody therapy, targeted therapy, T cell therapy, RNA / DNA therapy, or a combination thereof. The prior treatment may be a prior treatment that targets an oncogenic protein (e.g., antibody therapy, targeted therapy, T cell therapy, or RNA / DNA therapy). In embodiments where the oncogenic protein is HER2 / neu, the antibody may target any epitope on the HER2 / neu protein. Examples of such antibodies include, but are not limited to, trastuzumab (Herceptin®), ado-trastuzumab emtansine (Kadcyla™), fam-trastuzumab deruxtecan-nxki (Enhertu®), trastuzumab biosimilar, pertuzumab (Perjeta®), or derivatives thereof. In some embodiments, the antibody or derivative thereof is an antibody drug conjugate. Examples of antibody drug conjugates include, but are not limited to, antibodies having one or more linked molecules (e.g., chemotherapeutic agents). In embodiments where the cancer is a cancer comprising HER2 / neu expressing cells with HER2 / neu moderate expression or HER2 / neu low expression, the prior treatment comprises an antibody therapy targeting HER2 / neu. In embodiments where the cancer is a cancer that contains HER2 / neu expressing cells that are HER2 / neu positive, the subject has not received prior treatment for the cancer including antibody therapy that targets HER2 / neu.

[0039] The method for treating a subject can be used to reverse immune suppression in the subject, to induce a stronger immune response in the subject, to reduce the recurrence rate of the cancer in the subject, to increase disease-free survival in the subject, or combinations thereof. The method can include a step of determining the immune status of the subject. The immune status of the subject can be determined by any immune response assay to the peptide alone, the adjuvant alone, or the combination of both the peptide and the adjuvant. Examples of immune response assays include, but are not limited to, delayed type hypersensitivity (DTH) immune response, injection site reaction (ISR), ELISPOT assay, antibody titer assay, and T cell assay. The immune status of the subject can be determined before the start of the treatment, at the start of the treatment, after the start of the treatment, or any combination thereof. In one embodiment, the immune status of the subject is compared to that of a healthy or normal patient. A subject with a reduced immune response (e.g., reduced ISR) compared to a healthy or normal subject is a suitable recipient of the treatment method. In one embodiment, a healthy or normal immune state has an ISR of 50 millimeters (mm) or more to a peptide (e.g., GP2) or adjuvant (e.g., GM-CSF). In one embodiment, a hypoimmune state or suppressed immune state has an ISR of less than 50 mm to a peptide (e.g., GP2) or adjuvant (e.g., GM-CSF). In one embodiment, a hypoimmune state has an ISR of at least 40 mm less than the ISR of a healthy or normal immune state. A successful outcome of the treatment is restoration of the subject's immune system to a healthy or normal immune state. The treatment may also result in a stronger immune response in the subject, a reduced recurrence rate of the cancer in the subject, increased disease-free survival in the subject, or a combination thereof.

[0040] The present disclosure also provides the use of adjuvant for determining the immune status of a subject.The present invention is defined in claims.However, the following provides a non-exhaustive list of non-limiting exemplary aspects.Any one or more of the features of these aspects can be combined with any one or more of the features of another example, embodiment, or aspect described herein.

[0041] (Exemplary embodiment)

[0042] Embodiment 1. A method for treating a patient, comprising administering to a subject who has or has had cancer and has been treated for said cancer a peptide, which reverses immune suppression, produces a stronger immune response, reduces cancer recurrence rates, and / or increases disease-free survival.

[0043] The method of claim 1, wherein the peptide is at least 6 amino acids.

[0044] Embodiment 3 The method of embodiment 1 or 2, wherein the peptide is a peptide of an oncogenic protein.

[0045] Aspect 4. The method according to any one of Aspects 1 to 3, wherein the oncogenic protein is HER2 / neu.

[0046] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the prior treatment for the cancer comprises surgery, chemotherapy, antibody therapy, targeted therapy, T cell therapy, RNA / DNA therapy, or any other therapy that targets the oncogenic protein, or a combination thereof.

[0047] Aspect 6. The method of any one of Aspects 1 to 5, wherein the antibody therapy, targeted therapy, RNA / DNA therapy, or T cell therapy targets an oncogenic protein.

