Vaccine composition of cells expressing a lentiviral vector and method of use

Abstract

A vector construct that is a lentiviral construct containing DNA encoding GM-CSF is described. Also described is a vaccine composition comprising K562 cells transfected with this vector construct and possibly including U266 and H929. Methods for using the vaccine composition in a method of immunizing against plasma cell disorders including multiple myeloma and related disorders are described.

Description

Technical Field

[0001] The present invention relates to compositions and methods useful for vaccination against plasma cell disorders, including multiple myeloma, comprising a lentiviral vector construct encoding GM-CSF.

Background Art

[0002] The emergence of new therapeutic agents with low toxicity and high tumor specificity has significantly improved the clinical outcomes of patients with multiple myeloma (MM). Specifically, the combination of three drugs, a proteasome inhibitor, an immunomodulatory drug (IMiD), and a steroid, results in improved response rates and more durable remissions (Attal et al.; N. Engl. J. Med. (2017) 376:1311 - 20; Durie et al.; Lancet (2017) 389:519 - 27). This leads to improvements in progression - free survival (PFS) and overall survival (OS) (Richardson et al.; Hematology (2014) 255 - 61). However, a significant proportion of patients ultimately become resistant to such drugs and relapse. Approaches aimed at deepening and prolonging such responses include long - term maintenance therapies, with or without consolidation regimens. In particular, lenalidomide (Len) maintenance in both transplant and non - transplant settings has shown significant clinical benefits of such approaches (McCarthy et al.; J Clin Oncol (2017) 35:3279 - 89). The introduction of more effective treatments for MM has the potential for most of the overall improvement in clinical outcomes to be due to the ability to achieve deeper responses. In fact, minimal residual disease (MRD) assessment by either flow cytometry or next - generation sequencing can now detect as few as one cell in a million (ClonoSEQ Cleared for Residual Cancer Testing. Cancer Discov (2018) 8:OF6 - OF6).

[0003] Increasing literature shows a strong correlation between the depth of response and improvement in clinical outcomes, such that minimal residual disease negativity is now considered potentially an approvable clinical trial endpoint (Perrot et al.; Blood (2018) 132:2456-64). However, definitive clinical guidance and safe treatment options for the management of patients with minimal residual disease positive multiple myeloma are eagerly awaited in the art. Immunotherapy exploits the ability of the immune system to specifically recognize and eliminate cancer cells. Indeed, immune checkpoint blockade (Hargadon et al.; Int. Immunopharmacol. (2018) 62:29-39) and genetically modified T cells with chimeric antigen receptors (CAR-T) (Raje et al.; N. Engl. J. Med. (2019) 380:1726-37) have demonstrated clinical efficacy in hematological malignancies and, more restrictively, in solid tumors. In contrast, cancer vaccines to date have not shown similar benefits (Hu et al.; Nat. Rev. Immunol. (2018) 18:168-82).

[0004] The generation of a proliferative vaccine-specific immune response depends on the diversity and abundance of tumor-associated antigens, effective adjuvants, and co-administered immunostimulatory therapies. The established cell line K562 is genetically modified to produce granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF is a key immunostimulatory factor known to improve efficient antigen presentation (Borrello et al.; Hum. Gene Ther. (1999) 10:1983-91; Borrello et al.; Cytokine Growth Factor Rev. (2002) 13:185-93). Several factors, such as inadequate tumor antigen selection, choice of vaccine adjuvant, and the absence of an immune-modulatory therapy to combine with, can contribute to the lack of clinical efficacy. However, the disease burden has the potential to have a significant impact on clinical outcomes. Although lenalidomide's ability to increase vaccine-specific cellular and humoral immunity has been demonstrated (Noonan et al.; Clin. Cancer Res. (2012) 18:1426-34), in MRD-positive multiple myeloma, where the disease burden is very low, it was not known that vaccination with a multiple myeloma vaccine in combination with lenalidomide is a powerful therapeutic approach that enhances treatment efficacy without additional toxicity. Effective therapies for the treatment of MRD-positive MM and other plasma cell disorders are currently lacking, and there is also no common consensus on the management of this increasingly growing patient population. The present specification describes the first study that uses a vaccine composition containing a newly designed lentiviral vector to successfully treat patients with a burden due to minimal disease, not only improving the disease response but also preventing disease progression.

SUMMARY OF THE INVENTION

[0005] According to a first aspect of the present invention, there is described a composition for use in eliciting an immune response against a plasma cell disorder in a subject, comprising an effective amount of U266, H929, and K562 cells. According to another aspect of the present invention, there is described a lentiviral vector construct comprising a DNA sequence encoding GM-CSF. According to another aspect of the present invention, there is described the above lentiviral vector construct further comprising a DNA sequence encoding EF1a. According to another aspect of the present invention, there is described the above lentiviral vector construct, wherein the DNA sequence encoding EF1a is shown in SEQ ID NO: 2.

[0006] According to another aspect of the present invention, there is described the above lentiviral vector construct, wherein the amino acid sequence of GM-CSF is shown in SEQ ID NO: 3. According to another aspect of the present invention, there is provided the above-described lentiviral vector construct comprising the structure of LTR-EF1a-GMCSF-LTR. According to another aspect of the present invention, there is provided the above-described lentiviral vector construct, wherein the DNA encoding the LTR is as set forth in SEQ ID NO: 1. According to another aspect of the present invention, there is provided the above-described lentiviral vector construct, wherein the K562 cells contain the above-described vector construct. According to another aspect of the present invention, there is provided the above-described lentiviral vector construct, wherein the composition is a vaccine. According to another aspect of the present invention, there is provided the above-described lentiviral vector construct, wherein the vaccine is allogeneic. According to another aspect of the present invention, there is provided the above-described lentiviral vector construct, which further contains U266 and H929 cells.

[0007] According to another aspect of the present invention, the DNA sequence encoding GM-CSF can produce GM-CSF in an amount of up to about 1500 ng / 1×10 6 cells. There is provided the above-described lentiviral vector construct. According to another aspect of the present invention, there is provided the above-described composition, which is a vaccine. According to another aspect of the present invention, there is provided the above-described composition, wherein the vaccine is allogeneic. According to another aspect of the present invention, there is provided the above-described composition, wherein the K562 cells express GM-CSF. According to another aspect of the present invention, there is provided the above-described composition, wherein the K562 cells are transfected with a vector construct encoding GM-CSF. According to another aspect of the present invention, the gene encoding GM-CSF can produce GM-CSF in an amount of up to about 1500 ng / 1×10 6 cells. There is provided the above-described composition. According to another aspect of the present invention, there is provided the above-described composition, wherein the amount of GM-CSF produced is about 35 - 1200 ng / 1×10 6 cells.

[0008] According to another aspect of the present invention, there is provided the above composition, wherein GM-CSF is of human origin. According to another aspect of the present invention, there is provided the above composition, wherein the ratio of the combination of U266 and H929 cells to K562 cells is about 20:1. According to another aspect of the present invention, there is provided the above composition, wherein U266 and H929 cells are present in equal amounts. According to another aspect of the present invention, there is provided the above composition, wherein U266 and H929 cells are present in unequal amounts. According to another aspect of the present invention, there is provided the above composition, which induces an immune response in the subject when administered to the subject. According to another aspect of the present invention, there is provided the above composition, wherein a complete remission of the plasma cell disorder is induced in the subject by the immune response. According to another aspect of the present invention, there is provided the above composition, which prolongs the progression-free survival period in the subject.

[0009] According to another aspect of the present invention, there is provided the above composition, wherein the complete remission is determined as undetectable M spike and positive immunofixation electrophoresis. According to another aspect of the present invention, there is provided the above composition, wherein the subject is human. According to another aspect of the present invention, there is provided a method for inducing complete remission in a subject having multiple myeloma, comprising the step of administering the above composition to the subject. According to another aspect of the present invention, there is provided the above method, wherein the step of administering also includes the step of giving lenalidomide to the subject. According to another aspect of the present invention, there is provided the above method, wherein lenalidomide is given to the subject before, during, and / or after the step of administering. According to another aspect of the present invention, there is provided the above method, wherein the composition is a vaccine. According to another aspect of the present invention, there is provided the above method, wherein the vaccine is allogeneic. According to another aspect of the present invention, there is provided the above method, wherein K562 cells express the GM-CSF gene. According to another aspect of the present invention, there is provided the above method, wherein the K562 cells are transfected with a gene encoding GM-CSF.

