DNA-encoded bispecific t-cell engagers targeting cancer antigens and methods of use in cancer therapeutic agents

Abstract

To provide compositions comprising a recombinant nucleic acid sequence encoding a bispecific immune cell engaging antibody (DICE), a recombinant nucleic acid sequence encoding a bispecific T cell engaging (DBiTE) antibody, a fragment thereof, a variant thereof, or a combination thereof.SOLUTION: The invention provides a nucleic acid molecule encoding one or more synthetic DNA encoded bispecific immune cell engagers, where the more synthetic DNA encoded bispecific immune cell engagers comprise at least one antigen binding domain and at least one immune cell engaging domain.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Application No. 62 / 798,626, filed on January 30, 2019, and U.S. Provisional Application No. 62 / 827,265, filed on April 1, 2019, each of which is hereby incorporated by reference in its entirety.

[0002] The present invention relates to compositions comprising recombinant nucleic acid sequences for generating one or more synthetic DNA - encoded bispecific T - cell engagers (BiTE) and functional fragments thereof in vivo, and methods for preventing and / or treating cancer in a subject by administering such compositions.

Background Art

[0003] Monoclonal antibody therapy has been a game - changer in cancer therapeutics, but this treatment has several limitations, including the need for repeated administration, more limited stability, and cost. Further advances in monoclonal technology have been the development of bispecific T - cell engagers (BiTEs) that combine the specificity of monoclonal antibodies with the cytotoxic potential of T cells. BiTEs have shown promising results in clinical trials for leukemia (Viardot et al., 2016, Blood, 127(11):1410 - 6, Goebeler et al., 2016, J Clin Oncol, 34(10):1104 - 11). However, this therapy requires continuous intravenous infusion for 4 - 8 weeks per cycle (Zhu et al., 2016, Clin Pharmacokinet, 55(10):1271 - 88) and may have limitations in its production, so its applicability is limited. A longer - lived and simpler production method for antibody - based products could be an important new tool for cancer immunotherapy.

[0004] Accordingly, in the art, there is a need for longer-lived, simpler production, antibody-based products for cancer immunotherapy. The present invention meets this need. SUMMARY OF THE INVENTION

[0005] In one embodiment, the present invention relates to a nucleic acid molecule encoding one or more synthetic DNA-encoded bispecific immune cell engagers, wherein the synthetic DNA-encoded bispecific immune cell engager comprises at least one antigen-binding domain and at least one immune cell-engaging domain.

[0006] In one embodiment, the antigen-binding domain targets CD19, B cell maturation antigen (BCMA), CD33, fibroblast activation protein (FAP), follicle-stimulating hormone receptor (FSHR), epidermal growth factor receptor (EGFR), prostate-specific membrane antigen (PSMA), CD123 or Her2.

[0007] In one embodiment, the immune cell-engaging domain targets T cells, antigen-presenting cells, natural killer (NK) cells, neutrophils, or macrophages.

[0008] In one embodiment, the immune cell-engaging domain targets CD3, T cell receptor (TCR), CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5, CD40, FcgR, FceR, FcaR, or CD95. In one embodiment, the immune cell-engaging domain targets CD3.

[0009] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding one or more sequences selected from the following: a) an amino acid sequence having at least about 90% identity over the full length of the amino acid sequence to an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74 or SEQ ID NO: 76; b) an amino acid sequence having at least about 90% identity over at least 65% of the amino acid sequence to an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74 or SEQ ID NO: 76; c) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74 or SEQ ID NO: 76, and d) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,A fragment of an amino acid sequence comprising at least 65% of an amino acid sequence selected from SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74 or SEQ ID NO: 76.

[0010] In one embodiment, the nucleic acid molecule comprises: a) a nucleotide sequence having at least about 90% identity over the full length of the nucleic acid sequence to a nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, and SEQ ID NO: 75; b) a fragment of a nucleotide sequence having at least about 90% identity over at least 65% of the nucleic acid sequence to a nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, and SEQ ID NO: 75; c) the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or d) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37,A fragment of a nucleotide sequence comprising at least 65% of a nucleotide sequence selected from SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, and SEQ ID NO: 75.

[0011] In one embodiment, the nucleotide sequence is operably linked to a nucleic acid sequence encoding an IgE leader sequence.

[0012] In one embodiment, the nucleic acid molecule comprises an expression vector.

[0013] In one embodiment, the invention relates to a composition comprising a nucleic acid molecule encoding one or more synthetic DNA-encoded bispecific immune cell engagers, the synthetic DNA-encoded bispecific immune cell engager comprising at least one antigen-binding domain and at least one immune cell engaging domain. In one embodiment, the composition further comprises a pharmaceutically acceptable excipient.

[0014] In one embodiment, the invention relates to a method of preventing or treating a disease or disorder in a subject, the method comprising administering to the subject a nucleic acid molecule encoding one or more synthetic DNA-encoded bispecific immune cell engagers, or a composition comprising a nucleic acid molecule encoding one or more synthetic DNA-encoded bispecific immune cell engagers, the synthetic DNA-encoded bispecific immune cell engager comprising at least one antigen-binding domain and at least one immune cell engaging domain. In one embodiment, the disease is a benign tumor, cancer, or a cancer-related disease.

[0015] In one embodiment, the present invention relates to a nucleic acid molecule encoding one or more synthetic antibodies, the nucleic acid molecule comprising a nucleotide sequence encoding an anti-human epidermal growth factor receptor 2 (HER2) synthetic antibody, a nucleotide sequence encoding a fragment of the anti-HER2 synthetic antibody, a nucleotide sequence encoding a ScFv anti-HER2 synthetic antibody, or a nucleotide sequence encoding a fragment of the ScFv anti-HER2 synthetic antibody.

[0016] In one embodiment, the nucleotide sequence encodes an amino acid sequence having at least about 90% identity over the full length of the amino acid sequence relative to SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO: 66. In one embodiment, the nucleotide sequence encodes a fragment of an amino acid sequence having at least about 90% identity over at least 65% of the amino acid sequence relative to SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO: 66. In one embodiment, the nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO: 66. In one embodiment, the nucleotide sequence encodes a fragment of an amino acid sequence comprising at least 65% of SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO: 66.

[0017] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence having at least about 90% identity over the full length of SEQ ID NO: 61, SEQ ID NO: 63, or SEQ ID NO: 65. In one embodiment, the nucleic acid molecule comprises a fragment of a nucleotide sequence having at least about 90% identity over at least 65% of the nucleic acid sequence relative to the nucleotide sequence of SEQ ID NO: 61, SEQ ID NO: 63, or SEQ ID NO: 65. In one embodiment, the nucleic acid molecule comprises a selected nucleotide sequence of SEQ ID NO: 61, SEQ ID NO: 63, or SEQ ID NO: 65. In one embodiment, the nucleic acid molecule comprises a fragment of a nucleotide sequence comprising at least 65% of the nucleotide sequence of SEQ ID NO: 61, SEQ ID NO: 63, or SEQ ID NO: 65.

[0018] In one embodiment, the nucleotide sequence is operably linked to a nucleic acid sequence encoding an IgE leader sequence.

[0019] In one embodiment, the nucleic acid molecule comprises an expression vector.

[0020] In one embodiment, the present invention relates to a composition comprising a nucleic acid molecule encoding one or more synthetic antibodies, the nucleic acid molecule comprising a nucleotide sequence encoding an anti-human epidermal growth factor receptor 2 (HER2) synthetic antibody, a nucleotide sequence encoding a fragment of the anti-HER2 synthetic antibody, a nucleotide sequence encoding a ScFv anti-HER2 synthetic antibody, or a nucleotide sequence encoding a fragment of the ScFv anti-HER2 synthetic antibody. In one embodiment, the composition further comprises a pharmaceutically acceptable excipient.

[0021] In one embodiment, the present invention relates to a method of preventing or treating a disease in a subject, the method comprising administering to the subject a nucleic acid molecule encoding one or more synthetic antibodies, the nucleic acid molecule comprising a nucleotide sequence encoding an anti-human epidermal growth factor receptor 2 (HER2) synthetic antibody, a nucleotide sequence encoding a fragment of the anti-HER2 synthetic antibody, a nucleotide sequence encoding a ScFv anti-HER2 synthetic antibody, or a nucleotide sequence encoding a fragment of the ScFv anti-HER2 synthetic antibody.

[0022] In one embodiment, the present invention relates to a method of preventing or treating a disease in a subject, the method comprising administering to the subject a nucleic acid molecule encoding one or more synthetic antibodies, the nucleic acid molecule comprising a nucleotide sequence encoding an anti-human epidermal growth factor receptor 2 (HER2) synthetic antibody, a nucleotide sequence encoding a fragment of the anti-HER2 synthetic antibody, a nucleotide sequence encoding a ScFv anti-HER2 synthetic antibody, or a nucleotide sequence encoding a fragment of the ScFv anti-HER2 synthetic antibody. In one embodiment, the disease is a cancer associated with HER2 expression. In one embodiment, the disease is ovarian cancer or breast cancer.

[0023] In one embodiment, the present invention relates to a method for preventing or treating a disease in a subject, the method comprising administering to the subject a composition comprising a nucleic acid molecule encoding one or more synthetic antibodies, the nucleic acid molecule comprising a nucleotide sequence encoding an anti-human epidermal growth factor receptor 2 (HER2) synthetic antibody, a nucleotide sequence encoding a fragment of the anti-HER2 synthetic antibody, a nucleotide sequence encoding a ScFv anti-HER2 synthetic antibody, or a nucleotide sequence encoding a fragment of the ScFv anti-HER2 synthetic antibody. In one embodiment, the disease is cancer associated with HER2 expression. In one embodiment, the disease is ovarian cancer or breast cancer.

Brief Description of Drawings

[0024]

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Mode for Carrying Out the Invention

[0025] The present invention relates to a composition comprising a recombinant nucleic acid sequence encoding a bispecific immune cell engaging antibody (DICE), a recombinant nucleic acid sequence encoding a bispecific T cell engaging (DBiTE) antibody, fragments thereof, variants thereof, or combinations thereof. The composition can be administered to a subject in need thereof to promote in vivo expression and formation of DICE or DBiTE.

[0026] In one embodiment, DICE or DBiTE comprises at least one antigen-binding domain and at least one immune cell engaging domain. In one embodiment, the immune cell engaging domain is specific for an antigen expressed on the surface of an immune cell. Immune cells include, but are not limited to, T cells, antigen presenting cells, NK cells, neutrophils, and macrophages.

[0027] In various embodiments, the immune cell engaging domain comprises a nucleotide sequence encoding an antibody, fragment thereof, or variant thereof that is specific for binding to an immune cell specific receptor molecule. In one embodiment, the immune cell specific receptor molecule is a T cell surface antigen. In one embodiment, the T cell specific receptor molecule is one of CD3, TCR, CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5, CD40, FcgR, FceR, FcaR, and CD95.

[0028] In various embodiments, the antigen-binding domain comprises a nucleotide sequence encoding an antibody, fragment thereof, or variant thereof that is specific for binding to an antigen. In one embodiment, the antibody or fragment thereof is a DNA-encoded monoclonal antibody (DMAb) or fragment or variant thereof.

[0029] In one embodiment, the antigen-binding domain of DICE or DBiTE is specific for binding to a target antigen and for recruiting T cells to the target antigen. In one embodiment, the target antigen is a tumor antigen. In one embodiment, the antigen is CD19, B cell maturation antigen (BCMA), CD33, fibroblast activation protein (FAP), follicle-stimulating hormone receptor (FSHR), epidermal growth factor receptor (EGFR), prostate-specific membrane antigen (PSMA), CD123, and human epidermal growth factor receptor 2 (Her2). Thus, in one embodiment, the present invention provides a composition comprising one or more DICEs or DBiTEs, and a method for use in treating or preventing cancer or cancer-related diseases or disorders in a subject.

[0030] Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0031] The terms "comprise(s)", "include(s)", "having", "has", "can", "containing", and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising", "consisting of", and "consisting essentially of" the embodiments or elements presented herein, whether or not explicitly described.

[0032] "Antibody" can mean an antibody of class IgG, IgM, IgA, IgD, or IgE, or a fragment, Fab, F(ab’)2, Fd, a fragment or derivative thereof, as well as a single-chain antibody, and derivatives thereof. The antibody can be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity-purified antibody, or a mixture thereof, which exhibits sufficient binding specificity for a desired epitope or a sequence derived therefrom.

[0033] As used interchangeably herein, "antibody fragment" or "fragment of an antibody" refers to a portion of an intact antibody that contains an antigen-binding site or variable region. The portion does not include the constant heavy-chain domain of the Fc region of the intact antibody (i.e., CH2, CH3, or CH4 depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, Fab fragment, Fab’ fragment, Fab’-SH fragment, F(ab’)2 fragment, Fd fragment, Fv fragment, diabody, single-chain Fv (scFv) molecule, a single-chain polypeptide containing only one light-chain variable domain, a single-chain polypeptide containing three CDRs of the light-chain variable domain, a single-chain polypeptide containing only one heavy-chain variable region, and a single-chain polypeptide containing three CDRs of the heavy-chain variable region.

[0034] "Antigen" refers to a protein having the ability to generate an immune response in a host. The antigen can be recognized and bound by an antibody. The antigen can be of endogenous origin or from the external environment.

[0035] As used herein, "coding sequence" or "coding nucleic acid" can mean a nucleic acid (RNA or DNA molecule) that contains a nucleotide sequence encoding an antibody, as shown herein. The coding sequence can further include initiation and termination signals operably linked to control elements including a promoter, as well as a polyadenylation signal capable of inducing expression in a cell of an individual or mammalian to which the nucleic acid is administered. The coding sequence can further include a sequence encoding a signal peptide.

[0036] As used herein, "complementary" or "complementarity" can mean that a nucleic acid can form Watson-Crick (e.g., A-T / U and C-G) or Hoogsteen base pairs between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0037] As used herein, "constant current" defines the current that a tissue, or the cells that define the tissue, receives or experiences over the period during which an electrical pulse is delivered to the same tissue. The electrical pulse is delivered from an electroporation device described herein. The electroporation devices provided herein preferably have a feedback element with immediate feedback, such that this current remains at a constant number of amperes in the tissue over the lifetime of the electrical pulse. The feedback element can measure the resistance of the tissue (or cells) through the duration of the pulse and cause the electroporation device to vary its electrical energy output (e.g., increase the voltage), and thus the current in the same tissue remains constant through the electrical pulse (in the microsecond range) and between pulses. In some embodiments, the feedback element includes a controller.

[0038] As used herein, "current feedback" or "feedback" may be used interchangeably and may mean the active response of a provided electroporation device, which includes measuring the current in the tissue between electrodes and appropriately changing the energy output delivered by the EP device to maintain the current at a constant level. This constant level is preset by the user prior to the start of the pulse sequence or electrical treatment. The electrical circuit therein continuously monitors the current in the tissue between the electrodes, compares the monitored current (or the current in the tissue) with a preset current, and continuously adjusts the energy output to maintain the monitored current at the preset level. Feedback can be achieved by an electroporation component of the electroporation device, such as a controller, so that the current being monitored can be maintained at a preset level. Since the feedback loop is an analog closed-loop feedback, it can be immediate.

[0039] As used herein, "dispersed current" may mean the pattern of currents delivered from various needle electrode arrays of the electroporation devices described herein, and the pattern minimizes or preferably eliminates the generation of electroporation-related heat stress on any area of the tissue to be electroporated.

[0040] As used interchangeably herein, "electroporation", "electropermeabilization", or "electrokinetic enhancement" ("EP") may refer to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes, the presence of which enables biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cell membrane to the other.

[0041] As used herein, "endogenous antibody" may refer to an antibody produced in a subject to whom an effective dose of an antigen has been administered for the induction of a humoral immune response.

[0042] As used herein, "feedback mechanism" may refer to a process executed by either software or hardware (firmware), which process receives the impedance of a desired tissue (before, during, and / or after the delivery of energy pulses), compares it to a current value, preferably current, and adjusts the energy pulses delivered to achieve a preset value. The feedback mechanism may be executed by an analog closed-loop circuit.

[0043] "Fragment" may mean a polypeptide fragment of an antibody that is functional, i.e., capable of binding to a desired target and having the same intended effect as a full-length antibody. An antibody fragment may be 100% identical to the full length, except where at least one amino acid is missing from the N-terminus and / or C-terminus, and in each case, a signal peptide and / or methionine may or may not be present at position 1. The fragment may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the length of a particular full-length antibody, excluding any heterologous signal peptide that may be added. The fragment may comprise a fragment of a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the antibody, and may in addition comprise an N-terminal methionine or heterologous signal peptide that is not included when calculating the percent identity. The fragment may further comprise an N-terminal methionine and / or a signal peptide, such as an IgE or IgG signal peptide, etc. The N-terminal methionine and / or signal peptide may be linked to the antibody fragment.

[0044] A fragment of a nucleic acid sequence encoding an antibody can be 100% identical to the full length, except when it lacks at least one nucleotide from the 5’ end and / or the 3’ end, and in each case, there is or is not a sequence encoding a signal peptide and / or methionine at position 1. The fragment can include 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the length of a specific full-length coding sequence, excluding any heterologous signal peptide added. The fragment can include a fragment encoding a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the antibody, and additionally, optionally, can include a sequence encoding an N-terminal methionine or a heterologous signal peptide that is not included when calculating the percent identity. The fragment can further include a coding sequence for an N-terminal methionine and / or an immunoglobulin signal peptide, such as an IgE or IgG signal peptide. The coding sequence for the N-terminal methionine and / or the signal peptide can be ligated to the fragment of the coding sequence.

