EA045360B1 - METHODS AND COMPOSITIONS THAT INCREASE THE EFFECTIVENESS OF SUPERANTIGEN-MEDIATED IMMUNOTHERAPY OF MALIGNANT TUMORS - Google Patents

Description

Cross reference to related applications

This application claims the benefit of provisional patent application US 62/276955, filed January 10, 2016, the entire contents of which are hereby incorporated by reference in their entirety.

Field of technology to which the invention relates

The invention generally relates to methods and compositions for enhancing the activity of superantigen-mediated immunotherapy of malignant tumors, and more particularly relates to superantigen-mediated immunotherapy of malignant tumors with an immunostimulant that prevents malignant cells from escaping the immune system of an individual.

Prior Art

According to the American Cancer Society, more than one million people are diagnosed with cancer every year in the United States. A malignant tumor is a disease that results from the uncontrolled proliferation of cells that were once subject to natural control mechanisms but have transformed into malignant cells that continue to proliferate in an uncontrolled manner. In recent years, several types of immunotherapies have been developed that attempt to use an individual's immune system to seek out and destroy malignant cells. Such types of immunotherapies include, for example, types of immunotherapies that aim to enhance the body's natural defense mechanisms to fight cancer, using natural molecules produced in the body, or, alternatively, by introducing recombinant molecules engineered to improve, better target or restore immune system function.

Certain types of immunotherapy involve the administration of compounds that are known to be major enhancers of the immune system, such as cytokines such as IL-2 and interferon. Although the various types of immunotherapies developed to date have shown effectiveness, they may be associated with side effects, including, for example, off-target activities, allergic reactions to the administered active agents, including the possibility of cytokine storms, loss of activity due to stimulation by antibodies that bind and neutralize active agents, decreased blood cell count and fatigue.

Other types of immunotherapy use molecules called immune checkpoint inhibitors, which boost immune responses to cancer. Such checkpoint inhibitors act by inhibiting the ability of cancer cells to block immune checkpoint inhibitors, thereby resulting in increased activity of the therapy against the cancer. The first-generation immune checkpoint inhibitor ipilimumab (YERVOY®; Bristol-Myers Squibb) was approved by the US Food and Drug Administration in 2011 and is an IgG1 monoclonal antibody that can exploit ADCC-mediated cytotoxicity of regulatory T cells (Tregs). . Over the years, immunochemotherapy, a combination of immunotherapy and chemotherapy, has become important in the treatment of certain malignancies. For example, rituximab (RITUXAN®; Roche) is a CD-20-specific monoclonal antibody that depletes CD20-expressing cells and has become a standard component of the treatment of B-cell lymphomas, such as non-Hodgkin's lymphoma, using rituximab (R), cyclophosphamide (C ), hydroxydaunorubicin (H), oncovin (O) and prednisone (P), known as R-CHOP.

Recently, PD-1 inhibitors have been tested, such as nivolumab and pembrolizumab, which interfere with the inhibitory signals between PD-1 and PD-L1. Although these drugs cause increased long-term reactions in some patients, response rates for these drugs as monotherapy have been low, in the range of 21%, and complete response rates have been approximately 1% in some studies.

Although efforts are continually being made to combine different cancer therapies to improve patient outcomes, and efficacy benefits have been demonstrated for some combinations, safety issues have become a major concern as Combination medications may increase serious side effects. For example, grade 3 or 4 drug-related adverse events were reported in a significant number of patients who received anti-CTLA-4 antibody and anti-PD1 antibody in combination compared with patients who received anti-CTLA-4 antibody alone. Thus, despite the significant developments that have occurred in the fields of immunotherapy and oncology, there is still a need for safe and effective immunotherapies for the treatment of malignant tumors.

The essence of the invention

In recent years, various types of immunotherapies have been developed that attempt to use an individual's immune system to seek out and destroy malignant cells. Not

- 1 045360 Although the human immune system has the ability to destroy malignant cells, certain malignant cells have evolved the ability to turn off, suppress, or otherwise evade the host's immune system, allowing the malignant cells to continue to grow and proliferate uncontrollably.

The invention is based in part on the discovery that the directed immune response against a malignant tumor in an individual can be significantly enhanced by the combination of superantigen-based therapy with an immunostimulant that can (a) stimulate the activation of the T cell signaling cascade, (b) suppress the inhibitory T cell cascade between malignant cells and a T cell, (c) inhibits the transmission of inhibitory signals that lead to the expansion, activation and/or activity of T cells through the human immunoglobulin IgG4-mediated pathway, or (d) a combination of two or more of the above.

In one aspect, the invention relates to a method of treating a malignant tumor in a patient. The method includes administering to a patient (i) an effective amount of a superantigen conjugate, wherein the conjugate contains a superantigen covalently linked to a targeting moiety that binds to a first antigen preferentially expressed by cancer cells in the patient, and (ii) an effective amount of an immunostimulant (e.g., a checkpoint pathway inhibitor ), effective for at least one or more of (a) promoting activation of the T cell signaling cascade, (b) suppressing the inhibitory T cell signaling cascade between the malignant cells and the T cell, and/or (c) suppressing the inhibitory signaling cascade , which results in the expansion, activation and/or activity of T cells through the human immunoglobulin IgG4-mediated pathway (i.e., through the non-human IgG1-mediated immune response pathway), thereby enhancing the patient's immune response to malignant cells for treatment malignant tumor. Superantigen-based therapies can increase interferon secretion from T cells, which in turn can increase PD-L1 expression. However, it is not known to date whether this negative effect can be suppressed by a PD-1 inhibitor, which compensates for the positive effects of increased antigenicity. It is now discovered that superantigen conjugate-based therapies may likely influence PD1 inhibitor response rates or otherwise influence clinical outcome.

In some embodiments, the superantigen conjugate may be administered to a patient before, simultaneously with, or after an immunostimulant. In addition, the superantigen-immunostimulant conjugate can be administered together or sequentially with one or more additional agents that enhance the activity and/or selectivity of the therapeutic effect. Such agents include, for example, corticosteroids, additional immunomodulators, and such compounds designed to reduce the patient's potential immunoreactivity to the administered superantigen conjugate. For example, immunoreactivity to an administered superantigen can be reduced by co-administration, for example, with an anti-CD20 antibody and/or an anti-CD19 antibody, which reduces the formation of antibodies to the superantigen in the patient.

In some embodiments, the superantigen present in the superantigen conjugate binds to a T cell receptor on the surface of a T cell, such as a CD4+ and/or CD8+ T cell. The superantigen may be staphylococcal enterotoxin A or B, an immunologically reactive variant thereof, or an immunologically reactive fragment of staphylococcal enterotoxin A or B or an immunological variant thereof. The targeting moiety of the conjugate binds to an antigen preferentially expressed on the cancer cell, thereby linking the superantigen to the cancer cell. The targeting moiety can be used to direct the conjugate to cancer cells, preferably by binding to one or more different antigens, for example, one or more cell surface antigens expressed by cancer cells. In some embodiments, the cell surface antigen is, for example, oncofetal cancer antigen 5T4, which is present on cancer cells as well as certain cancer stem cells. Although a number of targeting moieties can be used to direct the superantigen to cancer cells expressing the first antigen, in some embodiments, the targeting moiety is an antibody, such as an anti-5T4 antibody. In some embodiments, the targeting fragment is a Fab fragment that binds 5T4. In some embodiments, the superantigen in the conjugate comprises amino acids 226-458 of SEQ ID NO: 7 (also residues 226,458 of SEQ ID NO: 8) or an immunologically reactive variant thereof, or an immunologically reactive fragment of each of the above.

In some embodiments, the immunostimulant is a checkpoint pathway inhibitor. For example, in some embodiments, the immunostimulant reduces the expression or activity of a programmed cell death ligand (PD-L), such as PD-L1 or PD-L2, expressed on the surface of cancer cells. PD-L is a ligand that binds to the programmed cell death receptor-1 (PD-1), which is expressed on T cells. Certain malignant cells express PD-L to reduce activation or

- 2 045360 T cell activity through the PD-1 checkpoint pathway in a manner that evades the patient's immune system. In some embodiments, the immunostimulant is an anti-PD-1 antibody that prevents a PD-L, such as PD-L1 or PD-L2, from binding to PD-1 expressed on the surface of a T cell. In some embodiments, the anti-PD-1 antibody is of or is based on a human IgG4 immunoglobulin isotype that induces significantly less antibody-dependent cell-mediated toxicity (ADCC) compared to the human IgG1 immunoglobulin isotype.

In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab and peibrolizumab.

Other PD-1 inhibitors include (i) anti-PD-1 antibodies, such as MK-3475 (Merck & Co), pidlizumab (CureTech), AMP-224 (AstraZeneca/Medimmune) and AMP-514 (AstraZeneca/Medimmune), and (ii) anti-PD-L1 antibodies, such as MPDL3280A (Genentech/Roche), MEDI-4736 (AstraZeneca/Medimmune) and MSB0010718C (EMD Serono/Merck KGA).

In some embodiments, other possible immunostimulants may be used, such as a 4-1BB (CD137) agonist (e.g., fully human anti-CD137 IgG4 antibody urelumab/BMS-663513), a LAG3 inhibitor (e.g., humanized anti-LAG3 IgG4 antibody LAG525, Novartis) ; an IDO inhibitor (eg, small molecule INCB024360, Incyte Corporation), a TGFOI inhibitor (eg, small molecule galunisertib, Eli Lilly) and other receptor or ligands that are found on T cells and/or tumor cells and that are suitable for pharmaceutical interaction-based intervention agonist/antagonist, but not via ADCC.

It should be understood that the method can be used to treat various malignant tumors, for example, a malignant tumor selected from the group consisting of breast cancer, cervical cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, cancer prostate, renal cell carcinoma and skin cancer.

In another aspect, the invention provides a pharmaceutical composition comprising (i) an effective amount of a superantigen conjugate, wherein the conjugate comprises a superantigen covalently linked to a targeting moiety that binds to a first antigen expressed by cancer cells in a patient, (ii) an effective amount of an immunostimulant effective to for at least one or more of the following: (a) stimulating the activation of the T cell signaling cascade, (b) suppressing the T cell inhibitory signaling cascade between the malignant cells and the T cell, and/or (c) suppressing the inhibitory signaling cascade that results in the expansion, activation and/or activity of T cells through a human immunoglobulin IgG4-mediated pathway (ie, through a non-human IgG1-mediated immune response pathway) in a patient, and (iii) a pharmaceutically acceptable excipient.

In some embodiments, the superantigen component of the conjugate binds to a T cell receptor expressed on the cell surface of the T cell. For example, in some embodiments, the superantigen comprises staphylococcal enterotoxin A or staphylococcal enterotoxin B, an immunologically reactive variant thereof, or an immunologically reactive fragment of staphylococcal enterotoxin A or B or a variant thereof. It should be understood that the targeting moiety in the conjugate may direct the conjugate to one or more antigens expressed in cancer cells. In some embodiments, the first antigen targeted by the conjugate is a cell surface antigen, for example, oncofetal cancer antigen 5T4. Although various targeting moieties can be used to direct the conjugate to an antigen expressed in cancer cells, in one embodiment, the targeting moiety is an antibody, such as an anti-5T4 antibody. In some embodiments, the targeting fragment comprises a Fab fragment that binds to the 5T4 antigen. In some embodiments, the superantigen in the conjugate comprises amino acid residues 226-458 of SEQ ID NO: 7 (as well as residues 226-458 of SEQ ID NO: 8) or an immunologically reactive variant thereof, or an immunologically reactive fragment of each of the above.

In some embodiments, the immunostimulant prevents PD-L (e.g., PD-L1 or PD-L2) expressed on cancer cells from binding to the PD-1 receptor expressed on the T cell, thereby reducing T cell activation and/or activity. cells. For example, the immunostimulant may be a PD-1 checkpoint pathway inhibitor, such as an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is of or is based on a human IgG4 immunoglobulin isotype that preferably induces significantly less ADCC compared to the human IgG1 immunoglobulin isotype. In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab and peibrolizumab.

These and other aspects and features of the invention are described in the following detailed description, figures and claims.

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Brief description of drawings

The above and other objects, features and advantages of the invention will become apparent from the following description of preferred embodiments, as illustrated in the accompanying drawings. Also, reference elements identify common features in the corresponding drawings. The figures are not necessarily to scale, but instead emphasis is placed on illustrating the principles of the present invention, wherein:

fig. 1 is a schematic representation of an exemplary treatment method of the invention using a superantigen-immunostimulant conjugate;

fig. 2 is a sequence alignment showing homologous regions A-E in certain wild-type and modified superantigens;

fig. 3 is an amino acid sequence corresponding to an exemplary superantigen conjugate;

fig. 4 is a bar graph illustrating the effect of ANYARA® and KEYTRUDA® alone or in combination on the viability of the non-small cell lung cancer (NSCLC) cell line, HCC827. Survival rates of HCC827 cells co-cultured with T cells were measured 48 hours after treatment with ANYARA® (0.1 mg/ml) and/or KEYTRUDA® (0.2 mg/ml) or media alone (control). n=3-6; mean ±SD *p<0.05, **p<0.02; ***p<0.005, as determined by two-tailed Student's t test;

fig. 5 is a bar graph illustrating the effect of ANYARA® and KEYTRUDA® alone or in combination on the viability of the NSCLC cell line, HCC827. Survival rates of HCC827 cells co-cultured with T cells were measured 48 hours after treatment with ANYARA® (10 mg/ml) and/or KEYTRUDA® (0.2 mg/ml) or media alone (control). n=3-6; mean ±SD. *p=0.005, **p<0.005; ***p<0.0005, as determined by two-tailed Student's t test; and figs. 6 is a bar graph illustrating the effect of C215Fab-SEA and anti-PD-1 mAb alone or in combination on tumor burden in the B16EpCAM lung tumor mouse model of melanoma. Mice were inoculated intravenously with B16-EpCAM melanoma cells and treated with C215Fab-SEA (0.5 mg/mouse) and/or anti-PD-1 mAb (200 mg/mouse). The control group was injected with PBS. On day 21, the mice were sacrificed, the lungs were removed, and the number of tumors was counted. n=7-8 mice/group; mean ±SEM. *p=0.05, **p<0.001, as determined by ANOVA.