[0010] According to another aspect of the present invention, there is provided the above method, wherein the GM-CSF gene is capable of expressing GM-CSF in an amount of up to about 1500 ng / 1×10 6 cells. According to another aspect of the present invention, there is provided the above method, wherein the GM-CSF gene is capable of expressing GM-CSF in an amount of about 35 - 1200 ng / 1×10 6 cells. According to another aspect of the present invention, there is provided the above method, wherein the amount of GM-CSF is produced on average every 24 hours. According to another aspect of the present invention, there is provided the above method, wherein the GM-CSF is of human origin. According to another aspect of the present invention, there is provided the above method, wherein the ratio of the combination of U266 and H929 cells to K562 cells is about 20:1. According to another aspect of the present invention, there is provided the above method, wherein the dosage of the composition is such that the ratio of tumor cells in the subject to K562 cells in the composition exceeds 2:1. According to another aspect of the present invention, there is provided the above method, wherein U266 and H929 cells are present in equal amounts in the composition.

[0011] According to another aspect of the present invention, there is provided the above method, wherein the U266 and H929 cells are present in the composition in an amount of about 5×10 7 cells, and the K562 cells are present in the composition in an amount of about 5×10 6 cells. According to another aspect of the present invention, there is provided the above method, wherein the plasma cell disorder is selected from the group consisting of MGUS, SMM, multiple myeloma, non-secretory multiple myeloma, indolent myeloma, light chain myeloma, plasma cell leukemia, and primary amyloidosis. According to another aspect of the present invention, there is provided the above method, wherein the plasma cell disorder is multiple myeloma. According to another aspect of the present invention, there is provided the above method, wherein the complete remission in the subject lasts for up to 5 years. According to another aspect of the present invention, there is provided the above method, wherein the complete remission is determined by measuring undetectable monoclonal spike and negative immunofixation electrophoresis. According to another aspect of the present invention, there is provided the above method, wherein the subject is positive for minimal residual disease. According to another aspect of the present invention, there is provided the above method, wherein the composition minimizes a non-specific immune response in the subject.

[0012] According to another aspect of the present invention, there is provided the above method, wherein the composition is administered to the subject in 1 to 5 doses at intervals of more than 1 day between each administration. According to another aspect of the present invention, there is provided the above method, wherein 2 to 4 doses are administered at intervals of more than 2 weeks between each administration. According to another aspect of the present invention, there is provided the above method, wherein 2 to 4 doses are administered at intervals of more than 4 weeks between each administration. According to another aspect of the present invention, there is provided the above method, wherein 4 doses are administered to the subject at intervals of about 1 month between each administration. According to another aspect of the present invention, there is provided the above method, wherein the first 3 administrations are at equal intervals. According to another aspect of the present invention, there is provided the above method, wherein all doses are administered within 1 year of each other. According to another aspect of the present invention, there is provided the above method, wherein at least one dose is administered between 7 and 18 days, inclusive, from the start of the lenalidomide course. According to another aspect of the present invention, there is provided the above method, wherein at least one dose is administered at about 15 days from the start of the lenalidomide course. According to another aspect of the present invention, there is provided a method of extending progression-free survival in a subject having multiple myeloma, comprising administering to the subject a composition according to claim 1 in combination with lenalidomide.

[0013] According to another aspect of the present invention, there is provided a method for inducing the proliferation of clonal T cells and an increase in myeloma-specific cytokine responses in a subject having multiple myeloma, comprising the step of administering to the subject a composition according to claim 1 in combination with lenalidomide. According to another aspect of the present invention, there is provided the above method, wherein the increase in the subject persists for up to 7 years after the step of administering. According to another aspect of the present invention, there is provided the above method, wherein the increase in the subject persists for up to 5 years after the step of administering. According to another aspect of the present invention, there is provided a method for inducing multiple myeloma-specific immunity in a subject, comprising the step of administering to the subject a composition according to claim 1 in combination with lenalidomide. According to another aspect of the present invention, there is provided the above method, wherein the subject is positive for minimal residual disease at the time of the step of administering. According to another aspect of the present invention, there is provided a method for preventing recurrence of multiple myeloma in a subject, comprising the step of administering to the subject a composition according to claim 1 in combination with lenalidomide.

[0014] According to another aspect of the present invention, there is provided the above method, wherein the subject is positive for minimal residual disease at the time of the step of administering. According to another aspect of the present invention, there is provided the above method, wherein the subject is human. Still other objects, features, and attendant advantages of the present invention will become apparent to those skilled in the art from a reading of the following detailed description of the embodiments, which is made in conjunction with the accompanying drawings.

[0015] Here, the invention of the present application will be described in more detail with reference to embodiments which are examples of compositions and methods shown as mere examples, and the accompanying drawings.

Brief Description of the Drawings

[0016]

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Modes for Carrying Out the Invention

[0017] Disclosed herein is a lentiviral vector construct encoding GM-CSF, which is useful for transfection of K562 cells. The vector construct includes a promoter sequence and two LTR sequences adjacent to the target protein that can be GM-CSF and that is expressed. K562 cells transfected with the lentiviral vector construct can be used to generate an allogeneic whole-cell GM-CSF-secreting multiple myeloma (MM) vaccine. This vaccine is used in combination with lenalidomide, which can also be referred to as "Len", and is administered to MM patients with a burden due to minimal residual disease defined as monoclonal spike undetectable but immunofixation electrophoresis (IFE) positive, and a method for demonstrating eradication of residual disease and conversion to complete remission (CR) is further described herein. The vaccine may also be effective against other plasma cell disorders characterized by an increase in monoclonal spike protein. Also described are the safety, response time, and immune surveillance of the vaccine and the MM-specific T cell response. To our knowledge, this is the first study that not only attempts to use a novel lentiviral vector construct encoding GM-CSF to treat patients with a burden due to minimal disease and further improve the efficacy of the disease, but also attempts to prevent disease progression.

[0018] As used herein and unless otherwise indicated, the term "about" is intended to mean ±5% of the value being modified. Thus, "about 100" means 95 - 105. In addition, the term "about" modifies terms in a series, such as "about 1, 2, 3, 4, or 5", and it should be understood that the term "about" modifies each member of the list such that "about 1, 2, 3, 4, or 5" is understood to mean "about 1, about 2, about 3, about 4, or about 5". The same applies to the term "at least" or other quantitative modifiers, for example, but not limited to, lists modified by terms such as "less than", "more than", etc. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[0019] As used herein, the terms "comprising" (and any form of comprising, such as "comprise", "comprises", and "comprised"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. As used herein, the terms "treat", "treated", or "treating" (treatment) mean both therapeutic treatments where the purpose is to slow down (reduce) an unwanted physiological symptom, disorder, or disease, or to obtain an advantageous or desired clinical outcome. For the purposes of the embodiments described herein, an advantageous or desired clinical outcome includes, but is not limited to, reduction of symptoms; decrease in the degree of a symptom, disorder, or disease; stabilization (i.e., non-worsening) of the state of a symptom, disorder, or disease; delay in the onset or slowing of the progression of a symptom, disorder, or disease; detectable or undetectable recovery or remission (partial or complete) of the state of a symptom, disorder, or disease; recovery of at least one measurable physical parameter not necessarily distinguishable by the patient; or enhancement or improvement of a symptom, disorder, or disease. Thus, "treatment of cancer" or "cancer treatment" or "treatment of multiple myeloma" or "multiple myeloma treatment" means an activity that alleviates or restores any primary event or secondary symptom or symptom associated with cancer, multiple myeloma, or any other symptom described herein. In some embodiments, the cancer to be treated is one of the cancers enumerated herein. In one embodiment, the cancer is multiple myeloma.

[0020] As used herein, the term "subject" can be used interchangeably with the term "patient". A subject can be a mammal, such as, for example, a dog, cat, monkey, horse, or cow. In some embodiments, the subject is a human. In some embodiments, the subject has been diagnosed with a blood cancer. In some embodiments, the subject has been diagnosed with multiple myeloma. In some embodiments, the subject is suspected of having multiple myeloma. As used herein, the term "expressing", when referring to cell surface receptors, such as, but not limited to, CD3, CD4, and CD8, can also refer to cells that are positive for those markers. For example, cells that express CD3 can also refer to CD3 positive (CD3 + ) cells. As used herein, the term "expressing" can also refer to a gene that is located intracellularly as part of chromosomal DNA or on another part of a vector. When a cell is induced to produce the protein encoded by the gene, the cell "expresses" the gene. The produced protein can be encapsulated within the cell or transported extracellularly.

