DNA-encoded multivalent antibodies targeting carbonic anhydrase 9 and methods of use in cancer therapeutics

Abstract

Disclosed herein are methods of treating or preventing renal cell carcinoma comprising administering compositions comprising a recombinant nucleic acid sequence encoding a therapeutic antibody targeting CA9, a fragment thereof, a variant thereof, or a combination thereof.

Description

Docket No.206193-0138-00WO DNA-ENCODED MULTIVALENT ANTIBODIES TARGETING CARBONIC ANHYDRASE 9 AND METHODS OF USE IN CANCER THERAPEUTICS CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63 / 652,846, filed May 29, 2024, and U.S. Provisional Application No.63 / 655,920, filed June 4, 2024, each of which is hereby incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under CA010815, awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO A "SEQUENCE LISTING” SUBMITTED AS AN XML FILE

[0003] The Sequence Listing written in the xml file: “206193-0138-00WO Sequence Listing.xml”; created on May 29, 2025, and 78,065 bytes in size, is hereby incorporated by reference herein in its entirety. BACKGROUND

[0004] Renal cell carcinoma (RCC) is responsible for 90% of kidney cancers, which stand as the 9th most common cancer in men and 14th most common cancer in women worldwide (Cancer Stat Facts: Kidney and Renal Pelvis Cancer. March 29, 2022). The clear cell subtype (ccRCC) comprises approximately 75% of cases, which in the U.S. has risen to 55,000 annually. Problematically, a third of patients are already metastatic upon diagnosis due to a mild symptomatic nature that slows diagnosis, and unlike earlier grades that are effectively treated with surgical resection, advanced lesions are inoperable (Cancer Stat Facts: Kidney and Renal Pelvis Cancer. March 29, 2022; Renal Cell Cancer Treatment Physican Data Query. National Cancer Institute). Intermediate- and poor-risk patients go on to acquire resistance to traditional chemotherapies, radiation, and targeted anti-angiogenics. With only 10-15% of these patients surviving 5 years, advanced ccRCC represents a cancerDocket No.206193-0138-00WO population in significant need of new treatment options (Hsieh., et al., Nature reviews. Disease primers, 2017.3: p.17009-17009).

[0005] Some patients with ccRCC have von Hippel-Lindau syndome (VHLS), which is caused by a mutation in the von Hippel-Lindau (VHL) gene. VHLS is the most common cause of hereditary renal cell carcinoma (RCC), specifically clear cell renal carcinoma (ccRCC). While renal cysts are estimated to occur in up to 63% of patients with VHLS, renal tumors are estimated to occur in 25–45% of patients with VHLS and have a mean age at presentation of 39 years. It should be noted that even cases of sporadic, non-familial ccRCC involve mutations or methylation of vhl in up to 91% of cases.

[0006] Immunotherapy has transformed the landscape of cancer care by empowering the immune system to detect and kill cancers, with particular potential for advanced ccRCC. Checkpoint inhibitors that unleash T cells upon tumors serve as a first-line therapy where they offer a relatively high response rate of 50-60%. However, beyond still leaving nearly half of patients nonresponsive, progression free survival is only extended to 1-2 years for responders due to discontinuations from acquired resistance and side effects (Hsieh., et al., Nature reviews. Disease primers, 2017.3: p.17009-17009). Resistant tumors subsequently acquire a more aggressive and metastatic phenotype and with no treatment options left, approximately 10,000 ccRCC patients die annually in the U.S. (Cancer Stat Facts: Kidney and Renal Pelvis Cancer. March 29, 2022). New therapies could leverage and expand this cancer’s immune responsivity by driving alternative mechanisms of action, like focusing immune activity directly to tumors through the select targeting of tumor-associated antigens (TAAs), to more effectively eradicate ccRCC.

[0007] Bispecific T cell engagers (BTEs) are a powerful, antibody-based immunotherapy designed to simultaneously bind immune and cancer cells to trigger cancer’s selective destruction. To accomplish this, they are equipped with multiple binding domains that recognize distinct epitopes or antigens, with most research directed at binding to the CD3 complex of T cells alongside a relevant TAA (Nie, et al., Antibody Therapeutics, 2020.3(1): p.18-62). The dual specificity forces a proximal relationship between target and T cells, with the goal of engaging the T cell-mediated cellular cytotoxicity (CMC) of cancer cells through the formation of artificial, MHC-independent synapses. While clinically successful, first- generation BTEs given their simplified, dual single-chain variable fragment (scFv) design have a half-life of ~2 hours (hrs) owing to the absence of an Fc region (Einsele et al., Cancer,Docket No.206193-0138-00WO 2020.126(14): p.3192-3201). Dramatic increases to circulation time have been made by numerous second-generation formats that reintroduce an Fc domain, such as the Persistent BTE (PBTE) that is equipped with a single-chain Fc domain (scFc) linked to the conventional BTE molecule (Wei et al., Frontiers in Immunology, 2022.13.). However, detailed comparative characterizations between first- and second-generation formats that shed light on potential trade-offs for clinically relevant formats like this one have yet to be reported. Nevertheless, the known benefits of PBTEs promoted the application of this format not only for additional targets, but to the creation of new structures that advance the scFc concept. For instance, PBTEs are still limited by their 1:1 valency that detracts from the higher avidity naturally afforded to antibodies. Increasing valency with additional binding domains has endowed other therapies with more functional affinity to wield greater potency and efficacy (Ellerman, Methods, 2019.154: p.102-117; Lee et al., Journal for ImmunoTherapy of Cancer, 2023.11(10): p. e007494). The longer residence times enabled by higher valency can also drive better tumor distribution and help distinguish between high- and low-target- expressing cells to better mitigate toxicity (Bacac, et al., Clinical Cancer Research, 2016. 22(13): p.3286-3297; Hernandez-Hoyos, et al., Mol Cancer Ther, 2016.15(9): p.2155-65; Adams et al., Clin Cancer Res, 2006.12(5): p.1599-605). Therefore, bispecific designs integrating both scFc and valency adaptations give rise to new and effective, antibody therapies for advanced ccRCC, if provided a highly targetable TAA by the disease.

[0008] Thus there is need in the art for the development of a highly focused CA9 targeting immunotherapy with high potency. The current invention satisfies this need. BRIEF SUMMARY OF THE INVENTION

[0009] In some embodiments, the invention relates to a method of treating or preventing renal cell carcinoma in a subject, comprising adminsitering to the subject a composition comprising one or more synthetic binding molecule, or a binding fragment thereof, or a nucleic acid molecule encoding comprising one or more synthetic binding molecule, or a binding fragment thereof, wherein the one or more synthetic binding molecule comprises at least one antigen binding domain specific for binding to carbonic anhydrase 9 (CA9), and at least one immune cell engaging domain.Docket No.206193-0138-00WO

[0010] In some embodiments, the immune cell engaging domain targets a T cell, an antigen presenting cell, a natural killer (NK) cell, a neutrophil or a macrophage.

[0011] In some embodiments, the immune cell engaging domain targets at least one T cell specific receptor molecule selected from CD3, the T cell receptor (TCR), CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5, CD40, FcgRs, FceRs, FcaRs and CD95. In some embodiments, the immune cell engaging domain targets CD3.

[0012] In some embodiments of the method, the synthetic binding molecule comprises: a CA9 heavy chain amino acid sequence of SEQ ID NO: 4, or a fragment thereof comprising at least the CDRs; a CA9 light chain amino acid sequence of SEQ ID NO: 2, or a fragment thereof comprising at least the CDRs; a CD3 heavy chain amino acid sequence of SEQ ID NO: 6, or a fragment thereof comprising at least the CDRs; and a CD3 light chain amino acid sequence of SEQ ID NO: 8, or a fragment thereof comprising at least the CDRs.

[0013] In some embodiments, the synthetic binding molecule further comprises: an Fc domain selected from SEQ ID NO: 29 or SEQ ID NO:30.

[0014] In some embodiments, the synthetic binding molecule further comprises: a second CA9 heavy chain amino acid sequence of SEQ ID NO: 4, or a fragment thereof comprising at least the CDRs; and a second CA9 light chain amino acid sequence of SEQ ID NO: 2, or a fragment thereof comprising at least the CDRs.

[0015] In some embodiments, the synthetic binding molecule comprises SEQ ID NO:10 or SEQ ID NO:12. In some embodiments, the synthetic binding molecule comprises SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 or SEQ ID NO:20. In some embodiments, the synthetic binding molecule comprises SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO:28.

[0016] In some embodiments, the method comprises administering a nucleic acid molecule comprising: the nucleotide sequence of SEQ ID NO:3 encoding the CA9 heavy chain, or a fragment thereof encoding at least the CDRs; the nucleotide sequence of SEQ ID NO:1 encoding the CA9 light chain, or a fragment thereof encoding at least the CDRs; the nucleotide sequence of SEQ ID NO:5 encoding the CD3 heavy chain, or a fragment thereof encoding at least the CDRs; and the nucleotide sequence of SEQ ID NO:7 encoding the CD3 light chain, or a fragment thereof encoding at least the CDRs.Docket No.206193-0138-00WO

[0017] In some embodiments, the nucleic acid molecule further comprises: a nucleotide sequence selected from SEQ ID NO: 31 and SEQ ID NO:32 encoding the Fc domain.

[0018] In some embodiments, the nucleic acid molecule further comprises: the nucleotide sequence of SEQ ID NO:3 encoding a second CA9 heavy chain, or a fragment thereof encoding at least the CDRs; the nucleotide sequence of SEQ ID NO:1 encoding a second CA9 light chain, or a fragment thereof encoding at least the CDRs.

[0019] In some embodiments, the nucleic acid molecule comprises SEQ ID NO:9 or SEQ ID NO:11. In some embodiments, the nucleic acid molecule comprises SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 or SEQ ID NO:19. In some embodiments, the nucleic acid molecule comprises SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27.

[0020] In some embodiments, the nucleotide sequence is operably linked to a nucleic acid sequence encoding an IgE leader sequence.

[0021] In some embodiments, the nucleic acid molecule comprises an expression vector.

[0022] In some embodiments, the composition comprises a pharmaceutically acceptable excipient.

[0023] In some embodiments, the renal cell carcinoma is metastatic.

[0024] In some embodiments, the subject has a von Hippel-Lindau (VHL) mutation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1A depicts the process of generating the PBTE format. An scFc was linked to the conventional BTE N-terminus. Figure 1B depicts the process of synthesizing the antibody. The antibody DNA was codon-optimized and inserted into a pVax1 expression vector for Expi293 cell expression and purification. Figure 1C depicts flow cytometry binding analyses against target cell lines revealed an interesting consequence of linking an scFc to the BTE to generate a PBTE. Doing so attenuated binding affinity to target cells, evidenced by right-shifted median fluorescent intensity (MFI) curves for ACHN-CA9, 293T- CA9, and T cells, with corresponding dissociation constant (KD) values declining approximately 2-fold for all cell types. Figure 1D depicts cytotoxicity impedance data of the primary RCC cell line 786-O and accompanying microscopic images that were taken at theDocket No.206193-0138-00WO terminal timepoint. At 30 nanomolar (nM), both the BTE and PBTE engaged T cell killing of the primary RCC cell line 786-O with over 50% cell death by the 24-hour timepoint. While the BTE maintained this activity at 200 picomolar (pM), PBTE activity declined nearly 2- fold. Figure 1E depicts cytotoxicity impedance data of the primary cell line A-498 and accompanying microscopic images that were taken at the terminal timepoint. Trending reductions in potency for the PBTE were similarly observed against the primary cell line A- 498.

[0026] Figure 2A depicts the full panel of single-chain formats to undergo further testing in unison for assessment of the functional effects of compounding domain appendages. Figure 2B depicts Western blotting results showing bands at appropriate MWs for each antibody. Figure 2C depicts flow cytometry data showing pronounced binding of each antibody at 500 nM as represented by 3-log population shifts for all formats, unlike the irrelevant-IgG1, negative control.

[0027] Figure 3 depicts graphs displaying tumor volumes (mm3) measured over time for NSG mice harboring subcutaneous metastatic SKRC-52 tumors. On day zero, the mice were injected with 106donor T cells (IP) and either a single 10 ug dose of synDNA or empty vector, as represented by the arrow (n=4-5). Mice are line-plotted individually while the bar graph displays tumor volumes (mm3) at day 30 as mean ± SEM.

[0028] Figure 4 depicts a line graph displaying tumor volumes (mm3) measured over time for NSG mice harboring subcutaneous xenografts of A-498 tumors. On day zero, the mice were injected with human T cells (IP) and either a single 100 μg dose of dBTE expression vector or empty vector. The single 100 μg dose of dBTE expression vector elicited complete tumor control. DETAILED DESCRIPTION

[0029] The present invention relates to methods of treating or preventing renal cell carcinoma (RCC) in a subject. In some embodiments, the renal cell carcinoma is metastatic. In some embodiments, the subject has a Von Hippel-Lindau (VHL) mutation. In some embodiments, the method comprises administering compositions comprising a recombinant nucleic acid sequence encoding a multivalent T cell engaging antibody targeting CA9, a fragment thereof, a variant thereof, or a combination thereof. In one embodiment, theDocket No.206193-0138-00WO antibody is a bispecific T cell engager (BTE). In one embodiment, the antibody is a persistent bispecific T cell engager (PBTE). In one embodiment, the antibody is a persistent multivalent T cell engager (PMTE). In one embodiment, the antibody of the invention is an immune cell engaging therapeutic antibody. The composition can be administered to a subject in need thereof to facilitate in vivo expression and formation of the antibody.

[0030] In one embodiment, the CA9 antibody (BTE, PBTE, or PMTE) comprises at least one antigen binding domain specific for binding to CA9, 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.

[0031] In various embodiments, the immune cell engaging domain comprises a nucleotide sequence encoding an antibody, a fragment thereof, or a variant thereof specific for binding to a 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, FcgRs, FceRs, FcaRs and CD95.

[0032] In various embodiments, the antigen binding domain comprises a nucleotide sequence encoding an antibody, a fragment thereof, or a variant thereof specific for binding to CA9. In one embodiment, the antibody or fragment thereof is a DNA encoded monoclonal antibody (DMAb) or a fragment or variant thereof.

[0033] In one embodiment, the antibody (BTE, PBTE, or PMTE) is specific for binding CA9, and recruiting a T cell to a cell expressing CA9. In some embodiments, cells expressing CA9 are cancer cells. In some embodiments, cells expressing CA9 are renal cell carcinoma cells. In some embodiments, cells expressing CA9 are metastatic renal cell carcinoma cells. In some embodiments, cells expressing CA9 are renal cell carcinoma cells with a mutation- in the VHL gene. Definitions

[0034] Unless otherwise defined, 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 document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to thoseDocket No.206193-0138-00WO described herein can be used in practice or testing of the present invention. 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.

[0035] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants 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 references 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 explicitly set forth or not.

[0036] “Antibody” may mean an antibody of classes IgG, IgM, IgA, IgD or IgE, or fragments, fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single chain antibodies, and derivatives thereof. The antibody may be an antibody isolated from the serum sample of mammal, a polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom.

[0037] “Antibody fragment” or “fragment of an antibody” as used interchangeably herein refers to a portion of an intact antibody comprising the antigen-binding site or variable region. The portion does not include the constant heavy chain domains (i.e. CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single- chain polypeptides containing the three CDRs of the light-chain variable domain, single- chain polypeptides containing only one heavy chain variable region, single-chain polypeptides containing the three CDRs of the heavy chain variable region, a nanobody, or a long CDR3 Fv.