Detailed description

The present invention relates to methods and compositions for treating cancer in a patient. In particular, the invention is based in part on the discovery that a targeted immune response against a patient's malignancy can be significantly enhanced by combining superantigen-based therapy with an immunostimulant that can (a) stimulate activation of the T cell signaling cascade, (b) suppress T cell an inhibitory signaling cascade between malignant cells and a T cell, (c) inhibiting an inhibitory signaling cascade that leads to expansion, activation and/or activity of the T cell through the human immunoglobulin IgG4-mediated pathway, or (d) a combination of two or more of the above . It has been found that administration of a tumor-targeted superantigen (TTS; a form of immunotherapy), together with an immunostimulant (eg, a PD-1 inhibitor), can result in enhanced anti-tumor activity for both the superantigen and the immunostimulant when combined with each other (i.e., the agents act synergistically), resulting in an effect that is greater than the additive effect of each agent when administered separately.

In one aspect, the invention relates to a method of treating a malignant tumor in a patient. The method includes administering to a patient (i) an effective amount of a superantigen conjugate, wherein the conjugate contains a superantigen covalently linked to a targeting moiety that binds to a first antigen preferentially expressed by cancer cells in the patient, and (ii) an effective amount of an immunostimulant (e.g., a checkpoint pathway inhibitor ), effective for at least one or more of (a) promoting activation of the T cell signaling cascade, (b) suppressing the T cell inhibitory signaling cascade between the malignant cells and the T cell, and/or (c) suppressing the inhibitory signaling cascade which results in the expansion, activation and/or activity of a T cell through the human immunoglobulin IgG4-mediated pathway (i.e., through the non-human IgG1-mediated immune response pathway), such as to enhance the immune response in the patient against malignant cells for treatment malignant tumor.

In another aspect, the invention provides a pharmaceutical composition comprising (i) an effective amount of a superantigen conjugate, wherein the conjugate comprises a superantigen covalently linked to a targeting moiety that binds to a first antigen expressed by cancer cells in a patient, (ii) an effective amount of an immunostimulant effective to for at least one or more of (a) promoting activation of the T cell signaling cascade, (b) suppressing the T cell inhibitory signaling cascade between malignant cells and T

- 4 045360 cell and/or (c) suppressing the inhibitory signaling cascade that leads to T cell expansion, activation and/or activity through the human immunoglobulin IgG4-mediated pathway (i.e., through the non-human IgG1-mediated immune response pathway) in the patient, and (iii) a pharmaceutically acceptable excipient.

I. Definitions.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. For purposes of the present invention, the following terms are defined below.

As used herein, the singular terms may mean one or more. For example, a statement such as treatment with a superantigen and an immunostimulant may mean treatment with: one superantigen and one immunostimulant; more than one superantigen and one immunostimulant; one superantigen and more than one immunostimulant or more than one superantigen and more than one immunostimulant.

As used herein, unless otherwise indicated, the term antibody is understood to mean an intact antibody (e.g., an intact monoclonal antibody) or an antigen binding fragment of an antibody, including an intact antibody or an antigen binding fragment of an antibody (e.g., a phage display antibody, including a fully human antibody, semisynthetic antibody or fully synthetic antibody) that has been optimized, engineered, or chemically conjugated. Examples of antibodies that have been optimized are affinity mature antibodies. Examples of antibodies that have been engineered include Fc optimized antibodies, antibodies engineered to reduce immunogenicity, and multispecific antibodies (eg, bispecific antibodies). Examples of antigen binding fragments include Fab, Fab', F(ab')2, Fv, single chain antibodies (eg, scFv), mini antibodies and diabodies. An antibody conjugated to a toxin moiety is an example of a chemically conjugated antibody.

As used herein, the terms malignant tumor and malignant are understood to mean a physiological condition in mammals that is typically characterized by uncontrolled cellular growth. Examples of malignancy include, but are not limited to, melanoma, carcinoma, lymphoma, blastoma, sarcoma and leukemia or lymphoid malignancies. More specific examples of malignancies include squamous cell carcinoma (eg, epithelial squamous cell carcinoma), lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung, peritoneal malignancy, hepatocellular carcinoma, gastric cancer, including gastrointestinal cancer , pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, malignant brain tumor, retinoblastoma, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, cancer of the female external genitalia, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, testicular cancer, and head and neck cancer, cancer of the gums or tongue . A malignant tumor includes a malignant tumor or malignant cells, for example, a malignant tumor may include a collection of individual cancer cells or malignant cells, such as leukemia, or a tumor including a collection of associated cancer cells or malignant cells.

As used herein, the term immunogen is a molecule that causes (promotes, induces or leads to) an immune response. Such an immune response may include the formation of antibodies, the activation of certain cells, such as, for example, specific immunocompetent cells, or both. The immunogen can be obtained from many types of substances, such as, but not limited to, molecules from organisms, such as, for example, proteins, protein subunits, killed or inactivated whole cells or lysates, synthetic molecules, and a wide range of other agents such as biological, and non-biological. It should be understood that essentially any macromolecule (including natural macromolecules or macromolecules produced by recombinant DNA approaches), including virtually all proteins, can serve as immunogens.

As used herein, the term immunogenicity refers to the ability of an immunogen to elicit (promote, induce or lead to) an immune response. Different molecules may have different degrees of immunogenicity, and it is known that a molecule with an immunogenicity that is higher compared to another molecule, for example, is capable of eliciting (promoting, inducing or leading to) a higher immune response than an agent with lower immunogenicity.

As used herein, the term antigen refers to a molecule that is recognized by antibodies, specific immunocompetent cells, or both. Antigen can be obtained from many types of substances, such as, but not limited to, molecules from organisms such as, for example, proteins, protein subunits, nucleic acids, lipids, killed or inactivated

- 5 045360 cells or lysates, synthetic molecules, and a wide range of other agents, both biological and non-biological.

As used herein, the term antigenicity refers to the ability of an antigen to be recognized by antibodies, specific immunocompetent cells, or both.

As used herein, the term major histocompatibility complex or MHC refers to a specific cluster of genes, many of which encode evolutionarily related cell surface proteins involved in antigen presentation, which are important determinants of histocompatibility. MHC class I or MHC-I functions primarily in antigen presentation to CD8+ T lymphocytes (CD8+ T cells). MHC class II, or MHC-II, functions primarily in the presentation of antigen to CD4+ T lymphocytes (CD4+ T cells).

As used herein, the term derived, e.g., derived from, includes, but is not limited to, e.g., wild-type molecules obtained from biological hosts, such as bacteria, viruses, and eukaryotic cells and organisms, and modified molecules, e.g., chemically modified means or obtained in recombinant expression systems.

As used herein, the terms seropositive, serological reaction or seroreactivity are understood as the ability of an agent, such as a molecule, to interact with antibodies in the serum of a mammal, such as, but not limited to, a human. This includes interactions with all types of antibodies, including, for example, antibodies specific to molecules and nonspecific antibodies that bind to molecules, regardless of whether the antibodies inactivate or neutralize the agent. As is known in the art, different agents may have different seroreactivity relative to each other, where an agent with lower seroreactivity compared to another, for example, will interact with multiple antibodies and/or have lower affinity and/or avidity for antibodies compared to with a product with higher seroreactivity. This may also include the ability of the agent to elicit an immune response to produce antibodies in an animal, such as a mammal, such as a human.

As used herein, the terms soluble T cell receptor or soluble TCR are understood to mean a soluble T cell receptor containing full-length (e.g., membrane-bound) receptor chains, except that the transmembrane region of the receptor chains is removed or mutated, thereby that this receptor, when expressed by a cell, will not insert into, pass through, or otherwise be associated with a membrane. A soluble T cell receptor may contain only the extracellular domains or extracellular fragments of the domains of the wild-type receptor (eg, does not contain the transmembrane and cytoplasmic domains).

As used herein, the term superantigen is understood as a class of molecules that stimulate a subset of T cells by binding to MHC class II molecules and Ve domains of T cell receptors, thereby activating T cells expressing specific segments of the Ve gene. The term includes wild-type superantigens, natural superantigens, such as superantigens isolated from certain bacteria or expressed by unmodified genes from the same bacteria, as well as modified superantigens, where, for example, the DNA sequence encoding the superantigen has been modified, for example by genetic engineering, for example, to produce a fusion protein with a targeting moiety, and/or alter certain properties of the superantigen, such as, but not limited to, its MHC class II binding (eg, to reduce affinity) and/or its seroreactivity, and/or its immunogenicity, and/ or antigenicity (for example, to reduce its seroreactivity). The definition includes wild-type and modified superantigens and any immunologically reactive variants thereof and/or fragments thereof described herein or in the following US patents and patent applications: patents

USA No. 5858363, 6197299, 6514498, 6713284, 6692746, 6632640, 6632441, 6447777, 6399332, 6340461,

6338845, 6251385, 6221351, 6180097, 6126945, 6042837, 6713284, 6632640, 6632441, 5859207, 5728388,

5545716, 5519114, 6926694, 7125554, 7226595, 7226601, 7094603, 7087235, 6835818, 7198398, 6774218,

6913755, 6969616 and 6713284, US Patent Application Nos. 2003/0157113, 2003/0124142, 2002/0177551, 2002/0141981, 2002/0115190, 2002/0051765 and 200 1/004 6501, and PCT international publication number WO/03/094846.

As used herein, the term targeting moiety refers to any structure, molecule or fragment that is capable of binding to cellular molecules, for example, cell surface molecules, preferably disease-specific molecules, such as an antigen, preferably expressed on a cancer (or malignant) cell . Exemplary targeting moieties include, but are not limited to, antibodies (including antigen binding moieties thereof) and the like, soluble T cell receptors, interleukins, hormones and growth factors.

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As used herein, the terms tumor-targeted superantigen or cancer-targeted superantigen are understood to mean a molecule containing one or more superantigens covalently linked (directly or indirectly) to one or more targeting moieties.

As used herein, the term T cell receptor is understood to mean a receptor that is specific to T cells, and includes the term as known in the art. The term also includes, for example, a receptor that contains a disulfide-linked heterodimer of highly variable α or β chains expressed on the cell membrane as a complex with CD3 invariant chains, and a receptor consisting of variable γ and δ chains expressed on the cell membrane. membrane in the form of a complex with CD3 in a subset of T cells.

As used herein, the terms therapeutically effective amount and effective amount are understood to mean an amount of an active agent, eg, a pharmaceutically active agent, or pharmaceutical composition, that has at least a certain effect in treating a disease or condition. The effective amount of pharmaceutically active agent(s) used to practice the present invention for therapeutic treatment varies depending on the route of administration, age, body weight and general health of the patient. These terms include, but are not limited to, synergistic conditions, such as those described in the present invention, where one agent alone, such as a superantigen conjugate or an immunostimulant (for example, an anti-PD-1 antibody), may have little or no effect, but in combination with each other, for example, but not limited to, through sequential dosing, two or more agents act in such a way that a synergistic result occurs.

As used herein, the terms patient and patient refer to the body that is to be treated with the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (eg, mice, monkeys, horses, cattle, pigs, dogs, cats, etc.), and more preferably includes humans.

As used herein, the terms treat, treater and treatment are understood to mean treating a disease in a mammal, such as a human. It includes: (a) inhibiting the disease, for example, stopping its progression; and (b) alleviating the disease, eg, causing regression of the disease state; and (c) treating the disease. As used in relation to therapeutic treatment, the terms prevent or block are understood as completely preventing or blocking, or not completely preventing or blocking (eg, partially preventing or blocking) a given act, action, activity or event.

As used herein, the term inhibits cancer growth is understood as a measurable slowing, stopping or reversing the progression of the growth rate of cancer cells or malignant cells in vitro or in vivo. Desirably, the growth rate is slowed by 20%, 30%, 50% or 70% or more, as determined using a suitable cell growth rate assay. Typically, reversing the progression of growth rate is accomplished by stimulating or accelerating necrotic or apoptotic cell death mechanisms in neoplastic cells, resulting in a decrease in neoplasia mass.

As used herein, the terms variant, variants, modified, altered, mutant, and the like. understood as proteins or peptides and/or other agents and/or compounds that differ from the reference protein, peptide or other compound. Options in this sense are described below and described in more detail elsewhere. For example, changes to the variant nucleic acid sequence may be silent, eg, they may not change the amino acids encoded by the nucleic acid sequence. If the changes are limited to silent changes of this type, the variant will encode a peptide with the same amino acid sequence as the reference peptide. Changes in the variant nucleic acid sequence may alter the amino acid sequence of the peptide encoded by the reference nucleic acid sequence. Such nucleic acid changes may result in amino acid substitutions, additions, deletions, fusions and/or truncations in the protein or peptide encoded by the reference sequence, as discussed below. Typically, amino acid sequence differences are limited such that the sequences of the reference and the variant are similar over their entire length and identical in many regions. The protein variant and the reference protein or peptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and/or truncations, which may be present in any combination. A variant may also be a fragment of a protein or peptide of the invention that differs from the reference protein or peptide sequence by being shorter than the reference sequence, particularly as a result of terminal or internal terminal deletions. Another variant of the protein or peptide of the invention also includes a protein or peptide that retains substantially the same function or activity as the reference protein or peptide. An option may also be: (i) an option in which

- 7 045360 one or more amino acid residues are replaced by a conserved or non-conservative amino acid residue, and such substituted amino acid residue may or may not be a residue encoded by the genetic code, or (ii) a variant in which one or more amino acid residues contain a substituent group , or (iii) an embodiment wherein the mature protein or peptide is fused to another compound, such as a compound to increase the half-life of the protein or peptide (eg, polyethylene glycol), or (iv) an embodiment wherein additional amino acids are fused to the mature protein or a peptide, such as a leader or secretory sequence or a sequence that is used to purify a mature protein or peptide. Variants can be produced by mutagenesis and/or alteration mechanisms such as chemical changes, fusions, adjuvants and the like, including those used for nucleic acids, amino acids, cells or organisms, and/or can be produced by recombinant methods.