[0021] As used herein, the term "vector construct" relates to a DNA or RNA molecule, such as a plasmid or virus, that can be used as a vehicle to transport a particular DNA segment into a host cell, as part of cloning or recombinant DNA techniques. The lentiviral vector constructs described herein are capable of serving in the constitutive replication and / or expression of the inserted target DNA sequence, and can enable the improved expression and production of the target DNA, as compared to simple plasmids, particularly those plasmids associated with selective expression using an antibiotic or other type of marker.

[0022] As used herein, the term "vaccine" refers to a product or composition that stimulates a subject's immune system to confer immunity against a particular disease or condition, thereby protecting the subject from that disease or condition. The vaccine can be part of a composition, which may or may not contain other components, including, but not limited to, an adjuvant. As used herein, the term "adjuvant" refers to a component that modifies the action of a principal component, e.g., a vaccine. An adjuvant, when used in a vaccine composition, can help to produce a more robust immune response in the subject to which the vaccine composition is administered. As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream or lymphatic system to other parts of the body. As used herein, the term "multiple myeloma" is defined as a cancer that occurs in white blood cells. In some embodiments, the white blood cells are white blood cells in the bone marrow. In some embodiments, multiple myeloma occurs in plasma cells.

[0023] As used herein, the term "plasma cell disorder" is as defined by the International Myeloma Working Group and includes blood cancers in which plasma cells become malignant and infiltrate the bone marrow. The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to the amount of a compound, formulation, substance, or composition described herein that is effective to achieve a particular biological result. Such results can include, but are not limited to, inhibition of cancer cell growth as determined by any means suitable in the art. As used herein, GM-CSF refers to granulocyte macrophage colony-stimulating factor, which is a known protein that is often used in cancer treatment. The first studies of GM-CSF, also called "Leukine" in the literature, included stimulation of macrophage and neutrophil growth to reduce the hematotoxicity associated with dose-intensive chemotherapy. When transfected into tumor cells and administered as a vaccine, the GM-CSF gene has demonstrated tumor regression and extended survival in both animal models and early clinical trials (Nemunaitis, Expert Rev Vaccines; 2005; 4(3): 259-74). As used herein, the term "GVAX" refers to a cancer vaccine composed of whole tumor cells genetically modified to secrete the immunostimulatory cytokine GM-CSF. One or more cell types may be included in the GVAX vaccine.

[0024] As used herein, the term "lenalidomide," also known by the trademark Revlimid, is a pharmaceutical used to treat multiple myeloma and myelodysplastic syndrome (MDS). It can be administered, without limitation, with steroids including dexamethasone. As used herein, the term "minimal residual disease" or "MRD" refers to the small number of cancer cells remaining in the body after treatment. The number of remaining cells is very small and often does not produce any physical signs or symptoms and cannot be detected by conventional methods, such as microscopic observation of cells and / or tracking of abnormal serum proteins in the blood. A positive test result for MRD means that residual disease has been detected. A negative result means that no residual disease has been detected. As described herein, MRD is used to measure the effectiveness of treatment and predict patients at risk of recurrence. If a patient is tested positive for MRD, it means that residual cancer cells still exist in the body after treatment. When MRD is detected, it is known as "MRD positive." If a patient is tested negative, it means that no residual cancer cells have been found. When MRD is not detected, it is known as "MRD negative."

[0025] Subjects who are candidates for administration of the vaccine compositions described herein may have received or may currently be receiving, without limitation, immunomodulatory drugs including thalidomide, lenalidomide, and pomalidomide, and proteasome inhibitors including, without limitation, bortezomib, carfilzomib, and ixazomib. Subjects who are candidates for administration of the vaccine compositions described herein may have a plasma cell disorder. Subjects with a plasma cell disorder can be identified by, but this is not a requirement for, an elevated serum level of the M spike protein, or "M spike." Subjects with a plasma cell disorder include, but are not limited to, subjects diagnosed with monoclonal gammopathy of undetermined significance ("MGUS"); smoldering multiple myeloma ("SMM"), non-secretory multiple myeloma, indolent myeloma, and multiple myeloma ("MM") including light chain myeloma; plasma cell leukemia; and primary amyloidosis.

[0026] Next-generation sequencing (NGS) technology and flow cytometry have enabled quantification of the burden due to "minimal residual disease" (MRD) in patients with MM. MRD negativity is a major prognostic factor for MM and, as it is now more achievable, has become a measurable endpoint for trials as well as treatment. However, guidance on the optimal treatment approach for MM patients who are in a state of complete remission (CR) but are still MRD positive is currently lacking. The vaccine compositions described herein can elicit a specific immune response against MM plasma cells that eradicates the burden due to minimal residual disease, thereby enabling conversion to a clinically meaningful outcome.

[0027] The vector construct may have, but is not limited to, the composition shown in FIG. 1. The promoter that can be used to assist GM-CSF protein expression is the EF1a promoter (SEQ ID NO: 2; FIG. 3), but any promoter that can increase the constitutive expression of GM-CSF protein in the selected host cell can be used. In addition, the vector construct may contain one or more long terminal repeats ("LTRs") (SEQ ID NO: 1; FIG. 2). LTRs are typically included in the vector construct as a pair of identical DNA sequences and can enable constitutive expression and integration of the target sequence into the host genome or chromosome when transcribed by a target sequence, such as GM-CSF (SEQ ID NO: 3; FIG. 4). The allogeneic GM-CSF-producing MM vaccine (MM-GVAX) described herein may include three or more distinct cell lines, including, but not limited to, the known heterologous MM cell lines H929 and U266, which are both publicly available from cell line depositories, such as ATCC (Manassas, VA; ATCC.org), and the also publicly available K562 cells. The K562 cell line can be transfected or transformed with the vector construct described herein, which contains a gene encoding GM-CSF in a constitutive expression-capable configuration. In addition, the vector construct can be composed of a promoter sequence and other known vector components, such as long terminal repeats ("LTRs"). The expression constructs that can be used include expression constructs containing typical known components operably linked to the GM-CSF coding sequence, such as components that enable optimal expression in the host cell, such as promoters, operators, origins of replication, etc. (see FIG. 1).

[0028] The amount of cells of each cell line in the vaccine composition is not limited and may be equal or unequal relative to the equal amount of each cell line. The ratio of the number of certain cell lines to the number of other cell lines may be equal, but is not limited to this ratio. The ratio of H929 and U266 may be 1:1, but is not limited to this ratio and may exist in unequal amounts. The ratio of the amount of H929 / U266 mixed cells to K562 / GM-CSF may be about 40:1 relative to K562 / GM-CSF, or may be about 35:1, 30:1, 25:1, 20:1, 15:1, or 10:1. In one embodiment, it is a ratio of about 20:1. Regardless of the ratio of the cell lines, in one embodiment, there is GM-CSF of about 50 - 1500 ng / 1×10 6 cells / 24 hours. The absolute amount of cells present in the vaccine is about 1×10 7 to about 1×10 9 cells for each of the H929 and U266 cells, and may include all amounts between 1, 5, 10, 50, or 100×10 7 cells. One embodiment includes the case where the composition has an equal amount of 5×10 7 cells for each of the H929 and U266 cells. The K562 / GM-CSF cells may be present in an amount of about 1×10 4 to about 1×10 7 cells, and may include all amounts between 1, 5, 10, 50, or 100×10 7 cells. One embodiment includes the case where the composition has an amount of 1×10 6 K562 / GF-CSF cells.