[0045] As used herein, the term “gene construct” refers to a DNA or RNA molecule that includes a nucleotide sequence encoding a protein, such as an antibody. The coding sequence includes start and stop signals operably linked to regulatory elements including a promoter and a polyadenylation signal capable of inducing expression in the cells of an individual to whom the nucleic acid molecule is administered. As used herein, the term “expressible form” refers to a gene construct that includes the necessary control elements operably linked to a coding sequence encoding a protein such that the coding sequence is expressed when present in the cells of an individual.

[0046] As used herein in the context of two or more nucleic acid or polypeptide sequences, "identical" or "identity" can mean having a specified percentage of residues that are the same over a specified region of the aligned sequences. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residues occur in both sequences, calculating the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the lengths of the two sequences are different or the alignment generates one or more overhangs and only a single sequence is included in the specified comparison region, the residues of the single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0047] As used herein, "impedance" can be used when discussing feedback mechanisms, can be converted to a current value according to Ohm's law, and thus allows comparison with a preset current.

[0048] As used herein, "immune response" can mean the activation of the host's immune system, e.g., the mammalian immune system, in response to the introduction of one or more nucleic acids and / or peptides. The immune response can be a cellular or humoral response, or both.

[0049] As used herein, "nucleic acid", "oligonucleotide", or "polynucleotide" can mean at least two nucleotides covalently linked together. A single-stranded depiction also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses probes that hybridize under stringent hybridization conditions.

[0050] A nucleic acid can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. A nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, and a nucleic acid can contain combinations of deoxyribo- and ribo-nucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. A nucleic acid can be obtained by chemical synthesis methods or by recombinant methods.

[0051] As used herein, "operably linked" can mean that the expression of a gene is under the control of a promoter that is thereby spatially connected thereto. A promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variations in this distance can be accommodated without loss of promoter function.

[0052] As used herein, "peptide", "protein", or "polypeptide" can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.

[0053] As used herein, "promoter" can mean a synthetic or naturally occurring molecule that can confer, activate, or enhance the expression of a nucleic acid in a cell. A promoter can include one or more specific transcriptional control sequences to further enhance its expression and / or to alter spatial and / or temporal expression. A promoter can also include distal enhancer or repressor elements that can be located several thousand base pairs from the transcription start site. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can control the expression of genetic components constitutively, or differentially in response to a cell, the tissue or organ in which expression occurs, or the developmental stage at which expression occurs, or external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter, and CMV IE promoter.

[0054] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of the proteins described herein. Signal peptide / leader sequence typically induces the localization of a protein. The signal peptide / leader sequence used herein preferably facilitates the secretion of the protein from the cell in which it is produced. The signal peptide / leader sequence is often cleaved from the rest of the protein and is often referred to as the mature protein after secretion from the cell. The signal peptide / leader sequence is linked at the N-terminus of the protein.

[0055] As used herein, "stringent hybridization conditions" can mean the average conditions under which a first nucleic acid sequence (e.g., a probe) will hybridize to a second nucleic acid sequence (e.g., a target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions can be selected to be about 5-10 °C lower than the thermal melting point (T m ) for a particular sequence at a defined ionic strength and pH. T m can be the temperature at which 50% of the probe complementary to the target hybridizes to the target sequence at equilibrium (under defined ionic strength, pH, and nucleic acid concentration) (since the target sequence is present in excess, at T m , 50% of the probe is occupied at equilibrium). Stringent conditions can be those where the salt concentration is less than about 1.0 M sodium ion concentration (or other salts) such as about 0.01-1.0 M sodium ion concentration at pH 7.0-8.3, and the temperature is at least about 30 °C for short probes (e.g., about 10-50 nucleotides) and at least about 60 °C for long probes (e.g., greater than about 50 nucleotides). Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2-10 times that of background hybridization. Exemplary stringent hybridization conditions include: incubation at 42 °C in 50% formamide, 5×SSC, and 1% SDS, or incubation at 65 °C in 5×SSC, 1% SDS, and washing at 65 °C in 0.2×SSC and 0.1% SDS.

[0056] As used herein, the terms "subject" and "patient" interchangeably refer to any vertebrate, including but not limited to mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice, non-human primates (e.g., monkeys such as cynomolgus monkeys or rhesus monkeys, chimpanzees, etc.), and humans). In some embodiments, the subject can be human or non-human. The subject or patient may be receiving other forms of treatment.

[0057] As used herein, "substantially complementary" means that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0058] As used herein, "substantially identical" means that when a first sequence is substantially complementary to the complement of a second sequence, the first and second sequences are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a region of 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, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more nucleotides or amino acids, or, with respect to nucleic acids.

[0059] As used herein, "synthetic antibody" refers to an antibody encoded by a recombinant nucleic acid sequence described herein and produced in a subject.

[0060] As used herein, "treat" or "treatment" can mean the protection of a subject from a disease through means that prevent, inhibit, suppress, or completely eliminate the disease. Preventing a disease includes administering an antibody of the invention to a subject prior to the onset of the disease. Inhibiting a disease includes administering an antibody of the invention to a subject after the induction of the disease but prior to its clinical appearance. Suppressing a disease includes administering an antibody of the invention to a subject after the clinical appearance of the disease.

[0061] As used herein with respect to a nucleic acid, "variant" can mean (i) a portion or fragment of a referenced nucleotide sequence, (ii) the complement of a referenced nucleotide sequence or a portion thereof, (iii) a nucleic acid that is substantially identical to the referenced nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, its complement, or a sequence that is substantially identical thereto.

[0062] A "variant" with respect to a peptide or polypeptide whose amino acid sequence differs by an insertion, deletion, or conservative substitution of an amino acid retains at least one biological activity. A variant can also mean a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitution of an amino acid, i.e., replacement of an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is typically recognized in the art as involving minor changes. These minor changes can be identified, in part, by considering the hydropathy index of the amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathy index of an amino acid is based on its hydrophobicity and charge considerations. It is known in the art that amino acids with similar hydropathy indices can be substituted and still retain protein function. In one embodiment, amino acids having a hydropathy index of ±2 are substituted. The hydrophilicity of an amino acid can also be used to identify substitutions that will result in a protein that retains its biological function. Consideration of the hydrophilicity of an amino acid in the context of a peptide allows calculation of a useful measure that has been reported to correlate well with the greatest local average hydrophilicity, antigenicity, and immunogenicity of that peptide. U.S. Patent No. 4,554,101 is hereby incorporated by reference in its entirety. Substitution of amino acids with similar hydrophilic values can result in a peptide that retains biological activity, e.g., immunogenicity, as understood in the art. The substitution can be carried out with amino acids having hydrophilic values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the particular side chain of that amino acid. Consistent with that observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, and in particular their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0063] A variant can be a nucleic acid sequence that is substantially identical over the full length of the complete gene sequence or a fragment thereof. The nucleic acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof. A variant can be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or a fragment thereof. The amino acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0064] As used herein, "vector" can mean a nucleic acid sequence containing an origin of replication. The vector can be a plasmid, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. The vector can be a DNA or RNA vector. The vector can be either a self-replicating extrachromosomal vector or a vector that integrates into the host genome.

[0065] For the recitation of numerical ranges herein, each number intervening therebetween with the same precision is explicitly contemplated. For example, for the range of 6-9, in addition to 6 and 9, the numbers 7 and 8 are contemplated, and for the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0066] Composition In one embodiment, the present invention relates to a composition comprising a recombinant nucleic acid sequence encoding DICE or DBiTE, fragments thereof, variants thereof, or combinations thereof. The composition can result in the generation of synthetic DNA-encoded bispecific immune cell engagers in a subject when administered to a subject in need thereof.

[0067] In one embodiment, DICE or DBiTE comprises at least one antigen-binding domain and at least one immune cell-engaging domain. In one embodiment, the immune cell-engaging domain is specific for an antigen expressed on the surface of an immune cell. Immune cells include, but are not limited to, T cells, antigen-presenting cells, NK cells, neutrophils, and macrophages.

[0068] In various embodiments, the immune cell-engaging domain comprises a nucleotide sequence encoding an antibody, fragment thereof, or variant thereof that is specific for binding to an immune cell-specific receptor molecule. In one embodiment, the immune cell-specific receptor molecule is a T cell surface antigen. In one embodiment, the T cell-specific receptor molecule is one of CD3, TCR, CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5, CD40, FcgR, FceR, FcaR, and CD95.

[0069] In various embodiments, the antigen-binding domain comprises an antibody, fragment thereof, or variant thereof that is specific for binding to an antigen. In one embodiment, the antigen is a tumor antigen. In one embodiment, the antigen is CD19, B cell maturation antigen (BCMA), CD33, fibroblast activation protein (FAP), follicle-stimulating hormone receptor (FSHR), epidermal growth factor receptor (EGFR), prostate-specific membrane antigen (PSMA), CD123, or human epidermal growth factor receptor 2 (Her2).

[0070] In one embodiment, the nucleotide sequence encoding CD19DBiTE encodes the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or a fragment or variant thereof. In one embodiment, the nucleotide sequence encoding CD19DBiTE comprises the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or a fragment or variant thereof.

[0071] In one embodiment, the nucleotide sequence encoding BCMADBiTE encodes the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12, or a fragment or variant thereof. In one embodiment, the nucleotide sequence encoding BCMADBiTE comprises the nucleotide sequence of SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11, or a fragment or variant thereof.

[0072] In one embodiment, the nucleotide sequence encoding CD33DBiTE encodes the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18, or a fragment or variant thereof. In one embodiment, the nucleotide sequence encoding CD33DBiTE comprises the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17, or a fragment or variant thereof.

[0073] In one embodiment, the nucleotide sequence encoding FAPBiTE encodes the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 24, or a fragment or variant thereof. In one embodiment, the nucleotide sequence encoding FAPDBiTE comprises the nucleotide sequence of SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 23, or a fragment or variant thereof.

[0074] In one embodiment, the nucleotide sequence encoding FSHRDBiTE encodes the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30, or a fragment or variant thereof. In one embodiment, the nucleotide sequence encoding FSHRDBiTE comprises the nucleotide sequence of SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 29, or a fragment or variant thereof.

[0075] In one embodiment, the nucleotide sequence encoding EGFRDBiTE encodes the amino acid sequence of SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36, or a fragment or variant thereof. In one embodiment, the nucleotide sequence encoding EGFRDBiTE comprises the nucleotide sequence of SEQ ID NO: 31, SEQ ID NO: 33, or SEQ ID NO: 35, or a fragment or variant thereof.

[0076] In one embodiment, the nucleotide sequence encoding PSMADBiTE encodes the amino acid sequence of SEQ ID NO: 38, SEQ ID NO: 40, or SEQ ID NO: 42, or a fragment or variant thereof. In one embodiment, the nucleotide sequence encoding PSMADBiTE comprises the nucleotide sequence of SEQ ID NO: 37, SEQ ID NO: 41, or SEQ ID NO: 43, or a fragment or variant thereof.

[0077] In one embodiment, the nucleotide sequence encoding CD123DBiTE encodes the amino acid sequence of SEQ ID NO: 44, SEQ ID NO: 46, or SEQ ID NO: 48, or a fragment or variant thereof. In one embodiment, the nucleotide sequence encoding CD123DBiTE comprises the nucleotide sequence of SEQ ID NO: 43, SEQ ID NO: 45, or SEQ ID NO: 47, or a fragment or variant thereof.

[0078] In one embodiment, the nucleotide sequence encoding HER2DBiTE encodes the amino acid sequence of SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 58, or SEQ ID NO: 60, or a fragment or variant thereof. In one embodiment, the nucleotide sequence encoding HER2DBiTE comprises the nucleotide sequence of SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 67, or a fragment or variant thereof.

[0079] In one embodiment, the nucleotide sequence encoding EGFRvIII2DICE encodes the amino acid sequence of SEQ ID NO: 70 or SEQ ID NO: 72, or a fragment or variant thereof. In one embodiment, the nucleotide sequence encoding EGFRvIII2DICE comprises the nucleotide sequence of SEQ ID NO: 69 or SEQ ID NO: 71, or a fragment or variant thereof.

[0080] In one embodiment, the nucleotide sequence encoding HER2DICE encodes the amino acid sequence of SEQ ID NO: 74, or SEQ ID NO: 76, or a fragment or variant thereof. In one embodiment, the nucleotide sequence encoding HER2DICE comprises the nucleotide sequence of SEQ ID NO: 73 or SEQ ID NO: 75, or a fragment or variant thereof.

[0081] In one embodiment, the composition comprises a nucleotide sequence encoding an anti-Her2 antibody (HER2DMAb). In one embodiment, the nucleotide sequence encoding HER2DMAb comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 62, SEQ ID NO: 64, or a fragment or variant thereof. In one embodiment, the nucleotide sequence encoding HER2DMAb comprises the nucleotide sequence of SEQ ID NO: 61, SEQ ID NO: 63, or a fragment or variant thereof.

[0082] In one embodiment, the composition comprises an scFv anti-Her2 antibody. In one embodiment, the nucleotide sequence encoding the scFv anti-Her2 antibody comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 66 or a fragment or variant thereof. In one embodiment, the nucleotide sequence encoding the scFv anti-Her2 antibody comprises the nucleotide sequence of SEQ ID NO: 65, or a fragment or variant thereof.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

[0083] In certain embodiments, the composition can treat, prevent, and / or protect against a disease or disorder associated with an antigen to which a synthetic antibody of the invention (e.g., a DMAb, ScFv antibody fragment, DICE, or DBiTE) binds. In one embodiment, the composition of the invention can treat, prevent, and / or protect against any disease, disorder, or condition associated with the expression of a target antigen. In certain embodiments, the composition can treat, prevent, and / or protect against cancer.

[0084] A synthetic antibody (e.g., a DMAb, ScFv antibody fragment, DICE, or DBiTE) can treat, prevent, and / or protect against a disease in a subject to whom the composition is administered. A synthetic antibody (e.g., a DMAb, ScFv antibody fragment, DICE, or DBiTE) can promote the survival period of a disease in a subject to whom the composition is administered. A synthetic antibody (e.g., a DMAb, ScFv antibody fragment, DICE, or DBiTE) can provide at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the survival period of a disease in a subject to whom the composition is administered. In other embodiments, a synthetic antibody (e.g., a DMAb, ScFv antibody fragment, DICE, or DBiTE) can provide at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80% of the survival period of a disease in a subject to whom the composition is administered.

[0085] The composition can result in the production of synthetic antibodies (e.g., DMAb, ScFv antibody fragments, DICE, or DBiTE) in a subject within at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 60 hours after administration of the composition to the subject. The composition can result in the production of synthetic antibodies (e.g., DMAb, ScFv antibody fragments, DICE, or DBiTE) in a subject within at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after administration of the composition to the subject. The composition can result in the production of synthetic antibodies (e.g., DMAb, ScFv antibody fragments, DICE, or DBiTE) in a subject within about 1 hour to about 6 days, about 1 hour to about 5 days, about 1 hour to about 4 days, about 1 hour to about 3 days, about 1 hour to about 2 days, about 1 hour to about 1 day, about 1 hour to about 72 hours, about 1 hour to about 60 hours, about 1 hour to about 48 hours, about 1 hour to about 36 hours, about 1 hour to about 24 hours, about 1 hour to about 12 hours, or about 1 hour to about 6 hours after administration of the composition to the subject.

[0086] When administered to a subject in need thereof, the composition can result in the production of synthetic antibodies (e.g., DMAb, ScFv antibody fragments, DICE, or DBiTE) in the subject more rapidly than the production of endogenous antibodies in a subject to whom an antigen is administered to induce a humoral immune response. The composition can result in the production of synthetic antibodies (e.g., DMAb, ScFv antibody fragments, DICE, or DBiTE) at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days before the production of endogenous antibodies in a subject to whom an antigen is administered to induce a humoral immune response.

[0087] The composition of the present invention can have the characteristics required of an effective composition, such as being safe so that the composition does not cause disease or death, being protective against disease, and providing ease of administration, few side effects, biological stability, and low cost per dose.

[0088] Recombinant nucleic acid sequence As described above, the composition may comprise a recombinant nucleic acid sequence. The recombinant nucleic acid sequence may encode a synthetic antibody (e.g., DMAb, ScFv antibody fragment, DICE or DBiTE), a fragment thereof, a variant thereof, or a combination thereof. The antibodies are described in more detail below.

[0089] The recombinant nucleic acid sequence may be a heterologous nucleic acid sequence. The recombinant nucleic acid sequence may comprise at least one heterologous nucleic acid sequence or one or more heterologous nucleic acid sequences.

[0090] The recombinant nucleic acid sequence may be an optimized nucleic acid sequence. Such optimization can increase or alter the immunogenicity of the antibody. Optimization can also improve transcription and / or translation. Optimization may include one or more of the following: a low GC content leader sequence to increase transcription, mRNA stability and codon optimization, addition of a kozak sequence (e.g., GCCACC) for increased translation, addition of an immunoglobulin (Ig) leader sequence encoding a signal peptide, and elimination of cis - acting sequence motifs (i.e., internal TATA box) as much as possible.