As used herein, the term sequential dosing and related terminology refers to the administration of at least one superantigen with at least one immunostimulant and includes staggered dosing of the agents (eg, spaced out over time) and changes in dosage amounts. This involves one agent being administered before the administration, overlapping the administration (partially or completely) or after the administration of the other agent. This term generally takes into account the best administration schedule to obtain a synergistic combination of at least one superantigen and at least one immunostimulant. With this dosing strategy (eg, sequential dosing), a synergistic effect of the combined administration of superantigen and immunostimulant can be achieved. In addition, the term sequential dosing and related terminology also includes the administration of at least one superantigen, one immunostimulant, and more or more optional additional compounds, such as, for example, a corticosteroid, an immunomodulator, and other agent designed to reduce possible immune activity to the superantigen conjugate administered to the patient.

As used herein, the terms systemic and systemically, with respect to administration, are understood to mean administration of an agent such that the agent is exposed to at least one system associated with the entire body, such as, but not limited to, the circulatory system, the immune system and the lymphatic system, not just a localized part of the body, such as, but not limited to, leading into a tumor. Thus, for example, a systemic therapy or systemically administered agent is a therapy or agent in which at least one system associated with the entire body is exposed to the therapy or agent, as opposed to simply the target tissue.

As used herein, the term parenteral administration includes any form of administration in which the compound is absorbed from the patient without involving intestinal absorption. Exemplary parenteral administrations that are used in the present invention include, but are not limited to, intramuscular, intravenous, intraperitoneal, or intra-articular administration.

Where the use of a term approximately precedes a quantitative value, the present invention also refers to the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term approximately refers to a change of ±10% of the nominal value unless otherwise stated or implied.

At various places throughout the specification, quantities are described in groups or ranges. It is specifically intended that the description include each and every distinct subcombination of members of such groups and ranges. For example, an integer in the range 0 to 4 0 is specifically intended to individually expand 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40, and an integer in the range of 1 to 20 are specifically intended to individually reveal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.

Throughout the specification, when compositions and kits are described as having, including, or containing specific components, or when processes and methods are described as having, including, or consisting of specific steps, it is contemplated that, in addition, there are compositions and kits of the present invention, which essentially contain or consist of the listed components, and that there are processes and methods of the present invention that essentially contain or consist of the listed processing steps and method.

In addition, it should be understood that elements and/or features of the composition or method described herein may be combined in various ways without departing from the spirit and scope of the present invention, whether express or implied herein. In other words, in this application, embodiments are described and illustrated in a manner that allows a clear and concise application to be written and illustrated, but it is intended and will be understood that the embodiments may be combined or separated in various ways without departing from the present teachings and the invention(s). For example, it should be understood that all the signs described and

- 8 045360 illustrated in the present description can be applied to all aspects of the invention(s) described and illustrated in the present description.

II. Superantigen conjugate A. Superantigens.

Superantigens are bacterial proteins, viral proteins, and human-engineered proteins capable of activating T lymphocytes, for example, in picomolar concentrations. Superantigens can also activate large subpopulations of T lymphocytes (T cells). Superantigens can bind to major histocompatibility complex I (MHCI) without being processed, and in particular can bind to conserved regions outside the antigen-binding groove on MHC class II molecules, avoiding much of the polymorphism in the conventional peptide binding site. Superantigens can also bind to the Ve chain of the T cell receptor (TCR) rather than binding to the hypervariable loops of the T cell receptor. Examples of bacterial superantigens include, but are not limited to, staphylococcal enterotoxin (SE), streptococcal pyrogenic exotoxin (SPE), Staphylococcus aureus toxic shock syndrome toxin (TSST-1), streptococcal mitogenic exotoxin (SME), streptococcal superantigen (SSA), staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin A (SEB) and staphylococcal enterotoxin E (SM).

Polynucleotide sequences encoding many superantigens have been isolated and cloned, and superantigens expressed by these sequences or modified (redesigned) polynucleotide sequences have been used in cancer therapy (see, ANYARA®, discussed below). The superantigens expressed by these polynucleotide sequences may be wild-type superantigens, modified superantigens, or wild-type superantigens or modified superantigens conjugated or fused to targeting moieties. Superantigens can be administered to a mammal, such as a human, directly, for example by injection, or they can be delivered, for example, by exposing the patient's blood to the superantigen outside the body, or, for example, by placing a gene encoding the superantigen within the mammal in which it is necessary to carry out treatment (eg, known gene therapy methods and vectors, such as, for example, through cells containing and capable of expressing the gene) and expression of the gene within the mammal.

Examples of superantigens and their administration to mammals are described in the following patents:

USA and patent applications: US patents No. 5858363, 6197299, 6514498, 6713284, 6692746, 6632640,

6632441, 6447777, 6399332, 6340461, 6338845, 6251385, 6221351, 6180097, 6126945, 6042837, 6713284,

6632640, 6632441, 5859207, 5728388, 5545716, 5519114, 6926694, 7125554, 7226595, 7226601, 7094603,

7087235, 6835818, 7198398, 6774218, 6913755, 6969616 and 6713284, US Patent Application Nos. 2003/0157113, 2003/0124142, 2002/0177551, 2002/0141 981, 2002/0115190 and 2002/0051765, and PCT international publication number WO/03 /094846.

B. Modified superantigens.

Within the scope of the present invention, superantigens can be designed in a variety of ways, including modifications that maintain or enhance the ability of the superantigen to stimulate T lymphocytes, and can, for example, change other aspects of the superantigen, such as, for example, its seroreactivity or immunogenicity. Modified superantigens include synthetic molecules that have superantigen activity (eg, the ability to activate subsets of T lymphocytes).

It is envisaged that various changes can be made to the polynucleotide sequences encoding the superantigen without appreciable loss of its biological value or activity, namely the induction of a T cell response, in order to lead to cytotoxicity of tumor cells. In addition, the affinity of the superantigen for the MHC class II molecule can be reduced with minimal impact on the cytotoxicity of the superantigen. This, for example, may help reduce toxicity that might otherwise occur if the superantigen retains its wild-type ability to bind to MHC class II antigens (as class II expressing cells such as immune system cells may then also be susceptible to influence of the response to superantigen).

Methods for modifying superantigens (e.g., polynucleotides and polypeptides), including the production of synthetic superantigens, are well known in the art and include, for example, PCR mutagenesis, alanine scanning mutagenesis, and site-directed mutagenesis (see US Pat. Nos. 5,220,007, 5,284,760, 5,354,670, 5,366,878 , 5389514, 5635377 and 5789166).

In some embodiments, the superantigen can be modified such that its seroreactivity is reduced relative to the reference wild-type superantigen, but its ability to activate T cells is maintained or increased relative to the wild type. One method for producing such modified superantigens involves replacing certain amino acids in certain regions from one superantigen with others. This is possible because many superantigens, including but not limited to SEA, SEE and SED, have a common

- 9 045360 sequence homology at certain regions that are associated with certain functions (Marrack and Kappler (1990) Science 248(4959): 1066; also see Fig. 2, which shows the region of homology for various wild-type and engineered superantigens). For example, in some embodiments of the present invention, a superantigen that has a desired T cell activation-inducing response but undesirable high seroreactivity is modified such that the resulting superantigen retains its ability to activate T cells but has reduced seroreactivity.

It is known and understood by those skilled in the art that human serum typically contains varying titers of antibodies to superantigens. For example, for staphylococcal superantigens, for example, the relative titers are TSST-1>SEB>SEC-1>SE3>SEC2>SEA>SED>SEE. As a result, the seroreactivity of, for example, SEE (staphylococcal enterotoxin E) is lower than the seroreactivity of, for example, SEA (staphylococcal enterotoxin A). Based on these data, one of ordinary skill in the art may prefer to administer a low titer superantigen, such as SEE, rather than a high titer superantigen, such as SEB (staphylococcal enterotoxin B). However, it has also been discovered that different superantigens have different T-cell activating properties relative to each other, and for wild-type superantigens, the best T-cell activating superantigens also tend to have undesirably high seroreactivity.

These relative titers in some cases correspond to possible problems with seroreactivity, such as problems with neutralizing antibodies. Thus, the use of a low titer superantigen, such as SEA or SEE, may be useful in reducing or eliminating seroreactivity of parenterally administered superantigens.

A low titre superantigen has low seroreactivity, as measured, for example, by signature antibodies to the superantigen in the general population. In some cases it may also have low immunogenicity. Such low titer superantigens can be modified to maintain their low titer as described herein.

Superantigen modification approaches can be used to create superantigens that have desired T cell activation properties and reduced seroreactivity, and in some cases also reduced immunogenicity. Given the fact that certain regions of superantigen homology are associated with seroreactivity, it is possible to design a recombinant superantigen that has the desired T cell activation and the desired seroreactivity and/or immunogenicity. In addition, the protein sequences and immunological cross-reactivity of superantigens or staphylococcal enterotoxins are divided into two related groups. One group consists of SEA, SEE and SED. The second group is SPEA, SEC and SEB. Thus, low-titer superantigens can be selected to reduce or eliminate cross-reactivity with high-titer or endogenous antibodies directed against staphylococcal enterotoxins.

Regions in superantigens that are believed to play a role in seroreactivity include, for example, region A, which contains amino acid residues 20, 21, 22, 23, 24, 25, 26 and 27; region B, which contains amino acid residues 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 and 49; region C, which contains amino acid residues 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 and 84; region D, which contains amino acid residues 187, 188, 189 and 190; and region E, which contains amino acid residues 217, 218, 219, 220, 221, 222, 223, 224, 225, 226 and 227 (see US Pat. No. 7,125,554, and FIG. 2 herein). Thus, it is envisaged that these regions can be mutated using, for example, amino acid substitution, to produce a superantigen with altered seroreactivity.

Polypeptide or amino acid sequences for the superantigens listed above can be obtained from any sequence data bank, for example, Protein Data Bank and/or GenBank. Exemplary GenBank accession numbers include, but are not limited to, SEE is P12993; SEA is P013163; SEB is P01552; SEC1 is P01553; SED is P20723 and SEH is AAA19777.

In some embodiments of the present invention, the wild-type SEE sequence (SEQ ID NO: 1) or the wild-type SEA sequence (SEQ ID NO: 2) can be modified such that the amino acids in any of the identified regions A-E (see, FIG. 2 ) are replaced with other amino acids. Such substitutions include, for example, K79, K81, K83 and D227 or K79, K81, K83, K84 and D227, or, for example, K79E, K81E, K83S and D227S or K79E, K81E, K83S, K84S and D227A. In some embodiments, the superantigen is SEA/E-120 (SEQ ID NO: 3; also see US Pat. No. 7,125,554) or SEA _d2 2 ₇ a (SEQ ID NO: 4; also see US Pat. No. 7,226,601).

1. Modified polynucleotides and polypeptides.

The biological functional equivalent of a polynucleotide encoding a natural or reference superantigen may comprise a polynucleotide that is designed to contain distinct sequences while simultaneously retaining the ability to encode the natural or reference superantigen. This can be done due to the degeneracy of the genetic code,

- 10 045360 for example, the presence of many codons that encode the same amino acids. In one example, it is possible to introduce a restriction enzyme-recognized sequence into a polynucleotide without interfering with the ability of such polynucleotide to encode a protein. Other polynucleotide sequences may encode superantigens that are different but functionally substantially equivalent in at least one biological property or activity (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 98 % biological property or activity, such as, but not limited to, the ability to induce a T cell response that results in tumor cell cytotoxicity) reference superantigen.

In another example, the polynucleotide may be (and encode) a superantigen that is functionally equivalent to the reference superantigen, although it may contain more significant changes. By definition, amino acids can be replaced by other amino acids in the protein structure without noticeable loss of bilateral binding ability to structures such as, for example, the antigen-binding regions of antibodies, binding sites on substrate molecules, receptors, etc. In addition, substitutions of conserved amino acids may not impair the biological activity of the protein; the resulting structural change is often not a change that affects the protein's ability to perform its intended function. Thus, it is contemplated that such various changes can be made to the sequence of genes and proteins described herein while still being consistent with the purposes of the present invention.

Amino acid substitutions can be designed to take advantage of the relative similarities of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, and/or the like. Analysis of the size, shape and/or type of amino acid side chain substituents reveals that arginine, lysine and/or histidine are positively charged residues; that alanine, glycine and/or series are of similar size, and/or that phenylalanine, tryptophan and/or tyrosine tend to be of similar shape. Thus, based on these discussions, arginine, lysine and/or histidine; alanine, glycine and/or serine, and/or phenylalanine, tryptophan and/or tyrosine are defined herein as biologically functional equivalents. In addition, unnatural amino acids can be introduced. Approaches for making amino acid substitutions with other natural and non-natural amino acids are described in US Patent No. 77 63253.

With respect to functional equivalents, it should be understood that, as implied in the definition of a biologically functional equivalent protein and/or polynucleotide, is the concept that there is a limited number of changes that can be made to a particular portion of the molecule while still maintaining the molecule with an acceptable level of equivalent biological activity. Thus, biologically functional equivalents are considered to be those proteins (and polynucleotides) wherein selected amino acids (or codons) can be replaced with others substantially without affecting the biological function. Functional activity includes the induction of a T-cell response, which leads to cytotoxicity of tumor cells.

It is further contemplated that a modified superantigen can be produced by replacing homologous regions of different proteins through domain swapping, which involves producing chimeric molecules using different, in this case related, polypeptides. By comparing different superantigen proteins to identify functionally related regions of these molecules (see, for example, Fig. 2), it is possible to replace related domains of these molecules in such a way as to determine the importance of these regions for superantigen function. These molecules may have an added value in that such chimeras may be different from natural molecules while possibly providing the same function.