[0029] The vaccine composition may contain components other than three or more cell lines, including other cell lines, adjuvants such as aluminum, such as aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate; squalene oil such as MF59; preservatives such as thimerosal or thimersal; stabilizers such as gelatin, sorbitol, sucrose, lactose, mannitol, glycerol, Medium 199, arginine hydrochloride, sodium glutamate, and urea; and emulsifiers such as polysorbate 80, sorbitan trioleate, and sodium citrate, but are not limited thereto. Other components commonly used in vaccine production may be present, including antibiotics, ovalbumin, yeast protein, latex, formaldehyde, glutaraldehyde; and regulatory substances such as acidity regulators such as sodium and / or potassium-based salts, disodium adipate, succinic acid, sodium hydroxide, histidine, sodium borate, tromethamine, and human serum albumin. Such components may be included in amounts typical of vaccine formulations, typically in very small amounts as long as their presence does not negatively affect the efficacy of the vaccine, but can be in any amount. Human serum albumin is typically used at 0-10%.

[0030] The allogeneic GM-CSF-producing MM vaccine (MM-GVAX) described herein can be administered to subjects diagnosed with plasma cell disorders such as multiple myeloma (MM). Candidate MM patients can be MRD positive or negative. Candidate MM patients have a low likelihood of disease burden. Candidate MM patients have the potential to achieve a near-stable complete remission (nCR), which is defined as the absence of an M spike in either serum or urine for at least 4 months and IFE positivity. The rate of conversion from nCR to true CR was 53.3%, and 8 patients improved clinically within an average period of 11.6 months from enrollment.

[0031] The method of administering the vaccine composition described herein is not particularly limited and may include oral, subcutaneous, intramuscular, intradermal, and intranasal routes. The vaccine composition can be administered once, twice, three times, four times, or five or more times. The time between administrations of the vaccine composition described herein is not limited and can be any time from 1 week to 4 months between administrations, for example, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, and any time in between. The time between multiple administrations may not be the same. In a specific example, the time between vaccine administrations is 1 month. In an example of an administration schedule, all vaccine doses are administered within a period including 1 year, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, or 5 months or less, and all time points in between. The generation of active tumor-specific T cells as a result of vaccine administration is affected by several factors, and specifically, the efficient transport of T cells to the tumor site is promoted by all of the presence of an appropriate antigen, effective antigen presentation, inhibition of the suppressive tumor microenvironment, and expression of appropriate chemokines. The vaccine compositions described herein take into account some of these key components.

[0032] First, the vaccine composition described herein acts as a source of tumor-associated antigens (TAAs) by virtue of the presence of two established, heterogeneous MM cell lines, H929 and U266. Such cell lines exhibit diverse patterns of somatic mutations that are often associated with high risk and MM relapse. Specifically, H929 encompasses the t(4;14) translocation and mutations in NRAS, while U266 has several mutations, including those in the BRAF and TP53 pathways (Moreaux et al.; Haematologica; 2011;96:574-82). Disease relapse occurs as a result of clonal evolution, which is known to sometimes cause more aggressive genetic mutations. The vaccine composition described herein is designed to stimulate the immune system against these antigens before some of these presumptively high-risk antigens emerge in the process of clonal evolution associated with disease worsening. Presentation of such high-risk antigens by the vaccine composition described herein has been shown to significantly affect the timing and / or aggressiveness of disease relapse.

[0033] Second, the vaccine composition described herein may include the genetically modified bystander GM-CSF-secreting cell line K562 / GM-CSF, which includes the lentiviral vector construct described herein, together with the two unmodified MM cell lines, H929 and U266. The GM-CSF gene used in the lentiviral vector used to transfect K562 cells is not limited and may be derived from any source, including humans. "Derived from" as used herein may mean that the GM-CSF is native to the manner or location in which it naturally occurs. GM-CSF is known to be a key immune adjuvant. Importantly, the use of the K562 / GM-CSF cell line allows titration of the amount of GM-CSF, thereby enabling delivery of the optimal dose in the vaccine compositions described herein. This GM-CSF dose is neither insufficient nor therapeutically excessive, such that even delivery of high doses of antigen may result in reduced efficacy due to induction of myeloid-derived suppressor cells (MDSC). It has been found that an effective vaccine requires a "therapeutic" dose of GM-CSF and a sufficient amount of antigen (Serafini et al.; Cancer Res. 2004; 64:6337-43). As described herein, K562 cells can express an amount of GM-CSF of about 50 ng to about 1500 ng / 1×10 6 cells as part of a lentiviral vector construct. This amount can be produced over a period of time or all at once. The period during which GM-CSF can be produced can be up to about 72 hours when measured by ELISA, but can be more or less than this depending on the need to maintain an effective amount of the vaccine composition. The above amount can be produced on average every 24 hours. It is also required that there be an excess of the antigen cell source, i.e., tumor cells, such that the tumor cell:bystander cell stoichiometry exceeds at least 2:1. The amount of GM-CSF can be measured by any known method, including but not limited to the enzyme-linked immunosorbent assay ("ELISA").

[0034] The vaccine composition can be irradiated using known methods that can inhibit the growth of tumor cell lines and induce immunogenic cell death to improve antigen delivery. The dose of the vaccine is typically in a 2:1 ratio to the tumor cells, and in particular, the ratio of tumor cells to K562 / GM-CSF cells is 2:1. The determination of the amount of tumor cells can be made by any known method, including but not limited to flow cytometry.

[0035] Third, although not limited, immunomodulatory drugs (IMiDs) including lenalidomide can significantly improve T cell responses in cancer patients and enhance the vaccine efficacy of the vaccine compositions described herein. IMiDs administrable by the vaccine compositions described herein include, but are not limited to, lenalidomide, thalidomide, and pomalidomide. Lenalidomide is a specific example. Lenalidomide (sometimes referred to as "Len" in the literature) can be used as a vaccine adjuvant or co-administered with the vaccine composition in the methods described herein. Lenalidomide can be administered at any time before the administration of the vaccine composition, co-administered with the vaccine composition, or administered after the vaccine composition. The dosage of lenalidomide can range from 2.5 to 25 mg / dose. The time before and after the administration of the vaccine composition is not limited and can include up to 10 years before and after, up to 4 years before and after, up to 3 years before and after, up to 2 years before and after, or up to 1 year before and after, and can be any time point between such time points, including, but not limited to, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1 month before and after. The administration of lenalidomide can be continuous or in several separate administrations. The administration of the vaccine compositions described herein has been shown to result in effective and sustained anti-MM immunity in combination with continuous administration of lenalidomide and low tumor burden.

[0036] Proliferation and persistence of T cell clone types by bulk analysis of the TCR repertoire can be observed in both blood and bone marrow (BM) after administration of the vaccine compositions described herein. Thus, both local proliferation of MM-specific clones induced by the vaccine compositions described herein in the bone marrow and the potential for infiltration by clone types derived from the periphery can result in the generation and maintenance of a long-term persistent immune response. Moreover, the increase in multifunctional cytokine-producing T cells in response to stimulation by both vaccine composition-related antigens and non-vaccine-related MM antigens supports the generation of a broad immune responsiveness to diverse heterologous shared MM antigens. Thus, the allogeneic whole-cell vaccines described herein overcome the barriers imposed by patient-specific tumor diversity to induce an effective T cell response against shared tumor-specific antigens and, moreover, can confer persistent anti-tumor immunity. The vaccine compositions described herein are shown to enhance the ability to detect T cell clone types that proliferated after vaccination and to characterize multifunctional cytokine T cell responses for up to 7 years after vaccination.

[0037] Thus, the vaccine compositions described herein enable the regression and maintenance of myeloma - monoclonal gammopathy of undetermined significance, known as "MGUS", an early multiple myeloma that is actually not cancer at all. MGUS is a benign condition indicated by low levels of M protein, low levels of abnormal plasma cells in the bone marrow, and the absence of active disease indicators. This condition has the potential to be disrupted by the continued activity of T cell-mediated immunity induced by the vaccine compositions described herein, and the presence of tissue-resident-like CD8 + T cell populations in the bone marrow of such patients is identified. Thus, patients diagnosed with MGUS but not progressing to multiple myeloma are also candidates for the vaccines described herein. Maintenance of MGUS patients by administration of the vaccines described herein may prevent progression to myeloma. Complete remission in patients with multiple myeloma can be achieved by administration of the vaccine composition according to the methods and dosing schedules described herein, as measured by patients with undetectable M spike protein and negative measurements in immunofixation electrophoresis, and thus, a method of inducing complete remission in such patients is feasible. Complete remission can persist in patients for up to 5 years, up to 6 years, or up to 7 years.