[0091] The recombinant nucleic acid sequence may comprise one or more recombinant nucleic acid constructs. The recombinant nucleic acid construct may comprise one or more components described in more detail below.

[0092] A recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence encoding a heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. A recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence encoding a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. A recombinant nucleic acid sequence construct can also include a heterologous nucleic acid sequence encoding a protease or peptidase cleavage site. A recombinant nucleic acid sequence construct can also include a heterologous nucleic acid sequence encoding an internal ribosome entry site (IRES). The IRES can be either a viral IRES or a eukaryotic IRES. A recombinant nucleic acid sequence construct can include one or more leader sequences where each leader sequence encodes a signal peptide. A recombinant nucleic acid sequence construct can include one or more promoters, one or more introns, one or more transcription termination regions, one or more start codons, one or more termination or stop codons, and / or one or more polyadenylation signals. A recombinant nucleic acid sequence construct can also include one or more linker or tag sequences. The tag sequence can encode a hemagglutinin (HA) tag.

[0093] Heavy chain polypeptide A recombinant nucleic acid sequence construct can include a heterologous nucleic acid encoding a heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The heavy chain polypeptide can include a variable heavy (VH) region and / or at least one constant heavy (CH) region. The at least one constant heavy region can include a constant heavy region 1 (CH1), a constant heavy region 2 (CH2), and a constant heavy region 3 (CH3), and / or a hinge region.

[0094] In some embodiments, the heavy chain polypeptide can include a VH region and a CH1 region. In other embodiments, the heavy chain polypeptide can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.

[0095] The heavy chain polypeptide may include a set of complementarity determining regions (the "CDRs"). The set of CDRs may include three hypervariable regions of the VH region. Proceeding from the N-terminus of the heavy chain polypeptide, these CDRs are designated "CDR1", "CDR2", and "CDR3", respectively. CDR1, CDR2, and CDR3 of the heavy chain polypeptide may contribute to antigen binding or recognition.

[0096] Light chain polypeptide The recombinant nucleic acid sequence construct may include a heterologous nucleic acid sequence encoding a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The light chain polypeptide may include a variable light chain (VL) region and / or a constant light chain (CL) region.

[0097] The light chain polypeptide may include a set of complementarity determining regions (the "CDRs"). The set of CDRs may include three hypervariable regions of the VL region. Proceeding from the N-terminus of the light chain polypeptide, these CDRs are designated "CDR1", "CDR2", and "CDR3", respectively. CDR1, CDR2, and CDR3 of the light chain polypeptide may contribute to antigen binding or recognition.

[0098] Protease cleavage site The recombinant nucleic acid sequence construct may include a heterologous nucleic acid sequence encoding a protease cleavage site. The protease cleavage site may be recognized by a protease or peptidase. The protease may be an endopeptidase or endoprotease, for example, but not limited to, furin, elastase, HtrA, calpain, trypsin, chymotrypsin, trypsin, and pepsin. The protease may be furin. In other embodiments, the protease may be a serine protease, threonine protease, cysteine protease, aspartic protease, metalloprotease, glutamic protease, or any protease that cleaves an internal peptide bond (i.e., does not cleave an N-terminal or C-terminal peptide bond).

[0099] The protease cleavage site may contain one or more amino acid sequences that promote or increase the efficiency of cleavage. The one or more amino acid sequences can promote or increase the efficiency of forming or generating a separate polypeptide. The one or more amino acid sequences may contain a 2A peptide sequence.

[0100] Linker sequence The recombinant nucleic acid sequence construct may contain one or more linker sequences. The linker sequence can spatially separate or ligate one or more of the components described herein. In other embodiments, the linker sequence can encode an amino acid sequence that spatially separates or ligates two or more polypeptides. In one embodiment, the linker sequence is a G4S linker sequence having the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 68).

[0101] Promoter The recombinant nucleic acid sequence construct may contain one or more promoters. The one or more promoters can be any promoter that can drive and regulate gene expression. Such a promoter is a cis-acting sequence element necessary for transcription via DNA-dependent RNA polymerase. The choice of promoter used to induce gene expression depends on the particular application. The promoter can be placed at approximately the same distance from the start of transcription in the recombinant nucleic acid sequence construct as from the transcription start site in its native setting. However, variations in this distance can be accommodated without loss of promoter function.

[0102] The promoter can be operably linked to a heterologous nucleic acid sequence encoding a heavy chain polypeptide and / or a light chain polypeptide. The promoter can be a promoter shown to be effective for expression in eukaryotic cells. A promoter operably linked to a coding sequence can be a promoter derived from Simian virus 40 (SV40), such as the CMV promoter, the SV40 early promoter, and the SV40 late promoter; a promoter of a Human immunodeficiency virus (HIV), such as the Mouse mammary tumor virus (MMTV) promoter, the Bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter; the Moloney virus promoter, the Avian leukosis virus (ALV) promoter, a Cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter; the Epstein-Barr virus (EBV) promoter, or the Rous sarcoma virus (RSV) promoter. The promoter can also be a promoter derived from a human gene, such as a human actin, human myosin, human hemoglobin, human muscle creatine, human polyhedrin, or human metallothionein.

[0103] The promoter can be a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is exposed to some specific external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development. The promoter can also be a tissue-specific promoter, such as a natural or synthetic muscle or skin-specific promoter. Examples of such promoters are described in U.S. Patent Application Publication No. 2004 / 0175727, the content of which is incorporated herein by reference in its entirety.

[0104] A promoter may be associated with an enhancer. The enhancer can be located upstream of the coding sequence. The enhancer can be a viral enhancer such as human actin, human myosin, human hemoglobin, human muscle creatine, or one from CMV, FMDV, RSV, or EBV. Polynucleotide function enhancement is described in U.S. Patent Nos. 5,593,972, 5,962,428, and W094 / 016737, the contents of each of which are hereby incorporated by reference in their entirety.

[0105] Transcription termination region A recombinant nucleic acid sequence construct may include one or more transcription termination regions. The transcription termination region can be downstream of the coding sequence to provide efficient termination. The transcription termination region can be obtained from the same gene as the promoter described above or from one or more different genes.

[0106] Start codon A recombinant nucleic acid sequence construct may include one or more start codons. The start codon can be located upstream of the coding sequence. The start codon can be in-frame with the coding sequence. The start codon can be associated with one or more signals necessary for efficient translation initiation, such as, but not limited to, a ribosome binding site.

[0107] Stop codon A recombinant nucleic acid sequence construct may include one or more termination or stop codons. The termination codon can be downstream of the coding sequence. The termination codon can be in-frame with the coding sequence. The termination codon can be associated with one or more signals necessary for efficient translation termination.

[0108] Polyadenylation signal The recombinant nucleic acid sequence construct may contain one or more polyadenylation signals. The polyadenylation signal may contain one or more signals necessary for efficient polyadenylation of the transcript. The polyadenylation signal can be located downstream of the coding sequence. The polyadenylation signal can be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal can be the polyadenylation signal from the pCEP4 plasmid (Invitrogen, San Diego, CA).

[0109] Leader sequence The recombinant nucleic acid sequence construct may contain one or more leader sequences. The leader sequence may encode a signal peptide. The signal peptide can be an immunoglobulin (Ig) signal peptide, such as, but not limited to, an IgG signal peptide and an IgE signal peptide.

[0110] Expression from the recombinant nucleic acid sequence construct As described above, the recombinant nucleic acid sequence construct may contain, among one or more components, a heterologous nucleic acid sequence encoding a heavy chain polypeptide and / or a heterologous nucleic acid sequence encoding a light chain polypeptide. Thus, the recombinant nucleic acid sequence construct can facilitate the expression of the heavy chain polypeptide and / or the light chain polypeptide.

[0111] When Arrangement 1 as described above is utilized, the first recombinant nucleic acid sequence construct can facilitate the expression of the heavy chain polypeptide, and the second recombinant nucleic acid sequence construct can facilitate the expression of the light chain polypeptide. When Arrangement 2 as described above is utilized, the recombinant nucleic acid sequence construct can facilitate the expression of the heavy chain polypeptide and the light chain polypeptide.

[0112] When expressed, for example, but not limited to, in cells, organisms, or mammals, the heavy chain polypeptide and the light chain polypeptide can assemble with synthetic antibodies (e.g., DMAb, ScFv antibody fragments, DICE, or DBiTE). In particular, the heavy chain polypeptide and the light chain polypeptide can interact with each other such that the assembly results in a synthetic antibody (e.g., DMAb, ScFv antibody fragments, DICE, or DBiTE) that can bind to an antigen. In other embodiments, the heavy chain polypeptide and the light chain polypeptide can interact with each other such that the assembly results in a synthetic antibody (e.g., DMAb, ScFv antibody fragments, DICE, or DBiTE) that is more immunogenic compared to an antibody that does not assemble as described herein. In yet other embodiments, the heavy chain polypeptide and the light chain polypeptide can interact with each other such that the assembly results in a synthetic antibody (e.g., DMAb, ScFv antibody fragments, DICE, or DBiTE) that can induce or elicit an immune response against an antigen.

[0113] Vector The recombinant nucleic acid sequence construct described above can be disposed in one or more vectors. The one or more vectors can include an origin of replication. The one or more vectors can be a plasmid, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. The one or more vectors can be either a self-replicating episomal vector or a vector that integrates into the host genome.

[0114] The one or more vectors can be an expression construct, which is generally a plasmid used to introduce a specific gene into a target cell. When the expression vector enters the cell, the heavy chain polypeptide and / or the light chain polypeptide encoded by the recombinant nucleic acid sequence construct are produced by the cellular transcription and translation machinery ribosome complex. The one or more vectors can express a large amount of stable messenger RNA and thus protein.

[0115] Expression vector One or more vectors can be circular plasmids or linear nucleic acids. Circular plasmids and linear nucleic acids can induce the expression of a specific nucleotide sequence in a suitable target cell. One or more vectors containing a recombinant nucleic acid sequence construct can be chimeric, meaning that at least one of its components is heterologous to at least one of the other components.

[0116] Plasmid One or more vectors can be plasmids. Plasmids can be useful for transfecting cells with a recombinant nucleic acid sequence construct. Plasmids can be useful for introducing a recombinant nucleic acid sequence construct into a target. Plasmids can also contain regulatory sequences that can be well-suited for gene expression in the cells into which the plasmid is administered.

[0117] Plasmids can also contain a mammalian origin of replication to maintain the plasmid episomally and produce multiple copies of the plasmid in the cell. The plasmid can be pVAX1, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which can contain an Epstein Barr virus origin of replication and a nuclear antigen EBNA-1 coding region, which can produce high-copy episomal replication without integration. The backbone of the plasmid can be pAV0242. The plasmid can be a replication-deficient adenovirus type 5 (Ad5) plasmid.

[0118] The plasmid can be pSE420 (Invitrogen, San Diego, Calif.), which can be used for protein production in Escherichia coli (E. coli). The plasmid can also be pYES2 (Invitrogen, San Diego, Calif.), which can be used for protein production in the Saccharomyces cerevisiae strain of yeast. The plasmid can also be of the MAXBAC™ Complete Baculovirus Expression System (Invitrogen, San Diego, Calif.), which can be used for protein production in insect cells. The plasmid can also be pcDNAI or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells.

[0119] RNA In one embodiment, the nucleic acid is an RNA molecule. In one embodiment, the RNA molecule is transcribed from a DNA sequence. Thus, in one embodiment, the invention provides an RNA molecule encoding one or more of the synthetic antibodies of the invention. The RNA can be a plus strand. Thus, in some embodiments, the RNA molecule can be translated by a cell without the need for any intervening replication step such as reverse transcription. The RNA molecules useful in the present invention can have a 5′ cap (e.g., 7-methylguanosine). This cap can improve in vivo translation of the RNA. The 5′ nucleotide of the RNA molecules useful in the present invention can have a 5′ triphosphate group. In capped RNA, this can be linked to 7-methylguanosine via a 5′-5′ bridge. The RNA molecule can have a 3′ poly-A tail. It can also contain a poly-A polymerase recognition sequence (e.g., AAUAAA) near its 3′ end. The RNA molecules useful in the present invention can be single-stranded. The RNA molecules useful in the present invention can include synthetic RNA. In some embodiments, the RNA molecule is a naked RNA molecule. In one embodiment, the RNA molecule is contained within a vector.

[0120] In one embodiment, the RNA has 5' and 3' UTRs. In one embodiment, the 5' UTR is 0 to 3000 nucleotides in length. The lengths of the 5' and 3' UTR sequences added to the coding region can be changed by different methods including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths necessary to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0121] The 5' and 3' UTRs can be the native, endogenous 5' and 3' UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into forward and reverse primers or by any other modification of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and / or translation efficiency of the RNA. For example, AU-rich elements in the 3' UTR sequence are known to be able to increase the stability of the RNA. Thus, the 3' UTR can be selected or designed to increase the stability of the transcribed RNA based on the properties of UTRs well known in the art.

[0122] In one embodiment, the 5’UTR may include the Kozak sequence of an endogenous gene. Alternatively, when a 5’UTR that is not endogenous to the gene of interest has been added by the above PCR, the consensus Kozak sequence can be redesigned by adding the 5’UTR sequence. The Kozak sequence can increase the efficiency of translation of some RNA transcripts, but does not appear to be required to enable efficient transcription for all RNAs. The requirements for the Kozak sequence of many RNAs are known in the art. In other embodiments, the 5’UTR may be derived from an RNA virus, and this RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3’ or 5’UTR to prevent exonucleolytic degradation of the RNA.

[0123] In one embodiment, the RNA has a cap on both the 5’ end and the 3’ poly(A) tail, which determines ribosome binding, initiation of translation, and the stability of the RNA in cells.

[0124] In one embodiment, the RNA is nucleoside-modified RNA. Nucleoside-modified RNA has certain advantages over unmodified RNA, including, for example, increased stability, low or absent innate immunogenicity, and improved translation.

[0125] Circular and linear vectors One or more vectors can be circular plasmids (e.g., autonomously replicating plasmids having an origin of replication) that transform target cells by integration into the cell genome or can exist episomally. The vector can be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing a heavy chain polypeptide and / or a light chain polypeptide encoded by a recombinant nucleic acid sequence construct.

[0126] Also provided herein are linear nucleic acids, or linear expression cassettes (“LECs”), that can be efficiently delivered to a subject via electroporation and that can express a heavy chain polypeptide and / or a light chain polypeptide encoded by a recombinant nucleic acid sequence construct. An LEC can be any linear DNA that is free of a phosphate backbone. An LEC may or may not contain any antibiotic resistance gene and / or phosphate backbone. An LEC may or may not contain other nucleic acid sequences not related to the desired gene expression.

[0127] An LEC can be derived from any plasmid that can be linearized. The plasmid may or may not be able to express a heavy chain polypeptide and / or a light chain polypeptide encoded by a recombinant nucleic acid sequence construct. The plasmid can be pNP (Puerto Rico / 34) or pM2 (New Caledonia / 99). The plasmid can be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector that can express a heavy chain polypeptide and / or a light chain polypeptide encoded by a recombinant nucleic acid sequence construct.

[0128] An LEC can be pcrM2. An LEC can be pcrNP. pcrNP and pcrMR can be derived from pNP (Puerto Rico / 34) and pM2 (New Caledonia / 99), respectively.

[0129] Method for preparing a vector Provided herein is a method for preparing one or more vectors in which a recombinant nucleic acid sequence construct is disposed. After the final subcloning step, the vector can be used to inoculate a cell culture in a large-scale fermentation tank using methods known in the art.

[0130] In other embodiments, after the final subcloning step, the vector can be used in one or more electroporation (EP) devices. The EP devices are described in more detail below.

[0131] One or more vectors can be formulated or manufactured using combinations of known devices and techniques, but preferably they are manufactured using the plasmid manufacturing techniques described in U.S. Provisional Patent Application No. 60 / 939,792, filed May 23, 2007, which has been allowed and is co-pending. In some examples, the DNA plasmids described herein can be formulated at a concentration of 10 mg / mL or greater. The manufacturing techniques also include various devices and protocols generally known to those of skill in the art, including, or incorporated in addition to, those described in U.S. Provisional Patent Application No. 60 / 939,792, including those described in U.S. Patent No. 7,238,522, a licensed patent issued on July 3, 2007. The above applications and patents, U.S. Provisional Patent Application No. 60 / 939,792 and U.S. Patent No. 7,238,522, respectively, are incorporated herein by reference in their entirety.

[0132] Antibody In some embodiments, the invention relates to a recombinant nucleic acid sequence encoding an antibody, a fragment thereof, a variant thereof, or a combination thereof. The antibody can bind or react with an antigen described in more detail below. In some embodiments, the antibody is a DNA-encoded monoclonal antibody (DMAb), a fragment thereof, or a variant thereof. In some embodiments, the fragment is a ScFv fragment. In some embodiments, the antibody is a DNA-encoded bispecific T cell engager (BiTE), a fragment thereof, or a variant thereof.