In some embodiments, the superantigen comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to a reference superantigen sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, wherein the superantigen optionally retains at least 50%, 60%, 70%, 80%, 90%, 95%, 98 %, 99% or 100% of the biological activity or properties of the reference superantigen.

In some embodiments, the superantigen comprises an amino acid sequence that is encoded by a nucleic acid that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the nucleic acid encoding the superantigen, selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, wherein the superantigen optionally retains at least 50%, 60%, 70%, 80%, 90 %, 95%, 98%, 99% or 100% of the biological activity or property of the reference superantigen.

Sequence identity can be determined in various ways that are known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis using the algorithm used in blastp, blastn, blastx, tblastn and tblastx (Karlin et al. (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Altschul, (1993) J Mol Evol 36, 290-300 Altschul et al (1997) Nucleic Acids Res 25:3389

- 11 045360

3402, incorporated by reference) are adapted for sequence similarity searching. For a discussion of the basic problems of searching sequence databases, see Altschul et al. (1994) Nature Genetics 6:119-129, which is incorporated by reference in its entirety. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to obtain maximum alignment over the entire length of the full-length sequences being compared. Search options for histogram, descriptions, alignments, expectations (i.e., the statistical significance threshold for reporting matches to database sequences), threshold, matrix, and filter are in the default settings. The default scoring matrix used in blastp, blastx, tblastn and tblastx is the BLOSUM62 matrix (Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919, incorporated by reference in its entirety). The four parameters of blastn can be selected as follows: B Q = 10 (penalty for creating a gap); R=10 (fine for extending the pass); wink=1 (forms hint words in each position of wink.sup.th upon request); and gapw=16 (sets the width of the window in which gap alignments are received). Equivalent Blastp parameter settings could be Q=9; R=2; wink=1 and gapw=32. Searches can also be performed using the NCBI (National Center for Biotechnology Information) BLAST Advanced Option (e.g., -G, gap penalty [integer]: default=5 for nucleotides/ 11 for proteins; -E, extension penalty passes [integer]: default=2 for nucleotides/1 for proteins; -q, nucleotide mismatch penalty [integer]: default= -3; -r, nucleotide match reward [integer]: default= 1; -e, expected value [real number]: default=10; -W, word size [integer]: default=11 for nucleotides/28 for megablast/3 for proteins; -y, cutoff (X) for blast extension in bits: default=20 for blastn/7 for others; -X, X cutoff value for gap alignment (in bits): default=15 for all programs, not applicable for blastn; and -Z, final cutoff value X for alignment with releases (in bits): 50 for blastn, 25 for others). For pairwise protein alignments, ClustalW can also be used (default parameters could include, for example, a Blosum62 matrix and gap creation penalty=10 and gap extension penalty=0.1). Sequence comparisons using Bestfit, available in GCG version 10.0, use the DNA parameters GAP=50 (gap penalty) and LEN=3 (gap penalty), and the equivalent settings for protein comparisons are GAP=8 and LEN= 2.

C. Targeted superantigens.

To increase specificity, the superantigen is preferably conjugated to a targeting moiety to produce a targeting superantigen conjugate that binds to an antigen preferably expressed by the cancer cell, for example a cell surface antigen such as 5T4. The targeting moiety is a carrier that can be used to bind the superantigen to cancer cells, for example, the surface of cancer cells. The targeted superantigen conjugate must retain the ability to activate large numbers of T lymphocytes. For example, a targeting superantigen conjugate would activate large numbers of T cells and direct them to tissues containing a tumor-associated antigen bound to the targeting moiety. In such situations, the specific target cells are preferentially destroyed, leaving the remainder of the organism relatively undamaged. This type of therapy is desirable because non-specific agents against cancer, such as cytostatic chemotherapeutic drugs, are non-specific and kill large numbers of cells not associated with the tumors being treated. For example, studies with targeted superantigen conjugates have demonstrated that inflammation with cytotoxic T lymphocyte (CTL) infiltration into tumor tissue rapidly increases in response to the first injection of targeted superantigen (Dohlsten et al. (1995) Proc. Natl. Acad. Sci. USA 92 :9791-9795). Such inflammation with CTL infiltration into the tumor is one of the main effectors of antitumor therapy with targeted superantigens.

Tumor-targeted superantigens are immunotherapy against cancer and are therapeutic fusion proteins containing a targeting moiety conjugated to the superantigen (Dohlsten et al. (1991) Proc. Natl. Acad. Sci. USA 88:9287-9291; Dohlsten et al. (1994) Proc Natl Acad Sci USA 91:8945-8949).

The targeting moiety can in principle be any structure that is capable of binding to a cellular molecule, for example a cell surface molecule, and is preferably a disease-specific molecule. The targeting molecule (eg, antigen) against which the targeting moiety is directed is typically different from (a) the vp chain of the epitope to which the superantigen binds and (b) the MHC class II epitopes to which the superantigens bind. The targeting moiety can be selected from antibodies, including antigen binding fragments thereof, soluble T cell receptors, growth factors, interleukins (eg, interleukin-2), hormones, etc.

In certain preferred embodiments, the targeting moiety is an antibody (eg, Fab, F(ab) ₂ , Fv, single chain antibody, etc.). Antibodies are

- 12 045360 very versatile and useful cell-specific targeting fragments, since, as a rule, they can be prepared against any cell surface antigen of interest. Monoclonal antibodies are raised against cell surface receptors, tumor-associated antigens, and leukocyte lineage-specific markers such as CD antigens. Antibody variable region genes can be readily isolated from hybridoma cells by methods well known in the art. Exemplary tumor-associated antigens that can be used to generate a targeting fragment may include, but are not limited to, gp100, MelanA/MART, MAGE-A, MAGE (melanoma E antigen), MAGE-3, MAGE-4, MAGEA3, tyrosinase, TRP2 , NYESO-1, CEA (carcinoembryonic antigen), PSA, p53, mammaglobin-A, survivin, Muc1 (mucin1)/DF3, metallopanstimulin-1 (MPS-1), cytochrome P450 isoform 1B1, binding protein 90K/Mac- 2, EpCAM (MK-1), HSP-70, hTERT (TRT), LEA, LAGE-1/CAMEL, TAGE-1, GAGE, 5T4, gp70, SCP-1, c-myc, cyclin B1, MDM2, p62 , Kos, IMP1, RCAS1, TA90, OA1, CT-7, HOM-MEL-40/SSX-2, SSX-1, SSX-4, HOM-TES-14/SCP-1, HOM-TES-85, HDAC5 , MBD2, TRIP4, NY-CO-45, KNSL6, HIP1R, Seb4D, KIAA1416, IMP1, 90K/Mac-2 binding protein, MDM2, NY/ESO, and LMNA.

Exemplary cancer-directed antibodies may include, but are not limited to, anti-CD19 antibodies, anti-CD20 antibodies, anti-5T4 antibodies, anti-Ep-CAM antibodies, anti-Her2/neu antibodies, anti-EGFR antibodies, anti-CEA antibodies, anti-membrane-specific antibodies. prostate antigen (PSMA) and antibodies to IGF-1R. It should be understood that the superantigen can be conjugated to an immunologically reactive antibody fragment, such as C215Fab, 5T4Fab (see, WO8907947) or C242Fab (see, WO9301303).

Examples of tumor-targeting superantigens that can be used in the present invention include C215Fab-SEA (SEQ ID NO: 5), 5T4Fab-SEAD227A (SEQ ID NO: 6) and 5T4Fab-SEA/E120 (SEQ ID NO: 7, see Fig. 3).

In a preferred embodiment, the preferred conjugate is a superantigen conjugate known as naptumomab estafenatox/ANYARA®, which is a fusion protein of the Fab fragment of the anti-5T4 antibody and the superantigen SEA/E-120. ANYARA® contains two protein chains that collectively include an engineered staphylococcal enterotoxin superantigen (SEA/E-120) and a 5T4-targeted Fab containing modified 5T4 variable region sequences fused to mouse IgG1/K antibody constant region sequences C242. The first chain of the protein contains residues 1 to 458 of SEQ ID NO: 7 (also see SEQ ID NO: 8) and includes the chimeric 5T4 Fab 5T4 Fab heavy chain corresponding to residues 1 to 222 of SEQ ID NO: 7, and the superantigen SEA/ E-120, corresponding to residues 226 to 458 of SEQ ID NO: 7, covalently linked through a tripeptide linker GGP, corresponding to residues 223 to 225 of SEQ ID NO: 7. The second strand contains residues 459 to 672 of SEQ ID NO: 7 (also see ., SEQ ID NO: 9) and includes the light chain of the chimeric Fab 5T4. The two protein chains are held together by non-covalent interactions between the heavy and light chains of the Fab. Residues 1-458 SEQ ID NO: 7 correspond to residues 1-458 SEQ ID NO: 8, and residues 459-672 SEQ ID NO: 7 correspond to residues 1-214 SEQ ID NO: 9. ANYARA® contains proteins SEQ ID NOS: 8 and 9, held together by non-covalent interactions between Fab heavy chains and Fab light chains. ANYARA® induces T cell-mediated cytolysis of malignant cells at concentrations of approximately 10 pM, and the superantigen component of the conjugate is designed to have low binding to human and MHC class II antibodies.

It is contemplated that other targeting fragment antibodies can be designed, modified, expressed and purified using methods known in the art and are discussed in more detail below.

Another type of targeting moiety includes the soluble T cell receptor (TCR). Some forms of soluble TCR may contain either only extracellular domains or extracellular and cytoplasmic domains. Other modifications of the TCR may also be contemplated to produce a soluble TCR in which the transmembrane domains have been removed and/or altered such that the TCR is not membrane bound, as described in U.S. Application Publication No. U.S. 2002/119149, U.S. 2002/0142389, U.S. 2003/0144474 and U.S. 2003/0175212, and international publications No. WO2003020763, WO9960120 and WO9960119.

The targeting moiety can be conjugated to the superantigen using recombinant methods or chemical coupling of the targeting moiety to the superantigen.

1. Recombinant linker (fusion protein).

It is contemplated that a gene encoding a superantigen linked directly or indirectly (eg, via an amino acid-containing linker) to a targeting moiety can be produced and expressed using conventional recombinant DNA technologies. For example, the amino terminus of a modified superantigen can be linked to the carboxy terminus of a targeting moiety or vice versa. For antibodies or antibody fragments that can serve as targeting fragments, a light or heavy chain can be used to produce the fusion protein.

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For example, for a Fab fragment, the amino terminus of the modified superantigen can be linked to the first constant domain of the antibody heavy chain (CH1). In some cases, the modified superantigen can be linked to the Fab fragment by linking the VH and VL domain to the superantigen. Alternatively, a peptide linker can be used to link the superantigen and the targeting moiety to each other. In the case where a linker is used, the linker preferably contains hydrophilic amino acid residues such as Gln, Ser, Gly, Glu, Pro, His and Arg. Preferred linkers are peptide bridges consisting of 1-10 amino acid residues, more particularly 3-7 amino acid residues. An exemplary linker is the tripeptide GlyGlyPro-. These approaches were successfully used in the design and production of the ANYARA® superantigen conjugate.

2. Chemical bonding.

It is also contemplated that the superantigen can be linked to the targeting moiety by chemical linkage. A linker, for example a peptide linker, may be necessary to chemically bind the superantigen to the targeting moiety. The peptide linker is preferably hydrophilic and provides one or more reactive molecules selected from amides, thioesters, disulfides, etc. (See US Patent Nos. 5858363, 6197299 and 6514498). It is also contemplated that homo- or heterobifunctional cross-linking reagents may be used for chemical coupling. When chemically linking a superantigen to a targeting moiety, functional groups (eg, primary amino groups or carboxy groups) are often used, which are present at many positions in the compounds.

D. Expression of superantigens and superantigen conjugates.

When recombinant DNA technologies are used, the superantigen or superantigen-targeting fragment conjugate can be expressed using standard expression vectors and expression systems.

Expression vectors, which are genetically engineered to contain a nucleic acid sequence encoding a superantigen, are introduced (eg, transfected) into host cells to produce the superantigen (see, for example, Dohlsten et al. (1994), Forsberg et al. (1997) J Biol Chem 272:12430-12436, Erlandsson et al (2003 J Mol Biol 333:893905 and WO2003002143).

Host cells can be genetically engineered, for example, by transformation or transfection methods to introduce nucleic acid sequences and superantigen expression. Introduction of nucleic acid sequences into a host cell can be accomplished by calcium phosphate precipitation transfection, DEAE-dextran-mediated transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scraping injection, ballistic injection, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al. (1986) Basic Methods In Molecular Biology and Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, ^2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Representative examples of suitable host cells include bacterial cells such as streptococcal, staphylococcal, E. coli, streptomycetes and Bacillus subtilis cells; fungal cells such as yeast cells and aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells.

Examples of production systems for superantigens can be found, for example, in US patent No. 6962694.

E. Protein purification.

The superantigen and/or superantigen-targeting fragment conjugates are preferably purified before use, which can be accomplished using various purification protocols. After separation of the superantigen or superantigen-targeting fragment conjugate from other proteins, the protein of interest can be further purified by chromatographic and electrophoretic methods to obtain partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for obtaining purified peptide are ion exchange chromatography, size exclusion chromatography, affinity chromatography, polyacrylamide gel electrophoresis, isoelectric focusing. The term purified, as used herein, is intended to mean a composition separated from other components where the macromolecule of interest (eg, protein) is purified to any degree relative to its original state. Generally, the term purified refers to a macromolecule that has been fractionated to remove various other components and that substantially retains its biological activity. The term substantially purified refers to a composition in which the macromolecule of interest forms a major component of the composition, such as comprising about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the content of the composition .

Various methods for quantifying steppe are known to those skilled in the art.