[0038] Prolongation of progression-free survival in subjects with multiple myeloma can be achieved by administration of the vaccine composition according to the methods and dosing schedules described herein, as measured by determination of the time to diagnosis until progression, recurrence or relapse, and thus, a method of prolonging progression-free survival in such patients is feasible. Progression-free survival can be measured for up to 5 years, 6 years, or up to 7 years. Elevation of clonal T cell expression and myeloma-specific cytokine responses in patients with multiple myeloma can be achieved by administration of the vaccine composition according to the methods and dosing schedules described herein, and thus, a method of elevating clonal T cell expression and myeloma-specific cytokine responses in such patients is feasible. Induction of multiple myeloma-specific immunity in patients with multiple myeloma can be achieved by administration of the vaccine composition according to the methods and dosing schedules described herein, and thus, a method of inducing multiple myeloma-specific immunity in such patients is feasible. Prevention of multiple myeloma relapse in patients who had previously had a positive diagnosis of multiple myeloma but had achieved MRD negativity up to that point can be achieved by administration of the vaccine composition according to the methods and dosing schedules described herein, and thus, a method of preventing multiple myeloma relapse in such patients is feasible.

[0039] The basis for what can be achieved by such methods, and further indicators of such success, are described herein. Accordingly, evidence is provided herein for the existence of a population of bone marrow (tissue)-resident quiescent T cells that lack features of exhaustion and senescence and exhibit high PD1 levels and an effector memory-like phenotype. Such findings support the concept of the BM as a reservoir of antigen-experienced memory T cells and provide evidence for the putative mechanisms by which this occurs. The post-vaccination bone marrow CD8 + T cell clustering without a teacher enables the identification of subpopulations that are preferentially enriched in patients in long-term disease remission. DNAM1 low CD27 - CD8 + This population of T cells was substantially absent from the bone marrow of patients with early post-vaccination relapse. Thus, CD27 with a heterogeneous partial dysfunction phenotype defined by the combined expression of both exhaustion and activation markers - CD8 + T cells are identified as a source of MM-reactive lymphocytes. The abundance of these induced by the vaccine compositions described herein represents a positive prognostic significance in newly diagnosed multiple myeloma patients. Based on the MM-MGUS model of continuous immune surveillance, the reduction of the tumor-reactive CD8 + T cell subpopulation significantly contributes to immune evasion and clinically significant disease worsening. The evidence presented herein clearly demonstrates that the reduction of the potential tumor-reactive CD8 + T cell subpopulation occurs prior to clinically evident disease relapse and, moreover, that this persistence correlates with long-term disease remission (Figures 14 and 15).

[0040] Moreover, the evidence presented herein supports the conclusion that the mechanisms by which vaccination confers anti-tumor immunity also include the generation of additional MM-specific T cells and the expansion of a stem-like quiescent T RM population in the bone marrow. Furthermore, the CD8 whose reduction occurs prior to clinically evident disease relapse +Heterogeneous populations of T cells are identified. The phenotypic characterization of the immunophenotype of the BM resident memory T cells described herein provides further insight into the important role played by bone marrow T cells in maintaining MM-specific immunity for several years after vaccination with the vaccine compositions described herein. The data described herein strongly support a positive correlation between the depth of response of MRD and clinical outcome. The stable reappearance of monoclonal proteins that do not meet the disease progression criteria described herein provides evidence that the vaccine compositions described herein are effective in controlling progression and treating multiple myeloma by both increasing clinical response and / or establishing an MM-MGUS balance that significantly delays disease progression when administered in a disease-low burden state. Current therapies for MM, including IMiD, proteasome inhibitors, and autologous BM transplantation with high-dose melphalan, have dramatically increased the number of patients achieving deep clinical responses. The multiple myeloma vaccine described herein is shown to be an effective treatment for preventing disease recurrence in patients who have been shown to have a short duration of response even after failing to achieve complete eradication of the MM clone or achieving MRD negativity when combined with lenalidomide.

Examples

[0041] The embodiments and descriptions herein are further described in more detail below with reference to the following non-limiting examples. (Example 1) Patient Selection and Eligibility Eligible patients were at least 18 years old, had a diagnosis of multiple myeloma, and had adequate hematopoietic, liver, and renal function, along with a performance status of 0 to 2 according to the Eastern Cooperative Oncology Group (ECOG). Patients were eligible regardless of their prior treatment experience. Autologous hematopoietic stem cell transplantation was not likely to have occurred within the past 12 months, and prior allogeneic bone marrow transplantation was not possible. To enroll, patients had to sustain a remission approaching complete remission during at least 4 months of observation on a lenalidomide (Len)-containing regimen. The key exclusion criteria were disease progression after steroid discontinuation and were defined as a detectable M spike > 0.5 g / dL or conversion to a true complete remission (defined as the absence of an M spike and negative serum / urine immunofixation) during the observation period.

[0042] Fifteen patients with multiple myeloma (MM) who had achieved a near complete remission (nCR) stable for at least 4 months on a Len-containing regimen were enrolled in the study. nCR was defined as undetectable M spike and positive serum and / or urine immunofixation (IFE) (BladE et al. Br J Haematol 1998;102:1115 - 23). At diagnosis, 53% of the patients had stage I disease according to the International Staging System (ISS) (Palumbo et al. J Clin Oncol 2015;33:2863 - 9). Notably, none of the enrolled patients had features of high-risk MM as defined by the International Myeloma Working Group (IMWG) cytogenetic criteria. At the time of enrollment, Len was continued, but all other anti-MM therapies were discontinued. Patients received four MM-GVAX vaccinations at 1, 2, 3, and 6 months, in combination with the current dose of Len at a median dose of 15 mg (range 2.5 - 25 mg) (Figure 1) for at least 1 year. The median age at enrollment was 69 years (range 45 - 81 years).

[0043] (Example 2) Vaccine Efficacy All 15 patients were able to participate in the clinical efficacy evaluation 1 year after the administration of the first MM-GVAX dose. Eight of the 15 patients (53.3%) achieved disease efficacy consistent with true complete remission (CR), which was defined as the absence of M spike and negative IFE, and had a median time to CR achievement of 11.6 months from enrollment (range 1.4 - 13.9 months). This CR conversion rate of 53.3% (95% CI: 26.6 - 78.7%) was significantly higher than the null hypothesis of a CR conversion rate of 25% defined by the design (p = 0.011), demonstrating clinical activity and meeting the primary endpoint of this trial. Notably, only 6 of the 15 enrolled patients experienced disease progression, which was defined as the appearance of an M spike of at least 0.5 g / dL and / or an increase in free light chain content exceeding 10 mg / dL and confirmed by repeated measurements. Pt6, Pt7, and Pt9 experienced early disease recurrence within the first year of trial enrollment. Subsequently, a detectable M spike occurred in 7 patients (46.7%), which did not meet the criteria for disease recurrence. This was variably persistent over time and did not require any treatment modification. At the time of analysis, the estimated median overall survival (OS) was 11.5 years from MM diagnosis (95% CI: 5.9 - n / a years) and 7.8 years from enrollment (95% CI: 4.2 - 7.8 years, n = 6 / 15, 40%). Subgroup analysis of patients who achieved CR showed no statistically significant differences in PFS and OS compared to patients who maintained stable nCR or those in whom a measurable M spike occurred subsequently. This finding suggests that vaccination can induce an immune balance that enables long-term disease control even if malignant MM plasma cell clones are not completely eradicated.

[0044] (Example 3) Vaccine Formulation and Administration The cell lines used in the vaccine formulation were manufactured by the Cell Processing and Gene Therapy Facility of the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, which complies with GMP. U266 and H929 were initially obtained from ATCC.

[0045] The lentiviral vector is constructed as shown in Figure 1. The GM-CSF gene is ligated to the EF1a promoter (see Figure 3), and LTRs are placed adjacent to both sides of the resulting DNA construct. The LTRs have the same sequence.

[0046] Briefly, the K562 cell line is transduced with the lentiviral vector constructed as described above to generate a cell line capable of expressing and producing GM-CSF (K562-GMCSF). Briefly, the K652 cell line is grown in a closed system (Wave Xuri) under perfusion conditions until a sufficient number for transduction is produced. Then, using the closed system, the medium is removed, the lentiviral vector construct is added for 24 hours, and then the culture is washed to transduce the cells in the culture bag. The cells are grown again under perfusion in Xuri. Then, the cells are assayed for GM-CSF production per total fixed cell number. This data is verified by performing three assays simultaneously using the same number of cells at several dilutions to determine the total amount of GM-CSF produced every 24 hours.