[0133] In some embodiments, the antibody can comprise a set of heavy and light chain complementarity determining regions (the "CDRs"), which are each inserted between a set of heavy and light chain frameworks (the "FRs") that provide support for the CDRs and define the spatial relationship of the CDRs relative to one another. The set of CDRs can include three hypervariable regions of the heavy chain V region or the light chain V region. Proceeding from the N-terminus of the heavy chain or light chain, these regions are designated "CDR1," "CDR2," and "CDR3," respectively. Thus, the antigen-binding site can include six CDRs, a set of CDRs from each of the heavy chain V region and the light chain V region.

[0134] The proteolytic enzyme papain preferentially cleaves the IgG molecule to obtain several fragments, two of which (F(ab) fragments) each contain a covalent heterodimer containing an intact antigen-binding site. The enzyme pepsin can cleave the IgG molecule to provide several fragments including an F(ab')2 fragment that contains both antigen-binding sites. Thus, the antibody can be a Fab or an F(ab')2. The Fab can include a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of the Fab can include a VH region and a CH1 region. The light chain of the Fab can include a VL region and a CL region.

[0135] The antibody can be an immunoglobulin (Ig). The Ig can be, for example, IgA, IgM, IgD, IgE, and IgG. The immunoglobulin can include a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of the immunoglobulin can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of the immunoglobulin can include a VL region and a CL region.

[0136] The antibody can be a polyclonal antibody or a monoclonal antibody. The antibody can be a chimeric antibody, a single-chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. A humanized antibody can be an antibody derived from a non-human species that binds to a desired antigen and has one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule.

[0137] The antibody can be a bispecific antibody described in more detail below. The antibody can also be a bifunctional antibody described in more detail below.

[0138] As described above, the antibody can be generated in a subject when the composition is administered to the subject. The antibody can have a half-life within the subject. In some embodiments, the antibody may be modified to extend or shorten its half-life within the subject. Such modifications are described in more detail below.

[0139] The antibody can be defucosylated as described in more detail below.

[0140] ScFv antibody In one embodiment, the DMAb of the present invention is a ScFv DMAb. In one embodiment, the ScFv DMAb relates to a Fab fragment that does not include those of the CH1 and CL regions. Thus, in one embodiment, the ScFv DMAb relates to a Fab fragment DMAb that includes VH and VL. In one embodiment, the ScFv DMAb includes a linker between VH and VL. In one embodiment, the ScFv DMAb is a ScFv-Fc DMAb. In one embodiment, the ScFv-Fc DMAb includes VH, VL, and the CH2 and CH3 regions. In one embodiment, the ScFv-Fc DMAb includes a linker between VH and VL. In one embodiment, the ScFv DMAb of the present invention has modified expression, stability, half-life, antigen binding, heavy chain-light chain pairing, tissue penetration, or a combination thereof compared to the parental DMAb.

[0141] In one embodiment, the ScFv DMAb of the present invention has an expression that is at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, at least 2.9 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 8.5 times, at least 9 times, at least 9.5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, or more than 50 times higher than that of the parental DMAb.

[0142] In one embodiment, the ScFv DMAb of the present invention has an antigen binding that is at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, at least 2.9 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 8.5 times, at least 9 times, at least 9.5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, or more than 50 times higher than that of the parental DMAb.

[0143] In one embodiment, the ScFv DMAb of the present invention has a half-life that is at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, at least 2.9 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 8.5 times, at least 9 times, at least 9.5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, or more than 50 times longer than that of the parental DMAb.

[0144] In one embodiment, the ScFv DMAb of the present invention has a stability that is at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, at least 2.9 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 8.5 times, at least 9 times, at least 9.5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, or more than 50 times higher than that of the parental DMAb.

[0145] In one embodiment, the ScFv DMAb of the present invention has a tissue permeability that is at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, at least 2.9 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 8.5 times, at least 9 times, at least 9.5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, or more than 50 times that of the parental DMAb.

[0146] In one embodiment, the ScFv DMAb of the present invention has a heavy-chain / light-chain pairing that is at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, at least 2.9 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 8.5 times, at least 9 times, at least 9.5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, or more than 50 times that of the parental DMAb.

[0147] In one embodiment, the anti-HER2 scFv antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 66, or a fragment of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 66. In one embodiment, the anti-HER2 scFv antibody comprises the amino acids of SEQ ID NO: 66, or a fragment of the amino acid sequence of SEQ ID NO: 66. In one embodiment, the anti-HER2 scFv antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence encoded by SEQ ID NO: 65, or a fragment of an amino acid sequence that is at least 90% identical to the amino acid sequence encoded by one of SEQ ID NO: 65. In one embodiment, the anti-HER2 scFv antibody comprises the amino acid sequence encoded by SEQ ID NO: 65, or a fragment of the amino acid sequence encoded by SEQ ID NO: 65.

[0148] Monoclonal antibody In one embodiment, the present invention provides an anti-HER2 antibody. The antibody can be a full-length monoclonal antibody, and immunologically active fragments (e.g., Fab or (Fab)2 fragments), monoclonal antibody heavy chains, or monoclonal antibody light chains.

[0149] The antibody can include a heavy chain and a light chain complementarity determining region (「CDR」) set, which are inserted between a heavy chain and a light chain framework (「FR」) set that each provide support to the CDRs and define the spatial relationship of the CDRs with respect to each other. The CDR set can include three hypervariable regions of the heavy chain V region or the light chain V region. Proceeding from the N-terminus of the heavy chain or the light chain, these regions are designated 「CDR1」, 「CDR2」, and 「CDR3」, respectively. Thus, the antigen-binding site can include six CDRs, including a CDR set from each of the heavy chain V region and the light chain V region.

[0150] An antibody can be an immunoglobulin (Ig). The Ig can be, for example, IgA, IgM, IgD, IgE, and IgG. An immunoglobulin can include a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of an immunoglobulin can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of an immunoglobulin can include a VL region and a CL region.

[0151] In one embodiment, the anti-HER2 antibody is optimized for expression in humans. In one embodiment, the anti-HER2 antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 62, or a fragment of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 62. In one embodiment, the anti-HER2 antibody comprises the amino acids of SEQ ID NO: 62, or a fragment of the amino acid sequence of SEQ ID NO: 62. In one embodiment, the anti-HER2 antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence encoded by SEQ ID NO: 61, or a fragment of an amino acid sequence that is at least 90% identical to the amino acid sequence encoded by one of SEQ ID NOs: 61. In one embodiment, the anti-HER2 antibody comprises the amino acid sequence encoded by SEQ ID NO: 61, or a fragment of the amino acid sequence encoded by SEQ ID NO: 61.

[0152] In one embodiment, the anti-HER2 antibody is optimized for expression in mice. In one embodiment, the anti-HER2 antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 64, or a fragment of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 64. In one embodiment, the anti-HER2 antibody comprises the amino acids of SEQ ID NO: 64, or a fragment of the amino acid sequence of SEQ ID NO: 64. In one embodiment, the anti-HER2 antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence encoded by SEQ ID NO: 61, or a fragment of an amino acid sequence that is at least 90% identical to the amino acid sequence encoded by one of SEQ ID NOs: 63. In one embodiment, the anti-HER2 antibody comprises the amino acid sequence encoded by SEQ ID NO: 63, or a fragment of the amino acid sequence encoded by SEQ ID NO: 63.

[0153] Bispecific T cell engager As described above, the recombinant nucleic acid sequence may encode a bispecific T cell engager (BiTE), a fragment thereof, a variant thereof, or a combination thereof. The antigen targeting domain of the BiTE may bind or react with an antigen described in more detail below.

[0154] The antigen targeting domain of the BiTE may comprise an antibody, a fragment thereof, a variant thereof, or a combination thereof. The antigen targeting domain of the BiTE may comprise a set of heavy and light chain complementarity determining regions (the "CDRs"), which are inserted between a set of heavy and light chain frameworks (the "FRs") that each provide support to the CDRs and define the spatial relationship of the CDRs to each other. The set of CDRs may comprise three hypervariable regions of the heavy chain V region or the light chain V region. Proceeding from the N-terminus of the heavy chain or light chain, these regions are designated "CDR1", "CDR2", and "CDR3", respectively. Thus, the antigen binding domain may comprise six CDRs, a set of CDRs from each of the heavy chain V region and the light chain V region.

[0155] The proteolytic enzyme papain preferentially cleaves the IgG molecule to obtain several fragments, two of which (F(ab) fragments) each contain a covalent heterodimer containing an intact antigen-binding site. The enzyme pepsin can cleave the IgG molecule to provide several fragments including an F(ab’)2 fragment containing both antigen-binding sites. Thus, the antigen target domain of BiTE can be Fab or F(ab’)2. Fab can contain a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of Fab can contain a VH region and a CH1 region. The light chain of Fab can contain a VL region and a CL region.

[0156] The antigen target domain of BiTE can be an immunoglobulin (Ig). Ig can be, for example, IgA, IgM, IgD, IgE, and IgG. Immunoglobulins can contain heavy chain polypeptides and light chain polypeptides. The heavy chain polypeptide of an immunoglobulin can contain a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of an immunoglobulin can contain a VL region and a CL region.

[0157] The antigen target domain of BiTE can be a polyclonal or monoclonal antibody. The antibody can be a chimeric antibody, a single-chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. A humanized antibody can be an antibody derived from a non-human species that binds to a desired antigen and has one or more complementarity determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule.

[0158] In one embodiment, at least one of the antigen-binding domain and the immune cell engaging domain of the DBiTE of the present invention is a ScFv DNA-encoded monoclonal antibody (ScFv DMAb) as described in detail above.

[0159] Bispecific antibody The recombinant nucleic acid sequence may encode a bispecific antibody, a fragment thereof, a variant thereof, or a combination thereof. The bispecific antibody can bind or react with two antigens, for example, two antigens described in more detail below. The bispecific antibody can be composed of two fragments of the antibodies described herein, whereby the bispecific antibody can bind or react with two desired target molecules including antigens, ligands including ligands for receptors, receptors including ligand binding sites on the receptors, ligand-receptor complexes, and markers, as described in more detail below.

[0160] The present invention provides a novel bispecific antibody comprising a first antigen-binding site that specifically binds to a first target and a second antigen-binding site that specifically binds to a second target, and has particularly advantageous properties such as producibility, stability, binding affinity, biological activity, specific targeting of certain T cells, targeting efficiency, and reduction of toxicity. In some examples, there is a bispecific antibody that binds to the first target with high affinity and to the second target with low affinity. In other examples, there is a bispecific antibody that binds to the first target with low affinity and to the second target with high affinity. In other examples, there is a bispecific antibody that binds to the first target with a desired affinity and to the second target with a desired affinity.

[0161] In one embodiment, the bispecific antibody is a bivalent antibody comprising a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen, and b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen.

[0162] The bispecific antibody molecule according to the present invention can have two binding sites of any desired specificity. In some embodiments, one of the binding sites enables a tumor antigen. In some embodiments, the binding site contained in the Fab fragment is a binding site specific for a tumor antigen. In some embodiments, the binding site contained in the single-chain Fv fragment is a binding site specific for a tumor antigen such as CD19, BCMA, CD33, FAP, FSHR, EGFR, PSMA, CD123 or Her2.

[0163] In some embodiments, one of the binding sites of the antibody molecules according to the invention can bind to a T cell-specific receptor molecule and / or a natural killer (NK) cell-specific receptor molecule. The T cell-specific receptor allows a T cell to bind to an epitope / antigen presented by another cell called an antigen-presenting cell or APC, and, in the presence of additional signals, to be thereby activated and to respond thereto, which is the so-called "T cell receptor" (TCR). The T cell receptor is known to resemble the Fab fragment of a naturally occurring immunoglobulin. Generally, it is monovalent and includes an alpha chain and a beta chain, and in some embodiments, a gamma chain and a delta chain (see above). Thus, in some embodiments, the TCR is TCR (alpha / beta), and in some embodiments, the TCR is TCR (gamma / delta). The T cell receptor forms a complex with the CD3 T cell coreceptor. CD3 is a protein complex consisting of four different chains. In mammals, the complex includes the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains associate with a molecule known as the T cell receptor (TCR) and the ζ chain to generate activation signals within T lymphocytes. Thus, in some embodiments, the T cell-specific receptor is the CD3 T cell coreceptor. In some embodiments, the T cell-specific receptor is the protein CD28, which is also expressed on T cells. CD28 can provide a co-stimulatory signal required for T cell activation. CD28 plays an important role in T cell proliferation and survival, cytokine production, and type 2 T helper development. A further example of a T cell-specific receptor is CD134, also known as Ox40. CD134 / OX40 is expressed 24 to 72 hours after activation and can be taken to define a secondary co-stimulatory molecule. Another example of a T cell receptor is 4-1BB, which can bind to the 4-1BB ligand on an antigen-presenting cell (APC), thereby generating a co-stimulatory signal for the T cell. Another example of a receptor found mainly on T cells is CD5, which is also found at low levels on B cells.A further example of a receptor that modifies T cell function is CD95, also known as the Fas receptor, which mediates apoptosis signaling by Fas-ligand expressed on the surface of other cells. CD95 has been reported to regulate the TCR / CD3-driven signaling pathway in resting T lymphocytes.

[0164] An example of an NK cell-specific receptor molecule is CD16, the low-affinity Fc receptor, and NKG2D. An example of a receptor molecule present on the surface of both T cells and natural killer (NK) cells is CD2 and further members of the CD2-superfamily. CD2 can act as a co-stimulatory molecule on T cells and NK cells.

[0165] In some embodiments, the first binding site of the antibody molecule binds to a tumor antigen and the second binding site binds to a T cell-specific receptor molecule and / or a natural killer (NK) cell-specific receptor molecule.

[0166] In some embodiments, the first binding site of the antibody molecule binds to CD19, BCMA, CD33, FAP, FSHR, EGFR, PSMA, CD123 or Her2, and the second binding site binds to a T cell-specific receptor molecule and / or a natural killer (NK) cell-specific receptor molecule. In some embodiments, the first binding site of the antibody molecule binds to CD19, BCMA, CD33, FAP, FSHR, EGFR, PSMA, CD123, or Her2, and the second binding site binds to one of CD3, TCR, CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5, CD40, FcgR, FceR, FcaR, and CD95. In some embodiments, the first binding site of the antibody molecule binds to CD19, BCMA, CD33, FAP, FSHR, EGFR, PSMA, CD123, or Her2, and the second binding site binds to CD3.

[0167] In some embodiments, the first binding site of the antibody molecule binds to a T cell-specific receptor molecule and / or a natural killer (NK) cell-specific receptor molecule, and the second binding site binds to a tumor antigen. In some embodiments, the first binding site of the antibody binds to a T cell-specific receptor molecule and / or a natural killer (NK) cell-specific receptor molecule, and the second binding site binds to CD19, BCMA, CD33, FAP, FSHR, EGFR, PSMA, CD123, or Her2. In some embodiments, the first binding site of the antibody binds to one of CD3, TCR, CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5, CD40, FcgR, FceR, FcaR, and CD95, and the second binding site binds to CD19, BCMA, CD33, FAP, FSHR, EGFR, PSMA, CD123, or Her2. In some embodiments, the first binding site of the antibody binds to CD3, and the second binding site binds to CD19, BCMA, CD33, FAP, FSHR, EGFR, PSMA, CD123, or Her2.

[0168] In one embodiment, the bispecific antibody of the present invention comprises a DBiTE comprising one or more scFv antibody fragments described herein, thereby enabling the DBiTE to bind or react with a desired target molecule.

[0169] In one embodiment, the DBiTE comprises a nucleic acid molecule encoding a first scFv specific for binding to a target disease-specific antigen linked to a second scFv specific for binding to a T cell-specific receptor molecule. The linkage may arrange the first and second domains in either order. For example, in one embodiment, the nucleotide sequence encoding the scFv specific for binding to the target disease-specific antigen is oriented 5' (or upstream) to the nucleotide sequence encoding the scFv specific for binding to the T cell-specific receptor molecule. In another embodiment, the nucleotide sequence encoding the scFv specific for binding to the target disease-specific antigen is oriented 3' (or downstream) to the nucleotide sequence encoding the scFv specific for binding to the T cell-specific receptor molecule.

[0170] Bifunctional antibody The recombinant nucleic acid sequence may encode a bifunctional antibody, a fragment thereof, a variant thereof, or a combination thereof. The bifunctional antibody may bind or react with the antigens described below. The bifunctional antibody may also be modified to confer additional functionality to the antibody beyond antigen recognition and binding to the antigen. Such modifications may include, but are not limited to, binding to factor H or a fragment thereof. Factor H is a soluble regulator of complement activation and can thus contribute to the immune response via complement-mediated lysis (CML).

[0171] Prolongation of antibody half-life As described above, synthetic antibodies (e.g., DMAb, ScFv antibody fragments, DICE or DBiTE) may be modified to prolong or shorten the half-life of the antibody in a subject. The modification may prolong or shorten the half-life of the antibody in the subject's serum.

[0172] The modification may be present in the constant region of the antibody. The modification may be one or more amino acid substitutions within the constant region of the antibody that prolong the half-life of the antibody as compared to the half-life of an antibody that does not contain one or more amino acid substitutions. The modification may be one or more amino acid substitutions within the CH2 domain of the antibody that prolong the half-life of the antibody as compared to the half-life of an antibody that does not contain one or more amino acid substitutions.