- 14 045360 nor protein purification, including, for example, determining the specific activity of the active fraction and estimating the amount of this protein in the fraction by SDS-PAGE analysis, high performance liquid chromatography (HPLC) or any other fractionation method. Various methods suitable for use in protein purification include, for example, ammonium sulfate precipitation, PEG, antibodies, and the like. or by heat denaturation followed by centrifugation; chromatography steps such as ion exchange chromatography, gel filtration, reverse phase chromatography, hydroxyapatite chromatography, affinity chromatography; isoelectric focusing; gel electrophoresis and combinations of these and other techniques. It is contemplated that the order of the various purification steps may be varied, or that certain steps may be omitted, and still provide a suitable method for producing a substantially purified protein or peptide.

III. Immunostimulant.

It is contemplated that the effectiveness of the immunostimulant can be increased by administering the immunostimulant to the patient to be treated in conjunction with a superantigen conjugate containing the superantigen and a targeting moiety. Exemplary immunostimulants may, for example: (a) stimulate activation of the T cell signaling cascade, (b) suppress the T cell inhibitory signaling cascade between malignant cells and the T cell, (c) suppress the inhibitory signaling cascade that leads to proliferation, activation and /or T cell activity through a non-human IgG1-mediated immune response pathway, e.g., human immunoglobulin IgG4-mediated pathway, (d) a combination of (a) and (b), (e) a combination of (a) and (c), (f ) combination of (b) and (c) and (e) combination of (a), (b) and (c).

In some embodiments, the immunostimulant is a checkpoint pathway inhibitor. Various T cell checkpoint inhibitory pathways have now been identified, such as the PD-1 immune checkpoint pathway and the cytotoxic T lymphocyte antigen 4 (CTLA-4) immune checkpoint pathway.

Sundstedt et al. (Sundstedt et al. (2012) J. Immunother. 35:344-35) demonstrated that the combination of a tumor-targeting superantigen (TTS) with anti-CTLA4 IgG1 antibody in a mouse model of B16 melanoma was more active than the individual components. In this study, although the superantigen induced CD4+ and CD8+ T cell infiltration, large numbers of regulatory T cells (Treg) also accumulated in the tumor microenvironment. It is believed that increased levels of these suppressor regulatory cells are a direct consequence of TTS therapy, which may limit its effectiveness. The authors noted that it is likely that the extremely high increase in CTLA-4 levels secondary to TTS resulted in anti-CTLA-4 antibody being predominantly found in the Treg population. Without being limited by theory, it is contemplated that the IgG1 antibody CTLA-4 is cytotoxic to the Treg population, which is central to its anti-cancer activity when used in combination with TTS. In contrast, some embodiments of the present invention use antibodies that are not known to deplete Tregs, e.g., IgG4 antibodies directed to non-CTLA-4 checkpoints (e.g., anti-PD-1 IgG4 antibody inhibitors), and thus they represent a novel combination whose synergistic effects are mediated by other mechanisms of action.

PD-1 is a receptor present on the surface of T cells that serves as an immune checkpoint that inhibits or otherwise modulates T cell activity at the appropriate time, preventing an overactive immune response. However, malignant cells can take advantage of this checkpoint by expressing ligands such as PD-L1, PD-L2, etc., which interact with PD-1 on the surface of T cells, turning off or modulating T cell activity . Using this approach, a malignant tumor can evade the T cell-mediated immune response.

In the CTLA-4 pathway, interaction of CTLA-4 on a T cell with its ligands (eg, CD80, also known as B7-1, and CD86) on the surface of antigen-presenting cells (rather than malignant cells) results in T cell inhibition. As a result, the ligand that inhibits T cell activation or activity (eg, CD80 or CD86) is provided by the antigen presenting cell (a key cell type in the immune system) rather than by the malignant cell. Although CTLA-4 and PD-1 binding have similar negative effects on T cells, the timing of suppression, responsible signaling mechanisms, and anatomical locations of immune inhibition by these two immune checkpoints differs (American Journal of Clinical Oncology. Volume 39, Number 1, February 2016 ). In contrast to CTLA-4, which is limited to the early initial phase of T cell activation, PD-1 acts much later during the effector phase (Keir et al. (2008) Annu. Rev Immunol., 26:677-704). Thus, T cell inhibition mediated through the PD1 checkpoint pathway is very different from T cell inhibition mediated through the CTLA-4 checkpoint pathway.

In some embodiments, the immunostimulant prevents (in whole or in part) the antigen expressed by the cancer cells from inhibiting the T cell inhibitory signaling cascade between the cancer cell and the T cell. In one embodiment

- 15 045360 such an immunostimulant is a checkpoint inhibitor, for example a PD-1 inhibitor. Examples of such immunostimulants include, for example, anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies. Thus, in one embodiment, the superantigen conjugate is administered to a PD-1-based immunostimulant, which may include (1) a molecule (e.g., antibody or small molecule) that binds to a PD-1 ligand (e.g., PD-L1 or PD -L2), preventing binding of a PD-1 ligand to its cognate PD-1 on a T cell, and/or (2) a molecule (e.g., antibody or small molecule) that binds to PD-1 on a T cell, preventing binding PD ligand expressed by the malignant cell of interest.

Additionally, in some embodiments, the immunostimulant prevents (in whole or in part) the antigen expressed by the malignant cell from inhibiting the proliferation, activation, and/or activity of T cells through the human IgG4 (non-human IgG1) mediated immune response pathway, for example, rather than through ADCC pathway. It is contemplated that although the immune response enhanced by the superantigen-immunostimulant conjugate may include some ADCC activity, the major component(s) of the immune response do not involve ADCC activity. In contrast, some of the antibodies currently used in immunotherapy, such as ipilimumab (anti-CTLA-4 IgG1 monoclonal antibody), can kill target cells via ADCC via a signaling cascade via their Fc domain via Fc receptors on effector cells. Ipilimumab, like many other therapeutic antibodies, was designed as a human IgG1 immunoglobulin, and although ipilimumab blocks interactions between CTLA-4 and CD80 or CD86, its mechanism of action is believed to involve ADCC depletion of tumor-infiltrating regulatory T cells that express high levels of CTLA-4. 4 on the cell surface. (Mahoney et al. (2015) Nature Reviews, Drug Discovery 14: 561-584). Given that CTLA-4 is expressed at high levels in a subset of T cells (regulatory T cells) that act to negatively regulate T cell activation, when anti-CTLA-4 IgG1 antibody is administered, the number of regulatory T cells decreases due to ADCC.

In some embodiments, it is necessary to use immunostimulants whose mechanism of action is primarily to block inhibitory signals between cancer cells and T cells without significantly depleting T cell populations (eg, allowing T cell populations to proliferate). In order to achieve this, it is necessary to use an antibody, for example an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-PD-L2 antibody, which is or is based on the human IgG4 isotype. The human IgG4 isotype is preferred in certain circumstances because this antibody isotype causes little or no ADCC activity compared to the human IgG1 isotype (Mahoney et al. (2015) supra). Thus, in some embodiments, the anti-PD-1 antibody, anti-PD-L1 antibody, or anti-PD-L2 antibody is of or is based on the human IgG4 isotype. As a result, it is not possible to extrapolate the possible therapeutic activity of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody with the human IgG4 isotype based on the activity of an antibody with a different isotype, such as the IgG1 isotype, especially if that antibody is directed to a different antigen , for example, CTLA-4, such as human immunoglobulin IgG1 to CTLA-4. In addition, CTLA-4 and PD-1 are two T cell inhibitory receptors with independent, statically undetectable mechanisms of action. Despite their shared ability to block T cell activation, the two proteins have unique structures and have very different effects on immune responses against cancer cells.

Exemplary PD-1/PD-L1-based immunostimulants are described in US Patent Nos. 8,728,474, 8,952,136 and 9,073,994, and EP Patent No. 1,537,878B1, and include anti-PD-1 antibodies. Exemplary anti-PD-1 antibodies include nivolumab (Bristol-Myers Squibb Co.), peibrolizumab (KEYTRUDA®, Merck & Co.) and atezolizumab (formerly MPDL3280A), MEDI4736, avelumab, and PDR001.

Protein-based immunostimulants can be designed, expressed and purified using methods known to those skilled in the art, for example, as described herein above. Antibodies to PD-1 can be designed, expressed, purified, formulated and administered as described in US patent No. 8728474, 8952136 and 9073994.

Other immunostimulants (for example, antibodies and various small molecules) can target signal transduction pathways involving one or more of the following ligands: B7-H3 (found on prostate cells, kidney cells, non-small cell lung cancer, pancreas, among others) glands, stomach, ovary, colon and rectum); B7-H4 (found along with others on breast cells, kidney cells, ovaries, pancreas, melanoma cells); HHLA2 (found along with other cells of the breast, lung, thyroid gland, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney); galectins (found along with others on cells of non-small cell cancer of the lungs, rectum, colon and stomach); CD30 (found along with others on cells of Hodgkin lymphoma, large cell lymphoma); CD70 (found along with others on cells of non-Hodgkin lymphoma, kidney); ICOSL (found along with others on cells of glioblastoma, melanoma) and CD155 (found along with others on cells of the kidney, prostate gland, under

- 16 045360 gastric gland, glioblastoma). Likewise, other possible immunostimulants that may be used include, for example, a 4-1BB (CD137) agonist (e.g., fully human IgG4 anti-CD137 antibody urelumab/BMS-663513), a LAG3 inhibitor (e.g., humanized anti-LAG3 IgG4 antibody LAG525 , Novartis); an IDO inhibitor (eg, small molecule INCB024360, Incyte Corporation), a TGFOI inhibitor (eg, small molecule Galunisertib, Eli Lilly) and other receptor or ligands that occur on T cells and/or tumor cells that are subject to pharmaceutical intervention based on agonist/antagonist interactions rather than interactions via ADCC.

A. Obtaining an antibody.

Methods for producing antibodies are known in the art. For example, DNA molecules encoding light chain variable regions and heavy chain variable regions can be chemically synthesized using the CDR sequences and variable regions of antibodies of interest, for example, the antibody sequences provided in US Pat. No. 8,952,136 and the hybridoma deposit described in the patent US No. 9,073,994. Synthetic DNA molecules can be ligated with other suitable nucleotide sequences, including, for example, constant region coding sequences and expression control sequences, to produce conventional gene expression constructs encoding the desired antibodies. Producing certain gene constructs is routine in the field. Alternatively, the sequences provided herein can be cloned from hybridomas by conventional hybridization methods or polymerase chain reaction (PCR) methods using synthetic nucleic acid probes whose sequences are based on sequence information provided herein or prior art sequence information in in relation to the genes encoding the heavy and light chains of mouse antibodies in hybridoma cells.

Nucleic acids encoding the antibodies described herein can be introduced (ligated) into expression vectors, which can be introduced into host cells by conventional transfection or transformation methods. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, HeLa cells, neonatal hamster kidney (NHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (eg, Hep G2), and myeloma cells that do not otherwise produce IgG protein. Transformed host cells can be grown under conditions that allow the host cells to express genes that encode immunoglobulin light and/or heavy chain variable regions.

The specific expression and purification conditions vary depending on the expression system used. For example, if a gene is to be expressed in E. coli, it is first cloned into an expression vector by placing the constructed gene downstream of a suitable bacterial promoter, such as Trp or Tac, and a prokaryotic signal sequence.

The expressed secreted protein accumulates in refractive or inclusion bodies and can be collected after cell disruption by French press or sonication. The refractive bodies are then dissolved and unfolded and the proteins are digested by methods known in the art.

If a DNA construct encoding an antibody described herein is to be expressed in eukaryotic host cells, eg CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, secretion signal, IgG enhancers and various introns. Such an expression vector optionally contains sequences encoding all or part of the constant region, allowing expression of all and part of the heavy and/or light chain. In some embodiments, a separate expression vector contains heavy and light chain variable regions to be expressed.

The gene construct can be introduced into eukaryotic host cells by conventional methods. Host cells express V _L or VH fragments, VL-VH heterodimers, V _H -V _L or V _L -V _H single-chain polypeptides, complete immunoglobulin heavy or light chains or sections thereof, each of which can be attached to a fragment with a different function ( e.g. cytotoxicity). In some embodiments, a host cell is transfected with a single vector expressing a polypeptide expressing all or part of a heavy chain (eg, a heavy chain variable region) or all or part of a light chain (eg, a light chain variable region). In other embodiments, a host cell is transfected with a single vector encoding (a) a polypeptide comprising a heavy chain variable region and a polypeptide comprising a light chain variable region, or (b) a complete immunoglobulin heavy chain and a complete immunoglobulin light chain. In other embodiments, a host cell is cotransfected with more than one expression vector (e.g., one expression vector expressing a polypeptide containing all or part of a heavy chain or heavy chain variable region, and another expression vector expressing a polypeptide containing all or part of a light chain or light chain variable region).

- 17 045360

A method of producing an immunoglobulin heavy chain variable region-containing polypeptide or an immunoglobulin light chain variable region-containing polypeptide may comprise growing (cultivating) a host cell transfected with an expression vector under conditions that allow expression of the immunoglobulin heavy chain variable region-containing polypeptide, or a polypeptide containing an immunoglobulin light chain variable region. The heavy chain variable region-containing polypeptide or the light chain variable region-containing polypeptide can then be purified using methods well known in the art, for example, an affinity tag such as glutathione S-transferase (GST) and a histidine tag.

A method of producing a monoclonal antibody that binds to a target protein, for example, PD-1, PD-L1 or PD-L2, or an antigen binding fragment of an antibody, may involve growing a host cell transfected with: (a) an expression vector encoding a complete or partial heavy chain immunoglobulin, and a separate expression vector encoding a complete or incomplete light chain of an immunoglobulin; or (b) a single expression vector encoding both chains (eg, complete or partial chains), under conditions that allow expression of both chains. The intact antibody (or antigen binding fragment) can be collected and purified using methods well known in the art, for example, protein A, protein G, affinity tags such as glutathione S-transferase (GST), and histidine tags. One skilled in the art knows how to express the heavy chain and the light chain from a single expression vector or from two separate expression vectors.