[0047] Also, the cell line is irradiated with radiation, and the GMCSF production assay is repeated. The cell line is administered with the final vaccine formulation in the irradiated form. The same number (each 5×10 7 ) of MM cell lines U266 and H929 are 5×10 6It was mixed with the bystander cell line K562GM-CSF of cells. After irradiating the vaccine cells with radiation, they were cryopreserved and stored in liquid nitrogen until the day of use. On the vaccination day, the individual cells were thawed, mixed at an appropriate concentration, and aspirated into three syringes. The final vaccine syringes were maintained on ice until administration, and this administration was performed within 60 minutes after thawing. Patients were vaccinated four times (cycles 1, 2, 3, and 6 respectively) on the 14th day of the 1st, 2nd, 3rd, and 6th months from registration, and moreover, Len was continued for at least one year at the dose administered before registration. Len was administered for 21 days out of 28 days. For each visit with a single vaccine inoculation, intradermal MM-GVAX was administered to the three limbs of the patient with a total volume of 1 mL or less, and pneumococcal conjugate vaccine 13 (PCV-13, Prevnar (registered trademark)) was injected intramuscularly into one arm. At the end of the trial, the patients continued treatment with Len, and evidence of clinical efficacy was assumed. All subjects had samples of BM and PB collected for safety assessment, determination of disease response, and correlation analysis of biomarkers at immediately before treatment (baseline), on the 14th day of cycle 3 (C3D14, before the third vaccine administration), and one year after registration. In some patients, additional follow-up samples of BM and PB were collected at the time point more than one year after trial registration and up to 7 years.

[0048] (Example 4) The efficacy of the vaccine can be predicted by the burden by MRD To quantify and track the burden of MM disease, next-generation sequencing-based minimal residual disease (MRD) testing was performed in 7 patients (46.7%) for whom baseline MM DNA was available (Rawstron et al. “A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study”; Leukemia [Internet]. 2016;30:929-36. Available from nature.com / articles / leu2015313). The MRD incidence frequency of the B-cell repertoire was 10 -6 (10 -4 %) in the test samples of all subjects except one, so an arbitrary threshold of 10 -3 (10 -1 %) in the bone marrow was identified as one that would allow the assessment of the clinical significance of the disease burden above this threshold (high-level MRD + ) and below this threshold (low-level MRD + ).

[0049] Dominant IGH and IGK / L cancer clones were identified from the immunosequencing results in pre-treatment bone marrow using the following criteria: 1) The sequences must have an occurrence frequency exceeding 5%. 2) The sequences must be present at more than 0.1% of the total nucleated cells. 3) The sequences must be discontinuously distributed (4 or fewer sequences in the sequence frequency over the next 10 years). 4) The sample must have more than 200 putative templates. Such dominant clones identified in the bone marrow were followed over time to determine the occurrence frequency of cancer clones at subsequent time points after treatment. To account for somatic hypermutation (SHM), IGH clones with no more than 2 mismatches with the dominant clones in the bone marrow were also followed over time. The MRD occurrence frequency in each sample was measured as the occurrence frequency of cancer clones in all proliferative reconstructions of the loci examined. All high-level MRD + Patients experienced disease recurrence within 1 year of enrollment (median = 4.8 months, range: 2.8 - 9.5 months), while low-level MRD + Patients had a significantly longer PFS (median = 84.15 months, range: 51.9 - 97.3 months, p = 0.01). Detection of high-level MRD compared to low-level MRD was associated with an increased likelihood of clinical recurrence (hazard ratio, HR = 25.79, 95% CI: 2.17 - 306.4). Notably, Pt2 and Pt4 had prolonged clinical responses despite the variable occurrence of detectable M spikes, which did not meet any criteria for disease progression. In contrast, Pt12 and Pt13 achieved a true CR and maintained their responses over time. Thus, the burden of MRD at the time of vaccination allows prediction of long-term clinical outcomes.

[0050] (Example 5) Systemic myeloma immunity is induced by MM-GVAX vaccination To determine the impact of MM-GVAX vaccination on the entire T cell receptor (TCR) repertoire, deep sequencing analysis of the TCR chain Vbeta□ (TRBV) was performed on matched peripheral blood (PB) and bone marrow (BM) samples from all patients at baseline, before the third MM-GVAX administration (C3D14), and at 1 year. Immune sequencing of the CDR3 regions of the human TCRβ and IGH and IGK / L chains of T and B cells was performed on genomic DNA extracted from BM or PB samples using the immunoSEQ® assay (Adaptive Biotechnologies, Seattle, WA), respectively. The extracted genomic DNA was amplified by bias-controlled multiplex PCR and then subjected to high-throughput sequencing.By collapsing and filtering the arrays, the absolute abundance of each unique CDR3 region was identified and quantified and further analyzed as previously described (available from Robins et al. “Comprehensive assessment of T-cell receptor β-chain diversity in αβ T cells”; Blood [Internet]. 2009;114:4099-107. ashpublications.org / blood / article / 114 / 19 / 4099 / 26771; available from Carlson et al. “Using synthetic templates to design an unbiased multiplex PCR assay”; Nat Commun [Internet]. 2013;4:2680. nature.com / articles / ncomms3680; available from Robins et al. “Ultra-sensitive detection of rare T cell clones”; J Immunol Methods [Internet]. 2012;375:14-9. linkinghub.elsevier.com / retrieve / pii / S0022175911002468; available from Kirsch et al. “T-cell receptor profiling in cancer:; Mol Oncol [Internet]. 2015;9:2063-70. doi.wiley.com / 10.1016 / j.molonc.2015.09.003; available from DeWitt et al. “A Public Database of Memory and Naive B-Cell Receptor Sequences”; Turner SJ, editor. PLoS One [Internet]. 2016;11:e0160853. dx.plos.org / 10.1371 / journal.pone.0160853). Only proliferative reconstructions were used for repertoire analysis.

[0051] Before vaccination, the TCR clone type composition varied, ranging from minimal repertoire bias to significant oligoclonal expansion. Despite the hypothesis that the TCR repertoire should become asymmetric with respect to the increase in clonality upon vaccination, no major changes were observed in the relative proportions of TCR proliferative reconstitution in either compartment. Proliferative clonality varied greatly over time among different patients, but no vaccine-related pattern could be identified. Overall, proliferative clonality appeared to be relatively stable over time in most subjects and was not correlated with clinical outcomes. Considering the asymmetry of the minimal TCR repertoire observed upon vaccination, the changes in clone abundance before and after vaccination were examined by comparing the frequencies of occurrence of each clone. Notably, after the first two administrations of MM-GVAX (C3D14), T cell clones proliferated significantly in both PB and BM of all subjects, and such clones reached high frequencies and persisted after the first proliferation after vaccination (Figure 2). Despite the absence of an overall change in clonality associated with vaccination, clear evidence of clone proliferation and contraction was present in both PB and BM samples. Interestingly, many of the clones that proliferated significantly after vaccination were not observed before vaccination, suggesting either that clones that had not been detected until vaccination proliferated or that new clone types gradually increased (Figure 3). The ability of MM-GVAX to induce a broad systemic immune response was emphasized by the proliferation of T cell clones in both PB and BM. Most clones were present at comparable frequencies in PB and BM, but some clone types were preferentially enriched in BM (Figure 4). To better understand the clone dynamics associated with vaccination, the Morisita index was used to determine the similarity between TCR repertoires in such two compartments. In some patients (Pt7, Pt10, and Pt15), the TCR repertoires of BM and PB became even more different from each other after vaccination compared to the baseline, but in most subjects, there was little change in the similarity between the two compartments after vaccination (Figure 5).Further analysis of the expanded T cell clones in BM and PB after vaccination revealed diverse patterns of clonal expansion across patients. In some, the frequency of the expanded clone types increased in both PB and BM, suggesting parallel processes in both compartments. In contrast, in others, in either BM or PB, some clones expanded in one compartment but not the other, or expanded in both compartments but showed preferential enrichment. Collectively, these results indicate that MM-GVAX induced a systemic T cell response and measurable clonal expansion in both BM and PB.