[0048] Embodiment 7. The method of any one of embodiments 1 to 6, wherein said oncogenic protein is HER2 / neu and said antibody targets an epitope on said HER2 / neu protein.

[0049] Embodiment 8. The method of any of embodiments 1 to 7, wherein the antibody is trastuzumab, trastuzumab biosimilar, ado-trastuzumab emtansine, fam-trastuzumab deruxtecan-nxki, pertuzumab, or other antibody targeting an epitope on the HER2 / neu protein, or a derivative thereof.

[0050] Embodiment 9. The method of any of embodiments 1 to 8, wherein the antibody or derivative comprises a linked molecule or multiple linked molecules, e.g., an antibody drug conjugate, which may include a chemotherapeutic drug.

[0051] Aspect 10. The method according to any one of aspects 1 to 9, wherein the peptide is a HER2 / neu-derived peptide.

[0052] Aspect 11. The method according to any one of aspects 1 to 10, wherein the peptide is GP2 or a derivative thereof.

[0053] The method of any one of claims 1 to 11, wherein the cancer comprises HER2 / neu expressing cells.

[0054] Aspect 13. The method of any one of Aspects 1 to 12, wherein the HER2 / neu expressing cells are HER2 / neu 3+, HER2 / neu positive, HER2 / neu intermediate expressing, or HER2 / neu low expressing.

[0055] Aspect 14 The method according to any one of aspects 1 to 13, wherein the cancer is breast cancer.

[0056] Embodiment 15. The method of any one of embodiments 1 to 14, wherein the subject had or has a HER2 / neu positive cancer.

[0057] Aspect 16. The method of any one of aspects 1 to 15, wherein the cancer is a cancer comprising HER2 / neu expressing cells that have moderate HER2 / neu expression or low HER2 / neu expression, and the prior treatment comprises an antibody therapy targeting HER2 / neu.

[0058] Aspect 17. The method of any one of Aspects 1 to 16, wherein the cancer is a cancer that contains HER2 / neu expressing cells that are HER2 / neu positive, and the subject has not received prior treatment for the cancer comprising an antibody therapy that targets HER2 / neu.

[0059] Embodiment 18. The method of any one of embodiments 1 to 17, wherein the treatment further comprises administering an adjuvant.

[0060] Embodiment 19. The method of any one of embodiments 1 to 18, wherein the peptide and the adjuvant are administered together in a composition or administered independently at the same or similar time.

[0061] Aspect 20. The method of any one of aspects 1 to 19, wherein the step of determining immune suppression comprises assaying delayed type hypersensitivity (DTH) immune response or injection site reaction (ISR) or any other immune response assay to the peptide or to the adjuvant alone.

[0062] Aspect 21: The method according to any one of aspects 1 to 20, wherein the peptide is a peptide of an oncogenic protein.

[0063] Aspect 22 The method according to any one of aspects 1 to 21, wherein the oncogenic protein is HER2 / neu.

[0064] Aspect 23: The method according to any one of aspects 1 to 22, wherein the peptide of the oncoprotein is GP2.

[0065] Embodiment 24. The method of any one of embodiments 1 to 23, wherein the subject had or has breast cancer, ovarian cancer, gastric cancer, cervical cancer, or any other HER2 / neu expressing cancer.

[0066] Aspect 25. The method of any one of aspects 1 to 24, wherein the step of determining immune suppression comprises an immune response assay capable of detecting a subject's immune response to the peptide or to the adjuvant alone.

[0067] Embodiment 26. The method of any one of embodiments 1 to 25, wherein the immune response assay is selected from a DTH response, an ISR, an ELISPOT assay, an antibody titer assay, and a T cell assay, or a combination thereof.

[0068] Aspect 27 The method of any one of aspects 1 to 26, wherein the treatment comprises measuring an injection site response to a therapeutic peptide for cancer. Aspect 28. The method according to any one of aspects 1 to 27, wherein the adjuvant alone is injected intradermally and the ISR is measured as a biomarker for determining the immune status of the patient.

[0069] Embodiment 29. The method according to any of embodiments 1 to 28, wherein the ISR measurements are compared to patients with a healthy or normal immune status, and subjects with a hypoimmune status patients are treated to restore the immune system to a healthy or normal immune status.

[0070] Embodiment 30 The method according to any one of embodiments 1 to 29, wherein the subject is treated with the peptide.