[0173] In some embodiments, one or more amino acid substitutions within the constant region may include replacing a methionine residue within the constant region with a tyrosine residue, a serine residue within the constant region with a threonine residue, a threonine residue within the constant region with a glutamine residue, or any combination thereof, thereby prolonging the half-life of the antibody.

[0174] In other embodiments, one or more amino acid substitutions within the constant region may include replacing a methionine residue within the CH2 domain with a tyrosine residue, a serine residue within the CH2 domain with a threonine residue, a threonine residue within the CH2 domain with a glutamine residue, or any combination thereof, thereby extending the half-life of the antibody.

[0175] Defucosylation The recombinant nucleic acid sequence may encode a non-fucosylated antibody (i.e., a defucosylated or non-fucosylated antibody), a fragment thereof, a variant thereof, or a combination thereof. Fucosylation includes the addition of the sugar fucose to a molecule, e.g., the attachment of fucose to N-glycans, O-glycans, and glycolipids. Thus, in a defucosylated antibody, fucose is not attached to the carbohydrate chains of the constant region. This lack of fucosylation may then improve FcγRIIIa binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity by the antibody as compared to a fucosylated antibody. Accordingly, in some embodiments, a non-fucosylated antibody may exhibit increased ADCC activity as compared to a fucosylated antibody.

[0176] An antibody may be modified to prevent or inhibit fucosylation of the antibody. In some embodiments, such a modified antibody may exhibit increased ADCC activity as compared to an unmodified antibody. The modification may be in the heavy chain, the light chain, or a combination thereof. The modification may be one or more amino acid substitutions in the heavy chain, one or more amino acid substitutions in the light chain, or a combination thereof.

[0177] Antigen In one embodiment, the synthetic antibody (e.g., DMAb, ScFv antibody fragment, DICE or DBiTE) targets an antigen or a fragment or variant thereof. The antigen can be a nucleic acid sequence, an amino acid sequence, a polysaccharide, or a combination thereof. The nucleic acid sequence can be DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. The amino acid sequence can be a protein, a peptide, variants thereof, fragments thereof, or combinations thereof. The polysaccharide can be a nucleic acid-encoded polysaccharide.

[0178] The antigen can be a tumor antigen. The antigen can be associated with an increased risk of cancer development or progression. In one embodiment, the antigen can be CD19, BCMA, CD33, FAP, FSHR, EGFR, PSMA, CD123, or Her2.

[0179] In one embodiment, the synthetic DNA-encoded bispecific immune cell engager of the present invention targets two or more antigens. In one embodiment, at least one antigen of the bispecific antibody is a tumor antigen. In one embodiment, at least one antigen of the bispecific antibody is a T cell activation antigen.

[0180] Tumor antigen The antigen-binding domain of the synthetic antibody (e.g., DMAb, ScFv antibody fragment, DICE or DBiTE) of the present invention can interact with a tumor antigen. In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen common to a specific hyperproliferative disorder such as cancer.

[0181] The types of tumor antigens referred to in the present invention can be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and do not occur in other cells in the body. TAA antigens are not unique to tumor cells and instead are expressed on normal cells under conditions that cannot induce a state of immunological tolerance to the antigen. The expression of antigens on tumors can occur under conditions that allow the immune system to respond to the antigen. TAA may be an antigen that is expressed on normal cells during fetal development when the immune system is immature and unable to respond, or may be an antigen that is normally present at very low levels on normal cells but is expressed at much higher levels on tumor cells.

[0182] The antigens discussed herein are included merely by way of example. The list is not intended to be exclusive, and additional examples will be readily apparent to those skilled in the art.

[0183] Tumor antigens are proteins produced by tumor cells that induce an immune response, particularly a T cell-mediated immune response. The selection of the antigen-binding portion of the present invention depends on the specific type of cancer being treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostin, PSMA, Her2, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0184] Exemplary examples of tumor - associated surface antigens are CD10, CD19, CD20, CD22, CD33, CD123, B - cell maturation antigen (BCMA), Fms - like tyrosine kinase 3 (FLT - 3, CD135), chondroitin sulfate proteoglycan 4 (CSPG4, melanoma - associated chondroitin sulfate proteoglycan), epidermal growth factor receptor (EGFR), Her2, Her3, IGFR, CD133, IL3R, fibroblast activation protein (FAP), CDCP1, Derlin1, tenascin, frizzled1 - 10, vascular antigen VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), PDGFR - α (CD140a), PDGFR - beta (CD140b), endoglin, CLEC14, Tem1 - 8, and Tie2. Further examples may include A33, CAMPATH - 1 (CDw52), carcinoembryonic antigen (CEA), carbonic anhydrase IX (MN / CAIX), CD21, CD25, CD30, CD34, CD37, CD44v6, CD45, CD133, de2 - 7EGFR, EGFRvIII, EpCAM, Ep - CAM, folate - binding protein, G250, Fms - like tyrosine kinase 3 (FLT - 3, CD135), follicle - stimulating hormone receptor (FSHR), c - Kit (CD117), CSF1R (CD115), HLA - DR, IGFR, IL - 2 receptor, IL3R, MCSP (melanoma - associated cell - surface chondroitin sulfate proteoglycan), Muc - 1, prostate - specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate - specific antigen (PSA), and TAG - 72. Examples of antigens expressed on the extracellular matrix of tumors are tenascin and fibroblast activation protein (FAP).

[0185] In one embodiment, the tumor antigen is a hormone or a fragment thereof that can be used to target a specific receptor. Examples include, but are not limited to, FSH hormone, LH hormone, TSH hormone, or fragments thereof.

[0186] Non-limiting examples of TSA or TAA antigens include the following: MART-1 / MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-system antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, etc.; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu, etc.; unique tumor antigens resulting from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7, and other differentiation antigens. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0187] Aspects of the present invention include synthetic antibodies (e.g., DMAb, ScFv antibody fragments, DICE or DBiTE) capable of generating an immune response in a subject, or biological functional fragments or variants thereof, and a composition for enhancing an immune response against an antigen in a subject in need thereof. In some embodiments, the antigen is CD19, BCMA, CD33, FAP, FSHR, EGFR, PSMA, CD123, or Her2. In some embodiments, the synthetic antibody of the present invention is a DBiTE comprising an scFv targeting CD19, BCMA, CD33, FAP, FSHR, EGFR, PSMA, CD123 or Her2.

[0188] T cell-specific receptor In one embodiment, the DBiTE or DICE of the present invention comprises an scFv of a T cell-specific receptor. T cell-specific receptors include, but are not limited to, CD3, TCR, CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5, CD40, FcgR, FceR, FcaR, and CD95.

[0189] Excipients and other components of the composition The composition may further comprise a pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient can be a functional molecule such as a vehicle, carrier, or diluent. A pharmaceutically acceptable excipient can be a transfection enhancer, which can include surfactants such as immune-stimulating complexes (ISCOMs), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection enhancers.

[0190] The transfection promoter is a polyanion, a polycation containing poly-L-glutamic acid (LGS), or a lipid. The transfection promoter is poly-L-glutamic acid, and the poly-L-glutamic acid can be present in the composition at a concentration of less than 6 mg / ml. The transfection promoter can also include surfactants such as immune-stimulating complexes (ISCOMs), Freund's incomplete adjuvant, LPS analogs containing monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene, and hyaluronic acid can also be administered and used together with the composition. The composition can also include liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection promoters, such as transfection promoters that are lipids, lecithin liposomes, or DNA-liposome mixtures (see, for example, W09324640), or other liposomes known in the art. The transfection promoter is a polyanion, a polycation containing poly-L-glutamic acid (LGS), or a lipid. The concentration of the transfection agent in the composition is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

[0191] The composition can further include a gene promoter described in U.S. Patent Application No. 021,579, filed April 1, 1994, which is incorporated herein by reference in its entirety.

[0192] The composition may contain DNA in an amount of about 1 nanogram to 100 milligrams, about 1 microgram to about 10 milligrams, or preferably about 0.1 microgram to about 10 milligrams, or more preferably about 1 milligram to about 2 milligrams. In some preferred embodiments, the composition according to the present invention contains about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the composition may contain about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the composition may contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the composition may contain about 1 to about 350 micrograms of DNA. In some preferred embodiments, the composition may contain about 25 to about 250 micrograms, about 100 to about 200 micrograms, about 1 nanogram to 100 milligrams, about 1 microgram to about 10 milligrams, about 0.1 microgram to about 10 milligrams, about 1 milligram to about 2 milligrams, about 5 nanograms to about 1000 micrograms, about 10 nanograms to about 800 micrograms, about 0.1 to about 500 micrograms, about 1 to about 350 micrograms, about 25 to about 250 micrograms, about 100 to about 200 micrograms of DNA.

[0193] The composition may be formulated according to the mode of administration used. Injectable pharmaceutical compositions may be sterile, pyrogen-free, and particle-free. Isotonic formulations or solutions may be used. Additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The composition may contain a vasoconstrictor. Isotonic solutions may include phosphate buffered saline. The composition may further contain stabilizers including gelatin and albumin. The stabilizer may enable a formulation containing LGS or a polycation or polyanion to be stable for a long time at room temperature or ambient temperature.

[0194] Method for generating synthetic antibodies The present invention also relates to a method for generating synthetic antibodies. The method can include administering the composition to a subject in need thereof by using a delivery method described in more detail below. Thus, the synthetic antibodies are generated in the subject or in vivo when the composition is administered to the subject.

[0195] The method can also include introducing the composition into one or more cells, and thus, the synthetic antibodies can be generated or produced within the one or more cells. The method can further include introducing the composition into one or more tissues, such as, but not limited to, skin and muscle, and thus, the synthetic antibodies can be generated or produced in the one or more tissues.

[0196] Delivery methods of the composition The present invention also relates to a method for delivering a composition to a subject in need thereof. The delivery method can include administering the composition to the subject. Administration can include, but is not limited to, DNA injection, with and without in vivo electroporation, liposome-mediated delivery, and nanoparticle-facilitated delivery.

[0197] The mammal receiving the delivery of the composition can be a human, a primate, a non-human primate, a cow, cattle, a sheep, a goat, an antelope, a bison, a swine, a bison, a bovid, a deer, a guinea pig, an elephant, a llama, an alpaca, a mouse, a rat, and a chicken.

[0198] The composition can be administered by different routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, intrabuccal, intrathoracic, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, and intraarticular, or combinations thereof. For veterinary use, the composition can be administered as a suitably acceptable formulation following normal veterinary practice. A veterinarian can readily determine the most appropriate dosing regimen and route of administration for a particular animal. The composition may be administered by conventional syringes, needleless injection devices, "microprojectile bombardment gone gun", or other physical methods such as electroporation ("EP"), "hydrodynamic methods", or ultrasound.

[0199] Electroporation Administration of the composition via electroporation can be achieved using an electroporation device configured to deliver an energy pulse effective to form reversible pores within the cell membrane to a desired tissue of a mammal, and preferably, the energy pulse is a constant current similar to a pre-set current input by the user. The electroporation device can comprise an electroporation component and an electrode assembly or a handle assembly. The electroporation component can include one or more of the various elements of the electroporation device, including a controller, a current waveform generator, an impedance tester, a waveform logger, an input element, a status reporting element, a communication port, a memory component, a power source, and a power switch, and can be incorporated. Electroporation can be achieved using an in vivo electroporation device, such as the CELLECTRA EP System (Inovio Pharmaceuticals, Plymouth Meeting, PA) or the Elgen electroporator (Inovio Pharmaceuticals, Plymouth Meeting, PA), to facilitate transfection of cells by a plasmid.

[0200] An electroporation component can function as one element of an electroporation device, and other elements are separate elements (or components) that communicate with the electroporation component. The electroporation component can function as two or more elements of the electroporation device and can communicate with still other elements of an electroporation device separate from the electroporation component. Elements of an electroporation device that exist as part of one electromechanical device or mechanical device need not be limited since the elements can function as one device or as separate elements that communicate with each other. The electroporation component may be capable of delivering energy pulses that generate a constant current within a desired tissue and includes a feedback mechanism. The electrode assembly may include an electrode array having a plurality of spatially arranged electrodes, and the electrode assembly receives the energy pulse from the electroporation component and delivers it through the electrodes to the desired tissue. At least one of the plurality of electrodes is neutral during delivery of the energy pulse, measures the impedance of the desired tissue, and communicates that impedance to the electroporation component. The feedback mechanism can receive the measured impedance and adjust the energy pulse delivered by the electroporation component to maintain a constant current.

[0201] The plurality of electrodes can deliver energy pulses in a dispersed pattern. The plurality of electrodes can deliver energy pulses in a dispersed pattern through control of the electrodes under a programmed sequence, and the programmed sequence is input into the electroporation component by a user. The programmed array may include a plurality of pulses delivered in the array, and each pulse of the plurality of pulses is delivered by at least two active electrodes having one neutral electrode that measures impedance, and a subsequent pulse of the plurality of pulses is delivered by a different one of the at least two active electrodes having one neutral electrode that measures impedance.

[0202] The feedback mechanism can be implemented by either hardware or software. The feedback mechanism can be implemented by an analog closed-loop circuit. The feedback occurs every 50 μs, 20 μs, 10 μs, or 1 μs, but preferably is real-time feedback or immediate (i.e., substantially immediate as determined by available techniques for determining response time). The neutral electrode can measure the impedance within the desired tissue and transmit the impedance to the feedback mechanism, which responds to the impedance and adjusts the energy pulses to maintain a constant current at a value similar to a preset current. The feedback mechanism can maintain a constant current continuously and immediately during the delivery of the energy pulses.

[0203] Examples of electroporation devices and electroporation methods that can facilitate delivery of the compositions of the present invention include those described in U.S. Patent No. 7,245,963 to Draghia-Akli et al. and U.S. Patent Publication No. 2005 / 0052630 filed by Smith et al., the contents of which are incorporated herein by reference in their entirety. Other electroporation devices and electroporation methods that can be used to facilitate delivery of the composition are provided in co-owned U.S. Patent Application No. 11 / 874,072, filed October 17, 2007, claiming priority to U.S. Provisional Patent Application No. 60 / 852,149, filed October 17, 2006, and U.S. Provisional Patent Application No. 60 / 978,982, filed October 10, 2007, all of which are incorporated herein by reference in their entirety under 35 U.S.C. § 119(e).

[0204] U.S. Patent No. 7,245,963 by Draghia-Akli et al. describes a modular electrode system and their use for facilitating the introduction of biomolecules into cells of selected tissues of a living body or a plant. The modular electrode system can include a plurality of needle electrodes, an electrical connector providing an electrical link from a programmable constant current pulse controller to the plurality of needle electrodes, and a power source. An operator can grasp the plurality of needle electrodes attached to a support structure and firmly insert them into selected tissues of a living body or a plant. Then, the biomolecules are delivered to the selected tissues via a hypodermic needle. The programmable constant current pulse controller is activated and a constant current electrical pulse is applied to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of the biomolecules into the cells between the plurality of electrodes. The entire content of U.S. Patent No. 7,245,963 is incorporated herein by reference.

[0205] U.S. Patent Publication No. 2005 / 0052630, filed by Smith et al., describes an electroporation device that can be used to effectively facilitate the introduction of biomolecules into cells of selected tissues within a living body or a plant. The electroporation device includes an electrokinetic device (an "EKD device") whose operation is specified by software or firmware. The EKD device generates a series of programmable constant current pulse patterns between electrodes within an array based on user control and input of pulse parameters, and enables the storage and acquisition of current waveform data. The electroporation device also includes a replaceable electrode disk having an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The entire content of U.S. Patent Publication No. 2005 / 0052630 is incorporated herein by reference.

[0206] The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 0052630 can be adapted for deep penetration into not only tissues such as muscle, but also other tissues or organs. Due to the configuration of the electrode array, an injection needle (for delivering a selected biomolecule) is also fully inserted into the target organ, and the injection is administered perpendicular to the target tissue in an area pre-marked by the electrodes. The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent No. 2005 / 005263 are preferably 20 mm in length and 21 gauge.

[0207] In addition, in some embodiments incorporating an electroporation device and its use, the following patents are contemplated: U.S. Patent No. 5,273,525 issued on December 28, 1993, U.S. Patent No. 6,110,161 issued on August 29, 2000, U.S. Patent No. 6,261,281 issued on July 17, 2001, and U.S. Patent No. 6,958,060 issued on October 25, 2005, and U.S. Patent No. 6,939,862 issued on September 6, 2005, which are electroporation devices as described therein. Further, patents encompassing the subject matter provided in U.S. Patent No. 6,697,669 issued on February 24, 2004, regarding the delivery of DNA using any of a variety of devices, and U.S. Patent No. 7,328,064 issued on February 5, 2008, which focused on methods of injecting DNA, are contemplated herein. The above patents are hereby incorporated by reference in their entirety.

[0208] Treatment methods Also provided herein is a method of treating, protecting against, and / or preventing a disease in a subject in need thereof by generating a synthetic antibody (e.g., a DMAb, scFv fragment, or DBiTE) in the subject. The method can include administering a composition to the subject. Administration of the composition to the subject can be performed using the delivery methods described above.

[0209] In certain embodiments, the present invention provides methods for treating, protecting against, and / or preventing cancer. In one embodiment, the method treats, protects against, and / or prevents tumor growth. In one embodiment, the method treats, protects against, and / or prevents cancer progression. In one embodiment, the method treats, protects against, and / or prevents cancer metastasis.