[0052] (Example 6) Vaccination induces an MM-specific multifunctional T cell response in the bone marrow. The T cell response in BM was functionally characterized for both vaccine-related and -unrelated MM antigens. Samples from all patients and time points were stimulated in vitro with lysates of the MM-GVAX cell lines (U266 and H929), and analyzed for intracellular cytokine production. The responses of MM-GVAX-specific interferon γ (IFNγ) and TNFα were significantly increased at the time of vaccination in both CD8 + and CD4 + T cell subsets (Figure 6). The frequency of CD4 + or CD8 + T cells producing IFNγ and / or TNFα in response to vaccine-related and -unrelated MM antigens significantly increased after only two vaccinations and maintained a sustained increase for up to four years (p<0.0001, Figure 7). BM-derived mononuclear cells obtained at the indicated time points before and after vaccination were stimulated with either 2% human AB serum alone or AIM-V medium supplemented with lysates of SW780 (bladder cancer cell line) or U266 / H929 (MM-GVAX cell line). After 5 days, the cells were harvested, stained, and analyzed by flow cytometry for intracellular cytokine production.

[0053] Notably, MM-GVAX induced multifunctional CD4 + and CD8 +The frequency of T cells significantly increased. This was defined as the proportion of T cells producing IFNγ and TNFα simultaneously, even to a low degree, as well as single cytokine-producing T cells. Interestingly, the most prominent change was in the generation of CD8 + T cells (Figure 8). Pt6, Pt7, and Pt9, which relapsed early after vaccination, generated vaccine-specific T cell cytokine responses comparable to those of patients who achieved long-term disease remission. These results demonstrate that the MM-GVAX-induced immune response is multifunctional, directed towards a wide range of commonly shared MM-related antigens, and involves both CD8 + and CD4 + T cells. Such findings support data from other studies indicating that effective vaccine-induced antitumor immunity requires both CD8 + and CD4 + tumor-specific T cells (Bennett et al. “Induction of a CD8 + + Cytotoxic T Lymphocyte Response by Cross-priming Requires Cognate CD4 + + T Cell Help”; J Exp Med [Internet]. 1997;186:65-70. Available from rupress.org / jem / article / 186 / 1 / 65 / 7193; and Alspach E, Lussier et al.; “MHC-II neoantigens shape tumour immunity and response to immunotherapy”; Nature [Internet]. 2019;574:696-701. Available from nature.com / articles / s41586-019-1671-8).

[0054] (Example 7) Vaccine-induced MM-specific T cell immunity persists for several years after vaccination An important feature of adaptive immunity is its persistence over time and the ability to initiate an effective response upon re-encounter with an antigen. Therefore, MM-GVAX-specific responses in available samples collected several years after vaccination were detected and characterized. Samples of bone marrow and peripheral blood were collected at a pre-established time point, and mononuclear cells were enriched using a gradient with Lymphoprep (STEMCELL Technologies, Inc. (registered trademark)) and cryopreserved in cryopreservation medium (50% complete AIM-V medium, 40% complement-depleted human AB serum, and 10% DMSO). The samples were then thawed, washed twice each with pre-warmed (37°C) AIM-V supplemented with 0.02 mg / mL DNase and phosphate-buffered saline (PBS). Flow cytometry reagents were purchased from BioLegend, BD Biosciences, and Invitrogen. Monoclonal antibodies were titrated to their optimal concentrations prior to use. Surface staining was performed at 37°C for 20 minutes, and intracellular detection of cytokines was then performed by incubating the cells with specific mAb cocktails for 20 minutes at room temperature after fixing the cells according to the manufacturer's instructions with the CytoFix / CytoPerm kit (BD Biosciences). All data were acquired on a Gallios (registered trademark) flow cytometer (Beckmann-Coulter) equipped with three lasers (violet, 405 nm; blue, 488 nm; red, 633 nm) and capable of detecting ten parameters. Flow cytometry data were corrected using FlowJo with single-stained cell controls and compensation beads (BioLegend). After pre-processing by biexponential transformation and standard gating to remove aggregates and dead cells, CD3 + CD8 +T cells were subsequently exported from FlowJo and further analyzed using custom scripts that utilized Bioconductor libraries and R packages by R (version 4.0.1). Briefly, the data were analyzed using the FlowSOM algorithm for unsupervised clustering and visualized by UMAP. Differential discovery analysis was performed on R using the diffcyt framework and CATALYST workflow (available from Nowicka et al.; “CyTOF workflow: differential discovery in high-throughput high-dimensional cytometry datasets”; F1000Research [Internet]. 2019;6:748. f1000research.com / articles / 6-748 / v3). The data were then recognized as one new file per cluster and further analyzed by FlowJo to determine the frequency of positive cell appearance and mean fluorescence intensity (MFI) for each marker.

[0055] In three patients with available long-term samples, the proliferating clone types detected at C3D14 were still present up to 7 years after vaccination. Surprisingly, the frequency of appearance of proliferating clones in each compartment remained relatively stable in both BM and PB from 1 year after vaccination, suggesting the establishment of an immune equilibrium steady state (Figure 9). Intracellular cytokine staining of such BM samples stimulated with both MM-GVAX-related and non-related MM antigens showed persistent multifunctional CD4 + and CD8 + T cell responses up to 7 years after vaccination (Figure 10). In summary, the persistence of vaccine-specific clones and MM-specific multifunctional T cells in PB and BM for 7 years after vaccination, even in the presence of detectable disease, suggests the establishment of immune equilibrium, which may be the cause of the long-term disease control observed in this study.

[0056] (Example 8) T cells in the BM exhibit an effector phenotype and tissue-resident-like properties The phenotypic composition of BM T cells was examined for the expression of checkpoint molecules, costimulatory molecules, and chemokine receptors. The BM T cell composition was remarkably similar over multiple time points (data not shown). A CD69-expressing tissue-resident-like T cell population (T RM ) was identified as being consistently present in all BM samples. The proportion of CD69 + T RM showed little variation over time, but CD8 + T RM was dominant over its CD4 + counterpart (Figure 11). By classical phenotypic identification of T cell subsets, a higher proportion of CD4 + T cells exhibited a central memory phenotype (T CM ), while in CD8 + T cells, effector memory (T EM ) and effector (T EMRA ) subsets were typical. Interestingly, in CD8 + T cells, stem cell memory-like T cells (T + ) were present at a higher frequency compared to the CD4 SCM subset (Figure 12). BM T RM predominantly showed the T EM and T EMRA phenotypes, but TCM and TSCM-like TRM could only be detected to a low extent. CD69 + CD8 +T cells are typical of the memory population and have the characteristics of tissue residency. Interestingly, these lack exhaustion markers, such as TIGIT and TIM3, as well as the senescence marker (CD57), but express the activation markers CD27 and PD1. Interestingly, BM TRM express both CXCR4 and CXCR6, BM homing chemokine receptors, at higher levels, which is thought to be characteristic of tissue-resident T cells (available from Kumar et al., “Human Tissue- Resident Memory T Cells Are Defined by Core Transcriptional and Functional Signatures in Lymphoid and Mucosal Sites”; Cell Rep [Internet]. ElsevierCompany.; 2017;20:2921-34. dx.doi.org / 10.1016 / j.celrep.2017.08.078) (Figure 13). Low levels of monoclonal protein reappeared, but a significant proportion of patients maintained disease control. This reappearance persisted over time but did not meet the recurrence criteria. This stable low-level disease suggests the establishment of an immune equilibrium. Identification of the BM resident T cell population consistently present in this cohort supports previous findings that T RM has a fundamental role in maintaining immune equilibrium and tumor surveillance (Park et al., “Tissue-resident memory CD8 + + T cells promote melanoma-immune equilibrium in skin”; Nature [Internet]. 2019;565:366-71. nature.com / articles / s41586-018- 0812-9 available).