[0038] “Antigen” refers to proteins that have the ability to generate an immune response in a host. An antigen may be recognized and bound by an antibody. An antigen may originate from within the body or from the external environment.Docket No.206193-0138-00WO

[0039] “Coding sequence” or “encoding nucleic acid” as used herein may mean refers to the nucleic acid (RNA or DNA molecule) that comprise a nucleotide sequence which encodes an antibody as set forth herein. The coding sequence may further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to whom the nucleic acid is administered. The coding sequence may further include sequences that encode signal peptides.

[0040] “Complement” or “complementary” as used herein may mean a nucleic acid may mean Watson-Crick (e.g., A-T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.

[0041] “Constant current” as used herein to define a current that is received or experienced by a tissue, or cells defining said tissue, over the duration of an electrical pulse delivered to same tissue. The electrical pulse is delivered from the electroporation devices described herein. This current remains at a constant amperage in said tissue over the life of an electrical pulse because the electroporation device provided herein has a feedback element, preferably having instantaneous feedback. The feedback element can measure the resistance of the tissue (or cells) throughout the duration of the pulse and cause the electroporation device to alter its electrical energy output (e.g., increase voltage) so current in same tissue remains constant throughout the electrical pulse (on the order of microseconds), and from pulse to pulse. In some embodiments, the feedback element comprises a controller.

[0042] “Current feedback” or “feedback” as used herein may be used interchangeably and may mean the active response of the provided electroporation devices, which comprises measuring the current in tissue between electrodes and altering the energy output delivered by the EP device accordingly in order to maintain the current at a constant level. This constant level is preset by a user prior to initiation of a pulse sequence or electrical treatment. The feedback may be accomplished by the electroporation component, e.g., controller, of the electroporation device, as the electrical circuit therein is able to continuously monitor the current in tissue between electrodes and compare that monitored current (or current within tissue) to a preset current and continuously make energy-output adjustments to maintain the monitored current at preset levels. The feedback loop may be instantaneous as it is an analog closed-loop feedback.Docket No.206193-0138-00WO

[0043] “Decentralized current” as used herein may mean the pattern of electrical currents delivered from the various needle electrode arrays of the electroporation devices described herein, wherein the patterns minimize, or preferably eliminate, the occurrence of electroporation related heat stress on any area of tissue being electroporated.

[0044] “Electroporation,” “electro-permeabilization,” or “electro-kinetic enhancement” (“EP”) as used interchangeably herein may refer to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio- membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cellular membrane to the other.

[0045] “Endogenous antibody” as used herein may refer to an antibody that is generated in a subject that is administered an effective dose of an antigen for induction of a humoral immune response.

[0046] “Feedback mechanism” as used herein may refer to a process performed by either software or hardware (or firmware), which process receives and compares the impedance of the desired tissue (before, during, and / or after the delivery of pulse of energy) with a present value, preferably current, and adjusts the pulse of energy delivered to achieve the preset value. A feedback mechanism may be performed by an analog closed loop circuit.

[0047] “Fragment” may mean a polypeptide fragment of an antibody that has function, i.e., can bind to desired target and have the same intended effect as a full length antibody. A fragment of an antibody may be 100% identical to the full length except missing at least one amino acid from the N and / or C terminal, in each case with or without signal peptides and / or a methionine at position 1. Fragments 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 percent of the length of the particular full length antibody, excluding any heterologous signal peptide 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 additionally comprise an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity. Fragments may further comprise an N terminal methionine and / or a signal peptide such as anDocket No.206193-0138-00WO immunoglobulin signal peptide, for example an IgE or IgG signal peptide. The N terminal methionine and / or signal peptide may be linked to a fragment of an antibody.

[0048] A fragment of a nucleic acid sequence that encodes an antibody may be 100% identical to the full length except missing at least one nucleotide from the 5' and / or 3' end, in each case with or without sequences encoding signal peptides and / or a methionine at position 1. Fragments 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 percent of the length of the particular full length coding sequence, excluding any heterologous signal peptide added. The fragment may comprise a fragment that encode 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 comprise sequence encoding an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity. Fragments may further comprise coding sequences for an N terminal methionine and / or a signal peptide such as an immunoglobulin signal peptide, for example an IgE or IgG signal peptide. The coding sequence encoding the N terminal methionine and / or signal peptide may be linked to a fragment of coding sequence.

[0049] “Genetic construct” as used herein refers to the DNA or RNA molecules that comprise a nucleotide sequence which encodes a protein, such as an antibody. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to gene constructs that contain the necessary regulatory elements operable linked to a coding sequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed.

[0050] “Identical” or “identity” as used herein in the context of two or more nucleic acids or polypeptide sequences, may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may 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 residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by theDocket No.206193-0138-00WO total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of 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 may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0051] “Impedance” as used herein may be used when discussing the feedback mechanism and can be converted to a current value according to Ohm's law, thus enabling comparisons with the preset current.

[0052] “Immune response” as used herein may mean the activation of a host’s immune system, e.g., that of a mammal, in response to the introduction of one or more nucleic acids and / or peptides. The immune response can be in the form of a cellular or humoral response, or both.

[0053] “Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0054] Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.

[0055] “Operably linked” as used herein may mean that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter may be positionedDocket No.206193-0138-00WO 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.

[0056] A “peptide,” “protein,” or “polypeptide” as used herein can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.

[0057] “Promoter” as used herein may mean a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of same. A promoter may also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the 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 SV 40 late promoter and the CMV IE promoter.

[0058] “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 a protein set forth herein. Signal peptides / leader sequences typically direct localization of a protein. Signal peptides / leader sequences used herein preferably facilitate secretion of the protein from the cell in which it is produced. Signal peptides / leader sequences are often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell. Signal peptides / leader sequences are linked at the N terminus of the protein.

[0059] “Stringent hybridization conditions” as used herein may mean conditions under which a first nucleic acid sequence (e.g., probe) will hybridize to a second nucleic acid sequence (e.g., target), such as in a complex mixture of nucleic acids. Stringent conditions areDocket No.206193-0138-00WO sequence dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5-10°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm may be the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01- 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 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 may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65°C.

[0060] “Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc) and a human). In some embodiments, the subject may be a human or a non-human. The subject or patient may be undergoing other forms of treatment.

[0061] “Substantially complementary” as used herein may mean 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.

[0062] “Substantially identical” as used herein may mean that a first and second sequence 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, 1100Docket No.206193-0138-00WO or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.

[0063] “Synthetic antibody” as used herein refers to an antibody that is encoded by the recombinant nucleic acid sequence described herein and is generated in a subject.

[0064] “Treatment” or “treating,” as used herein can mean protecting of a subject from a disease through means of preventing, suppressing, repressing, or completely eliminating the disease. Preventing the disease involves administering an antibody of the present invention to a subject prior to onset of the disease. Suppressing the disease involves administering a antibody of the present invention to a subject after induction of the disease but before its clinical appearance. Repressing the disease involves administering an antibody of the present invention to a subject after clinical appearance of the disease.

[0065] “Variant” used herein with respect to a nucleic acid may mean (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.

[0066] “Variant” with respect to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol.157:105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatestDocket No.206193-0138-00WO local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No.4,554,101, incorporated fully herein by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hyrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

[0067] A variant may be a nucleic acid sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof. The nucleic acid sequence may 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 may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence may 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.

[0068] “Vector” as used herein may mean a nucleic acid sequence containing an origin of replication. A vector may be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be either a self-replicating extrachromosomal vector or a vector which integrates into a host genome.

[0069] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 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.Docket No.206193-0138-00WO Methods

[0070] The present invention provides methods of treating, preventing, or reducing metastasis of renal cell carcinoma in a subject. In some embodiments, the renal cell carcinoma is metastatic. In some embodiments, the subject has a von Hippel-Lindau (VHL) gene mutation. In some embodiments, the method comprises administering a composition comprising a synthetic binding molecule (e.g., BTE, PBTE, or PMTE) to the subject wherein the synthetic binding molecule comprises at least one antigen binding domain specific for binding to Carbonic Anhydrase 9 (CA9) and at least on immune cell targeting domain. Administration of the composition to the subject can be done using the method of delivery described below.

[0071] In certain embodiments, the invention provides a method of 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. In one embodiment, the method treats, protects against, and / or prevents mestastasis of renal cell carcinoma in a subject with a VHL mutation.

[0072] The method can promote survival of the disease in the subject administered the composition. The method can provide at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% survival of the disease in the subject administered the composition. In other embodiments, the method can provide at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80% survival of the disease in the subject administered the composition.

[0073] The method can result in the persistence of the synthetic antibody (e.g., PBTE or PMTE) in the 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 of administration of the synthetic binding molecule or nucleic acid molecule encoding the synthetic binding molecule to the subject. The method can result in persistence of the synthetic antibody (e.g., PBTE or PMTE) in the 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 of administration of the synthetic binding molecule or nucleic acid molecule encoding the synthetic binding molecule to the subject. The method can result in persistence of the synthetic antibody (e.g., PBTE orDocket No.206193-0138-00WO PMTE) in the 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 of administration of the synthetic binding molecule or nucleic acid molecule encoding the synthetic binding molecule to the subject.

[0074] The method can result in persistence of the synthetic antibody (e.g., PBTE or PMTE) in the subject for longer than an endogenous antibody in a subject who is administered an antigen to induce a humoral immune response.

[0075] The composition of the present invention can have features required of effective compositions such as being safe so that the composition does not cause illness or death; being protective against illness; and providing ease of administration, few side effects, biological stability and low cost per dose. Composition

[0076] In one embodiment, the present invention relates to methods of administering a composition comprising a synthetic binding molecule (e.g., BTE, PBTE, or PMTE), or nucleic acid molecule encoding a synthetic binding molecule to a subject in need thereof. In some embodiments the subject has or is at risk of developing renal cancer. In some embodiments, the synthetic binding molecule comprises at least one antigen binding domain specific for binding CA9, a fragment thereof, a variant thereof, or a combination thereof, and at least one immune cell engaging domain.

[0077] In one embodiment, the synthetic binding molecule (BTE, PBTE, or PMTE) comprises at least one antigen binding domain specific for binding to CA9, 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.

[0078] In some embodiments, the synthetic binding molecules may comprise at least one heavy chain and at least one light chain complementarity determining region (“CDR”) set, respectively interposed between at least one heavy chain and at least one light chainDocket No.206193-0138-00WO framework (“FR”) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other. The CDR set may contain three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3,” respectively. An antigen- binding site, therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Heavy Chain Polypeptide

[0079] The binding molecule of the invention can include a heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The heavy chain polypeptide can include a variable heavy chain (VH) region and / or at least one constant heavy chain (CH) region. The at least one constant heavy chain region can include a constant heavy chain region 1 (CH1), a constant heavy chain region 2 (CH2), and a constant heavy chain region 3 (CH3), and / or a hinge region. Light Chain Polypeptide

[0080] The binding molecule of the invention can include a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The light chain polypeptide can include a variable light chain (VL) region and / or a constant light chain (CL) region.

[0081] The light chain polypeptide can include a complementarity determining region (“CDR”) set. The CDR set can contain three hypervariable regions of the VL region. Proceeding from N-terminus of the light chain polypeptide, these CDRs are denoted “CDR1,” “CDR2,” and “CDR3,” respectively. CDR1, CDR2, and CDR3 of the light chain polypeptide can contribute to binding or recognition of the antigen. Linker Sequence

[0082] The binding molecule of the invention of the invention can include one or more linker sequences. The linker sequence can spatially separate or link the one or more components described herein. In other embodiments, the linker sequence can comprise an amino acid sequence that spatially separates or links two or more polypeptides. In one embodiment, the linker sequence is a G4S linker sequence. In one embodiment, the linkerDocket No.206193-0138-00WO sequence is a G4S linker sequence, having an amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO:35). Leader Sequence

[0083] The binding molecule of the invention of the invention can include one or more leader sequences. In one embodiment, the leader sequence is a signal peptide. The signal peptide can be an immunoglobulin (Ig) signal peptide, for example, but not limited to, an IgG signal peptide and a IgE signal peptide. Antigen Binding Domain

[0084] In one embodiment, the binding molecule is directed to an antigen or 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, a variant thereof, a fragment thereof, or a combination thereof. The amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof. The polysaccharide can be a nucleic acid encoded polysaccharide.

[0085] The antigen can be a tumor antigen. The antigen can be associated with increased risk of cancer development or progression. The antigen can be associated with increased risk of cancer metastasis. In one embodiment, the antigen can be CA9.

[0086] In one embodiment, a synthetic bispecific immune cell engager (PBTE or PMTE) of the invention targets two or more antigen binding domains. In one embodiment, each of the antigen binding domains is specific for binding to a tumor antigen. In one embodiment, the antigen can be CA9.

[0087] Aspects of the present invention include compositions for enhancing an immune response against CA9 in a subject in need thereof, comprising administering a synthetic binding molecule capable of recruiting an immune cell to a CA9 expressing cell in the subject. In some embodiments, the synthetic antibody of this invention is a PBTE or PMTE comprising at least one scFv targeting CA9.

[0088] In various embodiments, the antigen binding domain comprises an antibody, a fragment thereof, or a variant thereof specific for binding to CA9.

[0089] In one embodiment, the CA9 synthetic antibody (e.g., BTE, PBTE, or PMTE) comprises at least one amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 or a fragmentDocket No.206193-0138-00WO or variant thereof. In one embodiment, the fragment of SEQ ID NO:2 or SEQ ID NO:4 is a binding fragment comprising at least one, at least two, or all three CDR sequences of SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, the binding fragment comprises at least six CDR sequences of SEQ ID NO:2 and SEQ ID NO:4. Immune Cell Binding Domain

[0090] In some embodiments, one of the binding sites of an antibody molecule according to the invention is able to bind a T-cell specific receptor molecule and / or a natural killer cell (NK cell) specific receptor molecule. A T-cell specific receptor is the so called "T- cell receptor" (TCRs), which allows a T cell to bind to and, if additional signals are present, to be activated by and respond to an epitope / antigen presented by another cell called the antigen-presenting cell or APC. The T cell receptor is known to resemble a Fab fragment of a naturally occurring immunoglobulin. It is generally monovalent, encompassing .alpha.- and .beta.-chains, in some embodiments, it encompasses .gamma.-chains and .delta.-chains (supra). Accordingly, in some embodiments, the TCR is TCR (alpha / beta) and in some embodiments, it is TCR (gamma / delta). The T cell receptor forms a complex with the CD3 T- Cell co-receptor. CD3 is a protein complex and is composed of four distinct chains. In mammals, the complex contains a CD3γ chain, a CD36 chain, and two CD3E chains. These chains associate with a molecule known as the T cell receptor (TCR) and the ζ-chain to generate an activation signal in T lymphocytes. Hence, in some embodiments, a T-cell specific receptor is the CD3 T-Cell co-receptor. In some embodiments, a T-cell specific receptor is CD28, a protein that is also expressed on T cells. CD28 can provide co- stimulatory signals, which are required for T cell activation. CD28 plays important roles in T- cell proliferation and survival, cytokine production, and T-helper type-2 development. Yet a further example of a T-cell specific receptor is CD134, also termed Ox40. CD134 / OX40 is being expressed after 24 to 72 hours following activation and can be taken to define a secondary costimulatory molecule. Another example of a T-cell receptor is 4-1 BB capable of binding to 4-1 BB-Ligand on antigen presenting cells (APCs), whereby a costimulatory signal for the T cell is generated. Another example of a receptor predominantly found on T- cells is CD5, which is also found on B cells at low levels. A further example of a receptor modifying T cell functions is CD95, also known as the Fas receptor, which mediatesDocket No.206193-0138-00WO apoptotic signaling by Fas-ligand expressed on the surface of other cells. CD95 has been reported to modulate TCR / CD3-driven signaling pathways in resting T lymphocytes.