B. Antibody modifications.

Methods for reducing or eliminating the antigenicity of antibodies and antibody fragments are known in the art. When the antibodies are intended to be administered to humans, the antibodies are preferably humanized to reduce or eliminate antigenicity in humans. Preferably, the humanized antibody has the same or substantially the same affinity for the antigen as the non-humanized mouse antibody from which it was derived.

In one humanization approach, chimeric proteins are produced in which mouse immunoglobulin constant regions are replaced with human immunoglobulin constant regions. See, for example, Morrison et al. (1984) Proc. Nat. Acad. Sci. 81:6851-6855, Neuberger et al. (1984) Nature 312:604-608; US Patents 6893625 (Robinson), 5500362 (Robinson) and 4816567 (Cabilly).

In an approach known as CDR grafting, CDR light and heavy chain variable regions are transplanted into scaffolds from other species. For example, CDR mice can be transplanted into human FR. In some embodiments, the CDRs of the light and heavy chain variable regions of the anti-ErbB3 antibody are grafted into a human FR or human FR consensus. To obtain a human FR consensus, FRs from multiple human heavy chain or light chain amino acid sequences are aligned to identify a consensus amino acid sequence. CDR transplantation is described in US Patent Nos. 7,022,500 (Queen), 6,982,321 (Winter), 6,180,370 (Queen), 6,054,297 (Carter), 5,693,762 (Queen), 5,859,205 (Adair), 5,693,761 (Queen), 5,565,332 (Hoogenboom), 5,585,089 (Queen ), 5530101 (Queen), Jones et al. (1986) Nature 321: 522-525, Riechmann et al. (1988) Nature 332: 323-327, Verhoeyen et al. (1988) Science 239: 1534-1536 and Winter (1998) FEBS Lett 430: 92-94.

In an approach called SUPERHUMANIZATION™, human CDR sequences are selected from human germline genes based on the structural similarity of the human CDR to the mouse antibody CDR to be humanized. See, for example, US Patent No. 6881557 (Foote) and Tan et al. (2002) J. Immunol. 169:1119–1125.

Other methods to reduce immunogenicity include remodeling, over-chimerization, and veneering/surface modification. See, for example, Vaswami et al. (1998) Annals of Allergy, Asthma, & Immunol. 81:105; Roguska et al. (1996) Prot. Engineer 9:895-904 and US Patent No. 6072035 (Hardman). In the veneering/surface modification approach, surface accessible amino acid residues in a mouse antibody are replaced with amino acid residues more commonly found at the same positions in a human antibody. This type of antibody surface modification is described, for example, in US Pat. No. 5,639,641 (Pedersen).

Another approach to convert a mouse antibody into a form suitable for medical use in humans is known as ACTIVMAB™ technology (Vaccinex, Inc., Rochester, NY), which incorporates a vaccinia virus vector for expression of antibodies in mammalian cells. It is indicated that high levels of combinatorial diversity of IgG heavy and light chains can be obtained. See, for example, US Patent Nos. 6,706,477 (Zauderer), 6,800,442 (Zauderer), and 6,872,518 (Zauderer).

Another approach to converting a mouse antibody into a form suitable for use in humans is a technology commercially used by KaloBios Pharmaceuticals, Inc. (Palo Alto, CA). This technology uses a proprietary human acceptor library to generate a library of target epitopes for antibody selection.

Another approach to modifying a mouse antibody into a form suitable for medical use

- 18 045360 research in humans is HUMAN ENGINEERING™ technology, which is used commercially by HOMA (US) LLC. See, for example, PCT Publication No. WO 93/11794 and US Patent Nos. 5766886, 5770196, 5821123 and 5869619.

Any suitable approach, including any of the above approaches, can be used to reduce or eliminate the immunogenicity of an antibody in a person, including binding a fragment component of a superantigen conjugate described herein.

Methods for producing multispecific antibodies are known in the art. Multispecific antibodies include bispecific antibodies. Bispecific antibodies are antibodies that have binding specificity to at least two different epitopes. Exemplary bispecific antibodies bind to two different epitopes of the antigen of interest. Bispecific antibodies can be produced as full-length antibodies or antibody fragments (eg, bispecific antibodies and F(ab') ₂ diabodies), as described, for example, in Milstein et al., Nature 305:537-539 (1983), WO 93/ 08829, Traunecker et al., EMBO J., 10:3655-3659 (1991), WO 94/04690, Suresh et al. (1986) Methods in Enzymology 121:210, WO96/27011, Brennan et al. (1985) Science 229: 81, Shalaby et al. (1992) J. Exp. Med. 175: 217-225, Kostelny et al. (1992) J. Immunol. 148(5):15471553, Hollinger et al. (1993) PNAS, 90:6444-6448, Gruber et al. (1994) J. Immunol. 152:5368, Wu et al. (2007) Nat. Biotechnol. 25(11): 1290-1297, in US Patent Publication No. 2007/0071675 and Bostrom et al., Science 323:1640-1644 (2009).

IV. Formulations and pharmaceutical compositions.

The superantigen-immunostimulant conjugate can be administered to a patient together, sequentially or periodically, to treat a malignant tumor, for example, slow the growth rate of malignant cells, reduce the incidence or number of metastases, reduce tumor size, inhibit tumor growth, reduce blood supply to the tumor or malignant cells, promote immune response against malignant cells or a tumor, preventing or inhibiting the progression of a malignant tumor, for example, by at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100%. Alternatively, the superantigen conjugate and the immunostimulant may be administered to a patient together, sequentially or intermittently, to treat the cancer, e.g., to prolong the lifespan of the cancer patient, e.g., 3 months, 6 months, 9 months, 12 months, 1 year, 5 years or 10 years.

The superantigen and immunostimulant conjugate can be formulated separately or with each other using methods known to those skilled in the art. For example, for therapeutic use, the superantigen conjugate and/or immunostimulant is combined with a pharmaceutically acceptable carrier. As used herein, a pharmaceutically acceptable carrier means buffers, carriers and excipients suitable for use in contact with tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications in accordance with a reasonable benefit/risk ratio. The carrier(s) must be acceptable in the sense that they are compatible with the other ingredients of the formulation and are not harmful to the recipient. Pharmaceutically acceptable carriers include buffers, solvents, dispersing media, coatings, isotonic and absorption delaying agents, and the like, which are compatible with pharmaceutical administration. The use of such media and means for pharmaceutically active substances is known in the art.

Pharmaceutical compositions containing the superantigen and/or immunostimulant described herein may be provided in a single dosage form or in different dosage forms. The pharmaceutical composition or compositions must be formulated so that they are compatible with the intended route of administration. Examples of routes of administration include intravenous (IV), intramuscular, intradermal, inhalation, transdermal, topical, transmucosal and rectal administration. Alternatively, agents may be administered locally rather than systemically, for example by injecting the agent or agents directly into the site of action, often in a depot or sustained release formulation.

Suitable compositions can be prepared by methods well known in the pharmaceutical field. For example, see Remington's Pharmaceutical Sciences, ^18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline, fatty oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and tonicizers such as sodium chloride or dextrose.

For intravenous administration, suitable vehicles include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier must be stable under the conditions of production and storage, and must be protected from the action of microorganisms. The carrier may be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerin, propylene glycol and liquid

- 19 045360 polyethylene glycol) and suitable mixtures thereof.

The pharmaceutical compositions are preferably sterile. Sterilization can be carried out, for example, by filtration through sterile filtration membranes. In the case where the composition is lyophilized, filter sterilization can be carried out before or after lyophilization and reconstitution.

The combined superantigen-immunostimulant conjugate of the present invention can be used alone or in combination with other compounds, such as carriers or other therapeutic compounds. The pharmaceutical compositions of the present invention contain an effective amount of one or more superantigen conjugates and one or more immunostimulants, for example, anti-PD-1 antibodies, and may also contain additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases pharmaceutically or pharmacologically acceptable refer to substances, eg compositions, that do not cause an adverse, allergic or other undesirable reaction when administered to a mammal, such as, for example, a human. The preparation of a pharmaceutical composition that contains at least one superantigen-immunostimulant conjugate is known to those skilled in the art in light of the present disclosure and as illustrated in Remington's Pharmaceutical Sciences, ^18th Ed. Mack Printing Company, 1990, incorporated herein by reference. In addition, for human administration, it should be understood that the drugs must meet the standards of sterility, pyrogenicity, general safety and purity as required by the FDA Office of Biological Standards.

In a specific embodiment, the compositions of the invention contain a tumor-targeting superantigen in combination with an immunostimulant. Such combinations include, for example, any tumor-targeting superantigen and/or immunostimulant as described herein.

In a specific embodiment, the tumor-targeting superantigen comprises a bacterial superantigen, including, but not limited to, staphylococcal enterotoxin (SE), streptococcal pyrogenic exotoxin (SPE), Staphylococcus aureus toxic shock syndrome toxin (TSST-1), streptococcal mitogenic exotoxin ( SME), streptococcal superantigen (SSA), staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB) and staphylococcal enterotoxin E (SEE) conjugated to a targeting moiety. In another embodiment of the invention, the compositions contain tumor-targeting superantigens containing superantigens with the following Protein Data Bank and/or GenBank accession numbers include, but are not limited to, SEE is P12993; SEA is P013163; SEB is P01552; SEC1 is P01553; SED is P20723; and SEH is AAA19777, as well as variants thereof conjugated to a targeting moiety.

In some embodiments, the superantigen conjugate comprises a wild-type superantigen sequence or an engineered superantigen sequence, such as a wild-type SEE sequence (SEQ ID NO: 1) or a wild-type SEA sequence (SEQ ID NO: 2), either of which may be so modified so that the amino acids in any of the identified regions A-E (see, Fig. 2) are replaced with other amino acids. In some embodiments, the superantigen incorporated into the conjugate is SEA/E-120 (SEQ ID NO: 3) or SEA _d227A (SEQ ID NO: 4).

Competitive examples of targeting moieties that should be conjugated to superantigens include, for example, any molecule that is capable of binding to a cellular molecule and preferably a disease-specific molecule, such as a specific cancer cell molecule. The targeting moiety can be selected from antibodies, including antigen binding moieties, soluble T cell receptors, growth factors, interleukins, hormones, etc. Exemplary cancer-targeting glanders antibodies include, but are not limited to, anti-CD19 antibodies, anti-CD20 antibodies, anti-5T4 antibodies, anti-Ep-CAM antibodies, anti-Her-2/neu antibodies, anti-EGFR antibodies, anti-CEA antibodies, to prostate specific membrane antigen (PSMA) and antibodies to IGF-1R. In one embodiment, the superantigen can be conjugated to an immunologically reactive antibody fragment, such as C215Fab, 5T4Fab (see, WO8907947) or C242Fab (see, WO9301303).

Examples of such tumor-targeted superantigens include C215Fab-SEA (SEQ ID NO: 5), 5T4Fab-SEA _D227A (SEQ ID NO: 6) and 5T4Fab-SEA/E-120 (SEQ ID NO: 7). In a preferred embodiment, the superantigen conjugate is a 5T4 Fab-SEA/E-120, known in the art as naptumomab estaphenatox/ANYARA®, which contains two polypeptide sequences that together define a Fab fragment of an anti-5T4 antibody, wherein one of the polypeptide sequences further comprises superantigen SEA/E-120, namely SEQ ID NO: 8 (V _H Fab 5T4 chimeric chain linked by a three amino acid long linker to SEA/E-120) and SEQ ID NO: 9 (V _L Fab 5T4 chimeric chain).

In a preferred embodiment, the compositions of the invention contain the tumor-targeting superantigen 5T4Fab-SEA/E-120, known in the art as naptumomab

- 20 045360 estaphenatox/ANYARA®, in combination with a PD-1 inhibitor such as an anti-PD-1 antibody, e.g. nivolumab (Bristol-Myers Squibb Co.), peibrolizumab (KEYTRUDA®, Merck & Co.), MK-3475 (Merck & Co), pidlizumab (CureTech), AMP-224 (AstraZeneca/Medimmune) and AMP-514 (AstraZeneca/Medimmune), or an anti-PD-L1 antibody such as MPDL3280A (Genentech/Roche), MEDI-4736 (AstraZeneca /Medimmune) and MSB0010718C (EMD Serono/Merck KGA).

In a specific embodiment, the compositions contain the superantigen-targeting naptumomab estaphenatox (ANYARA®) in combination with one or more anti-PD-1 antibodies, including nivolumab (Bristol-Myers Squibb Co.), peibrolizumab (KEYTRUDA®, Merck & Co.), atezolizumab (formerly known as MPDL3280A), MEDI4736, avelumab and PDR001.

The compositions or dosage form containing the superantigen-immunostimulant conjugate may contain different types of carriers depending on whether they are to be administered in solid, liquid or aerosol form, and whether they need to be sterilized for such injection routes.

Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like. or combinations thereof. The composition may also contain various antioxidants to prevent oxidation of one or more components. In addition, the action of microorganisms can be prevented by the addition of preservatives such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

In some embodiments, the pharmaceutical compositions may contain, for example, at least about 0.1% active compound. In other embodiments, the active compound may be contained from about 2% to about 75% by weight units, or from about 25% to about 60%, for example, and any range therefrom. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, shelf life, as well as other pharmacological factors, and as different dosages and treatment regimens may be necessary. Such definitions are known and used by those skilled in the art.

The active agents are administered in an amount or amounts effective to reduce, reduce, inhibit, or otherwise suppress the growth or proliferation of malignant cells, induce apoptosis, inhibit angiogenesis of a malignant tumor or tumor, inhibit metastasis, or induce cytotoxicity in cells. The effective amount of active compound(s) used to practice the present invention for the therapeutic treatment of cancer varies depending on the route of administration, age, body weight and general health of the patient. These terms include synergistic conditions, such as those presented and described in the present invention, where one agent alone, such as a superantigen conjugate or an immunostimulant such as an anti-PD-1 antibody, may have little or no effect, but when combined together with each other, for example, but not limited to, through sequential dosing, two or more agents act to produce a synergistic effect.