[0057] (Example 9) Immune correlates with clinical outcomes after MM-GVAX vaccination BM CD8 after vaccination +To gain insights into the immune phenotypes of T cells and their associations with clinical outcomes, BM CD8 of C3D14 + T cells were analyzed by FlowSOM, an unsupervised clustering algorithm, and visualization of various T cell clusters was simplified using dimensionality reduction techniques such as Uniform Manifold Approximation and Projection (UMAP). Hierarchical metaclustering of FlowSOM clusters grouped CD8 + T cell subsets into similar immune phenotypes, and two subsets of CD8 + T cells, namely C1 and C2, were identified. These subsets were enriched in patients with long-term disease control (responders) compared to patients with early progressive disease (relapsers) within the first year after vaccination (Figure 14, *, p < 0.05; **, p < 0.01). Notably, clusters C1 and C2 were defined by low to absent expression of DNAM1 and absence of CD27, and contained relatively heterogeneous subsets including senescent, effector, and exhausted CD8 + T cells. Further characterization of cluster C2 identified a CD69 + CD57 - subset that moderately expressed PD1 and was enriched in CD8 + T cells that were resident in the BM and potentially involved in long-term MM control. Interestingly, cluster C1 was characterized by increased CD57 expression, and it was suggested that such effector - senescent cells could still be functional despite lacking proliferative capacity. Subsequently, the results obtained by the FlowSOM algorithm were reproduced by standard flow cytometry techniques, where manually gated CD27 - DNAM1low / - CD8 + T cells were enriched in vaccine responders (Figure 15). Such findings indicate that changes in the CD8 + T cell composition occur earlier than clinically evident disease relapse and may serve as a reservoir of tumor - specific T cells, potentially playing a role in establishing and maintaining the vaccine - induced immune balance, BM CD8 +Evidence is provided that subsets of T cells are identified.

[0058] Although the present invention has been described in detail with reference to this exemplary embodiment, it will be apparent to those skilled in the art that various changes can be made and equivalents can be utilized without departing from the scope of the present invention. Each of the foregoing documents is hereby incorporated by reference in its entirety.

Claims

1. A lentiviral vector construct comprising a DNA sequence encoding GM-CSF.

2. The lentiviral vector construct according to claim 1, further comprising a DNA sequence encoding EF1a.

3. The lentiviral vector construct according to claim 2, wherein the DNA sequence encoding EF1a is shown in SEQ ID NO:

2.

4. The lentiviral vector construct according to claim 1, wherein the amino acid sequence of GM-CSF is shown in SEQ ID NO:

3.

5. The lentiviral vector construct according to claim 1, comprising the structure of LTR-EF1a-GMCSF-LTR.

6. The lentiviral vector construct according to claim 5, wherein the DNA encoding LTR is shown in SEQ ID NO:

1.

7. A composition comprising K562 cells, wherein the K562 cells comprise the vector construct according to claim 1.

8. The composition according to claim 7, which is a vaccine.

9. The composition according to claim 8, wherein the vaccine is allogeneic.

10. The composition according to claim 1, further comprising U266 and H929 cells.

11. The DNA sequence encoding GM-CSF is capable of producing GM-CSF in an amount of up to about 1500 ng / 1 x 10 6 cells, the composition according to claim 7.

12. The amount of GM-CSF produced is about 35 to 1200 ng / 1 × 10 6 cells, the composition according to claim 11.

13. The composition according to claim 7, wherein GM-CSF is of human origin.

14. The composition according to claim 10, wherein the ratio of the combination of U266 and H929 cells to K562 cells is about 20:

1.

15. The composition according to claim 10, wherein U266 and H929 cells are present in equal amounts.

16. The composition according to claim 10, wherein U266 and H929 cells are present in unequal amounts.

17. The composition according to claim 7, which induces an immune response in the subject when administered to the subject.

18. The composition according to claim 17, wherein the complete remission of the plasma cell disorder is induced in the subject by the immune response.

19. The composition according to claim 17, which prolongs the progression-free survival period in the subject.

20. The composition according to claim 17, wherein the complete remission is determined as undetectable M spike and positive immunofixation electrophoresis.

21. The composition according to claim 17, wherein the subject is human.

22. A method for treating plasma cell disorder in a subject, comprising the step of administering the composition according to claim 7 to the subject.

23. The method according to claim 22, wherein the step of administering further comprises the step of administering lenalidomide to the subject.

24. The method according to claim 24, wherein said lenalidomide is administered to the subject before, during, and / or after said administering step. **Claim 25** The method according to claim 22, wherein the composition is a vaccine. **Claim 26** The method according to claim 25, wherein the vaccine is allogeneic. **Claim 27** The method according to claim 22, wherein the K562 cells express the GM-CSF gene. **Claim 28** The method according to claim 22, wherein the K562 cells are transfected with a vector construct encoding GM-CSF. **Claim 29** The method according to claim 28, wherein the GM-CSF gene is capable of expressing GM-CSF in an amount of up to about 1500 ng / 1×10 6 cells. **Claim 30** The amount of GM-CSF expressed is about 35 to 1200 ng / 1 × 10 6 cells, the method according to claim 29. **Claim 31** The method according to claim 29, wherein the amount of GM-CSF is produced on average every 24 hours. **Claim 27** The method according to claim 22, wherein the GM-CSF is of human origin. **Claim 28** A method for treating plasma cell disorders in a subject, comprising the step of administering the composition according to claim 10 to the subject. **Claim 29** The method according to claim 28, wherein the ratio of the combination of U266 and H929 cells to K562 cells is about 20:

1. **Claim 30** The method according to claim 28, wherein the dose of the composition is such that the ratio of tumor cells in the subject to K562 cells in the composition exceeds 2:

1. **Claim 31** The method according to claim 28, wherein the U266 and H929 cells are present in equal amounts in the composition. **Claim 32** The U266 and H929 cells are present in the composition in an amount of about 5×10 7 cells, and the K562 cells are present in the composition in an amount of about 5×10 6 cells, the method according to claim 28. **Claim 33** The method according to claim 28, wherein a remission approaching complete remission or complete remission is achieved in the subject. **Claim 34** The method according to claim 28, wherein the remission approaching complete remission or complete remission persists in the subject for up to 5 years. **Claim 35** The method according to claim 33, wherein the complete remission is determined by measuring undetectable monoclonal spike and negative immunofixation electrophoresis. **Claim 36** The method according to claim 28, wherein the subject is positive for minimal residual disease. **Claim 37** The method according to claim 28, wherein the composition minimizes a non-specific immune response in the subject. **Claim 38** The method according to claim 28, wherein the composition is administered to the subject in 1 to 5 doses at intervals of more than 1 day between each administration. **Claim 39** The method according to claim 38, wherein 2 to 4 doses are administered at intervals of more than 2 weeks between each administration. **Claim 40** The method according to claim 38, wherein the interval between each administration exceeds 4 weeks. **Claim 41** The method according to claim 38, wherein 4 doses are administered at intervals of about 1 month between each administration. **Claim 42** The method according to claim 38, wherein the first three administrations are at equal intervals.

43. The method according to claim 38, wherein all doses are administered within one year of each other.

44. The method according to claim 38, wherein at least one dose is administered between the 7th and 18th days and including these days from the start of the lenalidomide course.

45. The method according to claim 38, wherein at least one dose is administered at about the 15th day from the start of the lenalidomide course.

46. The method according to claim 38, wherein the plasma cell disorder is selected from the group consisting of MGUS, SMM, multiple myeloma, non-secretory multiple myeloma, indolent myeloma, light chain myeloma, plasma cell leukemia, and primary amyloidosis.

47. The method according to claim 38, wherein the plasma cell disorder is multiple myeloma.

48. A method of prolonging progression-free survival in a subject having multiple myeloma, comprising the step of administering to the subject the composition according to claim 10 in combination with lenalidomide.

49. A method of inducing the proliferation of clonal T cells and an increase in the myeloma-specific cytokine response in a subject having multiple myeloma, comprising the step of administering to the subject the composition according to claim 10 in combination with lenalidomide.

50. The method according to claim 49, wherein the increase in the subject persists for up to 7 years after the step of administering.

51. The method according to claim 49, wherein the increase in the subject persists for up to 5 years after the step of administering.

52. A method of inducing multiple myeloma-specific immunity in a subject, comprising the step of administering to the subject the composition according to claim 10 in combination with lenalidomide.

53. The method according to claim 52, wherein the subject is positive for minimal residual disease at the time of the step of administering.

54. A method of preventing recurrence of multiple myeloma in a subject, comprising the step of administering to the subject the composition according to claim 10 in combination with lenalidomide.

55. The method according to claim 54, wherein the subject is positive for minimal residual disease at the time of the step of administering.

56. The method according to claim 22, wherein the subject is human.

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