[0091] An example of a NK cell specific receptor molecule is CD16, a low affinity Fc receptor and NKG2D. An example of a receptor molecule that is present on the surface of both T cells and natural killer (NK) cells is CD2 and further members of the CD2- superfamily. CD2 is able to act as a co-stimulatory molecule on T and NK cells.

[0092] In some embodiments, the synthetic binding molecule comprisies at least one binding site of the synthetic binding molecule binds a tumor antigen and at least one binding site that binds a T cell specific receptor molecule and / or a natural killer (NK) cell specific receptor molecule.

[0093] In some embodiments, at least one binding site of the synthetic binding molecule binds CA9, and at least one binding site of the synthetic binding molecule binds 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 CA9 and the second binding site binds one of CD3, TCR, CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5, CD40, FcgRs, FceRs, FcaRs and CD95. In some embodiments, at least one binding site of the synthetic binding molecule binds CA9 and at least one binding site of the synthetic binding molecule binds CD3.

[0094] In some embodiments, at least one binding site of the synthetic binding molecule binds a T cell specific receptor molecule and / or a natural killer (NK) cell specific receptor molecule and two or more bindings sites of the synthetic binding molecule a tumor antigen. In some embodiments, at least one binding site of the synthetic binding molecule binds a T cell specific receptor molecule and / or a natural killer (NK) cell specific receptor molecule and two or more bindings sites of the synthetic binding molecule bind CA9. In some embodiments, at least one binding site of the synthetic binding molecule binds one of CD3, TCR, CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5, CD40, FcgRs, FceRs, FcaRs and CD95, and the second binding site binds CA9. In some embodiments, at least one binding site of the synthetic binding molecule binds CD3, and two or more bindings sites of the synthetic binding molecule bind CA9.Docket No.206193-0138-00WO Arrangements

[0095] In one embodiment the synthetic binding molecule of the invention comprises a PBTE or PMTE, comprising an antibody Fc domain linked to one or more tandem scFv antibody fragments, thereby allowing the PBTE or PMTE to bind or react with the desired target molecules.

[0096] In one embodiment the PBTE or PMTE comprises at least one scFv specific for binding to a target disease-specific antigen linked to at least one scFv specific for binding to a T cell specific receptor molecule. The linkage may place the tandem scFv domains in any order, for example, in one embodiment, the scFv specific for binding to a target disease- specific antigen is oriented C-terminal to a scFv specific for binding to a T cell specific receptor molecule. In another embodiment, scFv specific for binding to a target disease- specific antigen is oriented N-terminal to a scFv specific for binding to a T cell specific receptor molecule. In some embodiments, each scFv has a heavy chain and a light chain. In some embodiments, the heavy chain of the disease-specific scFv is C-terminal to the light chain of the disease-specific scFv. In some embodiments, heavy chain of the disease-specific scFv is N-terminal to the light chain of the disease-specific scFv. In some embodiments, the heavy chain of the T cell specific receptor molecule scFV is C-terminal to the light chain of the T cell specific receptor molecule scFV. In some embodiments, the heavy chain of the T cell specific receptor molecule scFv is N-terminal to the light chain of the T cell specific receptor molecule scFv.

[0097] In one embodiment the PBTE or PMTE comprises at least one scFv specific for binding to a target disease-specific antigen linked to at least one scFv specific for binding to a T cell specific receptor molecule and an Fc domain. In one embodiment, the Fc domain is oriented C-terminal to the scFv domains. In another embodiment, the Fc domain is oriented N-terminal to the scFv domains. In various embodiments, the immune cell engaging domain comprises a nucleotide sequence encoding an antibody, a fragment thereof, or a variant thereof specific for binding to a 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, FcgRs, FceRs, FcaRs and CD95.Docket No.206193-0138-00WO Bispecific T Cell Engager

[0098] In some embodiments, the binding molecule of the invention can be a bispecific T cell engager (BTE), a fragment thereof, a variant thereof, or a combination thereof.

[0099] In one embodiment, the BTE comprises linked scFv binding molecules comprising an antigen binding domain linked to an immune cell engaging domain. In some embodiments, the BTE comprises a CA9 scFv comprising a heavy chain and light chain specific for binding to CA9 operably linked to a CD3 scFv comprising a heavy chain and light chain specific for binding to CD3. In some embodiments, the CA9 heavy chain comprises SEQ ID NO:4 and the CA9 light chain comprises SEQ ID NO:2. In some embodiments, the CD3 heavy chain comprises SEQ ID NO:6 and the CD3 light chain comprises SEQ ID NO:8. In some embodiments, the anti-CA9 heavy chain is C-terminal to the anti-CA9 light chain. In some embodiments, the anti-CA9 heavy chain is N-terminal to the anti-CA9 light chain. In some embodiments, the anti-CD3 heavy chain is C-terminal to the anti-CD3 light chain. In some embodiments, the anti-CD3 heavy chain is N-terminal to the anti-CD3 light chain.

[0100] In some embodiments, the BTE comprises SEQ ID NO:10 or SEQ ID NO:12. Persistent Bispecific T Cell Engager

[0101] In some embodiments, the binding molecule of the invention can be a persistent bispecific T cell engager (PBTE), a fragment thereof, a variant thereof, or a combination thereof.

[0102] In one embodiment, the PBTE comprises an antibody Fc domain linked to an antigen binding domain linked to an immune cell engaging domain. In some embodiments, the Fc domain comprises SEQ ID NO:33 or SEQ ID NO:34. In some embodiments, the Fc domain further comprises at least one stabilization motif. In some embodiments, the Fc domain comprises at least two copies of SEQ ID NO:33 or SEQ ID NO:34, connected by a linker. In some embodiments, the PBTE comprises the Fc domain of SEQ ID NO:29 or SEQ ID NO:30. In some embodiments, the PBTE comprises an antibody Fc domain linked to a CA9 scFv comprising a heavy chain and light chain specific for binding to CA9 operably linked to a CD3 scFv comprising a heavy chain and light chain specific for binding to CD3. In some embodiments, the CA9 heavy chain comprises SEQ ID NO:4 and the CA9 lightDocket No.206193-0138-00WO chain comprises SEQ ID NO:2. In some embodiments, the CD3 heavy chain comprises SEQ ID NO:6 and the CD3 light chain comprises SEQ ID NO:8. In some embodiments, the PBTE comprises an Fc domain operably linked to an anti-CA9 scFv comprising SEQ ID NO:4 and SEQ ID NO:2 and further operably linked to an anti-CD3 scFc comprising SEQ ID NO:6 and SEQ ID NO:8. In some embodiments, the anti-CA9 scFv is between the Fc domain and the anti-CD3 scFv. In some embodiments, the anti-CD3 scFv is between the Fc domain and the anti-CA9 scFv. In some embodiments, the anti-CA9 heavy chain is C-terminal to the anti- CA9 light chain. In some embodiments, the anti-CA9 heavy chain is N-terminal to the anti- CA9 light chain. In some embodiments, the anti-CD3 heavy chain is C-terminal to the anti- CD3 light chain. In some embodiments, the anti-CD3 heavy chain is N-terminal to the anti- CD3 light chain.

[0103] In some embodiments, the PBTE comprises SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 or SEQ ID NO:20. Persistent Multispecific T Cell Engager

[0104] In some embodiments, the binding molecule of the invention can be a persistent multispecific T cell engager (PMTE), a fragment thereof, a variant thereof, or a combination thereof.

[0105] In one embodiment, the PMTE comprises an antibody Fc domain linked to a first antigen binding domain linked to an immune cell engaging domain linked to a second antigen binding domain. In some embodiments, the Fc domain comprises SEQ ID NO:33 or SEQ ID NO:34. In some embodiments, the Fc domain further comprises at least one stabilization motif. In some embodiments, the Fc domain comprises at least two copies of SEQ ID NO:33 or SEQ ID NO:34, connected by a linker. In some embodiments, the PMTE comprises the Fc domain of SEQ ID NO:29 or SEQ ID NO:30. In some embodiments, the first and second antigen binding domain are both specific for binding to CA9. In some embodiments, the PMTE comprises an antibody Fc domain linked to a first CA9 scFv comprising a heavy chain and light chain specific for binding to CA9 operably linked to a CD3 scFv comprising a heavy chain and light chain specific for binding to CD3 linked to a second CA9 scFv comprising a heavy chain and light chain specific for binding to CA9. In some embodiments, the first CA9 heavy chain comprises SEQ ID NO:4 and the first CA9 light chain comprises SEQ ID NO:2. In some embodiments, the CD3 heavy chain comprisesDocket No.206193-0138-00WO SEQ ID NO:6 and the CD3 light chain comprises SEQ ID NO:8. In some embodiments, the second CA9 heavy chain comprises SEQ ID NO:4 and the second CA9 light chain comprises SEQ ID NO:2. In some embodiments, the PMTE comprises an Fc domain operably linked to a first anti-CA9 scFv comprising SEQ ID NO:4 and SEQ ID NO:2, operably linked to an anti-CD3 scFc comprising SEQ ID NO:6 and SEQ ID NO:8, operably linked to a second anti-CA9 scFv comprising SEQ ID NO:4 and SEQ ID NO:2. In some embodiments, the anti- CD3 scFv is between the first and second anti-CA9 scFv. In some embodiments, the heavy chain of the first anti-CA9 scFv is C-terminal to the light chain. In some embodiments, the heavy chain of the first anti-CA9 scFv is N-terminal to the light chain. In some embodiments, the anti-CD3 heavy chain is C-terminal to the anti-CD3 light chain. In some embodiments, the anti-CD3 heavy chain is N-terminal to the anti-CD3 light chain. In some embodiments, the heavy chain of the second anti-CA9 scFv is C-terminal to the light chain. In some embodiments, the heavy chain of the second anti-CA9 scFv is N-terminal to the light chain.

[0106] In some embodiments, the PMTE comprises SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO:28. Multispecific Antibody

[0107] The persistent multivalent T cell engager of the invention can be a multivalent antibody, a fragment thereof, a variant thereof, or a combination thereof. The multivalent antibody can bind or react with at least three target molecules, for example, an immune cell and at least two different antigens. The multivalent antibody can be comprised of at least three tandem linked scFvs, thereby allowing the multivalent antibody to bind or react with at least three desired target molecules.

[0108] The invention provides novel multivalent antibodies 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 a third antigen-binding site that specifically binds to a third target, with particularly advantageous properties such as producibility, stability, binding affinity, biological activity, specific targeting of certain T cells, targeting efficiency and reduced toxicity. In some instances, there are multivalent antibodies, wherein the multivalent antibody binds to each target with different affinity. In some instances, there are multivalent antibodies, wherein the multivalent antibody binds to each target with the same affinity.Docket No.206193-0138-00WO

[0109] A multispecific binding molecule according to the invention may have at least three binding sites of any desired specificity. In some embodiments, one of the binding sites is capable of binding a tumor antigen. In some embodiments, the binding site included in the single chain Fv fragment is a binding site specific for a CA9 tumor antigen.

[0110] In one embodiment the multispecific binding molecule, comprises a first scFv specific for binding to a target disease-specific antigen linked to a scFv specific for binding to a T cell specific receptor molecule linked to a second scFv specific for binding to a target disease-specific antigen. The linkage may place the first and second domains in any order, for example, in one embodiment, a an scFv specific for binding to a target disease-specific antigen is oriented C-Terminal to a scFv specific for binding to a T cell specific receptor molecule. In another embodiment, a nucleotide sequence encoding a scFv specific for binding to a target disease-specific antigen is oriented N-Terminal to a nucleotide sequence encoding a scFv specific for binding to a T cell specific receptor molecule. Extension of Antibody Half-Life

[0111] As described above, the synthetic antibody (e.g., BTE, PBTE, or PMTE) may be modified to extend or shorten the half-life of the antibody in the subject. The modification may extend or shorten the half-life of the antibody in the serum of the subject.

[0112] The modification may be present in a constant region of the antibody. The modification may be one or more amino acid substitutions in a constant region of the antibody that extend the half-life of the antibody as compared to a half-life of an antibody not containing the one or more amino acid substitutions. The modification may be one or more amino acid substitutions in the CH2 domain of the antibody that extend the half-life of the antibody as compared to a half-life of an antibody not containing the one or more amino acid substitutions.

[0113] In some embodiments, the one or more amino acid substitutions in the constant region may include replacing a methionine residue in the constant region with a tyrosine residue, a serine residue in the constant region with a threonine residue, a threonine residue in the constant region with a glutamate residue, or any combination thereof, thereby extending the half-life of the antibody.

[0114] In other embodiments, the one or more amino acid substitutions in the constant region may include replacing a methionine residue in the CH2 domain with a tyrosineDocket No.206193-0138-00WO residue, a serine residue in the CH2 domain with a threonine residue, a threonine residue in the CH2 domain with a glutamate residue, or any combination thereof, thereby extending the half-life of the antibody.

[0115] The composition can result in the generation of the synthetic antibody (e.g., BTE, PBTE, or PMTE) in the 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 of administration of the composition to the subject. The composition can result in generation of the synthetic antibody (e.g., BTE, PBTE, or PMTE) in the 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 of administration of the composition to the subject. The composition can result in generation of the synthetic antibody (e.g., BTE, PBTE, or PMTE) in the 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 of administration of the composition to the subject.

[0116] The composition, when administered to the subject in need thereof, can result in the generation of the synthetic antibody (e.g., BTE, PBTE, PMTE) in the subject more quickly than the generation of an endogenous antibody in a subject who is administered an antigen to induce a humoral immune response. The composition can result in the generation of the synthetic antibody (e.g., BTE, PBTE, PMTE) 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 generation of the endogenous antibody in the subject who was administered an antigen to induce a humoral immune response.

[0117] The composition of the present invention can have features required of effective compositions such as being safe so that the composition does not cause illness or death; being protective against illness; and providing ease of administration, few side effects, biological stability and low cost per dose.

[0118] In one embodiment, the CA9 synthetic antibody (e.g., BTE, PBTE, or PMTE) comprises at least one nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, 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 IDDocket No.206193-0138-00WO NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27 or a fragment or variant thereof. In one embodiment, the fragment of SEQ ID NO:1, SEQ ID NO:3, 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 or SEQ ID NO:27 encodes a binding fragment of an antibodyof the invention, and comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or all twelve CDR coding sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7, encoding at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or all twelve CDR sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8. In some embodiments, the fragment of SEQ ID NO:1, SEQ ID NO:3, 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 or SEQ ID NO:27 encodes a binding fragment of a DBTE of the invention comprising at least three CDR sequences of SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, the fragment of SEQ ID NO:1, SEQ ID NO:3, 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 or SEQ ID NO:27 encodes a binding fragment of a DBTE of the invention comprising at least six CDR sequences of SEQ ID NO:2 and SEQ ID NO:4. Substrates

[0119] In one embodiment, the present invention provides a scaffold, substrate, or device comprising a bispecific immune cell engager (e.g., PBTE or PMTE), fragment thereof, or nucleic acid molecule encoding the same. For example, in some embodiments, the present invention provides a tissue engineering scaffold, including but not limited to, a hydrogel, electrospun scaffold, polymeric matrix, or the like, comprising the modulator. In certain embodiments, a bispecific immune cell engager, fragment thereof, or nucleic acid molecule encoding the same, may be coated along the surface of the scaffold, substrate, or device. In certain embodiments, the bispecific immune cell engager, fragment thereof, or nucleic acid molecule encoding the same is encapsulated within the scaffold, substrate, or deviceDocket No.206193-0138-00WO Recombinant Nucleic Acid Sequence

[0120] In one embodiment the invention comprises a BTE, PBTE, or PMTE, comprising one or more scFv antibody fragments as described herein, thereby allowing the BTE, PBTE, or PMTE to bind or react with the desired target molecules.