[0071] Embodiment 31 The method according to any one of embodiments 1 to 30, wherein the ISR in a healthy or normal immune state is 50 mm or greater in diameter.

[0072] Embodiment 32 The method of any one of embodiments 1 to 31, wherein the hypoimmune state ISR is less than 50 mm in diameter.

[0073] Embodiment 33. The method of any one of embodiments 1 to 32, wherein the hypoimmune state ISR is 40 mm smaller in diameter than the healthy or normal immune state ISR.

[0074] Embodiment 34 The method of any one of embodiments 1 to 34, wherein the adjuvant comprises granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0075] Embodiment 35. A method according to any one of embodiments 1 to 34, wherein the GM-CSF is derivatized, non-glycosylated or partially non-glycosylated GM-CSF, or a truncated GM-CSF.

[0076] Embodiment 36 The method according to any one of embodiments 1 to 35, wherein the GM-CSF is recombinant GM-CSF.

[0077] Embodiment 37 The method of any one of embodiments 1 to 36, wherein the GM-CSF is sargramostim. EXAMPLES

[0078] (Example) The present disclosure is illustrated by the following examples. It should be understood that the specific examples, materials, amounts, and procedures should be interpreted broadly in accordance with the scope and spirit of the disclosure presented herein.

[0079] Example 1

[0080] BACKGROUND: Delayed-type hypersensitivity (DTH) skin tests were analyzed in a randomized, active-controlled, single-blind, multicenter, phase IIb study (NCT00524277) investigating GLSI-100 (GP2+GM-CSF) administered in the adjuvant setting to patients with node-positive and high-risk node-negative breast cancer whose tumors expressed any HER2 (immunohistochemistry [IHC] 1-3+). The study enrolled HLA-A*02 patients randomized to receive GLSI-100 versus GM-CSF alone. The primary objective of the study was to determine whether treatment with GLSI-100, a HER2-derived peptide, reduced recurrence rates.

[0081] (Injection site reaction analysis in a Phase IIb study of GP2)

[0082] Methods: Patients were randomized to receive GLSI-100 (500 micrograms (mcg) GP2 and 125 mcg GM-CSF) or control (GM-CSF) by six intradermal injections every 3 to 4 weeks for an initial 6 month period, followed by four booster injections every 6 months. The size of the injection site reactions, which occurred in almost all patients, was assessed for the 10 doses administered by measuring the maximum perpendicular diameter and the resulting orthogonal mean. DTH responses were assessed in a similar manner at baseline and after 6 months of treatment, and were shown to increase over time.

[0083] Results: The study enrolled 180 patients across 16 clinical sites, including both HER2 3+ positive and low and intermediate HER2 expressors (1-2+). After 5 years of follow-up, the 5-year disease-free survival (DFS) rate, assessed by Kaplan-Meier method, was 100% in 46 HER2 3+ patients treated with GLSI-100 who completed the primary immunization series (PIS) versus 89.4% (95% confidence interval (CI): 76.2, 95.5%) in 50 placebo patients treated with GM-CSF (p=0.0338) (Figure 1). GLSI-100 was shown to be well tolerated without any serious adverse events (SAEs) considered related to the study drug, and elicited robust immune responses as measured by topical skin tests and immunological assays. Injection site reactions were common and occurred in nearly 100% of patients treated with either GLSI-100 or GM-CSF alone. Patients treated with GLSI-100 had statistically significantly greater injection site reactions compared to the magnitude of reactions for patients treated with GM-CSF alone (p<0.05).

[0084] As shown in Figures 2 and 3, DTH responses at baseline and 6 months were consistently correlated with injection site responses, suggesting that injection site responses may be interchangeable with immune response data (baseline ρ=0.6, p<0.001; 6 months ρ=0.4, p=0.009).