[0210] In one embodiment, the present invention provides a method for preventing the growth of benign tumors such as, but not limited to, uterine fibroids. The method includes administering to a subject diagnosed with a benign tumor one or more of the compositions of the present invention in an effective amount.

[0211] When a synthetic antibody (e.g., a DMAb, scFv fragment, or DBiTE) is generated in a subject, the synthetic antibody (e.g., a DMAb, scFv fragment, or DBiTE) can bind to or react with an antigen. Such binding can neutralize the antigen, block recognition of the antigen by another molecule, such as a protein or nucleic acid, induce or elicit an immune response against the antigen, thereby treating, protecting against, and / or preventing a disease associated with the antigen in the subject.

[0212] The composition dosage can be from 1 μg to 10 mg of active ingredient / kg body weight / dose and can be from 20 μg to 10 mg of ingredient / kg body weight / dose. The composition can be administered once a day, twice a day, three times a day, four times a day, five times a day, six times a day, seven times a day, eight times a day, nine times a day, ten times a day, eleven times a day, twelve times a day, thirteen times a day, fourteen times a day, fifteen times a day, sixteen times a day, seventeen times a day, eighteen times a day, nineteen times a day, twenty times a day, twenty-one times a day, twenty-two times a day, twenty-three times a day, twenty-four times a day, twenty-five times a day, twenty-six times a day, twenty-seven times a day, twenty-eight times a day, twenty-nine times a day, thirty times a day, or thirty-one times a day. The number of composition dosages for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0213] Cancer treatment The present invention provides a method for treating or preventing cancer, or a method for treating and preventing tumor growth or metastasis. Related aspects of the present invention provide a method for preventing, assisting in prevention, and / or reducing hyperplasia or metastasis of tumor cells in an individual.

[0214] One aspect of the present invention provides a method for inhibiting metastasis in an individual in need thereof, the method comprising administering to the individual an effective amount of a composition of the present invention. The present invention further provides a method for inhibiting metastasis in an individual in need thereof, the method comprising administering to the individual an effective amount of any one of the compositions described herein that inhibits metastasis.

[0215] In some embodiments for treating or preventing cancer in an individual in need thereof, or for treating and preventing tumor metastasis, a second agent such as an anti-tumor agent is administered to the individual. In some embodiments, the second agent comprises a second metastasis inhibitor such as a plasminogen antagonist or an adenosine deaminase antagonist. In other embodiments, the second agent is an angiogenesis inhibitor.

[0216] The compositions of the present invention can be used to prevent, abate, minimize, control, and / or lessen cancer in humans and animals. The compositions of the present invention can also be used to slow the rate of primary tumor growth. The compositions of the present invention can be used to halt the spread of cancer cells when administered to a subject in need of treatment. Thus, the compositions of the present invention can be administered as part of a combination therapy with one or more other drugs or agents. When used as part of a combination therapy, the reduction in metastasis and the reduction in primary tumor growth obtained by the compositions of the present invention enable a more effective and efficient use of any pharmaceutical or drug therapy used to treat the patient. In addition, the control of metastasis by the compositions of the present invention provides a greater ability to localize the disease in one location in the subject.

[0217] In one embodiment, the present invention provides a method for preventing the metastasis of malignant tumors or other cancer cells, as well as a method for reducing the tumor growth rate. The method includes administering an effective amount of one or more of the compositions of the present invention to a subject diagnosed with a malignant tumor or cancerous cells, or a subject having a tumor or cancer cells.

[0218] The following are non-limiting examples of cancers that can be treated by the methods and compositions of the present invention: acute lymphoblastic; acute myeloid leukemia; adrenocortical carcinoma; adrenocortical carcinoma, childhood; appendiceal cancer; basal cell carcinoma; extrahepatic bile duct cancer; bladder cancer; bone cancer; osteosarcoma and malignant fibrous histiocytoma; brainstem glioma, childhood; brain tumor, adult; brain tumor, brainstem glioma, childhood; brain tumor, central nervous system atypical teratoid / rhabdoid tumor, childhood; central nervous system germ cell tumor; cerebellar astrocytoma; cerebral astrocytoma / malignant glioma; craniopharyngioma; ependymoblastoma; ependymoma; pineal parenchymal tumor of intermediate differentiation; supratentorial primitive neuroectodermal tumor and pineoblastoma; visual pathway and hypothalamic glioma; brain and spinal cord tumors; breast cancer; bronchial tumor; Burkitt lymphoma; carcinoid tumor; carcinoid tumor, gastrointestinal; central nervous system atypical teratoid / rhabdoid tumor; central nervous system germ cell tumor; central nervous system lymphoma; cerebellar astrocytoma cerebral astrocytoma / malignant glioma, childhood; cervical cancer; chordoma, childhood; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; esophageal cancer; Ewing family of tumors; extragonadal germ cell tumor; extrahepatic bile duct cancer; eye cancer, intraocular melanoma; eye cancer, retinoblastoma; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor (GIST); germ cell tumor, extracranial; germ cell tumor, extragonadal; germ cell tumor, ovarian; gestational trophoblastic tumor; glioma; glioma, pediatric brainstem; glioma, pediatric cerebellar astrocytoma; glioma, pediatric visual pathway and hypothalamic; hairy cell leukemia; head and neck cancer; hepatocellular (liver) cancer; histiocytosis, Langerhans cell; Hodgkin lymphoma; hypopharyngeal cancer; hypothalamic and visual pathway glioma; intraocular melanoma; islet cell tumor; kidney (renal cell) cancer; Langerhans cell histiocytosis; laryngeal cancer; leukemia, acute lymphoblastic; leukemia, acute myeloid; leukemia, chronic lymphocytic; leukemia, chronic myeloid; leukemia, hairy cell; lip and oral cavity cancer; liver cancer; lung cancer, non-small cell; lung cancer, small cell; lymphoma, AIDS-related; lymphoma, Burkitt; lymphoma, cutaneous T-cell; lymphoma, Hodgkin; lymphoma, non-Hodgkin; lymphoma, primary central nervous system; macroglobulinemia, Waldenström; malignant fibrous histiocytoma of bone and osteosarcoma; medulloblastoma; melanoma; melanoma, intraocular (eye); Merkel cell carcinoma; mesothelioma;Metastatic cervical squamous cell carcinoma with occult primary; Oral cancer; Multiple endocrine neoplasia syndromes, (pediatric); Multiple myeloma / plasma cell neoplasms; Mycosis; Mycetoma; Myelodysplastic syndromes; Myelodysplastic / myeloproliferative diseases; Myeloid leukemia, chronic; Myeloid leukemia, adult acute; Myeloid leukemia, pediatric acute; Myeloma, multiple; Myeloproliferative diseases, chronic; Nasal and paranasal cavity cancer; Nasopharyngeal cancer; Neuroblastoma; Non-small cell lung cancer; Oral cancer; Oral cavity cancer; Oropharyngeal cancer; Osteosarcoma and malignant fibrous histiocytoma of bone; Ovarian cancer; Ovarian epithelial cancer; Ovarian germ cell tumors; Ovarian low malignant potential tumors; Pancreatic cancer; Pancreatic cancer, islet cell tumors; Papillomatosis; Parathyroid cancer; Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pineal parenchymal tumors of intermediate differentiation; Pineoblastoma and supratentorial primitive neuroectodermal tumors; Pituitary tumors; Plasma cell neoplasms / multiple myeloma; Pleuropulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Renal cell (kidney) cancer; Renal pelvis and ureter, transitional cell carcinoma; Respiratory carcinomas involving the NUT gene on chromosome 15; Retinoblastoma; Rhabdomyosarcoma; Salivary gland cancer; Sarcomas, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, soft tissue; Sarcoma, uterine; Sézary syndrome; Skin cancer (non-melanoma); Skin cancer (melanoma); Skin cancer, Merkel cell carcinoma; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma, metastatic cervical squamous cell carcinoma with occult primary; Stomach (gastric) cancer; Supratentorial primitive neuroectodermal tumors; T-cell lymphoma, cutaneous; Testicular cancer; Throat cancer; Thymoma and thymic carcinoma; Thyroid cancer; Transitional cell carcinoma of the renal pelvis and ureter; Trophoblastic tumors, gestational; Urethral cancer; Uterine cancer, endometrial; Uterine sarcoma; Vaginal cancer; Vulvar cancer; Waldenström macroglobulinemia, as well as Wilms tumor.;

[0219] In one embodiment, the present invention provides a method for treating cancer metastasis, comprising treating a subject with a complementary therapy for cancer, such as surgery, chemotherapy, chemotherapeutic agents, radiation therapy, or hormone therapy, or combinations thereof, before, simultaneously with, or after treatment with the composition of the present invention.

[0220] As chemotherapeutic agents, cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramustine sodium phosphate, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, recombinant interferon alpha-2a, paclitaxel, teniposide, and streptozocin), cytotoxic alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethyl sulfonate), alkylating agents (e.g., azaleic acid, AZQ, BCNU, busulfan, bisulfan, carboxyphthalato platinum, CBDCA, CCNU, CHIP, chlorambucil, chloroazotocin, cisplatin, chromeson, cyanomorpholino doxorubicin, cyclodizon, cyclophosphamide, dianhydrogalactitol, fluorodopan, hepsulfam, hicanthone, ifosfamide, melphalan, methyl CCNU, mitomycin C, mitozolomide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teloxiron, tetraplatin, thiotepa, triethylenemelamine, uracil nitrogen mustard, and Yoshi-864), antimitotic agents (e.g., allocolchicine, halicondrin M, colchicine, colchicine derivatives, dolastatin 10, maytansine, lysocine, paclitaxel derivatives, paclitaxel, thiocolchicine, tritylcysteine, vinblastine sulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mitramycin, mitomycin, daunorubicin, VP-16-213, VM-26, navelbine, and taxotere), biological agents (e.g., alpha interferon, BCG, G-CSF, GM-CSF, and interleukin-2),Topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives, and morpholinodoxorubicin), topoisomerase II inhibitors (e.g., mitoxantrone, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyl daunomycin, oxantrazole, rubidazone, VM-26 and VP-16), and compounds (e.g., hydroxyurea, procarbazine, o,p’-DDD, dacarbazine, CCNU, BCNU, cis-diamine dichloroplatinum, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, Gliadel and porfimer sodium) are included.

[0221] An antiproliferative agent is a compound that reduces cell proliferation. Antiproliferative agents include alkylating agents, antimetabolites, enzymes, biological response modifiers, other agents, hormones and antagonists, androgen inhibitors (e.g., flutamide and leuprolide acetate), antiestrogens (e.g., tamoxifen citrate and its analogs, toremifene, droloxifene, and raloxifene), and additional examples of specific antiproliferative agents include, but are not limited to, levamisole, gallium nitrate, granisetron, strontium-89 chloride, filgrastim, pilocarpine, dexrazoxane, and ondansetron.

[0222] The compounds of the present invention can be administered alone or in combination with other anti-tumor agents including cytotoxic / anti-tumor agents and anti-angiogenic agents. Cytotoxic / anti-tumor agents are defined as agents that attack and kill cancer cells. Some cytotoxic / anti-tumor agents are alkylating agents that alkylate the genetic material of tumor cells, such as cisplatin, cyclophosphamide, nitrogen mustard, trimethylenethiophosphoramide, carmustine, busulfan, chlorambucil, bendamustine, uracil mustard, chromafazine, and dacarbazine. Other cytotoxic / anti-tumor agents are antimetabolites of tumor cells, such as cytosine arabinoside, fluorouracil, methotrexate, mercaptopurine, azathioprine, and procarbazine. Other cytotoxic / anti-tumor agents are antibiotics, such as doxorubicin, bleomycin, dactinomycin, daunorubicin, mitomycin, mitomycin C, and daunomycin. There are numerous commercially available liposomal formulations for these compounds. Still other cytotoxic / anti-tumor agents are mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine, and etoposide. Other cytotoxic / anti-tumor agents include taxol and its derivatives, L-asparaginase, anti-tumor antibodies, dacarbazine, azacitidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.

[0223] Anti-angiogenic agents are well known to those skilled in the art. Anti-angiogenic agents suitable for use in the methods and compositions of the present invention include anti-VEGF antibodies, including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligonucleotides. Other known inhibitors of angiogenesis include angiostatin, endostatin, interferon, interleukin 1 (including alpha and beta), interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinase-1 and -2 (TIMP-1 and -2). Small molecules including topoisomerase, such as razoxane, topoisomerase II inhibitors having anti-angiogenic activity, can also be used.