[0121] In one embodiment the BTE, PBTE, or PMTE 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, and an optional third scFv specific for binding to a target disease-specific antigen linked to the first or second scFv. The linkage may place the domains in any order, for example, in one embodiment, a nucleotide sequence encoding a scFv specific for binding to a target disease-specific antigen is oriented 5’ (or upstream) to a nucleotide sequence encoding a scFv specific for binding to a T cell specific receptor molecule, followed by a nucleotide sequence encoding a scFv specific for binding to a target disease-specific antigen.

[0122] As described above, the composition can comprise a recombinant nucleic acid sequence. The recombinant nucleic acid sequence can encode the synthetic antibody (BTE, PBTE, or PMTE), a fragment thereof, a variant thereof, or a combination thereof. The antibody is described in more detail below.

[0123] The recombinant nucleic acid sequence can be a heterologous nucleic acid sequence. The recombinant nucleic acid sequence can include at least one heterologous nucleic acid sequence or one or more heterologous nucleic acid sequences.

[0124] The recombinant nucleic acid sequence can 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 can include one or more of the following: low GC content leader sequence to increase transcription; mRNA stability and codon optimization; addition of a kozak sequence (e.g., GCC ACC) for increased translation; addition of an immunoglobulin (Ig) leader sequence encoding a signal peptide; and eliminating to the extent possible cis-acting sequence motifs (i.e., internal TATA boxes).

[0125] The recombinant nucleic acid sequence can include one or more recombinant nucleic acid sequence constructs. The recombinant nucleic acid sequence construct can include one or more components, which are described in more detail below.Docket No.206193-0138-00WO

[0126] The recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence that encodes a heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence that encodes a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct can also include a heterologous nucleic acid sequence that encodes a protease or peptidase cleavage site. The recombinant nucleic acid sequence construct can also include a heterologous nucleic acid sequence that encodes an internal ribosome entry site (IRES). An IRES may be either a viral IRES or an eukaryotic IRES. The recombinant nucleic acid sequence construct can include one or more leader sequences, in which each leader sequence encodes a signal peptide. The recombinant nucleic acid sequence construct can include one or more promoters, one or more introns, one or more transcription termination regions, one or more initiation codons, one or more termination or stop codons, and / or one or more polyadenylation signals. The recombinant nucleic acid sequence construct can also include one or more linker or tag sequences. The tag sequence can encode a histadine tag (6xHis tag). Nucleotide Sequence Encoding Bispecific T Cell Engager

[0127] In some embodiments, the nucleic acid molecule encodes a bispecific T cell engager (BTE), a fragment thereof, a variant thereof, or a combination thereof.

[0128] In one embodiment, the BTE comprises linked scFv binding molecules comprising an antigen binding domain linked to an immune cell engaging domain. In some embodiments, the BTE comprises a CA9 scFv comprising a heavy chain and light chain specific for binding to CA9 operably linked to a CD3 scFv comprising a heavy chain and light chain specific for binding to CD3. In some embodiments, the CA9 heavy chain comprises SEQ ID NO:4 and the CA9 light chain comprises SEQ ID NO:2. In some embodiments, the CD3 heavy chain comprises SEQ ID NO:6 and the CD3 light chain comprises SEQ ID NO:8. In some embodiments, the anti-CA9 heavy chain is C-terminal to the anti-CA9 light chain. In some embodiments, the anti-CA9 heavy chain is N-terminal to the anti-CA9 light chain. In some embodiments, the anti-CD3 heavy chain is C-terminal to the anti-CD3 light chain. In some embodiments, the anti-CD3 heavy chain is N-terminal to the anti-CD3 light chain.Docket No.206193-0138-00WO

[0129] In one embodiment, the nucleic acid molecule encoding the BTE comprises SEQ ID NO:3 encoding the CA9 heavy chain and SEQ ID NO:1 encoding the the CA9 light chain. In some embodiments, the nucleic acid molecule encoding the BTE comprises SEQ ID NO:5 encoding the CD3 heavy chain and SEQ ID NO:7 encoding the CD3 light chain. In some embodiments, the nucleotide sequence encoding the anti-CA9 heavy chain is 3’ to the nucleotide sequence encoding the anti-CA9 light chain. In some embodiments, the nucleotide sequence encoding the anti-CA9 heavy chain is 5’ to the nucleotide sequence encoding the anti-CA9 light chain. In some embodiments, the nucleotide sequence encoding the anti-CD3 heavy chain is 5’ to the nucleotide sequence encoding the anti-CD3 light chain. In some embodiments, the nucleotide sequence encoding the anti-CD3 heavy chain is 3’ to the nucleotide sequence encoding the anti-CD3 light chain.

[0130] In some embodiments, the nucleotide sequence encoding the BTE comprises SEQ ID NO:9 or SEQ ID NO:11. Nucleotide Sequence Encoding Persistent Bispecific T Cell Engager

[0131] In some embodiments, the nucleic acid molecule encodes a persistent bispecific T cell engager (PBTE), a fragment thereof, a variant thereof, or a combination thereof.

[0132] In one embodiment, the PBTE comprises an antibody Fc domain linked to an antigen binding domain linked to an immune cell engaging domain. In some embodiments, the Fc domain comprises SEQ ID NO:33 or SEQ ID NO:34. In some embodiments, the Fc domain further comprises at least one stabilization motif. In some embodiments, the Fc domain comprises at least two copies of SEQ ID NO:33 or SEQ ID NO:34, connected by a linker. In some embodiments, the PBTE comprises the Fc domain of SEQ ID NO:29 or SEQ ID NO:30. In some embodiments, the PBTE comprises an antibody Fc domain linked to a CA9 scFv comprising a heavy chain and light chain specific for binding to CA9 operably linked to a CD3 scFv comprising a heavy chain and light chain specific for binding to CD3. In some embodiments, the CA9 heavy chain comprises SEQ ID NO:4 and the CA9 light chain comprises SEQ ID NO:2. In some embodiments, the CD3 heavy chain comprises SEQ ID NO:6 and the CD3 light chain comprises SEQ ID NO:8. In some embodiments, the PBTE comprises an Fc domain operably linked to an anti-CA9 scFv comprising SEQ ID NO:4 and SEQ ID NO:2 and further operably linked to an anti-CD3 scFc comprising SEQ ID NO:6 and SEQ ID NO:8. In some embodiments, the anti-CA9 scFv is between the Fc domain and theDocket No.206193-0138-00WO anti-CD3 scFv. In some embodiments, the anti-CD3 scFv is between the Fc domain and the anti-CA9 scFv. In some embodiments, the anti-CA9 heavy chain is C-terminal to the anti- CA9 light chain. In some embodiments, the anti-CA9 heavy chain is N-terminal to the anti- CA9 light chain. In some embodiments, the anti-CD3 heavy chain is C-terminal to the anti- CD3 light chain. In some embodiments, the anti-CD3 heavy chain is N-terminal to the anti- CD3 light chain.

[0133] In one embodiment, the nucleic acid molecule encoding the PBTE comprises SEQ ID NO:3 encoding the CA9 heavy chain and SEQ ID NO:1 encoding the the CA9 light chain. In some embodiments, the nucleic acid molecule encoding the PBTE comprises SEQ ID NO:5 encoding the CD3 heavy chain and SEQ ID NO:7 encoding the CD3 light chain. In some embodiments, the nucleotide sequence encoding the anti-CA9 heavy chain is 3’ to the nucleotide sequence encoding the anti-CA9 light chain. In some embodiments, the nucleotide sequence encoding the anti-CA9 heavy chain is 5’ to the nucleotide sequence encoding the anti-CA9 light chain. In some embodiments, the nucleotide sequence encoding the anti-CD3 heavy chain is 5’ to the nucleotide sequence encoding the anti-CD3 light chain. In some embodiments, the nucleotide sequence encoding the anti-CD3 heavy chain is 3’ to the nucleotide sequence encoding the anti-CD3 light chain. In some embodiments, the nucleotide sequence encoding the PBTE comprises the nucleotide sequence encoding an Fc domain operably linked to a nucleotide sequences encoding the an anti-CA9 scFv comprising SEQ ID NO:3 and SEQ ID NO:1 and further operably linked to nucleotide sequences encoding the an anti-CD3 scFc comprising SEQ ID NO:5 and SEQ ID NO:7. In some embodiments, the nucleotide sequence encoding the anti-CA9 scFv is between the nucleotide sequence encoding the Fc domain and the nucleotide sequence encoding the anti-CD3 scFv. In some embodiments, the nucleotide sequence encoding the anti-CD3 scFv is between the nucleotide sequence encoding the Fc domain and the nucleotide sequence encoding the anti-CA9 scFv. In some embodiments, the Fc domain comprises SEQ ID NO:33 or SEQ ID NO:34. In some embodiments, the Fc domain further comprises at least one stabilization motif. In some embodiments, the Fc domain comprises at least two copies of SEQ ID NO:33 or SEQ ID NO:34, connected by a linker. In some embodiments, the PBTE comprises the Fc domain of SEQ ID NO:29 or SEQ ID NO:30.

[0134] In some embodiments, the nucleotide sequence encoding the PBTE comprises SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 or SEQ ID NO:19.Docket No.206193-0138-00WO Nucleotide Sequence Encoding Persistent Multispecific T Cell Engager

[0135] In some embodiments, the nucleic acid molecule encodes a persistent multispecific T cell engager (PMTE), a fragment thereof, a variant thereof, or a combination thereof.

[0136] In one embodiment, the PMTE comprises an antibody Fc domain linked to a first antigen binding domain linked to an immune cell engaging domain linked to a second antigen binding domain. In some embodiments, the Fc domain comprises SEQ ID NO:33 or SEQ ID NO:34. In some embodiments, the Fc domain further comprises at least one stabilization motif. In some embodiments, the Fc domain comprises at least two copies of SEQ ID NO:33 or SEQ ID NO:34, connected by a linker. In some embodiments, the PmTE comprises the Fc domain of SEQ ID NO:29 or SEQ ID NO:30. In some embodiments, the first and second antigen binding domain are both specific for binding to CA9. In some embodiments, the PMTE comprises an antibody Fc domain linked to a first CA9 scFv comprising a heavy chain and light chain specific for binding to CA9 operably linked to a CD3 scFv comprising a heavy chain and light chain specific for binding to CD3 linked to a second CA9 scFv comprising a heavy chain and light chain specific for binding to CA9. In some embodiments, the first CA9 heavy chain comprises SEQ ID NO:4 and the first CA9 light chain comprises SEQ ID NO:2. In some embodiments, the CD3 heavy chain comprises SEQ ID NO:6 and the CD3 light chain comprises SEQ ID NO:8. In some embodiments, the second CA9 heavy chain comprises SEQ ID NO:4 and the second CA9 light chain comprises SEQ ID NO:2. In some embodiments, the PMTE comprises an Fc domain operably linked to a first anti-CA9 scFv comprising SEQ ID NO:4 and SEQ ID NO:2, operably linked to an anti-CD3 scFc comprising SEQ ID NO:6 and SEQ ID NO:8, operably linked to a second anti-CA9 scFv comprising SEQ ID NO:4 and SEQ ID NO:2. In some embodiments, the anti- CD3 scFv is between the first and second anti-CA9 scFv. In some embodiments, the heavy chain of the first anti-CA9 scFv is C-terminal to the light chain. In some embodiments, the heavy chain of the first anti-CA9 scFv is N-terminal to the light chain. In some embodiments, the anti-CD3 heavy chain is C-terminal to the anti-CD3 light chain. In some embodiments, the anti-CD3 heavy chain is N-terminal to the anti-CD3 light chain. In some embodiments, the heavy chain of the second anti-CA9 scFv is C-terminal to the light chain. In some embodiments, the heavy chain of the second anti-CA9 scFv is N-terminal to the light chain.Docket No.206193-0138-00WO

[0137] In one embodiment, the nucleic acid molecule encoding the PMTE comprises SEQ ID NO:3 encoding the first CA9 heavy chain and SEQ ID NO:1 encoding the first CA9 light chain. In some embodiments, the nucleic acid molecule encoding the PMTE comprises SEQ ID NO:5 encoding the CD3 heavy chain and SEQ ID NO:7 encoding the CD3 light chain. In one embodiment, the nucleic acid molecule encoding the PMTE comprises SEQ ID NO:3 encoding the second CA9 heavy chain and SEQ ID NO:1 encoding the second CA9 light chain. In some embodiments, the nucleotide sequence encoding the anti-CA9 heavy chain is 3’ to the nucleotide sequence encoding the anti-CA9 light chain. In some embodiments, the nucleotide sequence encoding the anti-CA9 heavy chain is 5’ to the nucleotide sequence encoding the anti-CA9 light chain. In some embodiments, the nucleotide sequence encoding the anti-CD3 heavy chain is 5’ to the nucleotide sequence encoding the anti-CD3 light chain. In some embodiments, the nucleotide sequence encoding the anti-CD3 heavy chain is 3’ to the nucleotide sequence encoding the anti-CD3 light chain. In some embodiments, the nucleotide sequence encoding the PMTE comprises the nucleotide sequence encoding an Fc domain operably linked to a nucleotide sequences encoding a first anti-CA9 scFv comprising SEQ ID NO:3 and SEQ ID NO:1 and further operably linked to nucleotide sequences encoding the an anti-CD3 scFc comprising SEQ ID NO:5 and SEQ ID NO:7, and further operably linked to nucleotide sequences encoding a second anti-CA9 scFv comprising SEQ ID NO:3 and SEQ ID NO:1. In some embodiments, the nucleotide sequences encoding the first anti-CA9 scFv are between the nucleotide sequence encoding the Fc domain and the nucleotide sequences encoding the anti-CD3 scFv, and the nucleotide sequences encoding the anti-CD3 scFv are between the nucleotide sequences encoding the first and second anti-CA9 scFv. In some embodiments, the Fc domain comprises SEQ ID NO:33 or SEQ ID NO:34. In some embodiments, the Fc domain further comprises at least one stabilization motif. In some embodiments, the Fc domain comprises at least two copies of SEQ ID NO:33 or SEQ ID NO:34, connected by a linker. In some embodiments, the PMTE comprises the Fc domain of SEQ ID NO:29 or SEQ ID NO:30.

[0138] In some embodiments, the nucleic acid molecule encoding the PMTE comprises SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27.Docket No.206193-0138-00WO Promoter

[0139] The recombinant nucleic acid sequence construct can include one or more promoters. The one or more promoters may be any promoter that is capable of driving gene expression and regulating gene expression. Such a promoter is a cis-acting sequence element required for transcription via a DNA dependent RNA polymerase. Selection of the promoter used to direct gene expression depends on the particular application. The promoter may be positioned about the same distance from the transcription start in the recombinant nucleic acid sequence construct as it is from the transcription start site in its natural setting. However, variation in this distance may be accommodated without loss of promoter function.