[0085] As shown in Figure 4, after a single dose, the median orthogonal mean induration was 7.8 mm for GM-CSF patients and 34.0 mm for GLSI-100 patients (p=0.0036). Injection site reactions increased to 78 mm after the fourth injection, but remained above 50 mm for the duration of the injection series for patients treated with GLSI-100. The magnitude of injection site reactions for patients treated with GLSI-100 was similar across HER2 status. However, HER2 3+ control patients (HER2+ in Figure 4) (who were also treated with Herceptin®) had significantly smaller responses to GM-CSF than HER2 1-2+ control patients (HER2- in Figure 4) who did not receive Herceptin®, with an average difference of 43.1 mm across all 10 doses. Trastuzumab-treated HER2 3+ control patients appeared to have a suppressed immune state that was reversed by adding GP2 treatment, thereby increasing injection site reactions by an average of 39.2 mm across all 10 doses. Trastuzumab-treated HER2 3+ patients receiving GLSI-100 with an unsuppressed immune state (upper solid line in Figure 1 inset and upper solid line in Figure 4) were the patients with 100% disease-free survival during 5 years of follow-up (see Figure 1). Conclusion: Treatment of HER2 / neu positive patients (who received a standard course of trastuzumab after surgery) with GLSI-100 (GP2+GM-CSF) safely elicited a strong immune response evidenced by greater injection site reactions, reduced recurrence rates to 0% and increased disease-free survival to 100% during 5 years of follow-up. Injection site reactions in HER2 positive patients treated with GLSI-100 were significantly greater than those in the GM-CSF alone control group, providing further evidence of a specific GP2 immune response generated by treatment with GLSI-100. It was demonstrated that when GM-CSF was administered to control patients without peptides derived from the oncoprotein, the immune response to GM-CSF was lower, the immune status was suppressed, the recurrence rate was higher, and the disease-free survival rate was lower when compared to patients not treated with antibodies against the oncoprotein and who did not have high levels of expression of the oncoprotein, all of which were reversed by the addition of GP2.

[0086] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (e.g., nucleotide sequence entries in GenBank and RefSeq, and amino acid sequence entries in SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) are incorporated by reference in their entirety. Supplementary materials referenced in publications (e.g., supplementary tables, figures, materials and methods, and / or experimental data) are also incorporated by reference in their entirety. In the event of any discrepancy between the disclosure of this application and the disclosure of any document incorporated by reference herein, the disclosure of this application shall prevail. The detailed description and examples above are provided for clarity of understanding only. No unnecessary limitations should be understood therefrom. The present disclosure is not limited to the exact details shown and described, and variations obvious to those skilled in the art are included within the disclosure defined by the claims.

[0087] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims should be understood to be modified in all instances by the term "about". Thus, unless specifically indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending on the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0088] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, every numerical value inherently contains some range necessarily resulting from the standard deviation found in their respective testing measurements.

[0089] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading unless specifically specified to do so.

Claims

1. A composition for treating a patient, comprising a peptide, wherein the peptide is administered to a subject who has or has had cancer and has received prior treatment for the cancer, wherein the peptide reverses immunosuppression, elicits a stronger immune response, reduces cancer recurrence rates, and / or increases disease-free survival.

2. The composition according to claim 1, wherein the peptide comprises at least six amino acids.

3. The composition according to claim 1, wherein the peptide is a peptide of an oncogenic protein.

4. The composition according to claim 3, wherein the oncogenic protein is HER2 / neu.

5. The composition according to claim 4, wherein the peptide is a HER2 / neu-derived peptide.

6. The composition according to claim 5, wherein the peptide is GP2 or a derivative thereof.

7. The composition according to claim 4, wherein the cancer comprises HER2 / neu-expressing cells.

8. The composition according to claim 7, wherein the HER2 / neu-expressing cells are HER2 / neu 3+, HER2 / neu-positive, moderately expressing HER2 / neu, or lowly expressing HER2 / neu.

9. The composition according to claim 1, wherein the subject has or has HER2 / neu-positive cancer.

10. The composition according to claim 1, wherein determining the immunosuppression comprises a delayed-type hypersensitivity (DTH) immune response, an injection site reaction (ISR), or any other immune response assay to the peptide or adjuvant alone.

11. The composition according to claim 10, wherein the peptide is a peptide of an oncogenic protein.

12. The composition according to claim 11, wherein the oncogenic protein is HER2 / neu.

13. The composition according to claim 12, wherein the peptide of the oncogenic protein is GP2.

14. The composition according to claim 1, wherein determining the immunosuppression includes an immunoassay capable of detecting a target immune response to a peptide or adjuvant alone.