[0224] Other anti-cancer agents that can be used in combination with the composition of the present invention include, but are not limited to: ashibicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine, amphomycin, ametantrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, broxuridine, busulfan, calicheamicin, caracemide, carbetimer, carboplatin, carmustine, carboquone hydrochloride, carzelesin, cerivastatin, chlorambucil, cirolemycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, drostanolone propionate, duazomycin, edatrexate, efloxatin hydrochloride, elsamitrucin, enloplatin, empromate, epipropidine, epirubicin hydrochloride, erbulozole, esorubicin hydrochloride, estramustine, estramustine phosphate sodium, etanidazole, etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, fluorocytosine, fosquidone, fostriecin sodium, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, ilmofosine, interleukin II (including recombinant interleukin II, or rIL2), interferon alpha-2a, interferon alpha-2b, interferon alpha-n1, interferon alpha-n3, interferon beta-Ia, interferon gamma-Ib, iplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, ralitrexed hydrochloride,Romethrexol sodium, Romustine, Losoxantrone hydrochloride, Masoprocol, Maytansine, Mechlorethamine hydrochloride, Megestrol acetate, Melenegestrol acetate, Melphalan, Menogaril, Mercaptopurine, Methotrexate, Methotrexate sodium, Methoprine, Metsuredepa, Mitindomide, Mitocalcin, Mitochromin, Mitogirin, Mitomarcin, Mitomycin, Mitosper, Mitotan, Mitoxantrone hydrochloride, Mycophenolic acid, Nocodazole, Nogalamycin, Ormaplatin, Oxisuran, Paclitaxel, Pegaspargase, Periomycin, Pentamustine, Pepromycin sulfate, Perfosfamide, Pipobroman, Piposulfan, Pyroxantrone hydrochloride, Plicamycin, Promestane, Porfimer sodium, Porfiromycin, Prednimustine, Procarbazine hydrochloride, Puromycin, Puromycin hydrochloride, Pyrazofurin, Riboprine, Logretimide, Safingol, Safingol hydrochloride, Semustine, Simtrazene, Sparfosate sodium, Sparsomycin, Spirogermanium hydrochloride, SpiroMustine, Spiroplatin, Streptozocin, Streptozotocin, Slofenur, Talisomycin, Tegogalan sodium, Tegafur, Teloxantrone hydrochloride, Temoporfin, Teniposide, Teloxiron, Testolactone, Thiamiprine, Thioguanine, Thiotepa, Thiazofurin, Tirapazamine, Toremifene citrate, Trenbolone acetate, Trisciribine phosphate, Trimethoprim, Trimethoprim glucuronate, Triptorelin, Tributuzumab, Uracil mustard, Uredepa, Bapreotide, Verteporfin, Vinblastine sulfate, Vincristine sulfate, Vindesine, Vindesine sulfate, Vinepidine sulfate, Vinglycinate sulfate, Vinleurosine sulfate, Vinorelbine tartrate, Vinrosidine sulfate, Vinzolidine sulfate, Borozole, Zeniplatin, Dinostatin, Zorubicin hydrochloride. Other anticancer agents include, but are not limited to: 20-epi-1,25-dihydroxyvitamin D3, 5-ethynyluracil, Abiraterone, Aclarubicin, Acylfulvene, Adesipenol, Adzelesin, Aldesleukin, ALL-TK antagonist, Altretamine, Ambamustine, Amidox, Amifostine,Aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitor, antagonist D, antagonist G, antarelix, anti-dorsalizing morphogenetic protein-1, anti-androgen, prostate cancer, anti-estrogen, anti-neoplastic agent, antisense oligonucleotide, glycic acid aphidicolin, apoptosis gene regulator, apoptosis regulator, applinic acid, ara-CDP-DL-PTBA, arginine deaminase, aslacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivative, baranol, batimastat, BCR / ABL antagonist, benzochlorin, benzoyl staurosporine, beta-lactam derivative, beta-aretin, beta-claramycin B, betulinic acid, bFGF inhibitor, bicalutamide, bisantrene, bisaziridinyl spermine, bisnafide, bistratene A, bizelesin, breflate, broxuridine, budotitane, buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivative, canarypox IL-2, capecitabine, carboxamide-amino-triazole, carboxamide triazole, CaRest M3, CARN700, cartilage-derived inhibitor, carzelesin, casein kinase inhibitor (ICOS), castanospermine, cecropin B, cetrorelix, chlorine, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, clomifene analog, clotrimazole, corismycin A, corismycin B, combretastatin A4, combretastatin analog, conagenin, crambescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivative, curacin A, cyclopentane trione, cycloplatam, sipeamycin, cytarabine ocfosfate, cytolytic factor, cytostatic, daclizumab, decitabine, dehydrodimdenine B, deslorelin, dexamethasone, dexifosfamide, dexrazoxane, dexverapamil, diaziquone, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol,9-, dioxamycin, diphenylspirostatin,Docetaxel, Docosanol, Dolasetron, Doxifluridine, Droloxifene, Dronabinol, Duocarmycin SA, Ebselen, Ecromycin, Edelfosine, Edrecolomab, Efrotomycin, Element, Emitefur, Epirubicin, Epristeride, Estramustine Analog, Estrogen Agonist, Estrogen Antagonist, Ethanidazole, Etoposide Phosphate, Exemestane, Fadrozole, Fludarabine, Flavopiridol, Flesinoxan, Fluasterone, Fluorodaunomycin Hydrochloride, Formestane, Fotemustine, Gadolinium Texaphyrin, Gallium Nitrate, Galocitabine, Ganirelix, Gelatinase Inhibitor, Gemcitabine, Glutathione Inhibitor, Hepsulfamide, Heregulin, Hexamethylenebisacetamide, Hypericin, Ibandronic Acid, Idarubicin, Idoxifene, Idramantane, Ilmofosine, Ilomastat, Imidazoacridone, Imiquimod, Immunostimulatory Peptide, Insulin-like Growth Factor-1 Receptor Inhibitor, Interferon Agonist, Interferon, Interleukin, Yobenguan, Iododoxorubicin, Ipomoeanol, 4-, Iroplact, Iloprost, Isobenzazole, Isohomohalicondrin B, Itasetron, Jasplakinolide, Kahalalide F, Laminarin-N Triacetate, Lanreotide, Rayamycin, Lenograstim, Lentinan Sulfate, Leptostatin, Letrozole, Leukemia Inhibitory Factor, Leukocyte Alpha Interferon, Leuprorelin + Estrogen + Progesterone, Leuprolide, Levamisole, Rialoxazole, Linear Polyamine Analog, Lipophilic Disaccharide Peptide, Lipophilic Platinum Compound, Lysoclimide 7, Lobaplatin, Lumbricin, Lomustine, Lonidamine, Losoxantrone, Lovastatin, Roxifiban, Raltitrexed, Lutetium Texaphyrin, Lysophilin, Lytic Peptide, Mitansine, Mannostatin A, Marimastat, Masoprocol, Maspin, Matrilysin Inhibitor, Matrix Metalloproteinase Inhibitor, Menogaril, Melvalonate, Metecloric Acid, Methioninase, Metoclopramide, MIF Inhibitor,Mifepristone, Miltefosine, Milimostim, Mismatch double-stranded RNA, Mitoguazone, Mitolactol, Mitomycin analog, Mitonafide, Mitotoxin fibroblast growth factor-Saporin, Mitoxantrone, Mofarotene, Molgramostim, Monoclonal antibody, Human chorionic gonadotropin, Monophosphoryl lipid A + Mycobacterium cell wall sk, Mopidamol, Multidrug resistance gene inhibitor, Multiple tumor suppressor 1-based therapy, Mustard anticancer agent, Micaperoxide B, Mycobacterium cell wall extract, Miliappolone, N-acetyl dinarine, N-substituted benzamide, Napharelin, Nagrestip, Naloxone + Pentazocine, Napabine, Napterpin, Nartograstim, Nedaplatin, Nemorubicin, Neridronic acid, Neutral endopeptidase, Nilutamide, Nisamycin, Nitric oxide regulator, Nitroxide antioxidant, Nitrilin, O6-benzylguanine, Octreotide, Oxenone, Oligonucleotide, Onapristone, Ondansetron, Ondansetron, Oracin, Oral cytokine inducer, Ormaplatin, Osaterone, Oxaliplatin, Oxauromycin, Paclitaxel, Paclitaxel analog, Paclitaxel derivative, Parauramine, Palmitoyl lysophosphatidylcholine, Pamidronic acid, Panaxytriol, Panomifene, Parabactin, Pazelliptine, Pegaspargase, Perdesin, Pentosan polysulfate sodium, Pentostatin, Pentrozole, Perflubron, Perfosfamide, Perillyl alcohol, Phenazinomycin, Phenyl acetate, Phosphatase inhibitor, Picibanil, Pilocarpine hydrochloride, Pirarubicin, Pyrithioxime, Placetin A, Placetin B, Plasminogen activator inhibitor, Platinum complex, Platinum compound, Platinum triamine complex, Porfimer sodium, Porfiromycin, Prednisone, Propylbis-acridone, Prostaglandin J2, Proteasome inhibitor, Protein A-based immunomodulator, Protein kinase C inhibitor, Protein kinase C inhibitor, Microalgae, Protein tyrosine phosphatase inhibitor, Purine nucleoside phosphorylase inhibitor, Purpurin, Pyrazoloacridine, Pyridoxylated hemoglobin polyoxyethylene conjugate, Raf antagonist, Raltitrexed, Ramoseron,Ras farnesyl protein transferase inhibitor, Ras inhibitor, Ras-GAP inhibitor, demethylated retinoic peptide, rhenium Re186 etidronate, lysokinase, ribozyme, RII retinamide, logretimide, rohithicine, romurtide, roquinimex, rubiginone B1, ruboxyl, safingol, santopin, SarCNU, sarcophytol A, sargramostim, Sdi1 mimetic, semustine, aging-derived inhibitor 1, sense oligonucleotide, signal transduction inhibitor, signal transduction regulator, single-chain antigen-binding protein, sizofiran, sobuzoxane, sodium borocaptate, sodium phenylacetate, sorberol, somatomedin-binding protein, sonermin, sparfosic acid, spicamycin D, spiroxamine, sprengolentin, spongistatin 1, squalamine, stem cell inhibitor, stem cell division inhibitor, stipiamide, stromelysin inhibitor, sulfinosine, superactive intestinal peptide antagonist, sladistat, slamine, swine sonine, synthetic glycosaminoglycan, talimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium, tegafur, terlapirium, telomerase inhibitor, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine, taliblastine, thiocholin, thrombopoietin, thrombopoietin mimetic, timalphasin, thymopoietin receptor agonist, thymotrinan, thyroid-stimulating hormone, tin ethyl etiopurpurin, tirapazamine, titanocene dichloride, topsecentin, toremifene, totipotent stem cell factor, translation inhibitor, tretinoin, triacetyluridine, triciribine, trimethotrexate, tryptoreline, tropisetron, turosteride, tyrosine kinase inhibitor, tilostatin, UBC inhibitor, ubenimex, urogenital sinus-derived growth inhibitor, urokinase receptor antagonist, vapreotide, variolin B, vector system, erythrocyte gene therapy, veraresol, veramine, bergenin, verteporfin, vinorelbine, vinca sulfate, vitaxin, borozole, zanolteron, zinoplatin, dilascorb, and dinostatin stimulator. In one embodiment, the anticancer drug is 5-fluorouracil, taxol, or leucovorin.

[0225] In vitro and ex vivo generation of synthetic antibodies In one embodiment, synthetic antibodies (e.g., DMAb, ScFv fragments or DBiTE) are generated in vitro or ex vivo. For example, in one embodiment, a nucleic acid encoding a synthetic antibody (e.g., DMAb, ScFv fragment or DBiTE) can be introduced and expressed in in vitro or ex vivo cells. Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vector can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, the expression vector can be introduced into host cells by physical, chemical, or biological means.

[0226] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0227] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA vectors and RNA vectors. Viral vectors, and in particular retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from, for example, lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses. See, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0228] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0229] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with a lipid. Nucleic acids associated with lipids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, bound to liposomes via a linking molecule associated with both liposomes and oligonucleotides, trapped by liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained within micelles, or complexed with micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they can exist as bilayer structures as micelles or can have a "collapsed" structure. They can also be simply dispersed in solution and may form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include classes of compounds containing fat droplets that occur naturally within the cytoplasm, as well as long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Examples

[0230] The present invention is further illustrated in the following examples. It should be understood that these examples are given by way of illustration only while showing preferred embodiments of the present invention. From the above discussion and these examples, those skilled in the art can identify the essential features of the present invention and make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from the spirit and scope thereof. Accordingly, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description in addition to those shown and described herein. Such modifications are also intended to be within the scope of the appended claims.

[0231] Example 1 The research presented in this specification demonstrates the development of DNA-encoded bispecific T cell engagers (DBiTEs) targeting CD19, BCMA, CD33, FAP, FSHR, EGFR, PSMA, or CD123. The DBiTE constructs are highly expressed in vivo. Figure 1 shows the expression of BCMA DBiTE, CD33 DBiTE, and CD123 DBiTE. Figure 2 shows the expression of EGFRvIII DBiTE, FSHR DBiTE, PSMA DBiTE, and CD19 DBiTE. These novel DBiTEs represent new tools for cancer immunotherapy.

[0232] Figures 3 - 5 provide data showing that CD19 DBiTE functions against both B cell depletion and T cell activation. PBMCs from three independent donors were cultured in triplicate for 5 hours in the presence of supernatants of 5 μl of CD19 DBiTE or control DBiTE (EGFRvIII DBiTE). After incubation, the cells were stained for B cell and T cell markers and the potential cytolytic activity against early activation of B cells (CD19+ cells) and T cells was determined. Figure 4 provides data showing that all three donors showed depletion of B cells (CD19+ cells in the PBMC mixture) in the presence of CD19 DBiTE and not in the presence of the control DBiTE. Figure 5 provides data showing that all three donors showed an increase in the early activation marker CD69 in T cells in the presence of CD19 DBiTE and not in the presence of the control DBiTE.

[0233] Furthermore, experiments were conducted to demonstrate the cytotoxicity of BCMA DBiTE. BCMA DBiTE supernatant or CD33 DBiTE supernatant was incubated with the RPMI8226 cell line for 5 hours, and the derived T cells formed one donor at tumor-to-T cell ratios of 1:0, 1:3, and 1:7 (10,000 tumor cells per well). After 5 hours of incubation, BCMA dBiTE was able to lyse cells at ratios of 1:1, 1:3, and 1:7, but not in the absence of T cells, and no death occurred under any conditions in the presence of CD33 DBiTE.

[0234] Example 2 The studies presented in this specification demonstrate the development of DNA monoclonal antibodies and BiTEs targeting HER2 (HER2DMAb and HER2DBiTE), and their use as therapeutic agents for the treatment of ovarian and breast cancers. Both the DMAb and DBiTE constructs are highly expressed in vitro and in vivo for approximately 4 months. HER2DMAb binds to HER2 and induces HER2 signaling blockade and antibody-dependent cytotoxicity. HER2DBiTE effectively induces the cytotoxicity of T cells against HER2+ tumor cells. These novel DNA technologies represent new tools for further research in cancer immunotherapy.

[0235] Materials and methods are described here. Animals and cell lines C57Bl / 6 and Nu / J mice were purchased from Jackson labs. NSG mice were purchased from the Wistar Institute Animal Facility.

[0236] OVCAR3, SKOV3, and Brpkp110 cells were provided by J. R. Conejo-Garcia (Department of Immunology, Moffitt Cancer Center, FL). TOV-21G and RNG1 were provided by R. Zhang (The Wistar Institute). OVCAR3 tumors were generated as described above by injecting 3 million cells in PBS / matrigel (50 / 50) into the flank (Perales-Puchalt et al., 2017, Clin Cancer Res, 23(2):441-53). RD and 293T cells were purchased from ATCC.

[0237] Mice were injected with 100 μg of DNA resuspended in 80 μl of water into the tibialis anterior muscle (40 μl per leg) with 200 IU / ml of hyaluronidase (Sigma), and were treated by electroporation with a CELLECTRA device 1 minute after injection.

[0238] Design of HER2DMAb and HER2DBiTE HER2DMAb encoding the codon-optimized sequences of the heavy and light chains of the anti-HER2 monoclonal antibody pertuzumab was designed and generated. Both antibody chains were positioned in sequences separated by P2A and furin cleavage sites. The IgE leader sequence was replaced with the original leader sequence. HER2DBiTE was designed by encoding the codon-optimized scFv of HER2DMAb, followed by the scFv of the OKT3 anti-human CD3 antibody, and adding an IgE leader sequence. Both constructs were subcloned into a modified pVAX1 expression vector (Figures 6A and 11A).

[0239] An empty modified pVAX1 plasmid was used as a negative control.

[0240] In vitro DMAb expression One million 293T cells were seeded into each chamber of a 6-well plate. The next day, the cells were transfected with 1 μg of the HER2DMAb plasmid using Lipofectamin 2000 (Invitrogen). The supernatant was collected 48 hours after transfection.

[0241] Flow cytometry The anti-human antibodies used were directly fluorescent dye-conjugated. HER2 (24D2), CD45 (HI30), CD3 (HIT3A), CD69 (FN50), PD-1 (EH12.2H7), and secondary anti-human IgG APC (polyclonal) were obtained from Biolegend. Exclusion of live / dead was performed with 7AAD (Invitrogen) and Annexin V (Biolegend).

[0242] Immunoblot Protein extraction, denaturation, and Western blotting were performed as described previously (Perales-Puchalt et al., 2017, Clin Cancer Res, 23(2):441-53). Membranes were blotted with polyclonal anti-human IgG (H+L) (Bethyl) and anti-β-actin (a5441, Sigma-Aldrich). Images were captured with an ImageQuant LAS4000 (GE Healthcare Life Sciences).

[0243] For the signal transduction inhibition experiment, 200,000 OVCAR3 cells were seeded in 6-well plates and starved overnight in serum-free medium. The next day, 10 μg of purified HER2DMAb or PBS was added to the appropriate wells for 1 hour, followed by 10 ng / ml of HRG (Peprotech) for 30 minutes.

[0244] HER2 Binding ELISA ELISA plates were coated with 1 μg / ml of human HER2 recombinant protein (abcam) overnight at 4°C. Blocking was performed with PBST-10% FBS for 1 hour. Serum from HER2DMAb-expressing mice at different dilutions or controls (electroporated with empty pVax plasmid) was used as the primary antibody, and the incubation was performed at room temperature for 1 hour. The secondary antibody was goat anti-human IgG Fc HRP conjugate (Bethyl). After a 1-hour incubation, development was performed with SIGMAFAST OPD (Sigma Aldrich) and read at 450 nm.

[0245] DMAb Quantitative ELISA ELISA plates were coated with 1 μg / ml of goat anti-human IgG-Fc fragment antibody (Bethyl) overnight at 4°C. The next day, they were blocked with PBST-10% FBS for 1 hour at room temperature, washed, incubated with samples diluted in PBST-1% FBS for 1 hour at room temperature, washed, and incubated with HRP-conjugated goat anti-human kappa light chain antibody (Bethyl) at room temperature. After 1 hour of incubation, they were developed with SIGMAFAST OPD (Sigma Aldrich) and read at 450 nm. A standard curve was generated using purified human IgG / kappa (Bethyl).

[0246] CD3 and HER2 binding ELISA (DBiTE) ELISA plates were coated with 1 μg / ml of human HER2 recombinant protein (abcam) or human CD3 epsilon (Acrobiosystems) overnight at 4°C. They were blocked with PBST-10% FBS for 1 hour. Serum from HER2 DBiTE-expressing mice or controls (electroporated with empty pVax plasmid) was used as the primary antibody. They were incubated for 1 hour at room temperature. The secondary antibody was goat anti-human IgG H+L HRP conjugate (Bethyl). After 1 hour of incubation, the plates were developed with SIGMAFAST OPD (Sigma Aldrich) and read at 450 nm.

[0247] Detection of anti-HER2 DMAb and HER2 DBiTE antibodies ELISA plates were coated with 1 μg / ml of purified HER2 DMAb or HER2 DBiTE overnight at 4°C. The next day, the plates were blocked with PBST-10% FBS for 1 hour at room temperature, washed, incubated with samples diluted in PBST-1% FBS for 1 hour at room temperature, washed, and incubated with HRP-conjugated goat anti-mouse IgG antibody (Abcam) at room temperature. After 1 hour of incubation, the plates were developed with SIGMAFAST OPD (Sigma Aldrich).

[0248] Detection of T cell activation and apoptosis by HER2 DBiTE A 96-well plate was seeded with 5,000 OVCAR3 overnight at 4°C. The next day, serum from HER2 DBiTE-expressing mice or pVax control (diluted 1:20 in PBS, 100 μl) and 50,000 T cells were added and the plate was incubated at 37°C. After 24 hours, supernatant was taken for IFNγ ELISA and fresh supernatant was added. After 72 hours, flow cytometry was performed to measure T cell apoptosis and activation (CD3, CD69, PD-1, annexin V). For cell counts, 5,000 OVCAR3 were seeded with 100,000 T cells and viable T cell numbers were counted using the dead cell exclusion dye Trypan Blue (ThermoFisher) and the Countess II automated cell counter (ThermoFisher).

[0249] Interferon gamma ELISA Determination of human interferon gamma from the supernatant was performed using Human IFNg ELISA MAX (Biolegend) according to the manufacturer's instructions.