[0140] The promoter may be operably linked to the heterologous nucleic acid sequence encoding the heavy chain polypeptide and / or light chain polypeptide. The promoter may be a promoter shown effective for expression in eukaryotic cells. The promoter operably linked to the coding sequence may be a CMV promoter, a promoter from simian virus 40 (SV40), such as SV40 early promoter and SV40 later promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, human polyhedrin, or human metalothionein.

[0141] The promoter can be a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some particular 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 may also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in US patent application publication no. US20040175727, the contents of which are incorporated herein in its entirety.

[0142] The promoter can be associated with an enhancer. The enhancer can be located upstream of the coding sequence. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, FMDV,Docket No.206193-0138-00WO RSV or EBV. Polynucleotide function enhances are described in U.S. Patent Nos.5,593,972, 5,962,428, and W094 / 016737, the contents of each are fully incorporated by reference. Transcription Termination Region

[0143] The recombinant nucleic acid sequence construct can include one or more transcription termination regions. The transcription termination region can be downstream of the coding sequence to provide for efficient termination. The transcription termination region can be obtained from the same gene as the promoter described above or can be obtained from one or more different genes. Initiation Codon

[0144] The recombinant nucleic acid sequence construct can include one or more initiation codons. The initiation codon can be located upstream of the coding sequence. The initiation codon can be in frame with the coding sequence. The initiation codon can be associated with one or more signals required for efficient translation initiation, for example, but not limited to, a ribosome binding site. Termination Codon

[0145] The recombinant nucleic acid sequence construct can 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 required for efficient translation termination. Polyadenylation Signal

[0146] The recombinant nucleic acid sequence construct can include one or more polyadenylation signals. The polyadenylation signal can include one or more signals required for efficient polyadenylation of the transcript. The polyadenylation signal can be positioned downstream of the coding sequence. The polyadenylation signal may be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human β- globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 plasmid (Invitrogen, San Diego, CA).Docket No.206193-0138-00WO Leader Sequence

[0147] The recombinant nucleic acid sequence construct can include one or more leader sequences. The leader sequence can encode a signal peptide. The signal peptide can be an immunoglobulin (Ig) signal peptide, for example, but not limited to, an IgG signal peptide and a IgE signal peptide. Expression from the Recombinant Nucleic Acid Sequence Construct

[0148] As described above, the recombinant nucleic acid sequence construct can include, amongst the one or more components, the heterologous nucleic acid sequence encoding the heavy chain polypeptide and / or the heterologous nucleic acid sequence encoding the light chain polypeptide. Accordingly, the recombinant nucleic acid sequence construct can facilitate expression of the PBTE or PMTE.

[0149] Upon expression, for example, but not limited to, in a cell, organism, or mammal, the heavy chain polypeptides and the light chain polypeptides can assemble into the synthetic antibody (BTE, PBTE, or PMTE). In particular, the heavy chain polypeptide and the light chain polypeptide can interact with one another such that assembly results in the synthetic antibody (BTE, PBTE, or PMTE) being capable of binding the antigen. Vector

[0150] The recombinant nucleic acid sequence construct described above can be placed in one or more vectors. The one or more vectors can contain 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-replication extra chromosomal vector, or a vector which integrates into a host genome.

[0151] The one or more vectors can be a heterologous expression construct, which is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the heavy chain polypeptide and / or light chain polypeptide that are encoded by the recombinant nucleic acid sequence construct is produced by the cellular-transcription and translation machinery ribosomal complexes. The one or more vectors can express large amounts of stable messenger RNA, and therefore proteins.Docket No.206193-0138-00WO Expression Vector

[0152] The one or more vectors can be a circular plasmid or a linear nucleic acid. The circular plasmid and linear nucleic acid are capable of directing expression of a particular nucleotide sequence in an appropriate subject cell. The one or more vectors comprising the recombinant nucleic acid sequence construct may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. Plasmid

[0153] The one or more vectors can be a plasmid. The plasmid may be useful for transfecting cells with the recombinant nucleic acid sequence construct. The plasmid may be useful for introducing the recombinant nucleic acid sequence construct into the subject. The plasmid may also comprise a regulatory sequence, which may be well suited for gene expression in a cell into which the plasmid is administered.

[0154] The plasmid may also comprise a mammalian origin of replication in order to maintain the plasmid extrachromosomally and produce multiple copies of the plasmid in a cell. The plasmid may be pVAX1, pCEP4 or pREP4 from Invitrogen (San Diego, CA), which may comprise the Epstein Barr virus origin of replication and nuclear antigen EBNA-1 coding region, which may produce high copy episomal replication without integration. The backbone of the plasmid may be pAV0242. The plasmid may be a replication defective adenovirus type 5 (Ad5) plasmid.

[0155] The plasmid may be pSE420 (Invitrogen, San Diego, Calif.), which may be used for protein production in Escherichia coli (E.coli). The plasmid may also be p YES2 (Invitrogen, San Diego, Calif.), which may be used for protein production in Saccharomyces cerevisiae strains of yeast. The plasmid may also be of the MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, Calif.), which may be used for protein production in insect cells. The plasmid may also be pcDNAI or pcDNA3 (Invitrogen, San Diego, Calif.), which may be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells. RNA

[0156] In one embodiment, the nucleic acid is an RNA molecule. In one embodiment, the RNA molecule is transcribed from a DNA sequence. Accordingly, in one embodiment,Docket No.206193-0138-00WO the invention provides an RNA molecule encoding one or more of the synthetic antibodies of the invention. The RNA may be plus-stranded. Accordingly, in some embodiments, the RNA molecule can be translated by cells without needing any intervening replication steps such as reverse transcription. A RNA molecule useful with the invention may have a 5′ cap (e.g. a 7- methylguanosine). This cap can enhance in vivo translation of the RNA. The 5′ nucleotide of a RNA molecule useful with the invention may have a 5′ triphosphate group. In a capped RNA this may be linked to a 7-methylguanosine via a 5′-to-5′ bridge. A RNA molecule may have a 3′ poly-A tail. It may also include a poly-A polymerase recognition sequence (e.g. AAUAAA) near its 3′ end. A RNA molecule useful with the invention may be single- stranded. A RNA molecule useful with the invention may comprise synthetic RNA. In some embodiments, the RNA molecule is a naked RNA molecule. In one embodiment, the RNA molecule is comprised within a vector.

[0157] In one embodiment, the RNA has 5' and 3' UTRs. In one embodiment, the 5' UTR is between zero and 3000 nucleotides in length. The length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.

[0158] The 5' and 3' UTRs can be the naturally occurring, 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 the forward and reverse primers or by any other modifications 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, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of RNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

[0159] In one embodiment, the 5' UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5' UTR that is not endogenous to the gene of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficientDocket No.206193-0138-00WO translation. The requirement for Kozak sequences for many RNAs is known in the art. In other embodiments, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the RNA.

[0160] In one embodiment, the RNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability of RNA in the cell.

[0161] In one embodiment, the RNA is a nucleoside-modified RNA. Nucleoside- modified RNA have particular advantages over non-modified RNA, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation. Circular and Linear Vector

[0162] The one or more vectors may be circular plasmid, which may transform a target cell by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication). The vector can be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing the heavy chain polypeptide and / or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.

[0163] Also provided herein is a linear nucleic acid, or linear expression cassette (“LEC”), that is capable of being efficiently delivered to a subject via electroporation and expressing the heavy chain polypeptide and / or light chain polypeptide encoded by the recombinant nucleic acid sequence construct. The LEC may be any linear DNA devoid of any phosphate backbone. The LEC may not contain any antibiotic resistance genes and / or a phosphate backbone. The LEC may not contain other nucleic acid sequences unrelated to the desired gene expression.

[0164] The LEC may be derived from any plasmid capable of being linearized. The plasmid may be capable of expressing the heavy chain polypeptide and / or light chain polypeptide encoded by the recombinant nucleic acid sequence construct. The plasmid can be pNP (Puerto Rico / 34) or pM2 (New Caledonia / 99). The plasmid may be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing the heavy chain polypeptide and / or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.

[0165] The LEC can be pcrM2. The LEC can be pcrNP. pcrNP and pcrMR can be derived from pNP (Puerto Rico / 34) and pM2 (New Caledonia / 99), respectively.Docket No.206193-0138-00WO Pharmaceutical Compositions

[0166] In some embodiments, the invention provides compositions comprising the PBTE or PMTE, or fragments to variants thereof. In certain embodiments, the composition can treat, prevent, and or / protect against a disease or disorder associated with CA9 expression. In certain embodiments, the composition can treat, prevent, and or / protect against cancer associated with CA9 expression. In certain embodiments, the composition can treat, prevent, and or / protect against renal cell carcinoma.

[0167] The synthetic antibody (e.g., PBTE, or PMTE) can treat, prevent, and / or protect against disease in the subject administered the composition. The synthetic antibody (e.g., PBTE, or PMTE) of the invention can promote survival of the disease in the subject administered the composition. The synthetic antibody (e.g., PBTE or PMTE) can provide at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% survival of the disease in the subject administered the composition. In other embodiments, the synthetic antibody (e.g., PBTE, or PMTE) can provide at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80% survival of the disease in the subject administered the composition.

[0168] The composition can result in the persistence of the synthetic antibody (e.g., PBTE or PMTE) in the 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 of administration of the composition to the subject. The composition can result in persistence of the synthetic antibody (e.g., PBTE or PMTE) in the 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 of administration of the composition to the subject. The composition can result in persistence of the synthetic antibody (e.g., PBTE or PMTE) in the 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 of administration of the composition to the subject.Docket No.206193-0138-00WO

[0169] The composition, when administered to the subject in need thereof, can result in persistence of the synthetic antibody (e.g., PBTE or PMTE) in the subject for longer than an endogenous antibody in a subject who is administered an antigen to induce a humoral immune response.

[0170] The composition of the present invention can have features required of effective compositions such as being safe so that the composition does not cause illness or death; being protective against illness; and providing ease of administration, few side effects, biological stability and low cost per dose.

[0171] The composition dose can be between 1 μg to 10 mg active component / kg body weight / time, and can be 20 μg to 10 mg component / kg body weight / time. The composition can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of composition doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Excipients and Other Components of the Composition

[0172] The composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be functional molecules such as vehicles, carriers, or diluents. The pharmaceutically acceptable excipient can be a transfection facilitating agent, which can include surface active agents, such as immune- stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog 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 facilitating agents.

[0173] The transfection facilitating agent is a polyanion, polycation, including poly-L- glutamate (LGS), or lipid. The transfection facilitating agent is poly-L-glutamate, and the poly-L-glutamate may be present in the composition at a concentration less than 6 mg / ml. The transfection facilitating agent may also include surface active agents such as immune- stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the composition. The composition may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as aDocket No.206193-0138-00WO DNA-liposome mixture (see for example W09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid. 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.

[0174] The composition may further comprise a genetic facilitator agent as described in U.S. Serial No.021,579 filed April 1, 1994, which is fully incorporated by reference.

[0175] The composition may comprise DNA at quantities of from 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 milligram. In some preferred embodiments, composition according to the present invention comprises about 5 nanogram to about 1000 micrograms of DNA. In some preferred embodiments, composition can contain about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the composition can contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the composition can contain about 1 to about 350 micrograms of DNA. In some preferred embodiments, the composition can contain about 25 to about 250 micrograms, from about 100 to about 200 microgram, from about 1 nanogram to 100 milligrams; from about 1 microgram to about 10 milligrams; from about 0.1 microgram to about 10 milligrams; from about 1 milligram to about 2 milligram, from about 5 nanogram to about 1000 micrograms, from about 10 nanograms to about 800 micrograms, from about 0.1 to about 500 micrograms, from about 1 to about 350 micrograms, from about 25 to about 250 micrograms, from about 100 to about 200 microgram of DNA.

[0176] The composition can be formulated according to the mode of administration to be used. An injectable pharmaceutical composition can be sterile, pyrogen free and particulate free. An isotonic formulation or solution can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The composition can comprise a vasoconstriction agent. The isotonic solutions can include phosphate buffered saline. The composition can further comprise stabilizers including gelatin and albumin. TheDocket No.206193-0138-00WO stabilizers can allow the formulation to be stable at room or ambient temperature for extended periods of time, including LGS or polycations or polyanions. Cancer Therapy

[0177] The invention provides methods of treating or preventing cancer, or of treating and preventing growth or metastasis of tumors. Related aspects of the invention provide methods of preventing, aiding in the prevention, and / or reducing metastasis of hyperplastic or tumor cells in an individual. In one embodiment, the cancer is renal cell carcinoma.

[0178] One aspect of the invention provides a method of inhibiting metastasis in an individual in need thereof, the method comprising administering to the individual an effective amount of a composition of the invention. The invention further provides a method of inhibiting metastasis in an individual in need thereof, the method comprising administering to the individual an effective metastasis-inhibiting amount of any one of the compositions described herein.

[0179] In some embodiments of treating or preventing cancer, or of treating and preventing metastasis of tumors in an individual in need thereof, a second agent is administered to the individual, such as an antineoplastic agent. In some embodiments, the second agent comprises a second metastasis-inhibiting agent, such as a plasminogen antagonist, or an adenosine deaminase antagonist. In other embodiments, the second agent is an angiogenesis inhibiting agent.

[0180] The compositions of the invention can be used to prevent, abate, minimize, control, and / or lessen cancer in humans and animals. The compositions of the invention can also be used to slow the rate of primary tumor growth. The compositions of the invention when administered to a subject in need of treatment can be used to stop the spread of cancer cells. As such, the compositions of the invention can be administered as part of a combination therapy with one or more drugs or other pharmaceutical agents. When used as part of the combination therapy, the decrease in metastasis and reduction in primary tumor growth afforded by the compositions of the invention allows for a more effective and efficient use of any pharmaceutical or drug therapy being used to treat the patient. In addition, control of metastasis by the compositions of the invention affords the subject a greater ability to concentrate the disease in one location.Docket No.206193-0138-00WO

[0181] In one embodiment, the invention provides methods for preventing metastasis of malignant tumors or other cancerous cells as well as to reduce the rate of tumor growth. The methods comprise administering an effective amount of one or more of the compositions of the invention to a subject diagnosed with a malignant tumor or cancerous cells or to a subject having a tumor or cancerous cells.

[0182] In one embodiment, the invention provides a method to treat cancer metastasis comprising treating the subject prior to, concurrently with, or subsequently to the treatment with a composition of the invention, with a complementary therapy for the cancer, such as surgery, chemotherapy, chemotherapeutic agent, radiation therapy, or hormonal therapy or a combination thereof.

[0183] Chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucine phosphate sodium, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alfa- 2a recombinant, paclitaxel, teniposide, and streptozoci), cytotoxic alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylesulfonic acid), alkylating agents (e.g., asaley, AZQ, BCNU, busulfan, bisulphan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cis-platinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, cyclophosphamide, dianhydrogalactitol, fluorodopan, hepsulfam, hycanthone, iphosphamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teroxirone, tetraplatin, thiotepa, triethylenemelamine, uracil nitrogen mustard, and Yoshi-864), antimitotic agents (e.g., allocolchicine, Halichondrin M, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, trityl cysteine, 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), biologicals (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., mitoxantron, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCL,Docket No.206193-0138-00WO daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26 and VP-16), and synthetics (e.g., hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis-diamminedichloroplatimun, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel and porfimer sodium).