15. The composition according to claim 14, wherein the immune response assay is selected from a DTH reaction, an ISR, an ELISPOT assay, an antibody titer assay, and a T cell assay, or a combination thereof.

16. The composition according to claim 14, wherein the test includes measuring the injection site response to a therapeutic peptide for cancer.

17. The composition according to claim 10, characterized in that the adjuvant alone is administered by intradermal injection, and the ISR is measured as a biomarker for determining the immune status of the patient.

18. The composition according to claim 17, characterized in that the measured ISR is compared with that of a healthy or normally immune patient, and a subject having a low immune state is treated to restore the immune system to a healthy or normally immune state.

19. The composition according to claim 18, characterized in that the subject is treated with the peptide.

20. The composition according to claim 18, wherein the ISR in a healthy or normal immune state has a diameter of 50 mm or more.

21. The composition according to claim 18, wherein the low-immunity ISR has a diameter of less than 50 mm.

22. The composition according to claim 18, wherein the low-immunity ISR has a diameter 40 mm smaller than the ISR of a healthy or normal immune state.

23. A composition for treating a cancer comprising HER2 / neu-expressing cells in a subject having or having had the cancer and having received prior treatment for the cancer, comprising a GP2 peptide, wherein the therapeutic benefit induced by any treatment including GM-CSF alone or no treatment is negative or below normal unless reversed and increased by adding the GP2 peptide to GM-CSF treatment.

24. The composition according to claim 23, wherein the therapeutic benefit includes a lifetime benefit of a better-functioning immune system to any immune disorder, bacterial or viral infection, or any other disease in which therapeutic and clinical benefits may be induced by a better-functioning immune system, and which also includes, but is not limited to, an increased immune response to any immunotherapy, reduced cancer recurrence of the preceding or new cancer, and / or increased disease-free or long-term survival from the preceding or new cancer.

25. The composition according to claim 1 or 23, wherein the prior treatment of the cancer comprises surgery, chemotherapy, antibody therapy, targeted therapy, T-cell therapy, RNA / DNA therapy, or any other treatment targeting oncogenic proteins, or a combination thereof.

26. The composition according to claim 25, wherein the antibody therapy, targeted therapy, RNA / DNA therapy, or T cell therapy targets an oncogenic protein.

27. ​​The composition according to claim 26, wherein the oncogenic protein is HER2 / neu and the antibody targets an epitope on the HER2 / neu protein.

28. The composition according to claim 27, wherein the antibody is trastuzumab, trastuzumab biosimilar, ado-trastuzumab emtansine, fam-trastuzumab deruxtecan-nxki, pertuzumab, or another antibody that targets the epitope on the HER2 / neu protein, or a derivative thereof.

29. The composition according to claim 28, wherein the antibody or derivative comprises a linked molecule or a plurality of linked molecules, or an antibody-drug conjugate, which may contain a chemotherapeutic agent.

30. The composition according to claim 1 or 23, wherein the cancer is a cancer comprising HER2 / neu-expressing cells having moderate or low HER2 / neu expression, and the prior treatment comprises antibody therapy targeting HER2 / neu.

31. The composition according to claim 1 or 23, wherein the cancer is a cancer comprising HER2 / neu-positive HER2 / neu-expressing cells, and the subject has not undergone prior treatment for the cancer, including antibody therapy targeting HER2 / neu.

32. The composition according to claim 1 or 23, wherein the composition further comprises an adjuvant.

33. The composition according to claim 32, characterized in that the peptide and the adjuvant are administered together in the composition, or independently at the same time or at similar time points.

34. The composition according to claim 1 or 23, or the composition according to claim 10 or 17, wherein the composition further comprises an adjuvant. A composition wherein the adjuvant contains granulocyte-macrophage colony-stimulating factor (GM-CSF).

35. The composition according to claim 34, wherein the GM-CSF is a derivatized GM-CSF, a non-glycosylated GM-CSF, a partially non-glycosylated GM-CSF, or a shortened GM-CSF.

36. The composition according to claim 34, wherein the GM-CSF is recombinant GM-CSF.

37. The composition according to claim 34, wherein the GM-CSF is salglamostim.

38. The composition according to claim 1 or 23, wherein the subject has or is breast cancer, ovarian cancer, gastric cancer, cervical cancer, or any other HER2 / neu-expressing cancer.

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