In certain non-limiting examples, the dose of the superantigen conjugate may also contain from about 1 microgram/kg/body weight, about 5 micrograms/kg/body weight, about 10 micrograms/kg/body weight, about 15 micrograms/kg/body weight, about 20 micrograms /kg/body weight, approximately 50 micrograms/kg/body weight, approximately 100 micrograms/kg/body weight, approximately 200 micrograms/kg/body weight, approximately 350 micrograms/kg/body weight, approximately 500 micrograms/kg/body weight , approximately 1 milligram/kg/body weight, approximately 5 milligrams/kg/body weight, approximately 10 milligrams/kg/body weight, approximately 50 milligrams/kg/body weight, approximately 100 milligrams/kg/body weight, approximately 200 milligrams/ kg/body weight, approximately 350 milligrams/kg/body weight, approximately 500 milligrams/kg/body weight, up to approximately 1000 mg/kg/body weight or more per administration, and any range resulting from the values given. In non-limiting examples of the range derived from the numbers listed herein, the range is from about 5 mg/kg/body weight to about 100 mg/kg/body weight, from about 5 micrograms/kg/body weight to about 500 milligrams/kg/body weight , from approximately 1 microgram/kg/body weight to approximately 100 milligrams/kg/body weight. Other exemplary dosage ranges may be administered ranging from about 1 microgram/kg/body weight to about 1000 microgram/kg/body weight, from about 1 microgram/kg/body weight to about 100 microgram/kg/body weight, from about 1 microgram/ kg/body weight to approximately 75 microgram/kg/body weight, approximately 1 microgram/kg/body weight to approximately 50 microgram/kg/body weight, approximately 1 microgram/kg/body weight to approximately 40 microgram/kg/body weight body, from approximately 1 microgram/kg/body weight to approximately 30 microgram/kg/body weight, from approximately 1 microgram/kg/body weight to approximately 20 micro

- 21 045360 grams/kg/body weight, from approximately 1 microgram/kg/body weight to approximately 15 micrograms/kg/body weight, from approximately 1 microgram/kg/body weight to approximately 10 micrograms/kg/body weight, from approximately 5 micrograms/kg/body weight to approximately 1000 micrograms/kg/body weight, from approximately 5 micrograms/kg/body weight to approximately 100 micrograms/kg/body weight, from approximately 5 micrograms/kg/body weight to approximately 75 micrograms/ kg/body weight, from about 5 micrograms/kg/body weight to about 50 micrograms/kg/body weight, from about 5 micrograms/kg/body weight to about 40 micrograms/kg/body weight, from about 5 micrograms/kg/ body weight to about 30 micrograms/kg/body weight, from about 5 micrograms/kg/body weight to about 20 micrograms/kg/body weight, from about 5 micrograms/kg/body weight to about 15 micrograms/kg/body weight, from about 5 micrograms/kg/body weight to about 10 micrograms/kg/body weight, from about 10 micrograms/kg/body weight to about 1000 micrograms/kg/body weight, from about 10 micrograms/kg/body weight to about 100 micrograms/kg/body weight, from about 10 micrograms/kg/body weight to about 75 micrograms/kg/body weight, from about 10 micrograms/kg/body weight to about 50 micrograms/kg/body weight, from about 10 micrograms/ kg/body weight to approximately 40 micrograms/kg/body weight, from approximately 10 micrograms/kg/body weight to approximately 30 micrograms/kg/body weight, from approximately 10 micrograms/kg/body weight to approximately 20 micrograms/kg/body weight body, from approximately 10 micrograms/kg/body weight to approximately 15 micrograms/kg/body weight, from approximately 15 micrograms/kg/body weight to approximately 1000 micrograms/kg/body weight, from approximately 15 micrograms/kg/body weight approximately up to 100 micrograms/kg/body weight, from approximately 15 micrograms/kg/body weight to approximately 75 micrograms/kg/body weight, from approximately 15 micrograms/kg/body weight to approximately 50 micrograms/kg/body weight, from approximately 15 microgram/kg/body weight to approximately 40 microgram/kg/body weight, approximately 15 microgram/kg/body weight to approximately 30 microgram/kg/body weight, approximately 15 microgram/kg/body weight to approximately 20 microgram/kg /body weight, from approximately 20 micrograms/kg/body weight to approximately 1000 micrograms/kg/body weight, from approximately 20 micrograms/kg/body weight to approximately 100 micrograms/kg/body weight, from approximately 20 micrograms/kg/weight body weight to approximately 75 micrograms/kg/body weight, approximately 20 micrograms/kg/body weight to approximately 50 micrograms/kg/body weight, approximately 20 micrograms/kg/body weight to approximately 40 micrograms/kg/body weight, approximately from 20 micrograms/kg/body weight to approximately 30 micrograms/kg/body weight, etc., based on the numbers described above.

In some embodiments, for example, but not limited to, administration of a superantigen conjugate, the effective amount or dose of superantigen conjugate that is administered is an amount in the range of 0.01 to 500 μg/kg of patient body weight, for example, 0.1 -500 µg/kg of the patient's body weight and, for example, 1-100 µg/kg of the patient's body weight.

It is believed that the effective amount or dose of the immunostimulant administered in combination with the superantigen conjugate is a dose that results in at least additive, but preferably synergistic, antitumor effects and does not interfere with or inhibit immune enhancement or T cell activation. If the immunostimulant is administered simultaneously with the superantigen conjugate, the immunostimulant can be administered at a low dose so that it does not interfere with the mechanism of action of the superantigen conjugate.

Typically, a therapeutically effective amount of an immunostimulant, such as an anti-PD1 antibody, is in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 1 mg/kg to 10 mg/kg.

For example, peibrolizumab (KEYTRUDA®) can be administered intermittently at a dose of 2 mg/kg as an intravenous infusion. The amount of immunostimulant administered depends on variables such as the type and severity of the disease or indication being treated, the general health of the patient, the in vivo activity of the superantigen-immunostimulant conjugate, the pharmaceutical formulation, and the route of administration. V. Treatment regimens and indications

Treatment regimens can also vary and generally depend on the type of tumor, location of the tumor, disease progression, and the patient's health and age. Certain tumor types may require more aggressive treatment protocols, but at the same time, patients may be unable to tolerate more aggressive treatment regimens. Often, the clinician may be best suited to make such decisions based on his or her experience in the field and the known efficacy and toxicity (if any) of the therapeutic formulations.

In a specific embodiment, the treatment methods of the invention comprise administering a tumor-targeted superantigen in combination with an immunostimulant to a patient in need thereof, for example a patient with a malignant tumor. Such combination treatments include, for example, the administration of any tumor-targeted superantigen and/or immunostimulant as described herein. In a specific embodiment of the invention, the tumor-targeting superantigen comprises a bacterial superantigen, including, but not limited to

- 22 045360 by reading them, staphylococcal enterotoxin (SE), streptococcal pyrogenic exotoxin (SPE), Staphylococcus aureus toxic shock syndrome toxin (TSST-1), streptococcal mitogenic exotoxin (SME), streptococcal superantigen (SSA), staphylococcal enterotoxin A (SEA) , staphylococcal enterotoxin B (SEB) and staphylococcal enterotoxin E (SEE) conjugated to a targeting moiety.

In some embodiments, the superantigen conjugate comprises a wild-type superantigen sequence or an engineered superantigen sequence, such as a wild-type SEE sequence (SEQ ID NO: 1) or a wild-type SEA sequence (SEQ ID NO: 2), either of which may be so modified that amino acids in any of the defined regions A-E (see, Fig. 2) are replaced by other amino acids. In some embodiments, the superantigen incorporated into the conjugate is SEA/E-120 (SEQ ID NO: 3) or SEA _d227A (SEQ ID NO: 4).

Specific examples of targeting moieties that are conjugated to superantigens include, for example, any molecule that is capable of binding to a cellular molecule and is preferably a disease-specific molecule, such as a cancer cell-specific molecule. The targeting moiety can be selected from antibodies, including antigen binding moieties, soluble T cell receptors, growth factors, interleukins, hormones, etc. Exemplary cancer-directed antibodies may include, but are not limited to, anti-CD19 antibodies, anti-CD20 antibodies, anti-5T4 antibodies, anti-Ep-CAM antibodies, anti-Her-2/neu antibodies, anti-EGFR antibodies, anti-CEA antibodies, anti- prostate specific membrane antigen (PSMA) and antibodies to IGF-1R. In one embodiment, the superantigen can be conjugated to an immunologically reactive antibody fragment, such as C215Fab, 5T4Fab (see, WO8907947) or C242Fab (see, WO9301303).

Examples of such tumor-targeted superantigens include C215Fab-SEA (SEQ ID NO: 5), 5T4Fab-SEA _D227A (SEQ ID NO: 6) and 5T4Fab-SEA/E-120 (SEQ ID NO: 7). In a preferred embodiment, the superantigen conjugate is a 5T4 Fab-SEA/E-120, known in the art as naptumomab estaphenatox/ANYARA®, which contains two polypeptide sequences that together define a Fab fragment of an anti-5T4 antibody, wherein one of the polypeptide sequences further comprises superantigen SEA/E-120, namely SEQ ID. NO: 8 (V _H Fab 5T4 chimeric chain linked by a three amino acid long linker to SEA/E-120) and SEQ ID. NO: 9 (chimeric chain V _L Fab 5T4).

In a preferred embodiment, the compositions of the invention contain the tumor-targeting superantigen 5T4Fab-SEA/E-120, known in the art as naptumomab estafenatox/ANYARA® in combination with a PD-1 inhibitor, such as an anti-PD-1 antibody, for example, nivolumab ( Bristol-Myers Squibb Co.), peibrolizumab (KEYTRUDA®, Merck & Co.), MK-3475 (Merck & Co.), pidilizumab (CureTech), AMP-224 (AstraZeneca/Medimmune) and AMP-514 (AstraZeneca/Medimmune) or anti-PD-L1 antibody such as MPDL3280A (Genentech/Roche), MEDI-4736 (AstraZeneca/Medimmune), and MSB0010718C (EMD Serono/Merck KGA).

In a specific embodiment, the compositions contain the superantigen-targeting naptumomab estaphenatox (ANYARA®) in combination with one or more anti-PD-1 antibodies, including nivolumab (Bristol-Myers Squibb Co.), peibrolizumab (KEYTRUDA®, Merck & Co.), atezolizumab (formerly known as MPDL3280A), MEDI4736, avelumab and PDR001.

In addition, the superantigen conjugate and the immunostimulant can be administered with each other or sequentially together with one or more additional agents that enhance the activity and/or selectivity of the therapeutic effect. Such agents include, for example, corticosteroids, additional immunomodulators, and such compounds designed to reduce the patient's potential immunoreactivity to the administered superantigen conjugate. For example, immunoreactivity to an administered superantigen can be reduced by co-administration, for example, with an anti-CD20 antibody and/or an anti-CD19 antibody, which reduces the formation of superantigen antibodies in the patient.

Preferably, patients to be treated have adequate bone marrow function (defined as absolute peripheral blood granulocyte count >2000/mm ³ and platelet count 100,000/mm ³ ), adequate liver function (bilirubin <1.5 mg/dL) and adequate liver function. liver (creatinine <1.5 mg/dl).

In some embodiments, the treatment regimen of the present invention may include contacting neoplasia or tumor cells with a superantigen conjugate and an immunostimulant simultaneously. This can be obtained by contacting the cell with one composition or pharmacological composition that contains both agents, or by contacting the cell with two separate compositions or compositions simultaneously, where one composition contains a superantigen conjugate and the other contains an immunostimulant.

Alternatively, the superantigen conjugate may precede or follow the immunity

- 23 045360 simulator with intervals ranging from minutes, days to weeks. In embodiments where the other immunostimulant and the superantigen conjugate are administered separately to a cell, it should be ensured that no significant time lapses between the times of each delivery so that the superantigen conjugate and the immunostimulant are still able to exert a predominantly combined effect on the cell. In such cases, it is envisaged that the cell can be brought into contact with both treatments approximately 12-72 hours after each other. In some situations, a significant increase in the period of time for treatment may be desirable, however, when between several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6) between appropriate administrations , 7 or 8).

Various combinations can be used where the superantigen conjugate is A and the immunostimulant is B:

A/B/A, B/A/B, B/B/A, A/A/B, A/B/B, B/A/A, A/B/B/B, B/A/B/ IN,

B/B/B/A, B/B/A/B, A/A/B/B, A/B/A/B, A/B/B/A, B/B/A/A, B/ A/B/A,

B/A/A/B, A/A/A/B, B/A/A/A, A/B/A/A and A/A/B/A.

It is further contemplated that the present invention may be used in conjunction with surgery. In the case of surgery, the present invention can be used preoperatively, for example, to remove an inoperable tumor to be resected. Alternatively, the present invention can be used during and/or after surgery to treat residual or metastatic disease. For example, the tumor bed after resection can be injected or perfused with a composition containing a tumor-targeted superantigen and/or an immunostimulant. Perfusion may continue after resection, for example by leaving a catheter implanted at the surgical site. Periodic postoperative treatment is also suggested. Any combination of therapy according to the invention with surgery is included within the scope of the invention.

If necessary, continuous administration can also be performed, for example, when the tumor is excised and the tumor bed is debrideed to remove residual microscopically visible disease. Delivery by syringe or catheterization is preferred. Such continuous perfusion can be carried out for a period of about 1-2 hours, about 2-6 hours, about 6-12 hours, about 12-24 hours, about 1-2 days, about 1-2 weeks or longer after the start of treatment. Generally, the dose of the therapeutic composition during continuous perfusion will be equivalent to the dose obtained through single or multiple injections, adjusted for the period of time during which the perfusion is performed. It is further contemplated that limb perfusion may be used to administer the therapeutic compositions of the present invention, particularly in the treatment of melanoma and sarcoma.

A typical course of treatment for the primary tumor or tumor bed after resection may involve multiple dosing. A typical treatment of the primary tumor may include 6 doses over a period of two weeks. The two-week treatment regimen can be repeated one, two, three, four, five, six or more times. During the course of treatment, the need to complete planned doses may be reconsidered.