[0184] Antiproliferative agents are compounds that decrease the proliferation of cells. Antiproliferative agents include alkylating agents, antimetabolites, enzymes, biological response modifiers, miscellaneous agents, hormones and antagonists, androgen inhibitors (e.g., flutamide and leuprolide acetate), antiestrogens (e.g., tamoxifen citrate and analogs thereof, toremifene, droloxifene and roloxifene), Additional examples of specific antiproliferative agents include, but are not limited to levamisole, gallium nitrate, granisetron, sargramostim strontium-89 chloride, filgrastim, pilocarpine, dexrazoxane, and ondansetron.

[0185] The compounds of the invention can be administered alone or in combination with other anti-tumor agents, including cytotoxic / antineoplastic agents and anti-angiogenic agents. Cytotoxic / anti-neoplastic agents are defined as agents which attack and kill cancer cells. Some cytotoxic / anti-neoplastic agents are alkylating agents, which alkylate the genetic material in tumor cells, e.g., cis-platin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, carmustine, busulfan, chlorambucil, belustine, uracil mustard, chlomaphazin, and dacabazine. Other cytotoxic / anti-neoplastic agents are antimetabolites for tumor cells, e.g., cytosine arabinoside, fluorouracil, methotrexate, mercaptopuirine, azathioprime, and procarbazine. Other cytotoxic / anti-neoplastic agents are antibiotics, e.g., doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin, mytomycin C, and daunomycin. There are numerous liposomal formulations commercially available for these compounds. Still other cytotoxic / anti-neoplastic agents are mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine and etoposide. Miscellaneous cytotoxic / anti- neoplastic agents include taxol and its derivatives, L-asparaginase, anti-tumor antibodies, dacarbazine, azacytidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.

[0186] Anti-angiogenic agents are well known to those of skill in the art. Suitable anti- angiogenic agents for use in the methods and compositions of the invention include anti- VEGF antibodies, including humanized and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides. Other known inhibitors of angiogenesis include angiostatin, endostatin, interferons, interleukin 1 (including alpha and beta) interleukin 12, retinoic acid,Docket No.206193-0138-00WO and tissue inhibitors of metalloproteinase-1 and -2. (TIMP-1 and -2). Small molecules, including topoisomerases such as razoxane, a topoisomerase II inhibitor with anti-angiogenic activity, can also be used.

[0187] Other anti-cancer agents that can be used in combination with the compositions of the invention include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; 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; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1; interferon alfa-n3; interferon beta-I a; interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;Docket No.206193-0138-00WO puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride. Other anti-cancer drugs include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-;Docket No.206193-0138-00WO dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives;Docket No.206193-0138-00WO palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum- triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. In one embodiment, the anti- cancer drug is 5-fluorouracil, taxol, or leucovorin.Docket No.206193-0138-00WO Method of Delivery of the Composition

[0188] The present invention also relates to a method of delivering the composition to the subject in need thereof. The method of delivery can include, administering the composition to the subject. In some embodiments, the present invention relates to administration of a PBTE or PMTE antibody of the invention, or a nucleic acid molecule encoding a PBTE or PMTE antibody of the invention. In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is an RNA molecule. In some embodiments, the nucleic acid molecule is an mRNA molecule.

[0189] Administration can include, but is not limited to, intravenous delivery of an antibody, DNA injection, liposome mediated delivery, and nanoparticle facilitated delivery.

[0190] The mammal receiving delivery of the composition may be human, primate, non-human primate, cow, cattle, sheep, goat, antelope, bison, water buffalo, bison, bovids, deer, hedgehogs, elephants, llama, alpaca, mice, rats, and chicken.

[0191] The composition may be administered by different routes including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal intrathecal, and intraarticular or combinations thereof. For veterinary use, the composition may be administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian can readily determine the dosing regimen and route of administration that is most appropriate for a particular animal. The composition may be administered by traditional syringes, needleless injection devices, "microprojectile bombardment gone guns", or other physical methods such as electroporation (“EP”), “hydrodynamic method”, or ultrasound. Delivery Vehicles

[0192] In one embodiment, the present invention provides a composition comprising a delivery vehicle comprising an anti-CA9 PBTE or PMTE, or nucleic acid molecule encoding the same, as described herein. In one embodiment, the nucleic acid molecule encoding the anti-CA9 immune cell engaging antibody comprises an mRNA molecule.

[0193] Exemplary delivery vehicles include, but are not limited to, microspheres, microparticles, nanoparticles, polymerosomes, liposomes, and micelles. For example, inDocket No.206193-0138-00WO some embodiments, the delivery vehicle is a lipid nanoparticle loaded with a nucleic acid molecule encoding a PBTE or PMTE of the invention. In one embodiment, the nucleic acid molecule encoding the PBTE or PMTE comprises an mRNA molecule. In one embodiment, the mRNA encoding the PBTE corresponds to, or is transcribed from, the DNA sequence set forth in SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 or SEQ ID NO:19. In one embodiment, the mRNA encoding the PMTE corresponds to, or is transcribed from, the DNA sequence set forth in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27.

[0194] In some embodiments, the delivery vehicle provides for controlled release, delayed release, or continual release of its loaded cargo. In some embodiments, the delivery vehicle comprises a targeting moiety that targets the delivery vehicle to a treatment site.

[0195] In certain instances, expressing a protein by delivering the encoding mRNA has many benefits over methods that use protein, plasmid DNA or viral vectors. During mRNA transfection, the coding sequence of the desired protein is the only substance delivered to cells, thus avoiding all the side effects associated with plasmid backbones, viral genes, and viral proteins. More importantly, unlike DNA- and viral-based vectors, the mRNA does not carry the risk of being incorporated into the genome and protein production starts immediately after mRNA delivery. For example, high levels of circulating proteins have been measured within 15 to 30 min of in vivo injection of the encoding mRNA. In certain embodiments, using mRNA rather than the protein also has many advantages. Half-lives of proteins in the circulation are often short, thus protein treatment would need frequent dosing, while mRNA provides a template for continuous protein production for several days. Purification of proteins is problematic and they can contain aggregates and other impurities that cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257- 265).

[0196] In order to confirm the presence of the mRNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Northern blotting and RT- PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunogenic means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.Docket No.206193-0138-00WO Nanoparticle Formulations

[0197] In one embodiment, the composition of the invention may comprise a nanoparticle, including but not limited to a lipid nanoparticle (LNP), comprising a CA9 antibody of the invention, or a LNP comprising a nucleic acid encoding a CA9 antibody of the invention. In some embodiments, the composition comprises or encodes all or part of a CA9 binding molecule of the invention, or an immunogenically functional equivalent thereof. In some embodiments, the composition comprises an mRNA molecule that encodes all or part of a CA9 binding molecule of the invention.

[0198] In one embodiment, the LNP comprises or encapsulates an RNA molecule encoding at least one amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28, or a fragment or variant thereof.

[0199] In one embodiment, the composition further comprises one or more additional immunostimulatory agents. Immunostimulatory agents include, but are not limited to, an additional antigen or antigen binding molecule, an immunomodulator, or an adjuvant. Generation of Synthetic Antibodies In Vitro and Ex Vivo

[0200] In one embodiment, the synthetic antibody (e.g., BTE, PBTE, or PMTE) is generated in vitro or ex vivo. For example, in one embodiment, a nucleic acid encoding a synthetic antibody (e.g., BTE, PBTE, or PMTE) can be introduced and expressed in an in vitro or ex vivo cell. Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0201] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising 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 the introduction of a polynucleotide into a host cell is calcium phosphate transfection.Docket No.206193-0138-00WO

[0202] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially 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 lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0203] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water 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).

[0204] In the case where 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 the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Examples

[0205] The present invention is further illustrated in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention,Docket No.206193-0138-00WO are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Example 1

[0206] 90% of RCC cases involve notable overexpression of the TAA, carbonic anhydrase 9 (CA9) (Bui, et al., Clin Cancer Res, 2003.9(2):p.802-11). Its high and homogenous expression is observed at both primary and metastatic sites and its expression patterns correlate with poor prognoses (Bui, et al., Clin Cancer Res, 2003.9(2):p.802-11; Jensen, et al., BJU International, 2008.101(s4): p.41-44; Genega, et al., Am J Clin Pathol, 2010.134(6): p.873-9). This contrasts with its otherwise scarce expression in normal tissue, as its aberrant upregulation arises from the inactivation of a specific regulatory von Hippel- Lindau tumor-suppressor gene (Mboge, et al.,. Metabolites, 2018.8(1)). Functioning as a modulator of extracellular pH by catalyzing the interconversion of carbon dioxide to protons and bicarbonate, CA9 serves cancer cells by acidifying the tumor microenvironment (TME). In doing so, it drives the cancer growth, metabolic adaptation, invasion, and metastasis associated with poor patient prognoses (Mboge, et al.,. Metabolites, 2018.8(1)). Eliminating CA9-expressing cells in cancer or inhibiting its enzymatic activity could thus dismantle these effects. Given its consistent expression in advanced ccRCC and the immune responsivity of this disease, CA9 represents an ideal antigen for targeted immunotherapies aimed at the isolated eradication of cancer and pH equilibration of the TME, wherein relief from acidic conditions could further facilitate immune function and tumor clearance ( Pastorekova, and Gillies, Cancer and Metastasis Reviews, 2019.38(1): p.65-77).

[0207] Despite the promising alignment of these parameters, there are no approved therapies targeting CA9 and clinical testing is largely confined to radio- and chemotherapies (Grinceviciene, S. and D. Matulis, 2019. p.335-349). Vaccination strategies have demonstrated limited clinical efficacy for RCC and the phase III failure of girentuximab, an IgG1 monoclonal antibody relying on an antibody-dependent cellular cytotoxicity (ADCC)Docket No.206193-0138-00WO mechanism of action, emphasizes the need for newer preclinical approaches to better harnessing the power of the immune system in targeting CA9 (Chamie, et al., JAMA Oncol, 2017.3(7): p.913-920.).

[0208] Nucleic acid medicines like synthetic DNA (synDNA) and messenger RNA (mRNA) represent innovative strategies for delivering self-synthesized, durably expressed biologics for cancer. In contrast to the manufacturing and infusion of recombinant medicines, synDNA leverages one’s own cellular machinery in expressing and continually disseminating anti-cancer biologics (Patel, et al., BioDrugs, 2020.34(3): p.273-293). Pronounced improvements to pharmacokinetic (PK) parameters and efficacy have been reported with in vivo DNA-launched, bispecific T cell engagers (dBTE) targeting HER2, IL13Ra2, FSHR, and EGFRViii in ovarian and brain cancers (Bhojnagarwala, et al., Mol Ther Oncolytics, 2022.26: p.289-301; Bordoloi, et al., JCI Insight, 2022.7(22); Park, et al., Mol Ther Oncolytics, 2023.28: p.249-263; Perales-Puchalt, et al., JCI Insight, 2019.4(8)). The physiological benefits stand alongside manufacturing ones that include reduced costs and long-term temperature stability, which could eliminate the requirement for cold-chain storage and expand patient access (Patel, et al., BioDrugs, 2020.34(3): p.273-293). PBTE displays reduced affinity and potency compared to BTE

[0209] Early testing began with only the BTE and PBTE formats bispecific for CA9 and CD3. To generate the PBTE format, an scFc was linked to the conventional BTE N- terminus (Figure 1A). The antibody DNA was codon-optimized and inserted into a pVax1 expression vector for Expi293 cell expression and purification (Figure 1B). Flow cytometry binding analyses against target cell lines revealed an interesting consequence of linking an scFc to the BTE to generate a PBTE. Doing so attenuated binding affinity to target cells, evidenced by right-shifted median fluorescent intensity (MFI) curves for ACHN-CA9, 293T- CA9, and T cells, with corresponding dissociation constant (KD) values declining approximately 2-fold for all cell types (Figure 1C). To next assess the effects of the PBTE’s binding affinity reductions on cytotoxic potency in vitro, we leveraged xCELLigence technology that proxies cell death as a function of electrical impedance by adherent, target cells. Increasing cell index over time is a measure of unprohibited proliferation, unlike low indices resulting from the cytotoxic removal of impeding target cells, indicative of an effective therapy. At 30 nanomolar (nM), both the BTE and PBTE engaged T cell killing ofDocket No.206193-0138-00WO the primary RCC cell line (with VHL mutation) 786-O with over 50% cell death by the 24- hour timepoint. While the BTE maintained this activity at 200 picomolar (pM), PBTE activity declined nearly 2-fold (Figure 1D). Trending reductions in potency for the PBTE were similarly observed against the primary cell line A-498 (with VHL mutation)and accompanying microscopic images were taken at the terminal timepoint (Figure 1E). PMTE overcompensates for PBTE’s reduced CA9 avidity

[0210] A second CA9 binding domain was linked to the PBTE to generate the PMTE, whose anti-CD3 domain was placed medially to maintain equidistantly tight synapses for either engagement arm, to render a maximally potent molecule (Li et al., Cancer Cell, 2017. 13;31(3):383-395; Bluemel et al., Cancer Immunol Immunother., 2010.59(8):1197-209). The full panel of single-chain formats to undergo further testing in unison for assessment of the functional effects of compounding domain appendages is visually represented (Figure 2A). Western blotting presented bands at appropriate MWs for each antibody (Figure 2B). synDNA-delivered PMTE sustains durable activity in vitro with superior tumor control in mice

[0211] To first model a primary RCC condition, subcutaneous xenografts of A-498 (VHL mutant) were implanted in NSG mice, after which the mice were infused with IP human T cells and a single 100 μg dose of dBTE expression vector which elicited complete tumor control (Figure 4). At a low dose of 10 μg for distinguishment, both the dBTE’s and dPBTE’s displayed limited tumor control with respective measurements of approximately 1100 mm3and 1800 mm3tumors by day 33, with a significant difference between the two formats. dPMTE treatment appeared to induce the early elimination of tumors and enforce their ongoing suppression with a terminal 80 mm3mean measurement. Conversely, control mice did not survive beyond day 30.