[0250] In vitro cytotoxicity 10,000 OVCAR3 cells per well were seeded in a 96-well plate and, 18 hours later, incubated for 4 hours with 500,000 human PBMCs from healthy donors (provided by the University of Pennsylvania Human Immunology Core) or 500,000 splenocytes from nude mice, in the presence or absence of HER2DMAb. After 4 hours, the supernatant was collected, the cells were trypsinized, stained for 7AAD (Invitrogen), annexin V (Biolegend), and anti-human CD45 (Biolegend), and a flow cytometry-based cytotoxicity assay was performed as described above (Perales-Puchalt et al., 2017, Clin Cancer Res, 23(2):441-53). Alternatively, OVCAR3 or MDA-MB-231 expressing luciferase was used and luciferase expression was measured after co-culture. For the BiTE killing assay, 10,000 OVCAR3-luciferase cells were incubated for 5 hours with T cells at different ratios, washed with PBS, lysed, and luciferase expression was measured.

[0251] Antibody-dependent cell phagocytosis Macrophages were differentiated from human monocytes by seeding 1 million monocytes per T25 with 50 ng / ml human M-CSF (Peprotech). The medium with cytokines was changed on days 3 and 6. On day 6, the macrophages were trypsinized, stained with CellTrace Violet (Invitrogen) according to the manufacturer's instructions, seeded at 50,000 / well in a 96-well plate, and left overnight with 20 ng / ml M-CSF. On day 7, the OVCAR3 cells were stained with CFSE (Invitrogen) and 10,000 OVCAR3 cells were seeded on top of the macrophages with HER2DMAb or pVax serum. After 24 hours, the cells were trypsinized and flow cytometry was performed. Phagocytosis was measured as double-positive stained cells.

[0252] Immunofluorescence Mouse tumors were frozen in OCT (TissueTek), and frozen sections were cut. The slides were then fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. The sections were blocked using 5% normal goat serum and subsequently stained with HER2 DMAb antibody and anti-human AF488 conjugate secondary (Invitrogen).

[0253] Slides were observed using a Leica TCS SP5 II confocal microscope and LAS software (Leica).

[0254] Statistics Differences between the means of experimental groups were calculated using two-sided unpaired Student's t-test or one-way ANOVA, where two categorical variables were measured. Repeated measures were analyzed using two-way ANOVA. Error bars represent the standard deviation of the mean. Survival rates were compared using the log-rank test. All statistical analyses were performed using Graph Pad Prism 7.0. p < 0.05 was considered statistically significant.

[0255] The results of the experiment are described here.

[0256] Design and expression of HER2 DNA-encoded monoclonal antibody (DMAb) DNA-encoded antibodies (DMAbs) have a number of advantages over conventional protein antibodies. First, DNA is more stable than protein. This higher stability reduces treatment costs and limits the need to maintain strict cold storage of antibody chains, which can limit the product half-life (Hernandez et al., 2018, Am J Manag Care, 24(2):109-12). Additionally, intracellular delivery of these antibody-encoding DNA plasmids achieves stable plasma antibody concentrations over extended periods, limits the need for multiple administrations, and provides a novel tool for cancer immunotherapy.

[0257] HER2DMAb was generated by encoding the codon and RNA optimization sequences of the heavy and light chains of pertuzumab into the pVAX1 plasmid expression vector (Figure 6A). These sequences were preceded by the IgE signal peptide, and the heavy and light chains were separated by P2A and furin cleavage sites. Antibody expression was tested in vitro by transfecting 293T cells with DNA encoding HER2DMAb or an irrelevant protein. After 48 hours, the supernatant was collected and western blot was performed. Bands corresponding to the heavy and light antibody chains were identified in the HER2DMAb-transfected 293T supernatant but not in the irrelevant protein control (Figure 6B). ELISA was used to determine the amount of human IgG, and it was observed that HER2DMAb was expressed by 293T at 5 - 6 μg / ml, which was verified using RD cells (Figure 7A).

[0258] After confirming in vitro expression, the expression of HER2DMAb was confirmed in vivo. 200 μg of HER2DMAb or an empty vector was injected into the anterior tibialis muscle of mice, followed by adaptive electroporation using the CELLECTRA 3P system (Tebas et al., 2017, N Engl J Med, Epub ahead of print). Similar to the in vitro system, the presence of human IgG was identified in the serum from HER2DMAb-injected mice but not in the control (Figure 6C), and the expression level in mouse serum reached 50 μg / ml, with an average of approximately 25 μg / ml (Figure 6D).

[0259] Next, the ability of DNA-encoded human IgG to bind to human HER2 was tested. Plates were coated with human HER2 protein and incubated with sera from HER2DMAb-treated mice or control sera. HER2DMAb from mouse sera bound to human HER2 in a dose-dependent manner (Figure 6E). To confirm HER2 binding when the protein is present on the cell surface, human HER2 was overexpressed in the mouse cell line Brpkp110. HER2DMAb bound to human HER2 by flow cytometry only when heterologously expressed (Figure 6F).

[0260] HER2 is expressed in human ovarian cancer cell lines. Pertuzumab, unlike trastuzumab, does not require HER2 overexpression in tumor cells for its antitumor activity (Agus et al., 2002, Cancer Cell, 2(2):127-37). In ovarian cancer, pertuzumab has shown a tendency to increase progression-free survival in combination therapy with gemcitabine and paclitaxel (Kurzeder et al., 2016, J Clin Oncol, 34(21):2516-25). HER2 is overexpressed in approximately 11.4% of ovarian cancers (histological score 2+ / 3+) (Bookman et al., 2003, J Clin Oncol, 21(2):283-90). To determine whether HER2 is also expressed in ovarian cancer cell lines, flow cytometry was performed using a commercially available 24D2 antibody (Figure 8A). Binding of HER2DMAb was verified by performing flow cytometry on these same cells (Figure 8B). To further validate the in vivo expression and potential targeting of ovarian cancer cell lines using HER2DMAb, OVCAR3 tumors were generated in mice and immunofluorescence was performed on tumor frozen sections. Positive binding was seen using sera from HER2DMAb-transfected mice but not control sera, confirming in vivo expression of HER2 and binding of HER2DMAb (Figure 8C).

[0261] HER2DMAb mediates HER2 signal transduction blockade and antibody-dependent cytotoxicity. Different mechanisms underlie the antitumor effects of anticancer antibodies. Pertuzumab acts by preventing HER2 heterodimerization and agonist-mediated signal transduction (Franklin et al., 2004, Cancer Cell, 5(4):317-28). As expected, HER2DMAb prevented heregulin-induced (HRG-induced) signal transduction of the HER2-HER3 agonist in OVCAR3 cells, as demonstrated by a decrease in Akt phosphorylation when compared to vehicle controls (Figure 9A).

[0262] Another mechanism by which the MAb has antitumor activity is through antibody-dependent cytotoxicity (ADCC). To study the ADCC potential of HER2DMAb, OVCAR3 cells were co-incubated with or without peripheral blood mononuclear cells (PBMCs) in the presence of serum from HER2DMAb-treated or empty vector-treated mice. HER2DMAb serum effectively killed ovarian cancer cells in the presence of PBMCs but not in their absence. In addition, no killing was observed under control serum conditions (Figure 9B and Figure 7B) or against HER2 cell lines such as MDA-MB-231 (Figure 7C). Similarly, HER2dMAb demonstrated antibody-dependent phagocytosis activity (Figure 7D).

[0263] HER2DMAb delays cancer progression in vivo. To determine the antitumor effect of HER2DMAb in vivo, mice were challenged with the OVCAR-3 ovarian cancer cell line. Nude mice do not have T cells but have enhanced NK and macrophage activities, and their splenocytes can lyse OVCAR3 in vitro in the presence of HER2dMAb (Figure 7E). When the tumors reached an average of 50 mm3, 100 μg of HER2DMAb or an empty vector was delivered intramuscularly by EP. HER2DMAb-injected animals showed a significant delay in tumor growth and an improvement in survival (Figure 9C). HER2DMAb antibody levels peaked at approximately 20 μg / ml two weeks after DMAb injection and remained at approximately 5-10 μg / ml for one month until the end of the experiment (Figure 9D). To verify the antitumor effect in an immunocompetent host that more closely mimics clinical administration, tumors were generated using the mouse human HER2 breast cancer cell line Brkpk110. This cell line was engineered to express HER2 levels similar to OVCAR3 (Figure 9E). Five days after tumor challenge, the mice were treated with HER2DMAb or an empty vector. HER2DMAb also delayed tumor progression in this aggressive breast cancer model (Figure 9F).

[0264] When the kinetics of HER2dMAb were studied, it was noted that there was a decrease in antibody expression over approximately 300 days. To investigate this phenomenon, the induction of antibodies against this human construct expressed in mice was evaluated. The generation of anti-HER2dMAb antibodies was observed in the serum after treatment with HER2dMAb (Figure 7F), which could contribute to its decline over time.

[0265] Generation, expression, and cytotoxicity of HER2BiTE Bispecific T cell engagers (BiTEs) have two binding antibody fragments (scFvs), one of which engages a tumor antigen and the other binds to T cells driving CD3 activation, thereby activating them. Despite high antitumor activity, these new tools have major limitations due to an in vivo elimination half-life of approximately 2.1 hours, so BiTE therapy progresses slowly. This short half-life imposes on BiTE therapy the need for continuous intravenous infusion using an infusion pump over 4 - 8 weeks per cycle. Recent experiments using RNA-expressing BiTEs have shown expression up to 6 days after IV injection, representing a considerable advance (Stadler et al., 2017, Nat Med, 23(7):815-7).

[0266] An optimized HER2 BiTE was generated by fusing the scFv of HER2 DmAb with the scFv of the stimulatory antibody anti-CD3 (OKT3) (Figure 11A). HER2 BiTE was efficiently expressed in vivo when injected into the mouse anterior tibialis muscle and electroporated (Figure 11B). The new HER2 DBiTE retained binding to HER2 and bound to CD3 (Figures 10A and 10B). Importantly, although the stimulation provided by UCHT1 has been reported to be able to kill T cells, when OKT3 cells were co-cultured with HER2 DBiTE, no increased apoptosis rate or difference in T cell numbers was observed compared to a mere control in the presence of HER2+ cells (Figures 10C and 10D). To determine the function of HER2 DBiTE expressed in vivo, sera from mice injected with HER2 DBiTE or an empty vector were cultured with HER2+ ovarian cancer cells and T cells. Sera from mice after HER2 DBiTE treatment showed T cell activation (Figures 10E - 10G), as well as efficient dose-dependent cytotoxicity against OVCAR3 and CAOV3 cells. No cytotoxicity was observed upon incubation with sera from empty vector-treated mice, or cytotoxicity was not observed in the absence of T cells (Figures 11C and 10H). Incubation of OVCAR3 with T cells at a ratio of 1:5 with 5% serum (5 μl in 100 μl) from treated mice indicated that the DBiTE showed strong activity for approximately 4 months (Figure 11D). Generation of anti-HER2 DBiTE antibodies, such as HER2 DBiTE, was observed, which could be part of the cause of the circulating levels over time (Figure 10I). To determine the in vivo DBiTE antitumor activity, NOD / SCID-γ (NSG) mice were challenged with OVCAR3. The mice were treated with a single dose of 200 μg of HER2 DBiTE or an empty vector 1 day after tumor transplantation. Two weeks after tumor inoculation, when the tumor was approximately 50 mm 3 in size, 10,000,000 PBMCs were intraperitoneally injected into each mouse. HER2 DBiTE treatment significantly affected tumor progression (Figure 11E), and tumor regression or tumor elimination was observed in 8 out of 10 tumors, while no tumor effect was observed in the control group (Figure 11F).

[0267] The in vivo effect of HER2 DBiTE was not observed in the absence of PBMCs (Figure 10J). HER2 DBiTE delivered by simple injection, which lasts for only a few seconds, was expressed in vivo for approximately four months and exhibited dramatic antitumor activity. Synthetic DNA delivery of DBiTE may reduce the burden generated by the short half-life of DBiTE therapy and provide a new application for this tool in cancer immunotherapy.

[0268] Collectively, the data demonstrate that DMAb can encode HER2 DMAb and HER2 DBiTE, enabling them to be expressed at high levels, durably in vivo, and drive potent antitumor activity. This approach provides a valuable new tool for the treatment of ovarian cancer and potentially other cancers.

[0269] Example 3 EGFRvIII-targeted DNA-encoded immune cell engager (DICE) generates in vivo expression of a bispecific antibody that induces T cell-mediated cytolytic activity against EGFRvIII-positive tumors and controls tumor growth in a GBM mouse model. The development of bispecific antibodies targeting T cells and tumor-associated antigens (TAAs) has expanded exponentially in recent years in both preclinical and clinical settings. In 2017, one bispecific antibody was approved for the treatment of acute lymphoblastic leukemia. However, due to its low molecular weight, the serum half-life of the antibody is only about 4 hours. As a result, treatment requires continuous IV injection of the antibody over several days, which can extend over several weeks. The suboptimal pharmacokinetic profile, along with other difficulties related to manufacturing and molecular stability, presents a major challenge in the development of bispecific antibodies. To address these issues, an optimized synthetic DNA-encoded immunocyte engager (DICE) designed to express bispecific antibodies in vivo has been developed. Mice receiving a single dose of HER2-DICE demonstrated long-term in vivo expression of the bispecific antibody and T cell-mediated cytolytic activity against HER2-expressing ovarian cell lines for over 120 days. In the same study, HER2-DICE not only controlled tumor progression but also promoted tumor clearance in many animals in an ovarian cancer mouse model. Using a similar strategy, a DICE targeting EGFRvIII, a TAA expressed in 30 - 50% of glioblastoma multiforme (GBM) patients, was developed. Supernatant samples from cells transfected with EGFRvIII-DICE in vitro showed strong target-specific binding affinity for both EGFRvIII and CD3 and induced T cell-mediated cytolytic activity against GBM cell lines overexpressing EGFRvIII. Co-culture of target cells and primary human T cells in the presence of EGFRvIII-DICE supernatant stimulated a robust T cell response and showed significant levels of IFNγ, TNFα, and CD107a in the cytotoxic T cell population. Finally, in a GBM mouse challenge model, treatment of NSG mice repopulated with human T cells with EGFRvIII-DICE resulted in control of tumor growth, which was not observed in the empty vector control group. These studies support that synthetic DNA delivery of bispecific antibodies can activate cytotoxic T cell function and generate potent and functional antibodies that can be investigated as an alternative approach for the development of bispecific antibodies for cancer immunotherapy.

[0270] The foregoing detailed description and the accompanying examples are merely illustrative and are not to be construed as limiting the scope of the invention, which is understood to be defined solely by the appended claims and their equivalents.

[0271] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Without limitation, such changes and modifications, including those related to chemical structures, substituents, derivatives, intermediates, compounds, compositions, formulations, or methods of use of the invention, can be made without departing from the spirit and scope thereof.

Claims

1. 1. A nucleic acid molecule encoding one or more synthetic DNA-encoded bispecific immune cell engagers, wherein said one or more synthetic DNA-encoded bispecific immune cell engagers comprise at least one antigen-binding domain and at least one immune cell engaging domain.

2. 2. The nucleic acid molecule of claim 1, wherein the antigen-binding domain targets at least one antigen selected from the group consisting of B-cell maturation antigen (BCMA), CD33, fibroblast activation protein (FAP), follicle-stimulating hormone receptor (FSHR), epidermal growth factor receptor (EGFR), prostate-specific membrane antigen (PSMA), CD123, and human epidermal growth factor receptor 2 (Her2).

3. 2. The nucleic acid molecule of claim 1, wherein the immune cell engaging domain targets a cell selected from the group consisting of a T cell, an antigen-presenting cell, a natural killer (NK) cell, a neutrophil, and a macrophage.

4. 2. The nucleic acid molecule of claim 1, wherein the immune cell engaging domain targets at least one T cell-specific receptor molecule selected from the group consisting of CD3, T cell receptor (TCR), CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5, CD40, FcgR, FceR, FcaR, and CD95.

5. The nucleic acid molecule of claim 4, wherein the immune cell engaging domain targets CD3.

6. a) an amino acid sequence having at least about 90% identity over the entire length of the amino acid sequence to an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74 or SEQ ID NO:76; b) a fragment of an amino acid sequence having at least about 90% identity over at least 65% of the amino acid sequence to an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74 or SEQ ID NO:76; c) an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74 or SEQ ID NO:76; d) a fragment of an amino acid sequence comprising at least 65% of an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74 or SEQ ID NO:

76.

7. a) a nucleotide sequence having at least about 90% identity over the entire length of the nucleic acid sequence to a nucleotide sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, and SEQ ID NO:75; b) a fragment of a nucleotide sequence having at least about 90% identity over at least 65% of the nucleic acid sequence to a nucleotide sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, and SEQ ID NO:75; c) a nucleotide sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, and SEQ ID NO:75; d) a fragment of a nucleotide sequence comprising at least 65% of a nucleotide sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, and SEQ ID NO:

75.

8. The nucleic acid molecule of any one of claims 1 to 7, wherein the nucleotide sequence is operably linked to a nucleic acid sequence encoding an IgE leader sequence.

9. The nucleic acid molecule according to any one of claims 1 to 8, wherein the nucleic acid molecule comprises an expression vector.

10. A composition comprising the nucleic acid molecule of any one of claims 1 to 9.

11. The composition of claim 10, further comprising a pharmaceutically acceptable excipient.

12. A nucleic acid molecule according to any one of claims 1 to 9 or a composition according to claim 10 or 11 for preventing a disease or disorder.

13. 13. The nucleic acid molecule or composition of claim 12, wherein the disease is selected from the group consisting of benign tumors, cancer, and cancer-related diseases.

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