Superantigen conjugate immunotherapy often results in rapid (within hours) and potent polyclonal activation of T lymphocytes. A superantigen conjugate treatment cycle may include 4 to 5 intravenous injections of superantigen conjugate per day. Such treatment cycles can be carried out at intervals of, for example, 4 to 6 weeks. Inflammation with CTL infiltration into the tumor is one of the main effectors of antitumor therapeutic superantigens. After a short period of massive CTL activation and differentiation, the T cell response rapidly declines (within 4-5 days) to baseline levels. Thus, the period of lymphocyte proliferation during which cytotoxic drugs may interfere with superantigen treatment is short and well defined. Only with the superantigen/immunostimulant therapy of the present invention is such a defined period of time for activity reliable, thus providing a new complex treatment based on a high dose cytotoxic agent/immunotherapy.

It is contemplated that a number of malignant tumors may be treated with the methods and compositions described herein, including, but not limited to, primary or metastatic melanoma, adenocarcinoma, squamous cell carcinoma, squamous adenocarcinoma, thioma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, multiple myeloma, neuroblastoma, NPC, bladder cancer, cervical cancer, etc.

In addition, the cancer that can be treated by the methods and compositions described herein, depending on the location in the body and/or the system being treated, can be, but is not limited to, bone cancer (for example, tumors of the family

- 24 045360

Ewing, osteosarcoma); brain cancer (eg, adult brain cancer, (eg, adult brain cancer, brainstem glioma (childhood), cerebral astrocytoma (childhood), cerebral astrocytoma/malignant glioma (childhood), ependymoma (childhood) , medulloblastoma (childhood), supratentorial primitive neuroectodermal tumors and pineoblastoma (childhood), optic nerve and hypothalamic glioma (childhood) and childhood brain cancer (other)); breast cancer (eg, breast cancer in women or in men); digestive system/gastrointestinal cancer (eg, anal cancer, bile duct cancer (extrahepatic), carcinoid tumor (gastrointestinal), colon cancer, esophageal cancer, gallbladder cancer, liver cancer (adult primary) , liver cancer (childhood), pancreatic cancer, small intestinal cancer, stomach cancer (gastric)); endocrine malignancies (eg, adrenocortical carcinoma, carcinoid tumor (gastrointestinal), islet cell carcinoma (pancreatic endocrine), paracancer thyroid gland, pheochromocytoma, pituitary tumor, thyroid cancer); eye cancer (eg, melanoma (intraocular), retinoblastoma); urogenital cancer (eg, bladder cancer, kidney cancer (renal cell), penile cancer, prostate cancer, renal pelvis and ureteral cancer (transitional cell), testicular cancer, urethral cancer, Wilms tumor and other childhood kidney tumors); germinal cell carcinoma (eg, extracranial germ cell tumor (childhood), extragonadal germ cell tumor, ovarian germ cell tumor, testicular cancer); gynecological cancer (eg, cervical cancer, endometrial cancer, gestational trophoblastic tumor, epithelial ovarian cancer, ovarian germ cell tumor, low-grade ovarian tumor, uterine sarcoma, vaginal cancer, cancer of the female external genitalia); head and neck cancer (eg, hypopharyngeal cancer, laryngeal cancer, lip and oral cavity cancer, metastatic squamous cell neck cancer of unknown origin, nasopharyngeal cancer, oropharyngeal cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, salivary gland cancer); lung cancer (eg, non-small cell lung cancer, small cell lung cancer); lymphoma (eg, AIDS-associated lymphoma, T-cell lymphoma, Hodgkin's lymphoma (adult), Hodgkin's lymphoma (childhood), Hodgkin's lymphoma during pregnancy, mycosis fungoides, non-Hodgkin's lymphoma (adult), non-Hodgkin's lymphoma (childhood) , non-Hodgkin's lymphoma during pregnancy, primary lymphoma of the central nervous system, Sézary syndrome, T-cell lymphoma (skin), Waldenström's macroglobulinemia); musculoskeletal (eg, Ewing family tumors, osteosarcoma/malignant fibrous hytiocytoma of bone, rhabdomyosarcoma (childhood), soft tissue sarcoma (adult), soft tissue sarcoma (childhood), uterine sarcoma); neurological (eg, adult brain tumor, childhood brain tumor (eg, brainstem glioma, cerebral astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors and pineoblastoma, optic nerve and hypothalamic region glioma, other brain tumors), neuroblastoma, primary pituitary lymphoma of the central nervous system); respiratory/thoracic (eg, non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, thyoma, and thymic carcinoma) and skin (eg, cutaneous T-cell lymphoma, Kaposi's sarcoma, melanoma, and skin cancer).

It should be understood that the method can be used to treat a variety of cancers, for example, a cancer selected from the group consisting of breast cancer, cervical cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, cancer prostate, renal cell carcinoma and skin cancer.

Additionally, a malignant tumor may include a tumor consisting of tumor cells. For example, tumor cells may include, but are not limited to, a melanoma cell, a bladder cancer cell, a breast cancer cell, a lung cancer cell, a colon cancer cell, a prostate cancer cell, a liver cancer cell, a pancreatic cancer cell, a cancer cell stomach cancer cell, testicular cancer cell, kidney cancer cell, ovarian cancer cell, lymphatic cancer cell, skin cancer cell, brain cancer cell, bone cancer cell or soft tissue cancer cell. Examples of solid tumors that can be treated according to the invention include, but are not limited to, sarcomas and carcinomas: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medullob

- 25 045360 lastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.

VI. Sets.

The invention further provides kits containing, for example, a first container containing a superantigen conjugate and a second container containing an immunostimulant, such as an anti-PD-1 antibody. Such a kit may also contain additional agents, such as, for example, a corticosteroid or other lipid modulator. The container means itself may be a syringe, pipette and/or other similar device from which the composition can be applied to specific areas of the body, injected into an animal and/or applied and/or mixed with other components of the kit.

The kits may contain a suitably aliquoted superantigen conjugate and/or immunostimulant and optionally a lipid and/or additional agent of the composition of the present invention. Kit components can be packaged in aqueous media or in lyophilized form. When the kit components are provided in one and/or more liquid solutions, the liquid solution is a sterile aqueous solution.

The practice of the invention will be more fully understood from the above examples, which are given herein for illustrative purposes only and should not be construed as limiting the invention in any way.

Examples

Example 1: Combination Treatment with ANYARA® and an Anti-PD-1 Antibody Inhibitor Against the N SCLC Tumor Cell Line

This example describes an in vitro study testing the anti-cancer effect of a combination of a tumor-targeting superantigen, ANYARA®, and an anti-PD-1 antibody, KEYTRUDA®, against the non-small cell lung cancer (NSCLC) tumor cell line HCC827.

Peripheral blood mononuclear cells (PBMCs) from healthy donors were incubated for 4 days with 10 ng/ml staphylococcal enterotoxin A (SEA). T cells were then isolated and incubated with IL-2 for another 1 day. 1 x 10 ⁴ HCC827 cells per well were incubated in 9b-well plates for 1 hour with T cells and KEYTRUDA® at a concentration of 0.2 mg/ml. The effector:target ratio (T cells:HCC827 cells) was 8:1. After 1 hour of incubation with or without KEYTRUDA®, ANYARA® was added to the wells at various concentrations (0, 0.1 mg/ml, and 10 mg/ml) and the plates were incubated for an additional 48 hours. At the end of treatment, the culture supernatant was removed, including suspended T cells and tumor cells, and washed the attached tumor cells once with culture medium. The viability of the remaining HCC827 was tested using the CC8 kit (Cell Counting Kit-8, Sigma Aldrich) according to the manufacturer's protocol. The viability of the control group was normalized to 100%. Malignant cell viability (%)=(OD value of treatment group/OD value of control group)x100.

As shown in FIG. 4 and fig. 5, the combination of ANYARA® with KEYTRUDA® had the greatest effect on the viability of HCC827 cells. Although ANYARA® alone at both concentrations reduced the viability of HCC827 cells, it was much less effective compared to the combination of ANYARA® with KEYTRUDA®. KEYTRUDA® at the tested concentration of 0.2 mg/ml had no effect on the viability of malignant cells. ANYARA® at a lower concentration of 0.1 mg/ml reduced the viability of HCC827 cells to 60±9.4% (p<0.05 compared to control), while the combination of ANYARA® with KEYTRUDA® reduced cell viability to 33±4 .9% (p<0.005 compared to control; p<0.05 compared to ANYARA®) (Fig. 4). ANYARA® at a higher concentration (10 mg/ml) had a more pronounced effect than at a lower concentration, reducing the viability of malignant cells to 40±6.6% (p<0.005 compared to control), but also at this concentration the combination ANYARA® with KEYTRUDA® was significantly more effective and reduced cell viability to 14±4.2% (p<0.0005 vs. control; p=0.005 vs. ANYARA®) (Figure 5).

Taken together, these results demonstrate the synergistic effect of the immunostimulant anti-PD-1 antibody (KEYTRUDA®) with a tumor-targeted superantigen (ANYARA®) and support the concept that administration of a tumor-targeted superantigen together with an immunostimulant can result in enhanced anti-tumor activity that is greater than the additive effect of each agent when administered separately.

Example 2: Combination treatment with a tumor-targeting superantigen and a mouse anti-PD-1 antibody inhibitor in a mouse model of B16 Melanoma.

This example describes a study testing the effect of the tumor-targeting superantigen C215Fab-SEA and the mouse anti-PD-1 antibody in the B16-EpCAM mouse melanoma model in vivo. Combination treatment of a tumor-targeting superantigen and an anti-PD-1 antibody was tested in a syngeneic tumor model using low-immunogenic B16 melanoma cells transfected with the human colon carcinoma antigen EpCAM, which is recognized by the anti-C215 antibody. On

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Claims (14)

The tumor-targeted superantigen C215Fab-SEA is a fusion protein that contains a tumor-reactive mAb (C215Fab) and the bacterial superantigen staphylococcal enterotoxin A (SEA). C215Fab-SEA was used instead of ANYARA® to facilitate in vivo mouse experiments. For the study, C57B1/6 mice were inoculated intravenously (iv) with 1.75 x 105 B16-EpCAM melanoma cells into the tail vein to induce tumors in the lungs. Mice were treated on days 5–8 with daily IV injection of C215Fab-SEA (0.5 mg/mouse) and/or intraperitoneal (IP) injections of anti-PD-1 mAb (200 mg/mouse) twice weekly. The control group was treated with PBS using the same route of administration and the same protocol as the combination treatment group. On day 21, the mice were sacrificed and the lungs were removed. After fixation in Bouin's solution for at least 24 hours, the number of tumors in the lungs was counted. The results of the study are shown in Fig. 6. As expected, due to the fact that B16 melanoma is a low-immunogenic tumor, PD-1 mAb monotherapy had no effect on the number of tumors in the lungs. C215Fab-SEA alone reduced the number of B16 tumors in the lungs by approximately 50%, from 217.1 ± 15.9 (mean ± SEM) tumors in control-treated mice to 98.8 ± 16.4 on day 21. C215Fab- SEA and anti-PD-1 mAb in combination reduced tumor burden by approximately 80%, a significant reduction (p<0.05) compared with monotherapy. These results further demonstrate the feasibility of combining a tumor-targeted superantigen with an immunostimulant to treat poorly immunogenic tumors. Incorporation by reference. The entire description of each of the patent and scientific documents set forth herein is incorporated by reference for all purposes. Equivalents. The invention can be embodied in other specific forms without going beyond the essence or its main characteristics. Thus, the above embodiments are to be considered in all respects as illustrative and not as limiting of the invention described herein. Thus, the scope of the invention is indicated by the appended claims and not by the above description, and all changes that come within the meaning and range of equivalence of the claims are intended to be included therein. Claim 1. A method of treating a malignant tumor in a patient, including: administering to the patient (i) an effective amount of a superantigen conjugate containing a superantigen containing amino acid residues 226-458 of SEQ ID NO: 7 or an immunologically reactive variant and/or fragment thereof covalently linked to a targeting moiety that binds to the 5T4 cancer antigen expressed by cancer cells from the patient; and (ii) an effective amount of an immunostimulant selected from an anti-PD-1 antibody and an anti-PD-L1 antibody so as to enhance an immune response in a patient against cancer cells to treat the cancer. 2. The method according to claim 1, where the superantigen is administered to the patient before, simultaneously or after the immunostimulant. 3. The method according to claim 1 or 2, where the superantigen binds to the T-cell receptor on the surface of the T cell. 4. The method according to any one of claims 1-3, where the targeting fragment is an antibody to 5T4. 5. The method according to claim 4, wherein the anti-5T4 antibody contains a Fab fragment that binds to the 5T4 malignant tumor antigen. 6. The method according to any one of claims 1 to 5, wherein the superantigen conjugate contains a first protein chain containing SEQ ID NO: 8, and a second protein chain containing SEQ ID NO: 9. 7. The method according to any one of claims 3 to 6, wherein a programmed cell death ligand (PD-L) is expressed on the surface of the malignant cells, which binds to programmed cell death protein 1 (PD-1) expressed by the T cell. 8. The method of claim 7, wherein the immunostimulant prevents PD-L from binding to PD-1 expressed on the surface of the T cell. 9. The method of claim 1, wherein the anti-PD-1 antibody or anti-PD-L1 antibody is of or is based on a human IgG4 isotype. 10. The method according to claim 1, where the immunostimulant is an antibody to PD-1. 11. The method according to claim 1 or 10, wherein the anti-PD-1 antibody is selected from the group consisting of nivolumab and pembrolizumab. 12. The method according to claim 1, where the immunostimulant is an antibody to PD-L1. 13. The method of claim 1 or 12, wherein the anti-PD-L1 antibody is selected from the group consisting of atezolizumab and MEDI4736. 14. The method according to any one of claims 1 to 13, where the malignant tumor is selected from the group consisting of breast cancer, cervical cancer, colorectal cancer, gastric cancer, non-small cell cancer -