[0212] To model metastatic ccRCC, SKRC-52 xenografts were implanted in NSG mice, after which the mice were infused with human T cells and administered a single, low dose of 10 μg dBTE or empty vector control. Control mice experienced rapid tumor growth with mice reaching the terminal volume between days 15-27. The dPBTE produced noticeable yet marginal tumor control with a 2000 mm3mean tumor volume by this day, with only one mouse surviving to day 33. The conventional dBTE trended towards higher activityDocket No.206193-0138-00WO with mouse tumors measuring 1250 mm3. In contrast, dPMTE treatment eliminated SKRC-52 tumors soon after administration, with 3 of the 5 mice remaining completely tumor free by day 33 and a terminal mean volume of 190 mm3. Day 30 statistics, powered by at least an n=2 per group, demonstrate the dPMTE’s significant tumor suppression compared to both the dBTE and dPBTE (Figure 3). Example 2: Sequences G37 VL DNA sequence- SEQ ID NO:1 cagagcgtgctgacacagcccccttccgtgtctggagcaccaggacagcgggtgaccatcagctgcacaggcagctcctctaacatcggcgccggct acgacgtgcactggtatcagcacctgccaggaaccgcaccaaagctgctgatctacggcaactctaatagacctagcggcgtgccagatcggtttagcg gatccaagtctggcaccagcgcctccctggcaatcacaggactgcaggcagaggacgagacagattacttctgccagtcctatgatagctccctgtctg cctgggtgtttggcggcggaaccaaggtgaccgtgctg G37 VL Amino acid sequence- SEQ ID NO:2 QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLPGTAPKLLIYGNSNRPSGVPDRFS GSKSGTSASLAITGLQAEDETDYFCQSYDSSLSAWVFGGGTKVTVL G37 VH DNA sequence- SEQ ID NO:3 caggtgcagctggtgcagagcggcggcggcgtggtgcagcctggcggctccctgagactgtcttgtgccgccagcggcttccctttttctagctacgca atgagctgggtgcggcaggcaccaggcaagggactggagtgggtgtccgccatctctgccaacggcggaaccacatactatgccgactccgtgaag ggcaggttcaccatctccagagataactctaagaatacactgtacctgcagatgaacagcctgagggcagaggacaccgccgtgtactattgcgccaac aatggcaattataggggcgcctttgatatctggggccagggcaccatggtgacagtgtccagc G37 VH Amino acid sequence- SEQ ID NO:4 QVQLVQSGGGVVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEWVSAISANGGTTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANNGNYRGAFDIWGQGTMVTVSS CD3 VH DNA sequence- SEQ ID NO:5 gaggtgcagctggtggagagcggcggcggcctggtgcagccaggcggcagcctgaggctgtcctgtgcagcaagcggatactccttc accggctatacaatgaattgggtgaggcaggcccctggcaagggcctggaatgggtggccctgatcaacccctacaagggcgtgtccaDocket No.206193-0138-00WO cctataatcagaagttcaaggaccgctttaccatctctgtggataagagcaagaacacagcctacctgcagatgaatagcctgagagccga ggacacagccgtgtactattgcgcacggagcggatactatggcgactccgattggtattttgacgtgtggggccagggcaccctggtgac agtgagcagc CD3 VH Amino acid sequence- SEQ ID NO:6 EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS CD3 VL DNA sequence- SEQ ID NO:7 gacatccagatgacccagtccccatctagcctgtctgccagcgtcggcgacagggtgaccatcacatgtcgcgcctctcaggatatcaggaactacctg aattggtatcagcagaagcccggcaaggcccctaagctgctgatctactatacatccaggctggagtctggagtgccaagcaggttctccggatctgga agcggaaccgactacaccctgacaatctcctctctgcagcccgaggatttcgccacatactattgtcagcagggcaataccctgccttggacatttggcca gggcaccaaggtggagatcaagagctcc CD3 VL Amino acid sequence- SEQ ID NO:8 DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSG SGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKSS G37_DBiTE DNA sequence- SEQ ID NO:9 atggactggacctggatcctgttcctggtggctgccgccaccagagtgcactctcagagcgtgctgacacagcccccttccgtgtctggagcaccagga cagcgggtgaccatcagctgcacaggcagctcctctaacatcggcgccggctacgacgtgcactggtatcagcacctgccaggaaccgcaccaaagc tgctgatctacggcaactctaatagacctagcggcgtgccagatcggtttagcggatccaagtctggcaccagcgcctccctggcaatcacaggactgc aggcagaggacgagacagattacttctgccagtcctatgatagctccctgtctgcctgggtgtttggcggcggaaccaaggtgaccgtgctgggcggcg gcggcagcggcggcggcggcagcggcggcggcggctcccaggtgcagctggtgcagagcggcggcggcgtggtgcagcctggcggctccctga gactgtcttgtgccgccagcggcttccctttttctagctacgcaatgagctgggtgcggcaggcaccaggcaagggactggagtgggtgtccgccatctc tgccaacggcggaaccacatactatgccgactccgtgaagggcaggttcaccatctccagagataactctaagaatacactgtacctgcagatgaacag cctgagggcagaggacaccgccgtgtactattgcgccaacaatggcaattataggggcgcctttgatatctggggccagggcaccatggtgacagtgtc cagcggcggcggcggctcggaggtgcagctggtggagagcggcggcggcctggtgcagccaggcggcagcctgaggctgtcctgtgcagcaagc ggatactccttcaccggctatacaatgaattgggtgaggcaggcccctggcaagggcctggaatgggtggccctgatcaacccctacaagggcgtgtc cacctataatcagaagttcaaggaccgctttaccatctctgtggataagagcaagaacacagcctacctgcagatgaatagcctgagagccgaggacac agccgtgtactattgcgcacggagcggatactatggcgactccgattggtattttgacgtgtggggccagggcaccctggtgacagtgagcagcggcgDocket No.206193-0138-00WO gcggcggcagcggcggcggcggcagcggcggcggcggctctgacatccagatgacccagtccccatctagcctgtctgccagcgtcggcgacagg gtgaccatcacatgtcgcgcctctcaggatatcaggaactacctgaattggtatcagcagaagcccggcaaggcccctaagctgctgatctactatacatc caggctggagtctggagtgccaagcaggttctccggatctggaagcggaaccgactacaccctgacaatctcctctctgcagcccgaggatttcgccac atactattgtcagcagggcaataccctgccttggacatttggccagggcaccaaggtggagatcaagagctcctgataa G37_DBiTE Amino acid sequence- SEQ ID NO:10 MDWTWILFLVAAATRVHSQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLPGTAP KLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDETDYFCQSYDSSLSAWVFGGGTKVTVL GGGGSGGGGSGGGGSQVQLVQSGGGVVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEW VSAISANGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANNGNYRGAFDIWG QGTMVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWV ALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDV WGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQ QKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGT KVEIKSS G37_DBiTE with His tag DNA sequence- SEQ ID NO:11 atggactggacctggatcctgttcctggtggctgccgccaccagagtgcactctcagagcgtgctgacacagcccccttccgtgtctggagcaccagga cagcgggtgaccatcagctgcacaggcagctcctctaacatcggcgccggctacgacgtgcactggtatcagcacctgccaggaaccgcaccaaagc tgctgatctacggcaactctaatagacctagcggcgtgccagatcggtttagcggatccaagtctggcaccagcgcctccctggcaatcacaggactgc aggcagaggacgagacagattacttctgccagtcctatgatagctccctgtctgcctgggtgtttggcggcggaaccaaggtgaccgtgctgggcggcg gcggcagcggcggcggcggcagcggcggcggcggctcccaggtgcagctggtgcagagcggcggcggcgtggtgcagcctggcggctccctga gactgtcttgtgccgccagcggcttccctttttctagctacgcaatgagctgggtgcggcaggcaccaggcaagggactggagtgggtgtccgccatctc tgccaacggcggaaccacatactatgccgactccgtgaagggcaggttcaccatctccagagataactctaagaatacactgtacctgcagatgaacag cctgagggcagaggacaccgccgtgtactattgcgccaacaatggcaattataggggcgcctttgatatctggggccagggcaccatggtgacagtgtc cagcggcggcggcggctcggaggtgcagctggtggagagcggcggcggcctggtgcagccaggcggcagcctgaggctgtcctgtgcagcaagc ggatactccttcaccggctatacaatgaattgggtgaggcaggcccctggcaagggcctggaatgggtggccctgatcaacccctacaagggcgtgtc cacctataatcagaagttcaaggaccgctttaccatctctgtggataagagcaagaacacagcctacctgcagatgaatagcctgagagccgaggacac agccgtgtactattgcgcacggagcggatactatggcgactccgattggtattttgacgtgtggggccagggcaccctggtgacagtgagcagcggcg gcggcggcagcggcggcggcggcagcggcggcggcggctctgacatccagatgacccagtccccatctagcctgtctgccagcgtcggcgacagg gtgaccatcacatgtcgcgcctctcaggatatcaggaactacctgaattggtatcagcagaagcccggcaaggcccctaagctgctgatctactatacatc caggctggagtctggagtgccaagcaggttctccggatctggaagcggaaccgactacaccctgacaatctcctctctgcagcccgaggatttcgccac atactattgtcagcagggcaataccctgccttggacatttggccagggcaccaaggtggagatcaagagctcccaccaccaccaccaccactgataa G37_DBiTE with His tagDocket No.206193-0138-00WO Amino acid sequence- SEQ ID NO:12 MDWTWILFLVAAATRVHSQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLPGTAP KLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDETDYFCQSYDSSLSAWVFGGGTKVTVL GGGGSGGGGSGGGGSQVQLVQSGGGVVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEW VSAISANGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANNGNYRGAFDIWG QGTMVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWV ALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDV WGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQ QKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGT KVEIKSSHHHHHH G37-DBiTE-Fc DNA sequence- SEQ ID NO:13 ATGGACTGGACCTGGATCCTGTTCCTGGTGGCTGCCGCCACCAGGGTGCACAGCGACAAGA CCCACACATGCCCTCCATGTCCAGCACCAGAGGCAGCCGGCGGACCTAGCGTGTTCCTGTTT CCCCCTAAGCCAAAGGATACCCTGATGATCTCTCGGACCCCTGAGGTGACATGCGTGGTGGT GGACGTGAGCCACGAGGCACCAGAGGTGAAGTTCAACTGGTACGTGGATGGCGTGGAGGTG CACAATGCCAAGACAAAGCCTTGCGAGGAGCAGTACGGCTCCACCTATCGCTGCGTGAGCG TGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCGCCGTGTCCAA TAAGGCCCTGCCTGCCCCAATCGAGAAGACCATCTCTAAGGCCAAGGGCCAGCCTAGGGAG CCACAGGTGTACACACTGCCACCCTCCAGAGAGGAGATGACCAAGAACCAGGTGTCTCTGA CATGTCTGGTGAAGGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGTCCAATGGCCA GCCTGAGAACAATTACAAGACCACACCTCCAGTGCTGGACTCTGATGGCAGCTTCTTTCTGT ATTCTAAGCTGACCGTGGATAAGAGCCGGTGGCAGCAGGGCAACGTGTTTAGCTGCTCCGT GATGCACGAGGCCCTGCACAATCACTACACACAGAAGTCTCTGAGCCTGTCCCCAGGCAAG GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC GGCGGCGGCGGCAGCGGCGGCGGCGGCTCGGATAAAACCCACACATGCCCTCCCTGTCCAG CACCTGAGGCAGCCGGCGGACCAAGCGTGTTCCTGTTTCCACCCAAGCCTAAAGACACCCTG ATGATCAGCAGGACCCCCGAAGTCACCTGCGTGGTCGTGGACGTGTCCCACGAGGCACCTG AAGTCAAGTTCAACTGGTACGTGGACGGAGTCGAAGTCCATAACGCCAAGACAAAGCCCTG TGAAGAGCAGTACGGCAGCACCTATAGATGCGTGAGCGTGCTGACAGTGCTGCATCAGGAT TGGCTGAATGGCAAGGAATACAAGTGCGCCGTGTCTAATAAGGCCCTGCCAGCCCCCATCG AGAAGACCATCTCCAAGGCAAAGGGACAGCCAAGGGAGCCTCAGGTGTACACACTGCCTCC AAGCCGCGAAGAAATGACTAAAAACCAGGTGTCCCTGACCTGCCTGGTCAAAGGCTTCTAT CCATCTGATATTGCCGTGGAGTGGGAGAGCAATGGCCAGCCCGAAAATAATTACAAGACCA CACCCCCTGTGCTGGACTCCGATGGCTCTTTCTTTCTGTATTCCAAACTGACTGTGGATAAGT CTCGCTGGCAGCAGGGAAACGTGTTTTCTTGTAGCGTGATGCATGAGGCCCTGCATAACCAC TACACACAGAAGTCCCTGTCTCTGAGCCCTGGCAAGGGCGGCGGCGGCAGCGGCGGCGGCGDocket No.206193-0138-00WO GCtctcagagcgtgctgacacagcccccttccgtgtctggagcaccaggacagcgggtgaccatcagctgcacaggcagctcctctaacatcggcgc cggctacgacgtgcactggtatcagcacctgccaggaaccgcaccaaagctgctgatctacggcaactctaatagacctagcggcgtgccagatcggtt tagcggatccaagtctggcaccagcgcctccctggcaatcacaggactgcaggcagaggacgagacagattacttctgccagtcctatgatagctccct gtctgcctgggtgtttggcggcggaaccaaggtgaccgtgctgggcggcggcggcagcggcggcggcggcagcggcggcggcggctcccaggtg cagctggtgcagagcggcggcggcgtggtgcagcctggcggctccctgagactgtcttgtgccgccagcggcttccctttttctagctacgcaatgagc tgggtgcggcaggcaccaggcaagggactggagtgggtgtccgccatctctgccaacggcggaaccacatactatgccgactccgtgaagggcagg ttcaccatctccagagataactctaagaatacactgtacctgcagatgaacagcctgagggcagaggacaccgccgtgtactattgcgccaacaatggc aattataggggcgcctttgatatctggggccagggcaccatggtgacagtgtccagcggcggcggcggctcggaggtgcagctggtggagagcggc ggcggcctggtgcagccaggcggcagcctgaggctgtcctgtgcagcaagcggatactccttcaccggctatacaatgaattgggtgaggcaggccc ctggcaagggcctggaatgggtggccctgatcaacccctacaagggcgtgtccacctataatcagaagttcaaggaccgctttaccatctctgtggataa gagcaagaacacagcctacctgcagatgaatagcctgagagccgaggacacagccgtgtactattgcgcacggagcggatactatggcgactccgatt ggtattttgacgtgtggggccagggcaccctggtgacagtgagcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggctctga catccagatgacccagtccccatctagcctgtctgccagcgtcggcgacagggtgaccatcacatgtcgcgcctctcaggatatcaggaactacctgaat tggtatcagcagaagcccggcaaggcccctaagctgctgatctactatacatccaggctggagtctggagtgccaagcaggttctccggatctggaagc ggaaccgactacaccctgacaatctcctctctgcagcccgaggatttcgccacatactattgtcagcagggcaataccctgccttggacatttggccagg gcaccaaggtggagatcaagagctcctgataa G37-DBiTE-Fc Amino acid sequence- SEQ ID NO:14 MDWTWILFLVAAATRVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEAPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEAPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGS GGGGSQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLPGTAPKLLIYGNSNRPSGV PDRFSGSKSGTSASLAITGLQAEDETDYFCQSYDSSLSAWVFGGGTKVTVLGGGGSGGGGSGGG GSQVQLVQSGGGVVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEWVSAISANGGTTYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANNGNYRGAFDIWGQGTMVTVSSGGGG SEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGG GGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYT SRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKSS G37-DBiTE-Fc with His tag DNA sequence- SEQ ID NO:15Docket No.206193-0138-00WO ATGGACTGGACCTGGATCCTGTTCCTGGTGGCTGCCGCCACCAGGGTGCACAGCGACAAGA CCCACACATGCCCTCCATGTCCAGCACCAGAGGCAGCCGGCGGACCTAGCGTGTTCCTGTTT CCCCCTAAGCCAAAGGATACCCTGATGATCTCTCGGACCCCTGAGGTGACATGCGTGGTGGT GGACGTGAGCCACGAGGCACCAGAGGTGAAGTTCAACTGGTACGTGGATGGCGTGGAGGTG CACAATGCCAAGACAAAGCCTTGCGAGGAGCAGTACGGCTCCACCTATCGCTGCGTGAGCG TGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCGCCGTGTCCAA TAAGGCCCTGCCTGCCCCAATCGAGAAGACCATCTCTAAGGCCAAGGGCCAGCCTAGGGAG CCACAGGTGTACACACTGCCACCCTCCAGAGAGGAGATGACCAAGAACCAGGTGTCTCTGA CATGTCTGGTGAAGGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGTCCAATGGCCA GCCTGAGAACAATTACAAGACCACACCTCCAGTGCTGGACTCTGATGGCAGCTTCTTTCTGT ATTCTAAGCTGACCGTGGATAAGAGCCGGTGGCAGCAGGGCAACGTGTTTAGCTGCTCCGT GATGCACGAGGCCCTGCACAATCACTACACACAGAAGTCTCTGAGCCTGTCCCCAGGCAAG GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC GGCGGCGGCGGCAGCGGCGGCGGCGGCTCGGATAAAACCCACACATGCCCTCCCTGTCCAG CACCTGAGGCAGCCGGCGGACCAAGCGTGTTCCTGTTTCCACCCAAGCCTAAAGACACCCTG ATGATCAGCAGGACCCCCGAAGTCACCTGCGTGGTCGTGGACGTGTCCCACGAGGCACCTG AAGTCAAGTTCAACTGGTACGTGGACGGAGTCGAAGTCCATAACGCCAAGACAAAGCCCTG TGAAGAGCAGTACGGCAGCACCTATAGATGCGTGAGCGTGCTGACAGTGCTGCATCAGGAT TGGCTGAATGGCAAGGAATACAAGTGCGCCGTGTCTAATAAGGCCCTGCCAGCCCCCATCG AGAAGACCATCTCCAAGGCAAAGGGACAGCCAAGGGAGCCTCAGGTGTACACACTGCCTCC AAGCCGCGAAGAAATGACTAAAAACCAGGTGTCCCTGACCTGCCTGGTCAAAGGCTTCTAT CCATCTGATATTGCCGTGGAGTGGGAGAGCAATGGCCAGCCCGAAAATAATTACAAGACCA CACCCCCTGTGCTGGACTCCGATGGCTCTTTCTTTCTGTATTCCAAACTGACTGTGGATAAGT CTCGCTGGCAGCAGGGAAACGTGTTTTCTTGTAGCGTGATGCATGAGGCCCTGCATAACCAC TACACACAGAAGTCCCTGTCTCTGAGCCCTGGCAAGGGCGGCGGCGGCAGCGGCGGCGGCG GCtctcagagcgtgctgacacagcccccttccgtgtctggagcaccaggacagcgggtgaccatcagctgcacaggcagctcctctaacatcggcgc cggctacgacgtgcactggtatcagcacctgccaggaaccgcaccaaagctgctgatctacggcaactctaatagacctagcggcgtgccagatcggtt tagcggatccaagtctggcaccagcgcctccctggcaatcacaggactgcaggcagaggacgagacagattacttctgccagtcctatgatagctccct gtctgcctgggtgtttggcggcggaaccaaggtgaccgtgctgggcggcggcggcagcggcggcggcggcagcggcggcggcggctcccaggtg cagctggtgcagagcggcggcggcgtggtgcagcctggcggctccctgagactgtcttgtgccgccagcggcttccctttttctagctacgcaatgagc tgggtgcggcaggcaccaggcaagggactggagtgggtgtccgccatctctgccaacggcggaaccacatactatgccgactccgtgaagggcagg ttcaccatctccagagataactctaagaatacactgtacctgcagatgaacagcctgagggcagaggacaccgccgtgtactattgcgccaacaatggc aattataggggcgcctttgatatctggggccagggcaccatggtgacagtgtccagcggcggcggcggctcggaggtgcagctggtggagagcggc ggcggcctggtgcagccaggcggcagcctgaggctgtcctgtgcagcaagcggatactccttcaccggctatacaatgaattgggtgaggcaggccc ctggcaagggcctggaatgggtggccctgatcaacccctacaagggcgtgtccacctataatcagaagttcaaggaccgctttaccatctctgtggataa gagcaagaacacagcctacctgcagatgaatagcctgagagccgaggacacagccgtgtactattgcgcacggagcggatactatggcgactccgatt ggtattttgacgtgtggggccagggcaccctggtgacagtgagcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggctctga catccagatgacccagtccccatctagcctgtctgccagcgtcggcgacagggtgaccatcacatgtcgcgcctctcaggatatcaggaactacctgaat tggtatcagcagaagcccggcaaggcccctaagctgctgatctactatacatccaggctggagtctggagtgccaagcaggttctccggatctggaagcDocket No.206193-0138-00WO ggaaccgactacaccctgacaatctcctctctgcagcccgaggatttcgccacatactattgtcagcagggcaataccctgccttggacatttggccagg gcaccaaggtggagatcaagagctcccaccaccaccaccaccactgataa G37-DBiTE-Fc with His tag Amino acid sequence- SEQ ID NO:16 MDWTWILFLVAAATRVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEAPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEAPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGS GGGGSQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLPGTAPKLLIYGNSNRPSGV PDRFSGSKSGTSASLAITGLQAEDETDYFCQSYDSSLSAWVFGGGTKVTVLGGGGSGGGGSGGG GSQVQLVQSGGGVVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEWVSAISANGGTTYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANNGNYRGAFDIWGQGTMVTVSSGGGG SEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGG GGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYT SRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKSSHHHHHH G37-DBiTE-Fc+ (with mutations for half life extension) DNA sequence- SEQ ID NO:17 atggactggacctggatcctgttcctggtggctgccgccaccagggtgcacagcgacaagacccacacatgccctccatgtccagcaccagaggcag ccggcggacctagcgtgttcctgtttccccctaagccaaaggataccctgatgatctctcggacccctgaggtgacatgcgtggtggtggacgtgagcca cgaggcaccagaggtgaagttcaactggtacgtggatggcgtggaggtgcacaatgccaagacaaagccttgcgaggagcagtacggctccacctat cgctgcgtgagcgtgctgacagtgctgcaccaggactggctgaacggcaaggagtataagtgcgccgtgtccaataaggccctgcctgccccaatcga gaagaccatctctaaggccaagggccagcctagggagccacaggtgtacacactgccaccctccagagaggagatgaccaagaaccaggtgtctctg acatgtctggtgaagggcttctatcccagcgacatcgccgtggagtgggagtccaatggccagcctgagaacaattacaagaccacacctccagtgctg gactctgatggcagcttctttctgtattctaagctgaccgtggataagagccggtggcagcagggcaacgtgtttagctgctccgtgctgcacgaggccct gcactctcactacacacagaagtctctgagcctgtccccaggcaagggcggcggcggcagcggcggcggcggcagcggcggcggcggcagcgg cggcggcggcagcggcggcggcggcagcggcggcggcggctcggataaaacccacacatgccctccctgtccagcacctgaggcagccggcgg accaagcgtgttcctgtttccacccaagcctaaagacaccctgatgatcagcaggacccccgaagtcacctgcgtggtcgtggacgtgtcccacgaggc acctgaagtcaagttcaactggtacgtggacggagtcgaagtccataacgccaagacaaagccctgtgaagagcagtacggcagcacctatagatgcg tgagcgtgctgacagtgctgcatcaggattggctgaatggcaaggaatacaagtgcgccgtgtctaataaggccctgccagcccccatcgagaagacc atctccaaggcaaagggacagccaagggagcctcaggtgtacacactgcctccaagccgcgaagaaatgactaaaaaccaggtgtccctgacctgcc tggtcaaaggcttctatccatctgatattgccgtggagtgggagagcaatggccagcccgaaaataattacaagaccacaccccctgtgctggactccgaDocket No.206193-0138-00WO tggctctttctttctgtattccaaactgactgtggataagtctcgctggcagcagggaaacgtgttttcttgtagcgtgctgcacgaggccctgcactctcact acacacagaagtccctgtctctgagccctggcaagggcggcggcggcagcggcggcggcggctctcagagcgtgctgacacagcccccttccgtgt ctggagcaccaggacagcgggtgaccatcagctgcacaggcagctcctctaacatcggcgccggctacgacgtgcactggtatcagcacctgccagg aaccgcaccaaagctgctgatctacggcaactctaatagacctagcggcgtgccagatcggtttagcggatccaagtctggcaccagcgcctccctggc aatcacaggactgcaggcagaggacgagacagattacttctgccagtcctatgatagctccctgtctgcctgggtgtttggcggcggaaccaaggtgac cgtgctgggcggcggcggcagcggcggcggcggcagcggcggcggcggctcccaggtgcagctggtgcagagcggcggcggcgtggtgcagc ctggcggctccctgagactgtcttgtgccgccagcggcttccctttttctagctacgcaatgagctgggtgcggcaggcaccaggcaagggactggagt gggtgtccgccatctctgccaacggcggaaccacatactatgccgactccgtgaagggcaggttcaccatctccagagataactctaagaatacactgta cctgcagatgaacagcctgagggcagaggacaccgccgtgtactattgcgccaacaatggcaattataggggcgcctttgatatctggggccagggca ccatggtgacagtgtccagcggcggcggcggctcggaggtgcagctggtggagagcggcggcggcctggtgcagccaggcggcagcctgaggct gtcctgtgcagcaagcggatactccttcaccggctatacaatgaattgggtgaggcaggcccctggcaagggcctggaatgggtggccctgatcaacc cctacaagggcgtgtccacctataatcagaagttcaaggaccgctttaccatctctgtggataagagcaagaacacagcctacctgcagatgaatagcct gagagccgaggacacagccgtgtactattgcgcacggagcggatactatggcgactccgattggtattttgacgtgtggggccagggcaccctggtga cagtgagcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggctctgacatccagatgacccagtccccatctagcctgtctgcc agcgtcggcgacagggtgaccatcacatgtcgcgcctctcaggatatcaggaactacctgaattggtatcagcagaagcccggcaaggcccctaagct gctgatctactatacatccaggctggagtctggagtgccaagcaggttctccggatctggaagcggaaccgactacaccctgacaatctcctctctgcag cccgaggatttcgccacatactattgtcagcagggcaataccctgccttggacatttggccagggcaccaaggtggagatcaagagctcctgataa G37-DBiTE-Fc+ (with mutations for half life extension) Amino acid sequence- SEQ ID NO:18 MDWTWILFLVAAATRVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEAPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGKGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEAPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGKGGGGSGG GGSQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLPGTAPKLLIYGNSNRPSGVPD RFSGSKSGTSASLAITGLQAEDETDYFCQSYDSSLSAWVFGGGTKVTVLGGGGSGGGGSGGGGS QVQLVQSGGGVVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEWVSAISANGGTTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANNGNYRGAFDIWGQGTMVTVSSGGGGSE VQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQK FKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTS RLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKSSDocket No.206193-0138-00WO G37-DBiTE-Fc+ with His tag (with mutations for half life extension) DNA sequence- SEQ ID NO:19 atggactggacctggatcctgttcctggtggctgccgccaccagggtgcacagcgacaagacccacacatgccctccatgtccagcaccagaggcag ccggcggacctagcgtgttcctgtttccccctaagccaaaggataccctgatgatctctcggacccctgaggtgacatgcgtggtggtggacgtgagcca cgaggcaccagaggtgaagttcaactggtacgtggatggcgtggaggtgcacaatgccaagacaaagccttgcgaggagcagtacggctccacctat cgctgcgtgagcgtgctgacagtgctgcaccaggactggctgaacggcaaggagtataagtgcgccgtgtccaataaggccctgcctgccccaatcga gaagaccatctctaaggccaagggccagcctagggagccacaggtgtacacactgccaccctccagagaggagatgaccaagaaccaggtgtctctg acatgtctggtgaagggcttctatcccagcgacatcgccgtggagtgggagtccaatggccagcctgagaacaattacaagaccacacctccagtgctg gactctgatggcagcttctttctgtattctaagctgaccgtggataagagccggtggcagcagggcaacgtgtttagctgctccgtgctgcacgaggccct gcactctcactacacacagaagtctctgagcctgtccccaggcaagggcggcggcggcagcggcggcggcggcagcggcggcggcggcagcgg cggcggcggcagcggcggcggcggcagcggcggcggcggctcggataaaacccacacatgccctccctgtccagcacctgaggcagccggcgg accaagcgtgttcctgtttccacccaagcctaaagacaccctgatgatcagcaggacccccgaagtcacctgcgtggtcgtggacgtgtcccacgaggc acctgaagtcaagttcaactggtacgtggacggagtcgaagtccataacgccaagacaaagccctgtgaagagcagtacggcagcacctatagatgcg tgagcgtgctgacagtgctgcatcaggattggctgaatggcaaggaatacaagtgcgccgtgtctaataaggccctgccagcccccatcgagaagacc atctccaaggcaaagggacagccaagggagcctcaggtgtacacactgcctccaagccgcgaagaaatgactaaaaaccaggtgtccctgacctgcc tggtcaaaggcttctatccatctgatattgccgtggagtgggagagcaatggccagcccgaaaataattacaagaccacaccccctgtgctggactccga tggctctttctttctgtattccaaactgactgtggataagtctcgctggcagcagggaaacgtgttttcttgtagcgtgctgcacgaggccctgcactctcact acacacagaagtccctgtctctgagccctggcaagggcggcggcggcagcggcggcggcggctctcagagcgtgctgacacagcccccttccgtgt ctggagcaccaggacagcgggtgaccatcagctgcacaggcagctcctctaacatcggcgccggctacgacgtgcactggtatcagcacctgccagg aaccgcaccaaagctgctgatctacggcaactctaatagacctagcggcgtgccagatcggtttagcggatccaagtctggcaccagcgcctccctggc aatcacaggactgcaggcagaggacgagacagattacttctgccagtcctatgatagctccctgtctgcctgggtgtttggcggcggaaccaaggtgac cgtgctgggcggcggcggcagcggcggcggcggcagcggcggcggcggctcccaggtgcagctggtgcagagcggcggcggcgtggtgcagc ctggcggctccctgagactgtcttgtgccgccagcggcttccctttttctagctacgcaatgagctgggtgcggcaggcaccaggcaagggactggagt gggtgtccgccatctctgccaacggcggaaccacatactatgccgactccgtgaagggcaggttcaccatctccagagataactctaagaatacactgta cctgcagatgaacagcctgagggcagaggacaccgccgtgtactattgcgccaacaatggcaattataggggcgcctttgatatctggggccagggca ccatggtgacagtgtccagcggcggcggcggctcggaggtgcagctggtggagagcggcggcggcctggtgcagccaggcggcagcctgaggct gtcctgtgcagcaagcggatactccttcaccggctatacaatgaattgggtgaggcaggcccctggcaagggcctggaatgggtggccctgatcaacc cctacaagggcgtgtccacctataatcagaagttcaaggaccgctttaccatctctgtggataagagcaagaacacagcctacctgcagatgaatagcct gagagccgaggacacagccgtgtactattgcgcacggagcggatactatggcgactccgattggtattttgacgtgtggggccagggcaccctggtga cagtgagcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggctctgacatccagatgacccagtccccatctagcctgtctgcc agcgtcggcgacagggtgaccatcacatgtcgcgcctctcaggatatcaggaactacctgaattggtatcagcagaagcccggcaaggcccctaagct gctgatctactatacatccaggctggagtctggagtgccaagcaggttctccggatctggaagcggaaccgactacaccctgacaatctcctctctgcagDocket No.206193-0138-00WO cccgaggatttcgccacatactattgtcagcagggcaataccctgccttggacatttggccagggcaccaaggtggagatcaagagctcccaccaccac caccaccactgataa  G37-DBiTE-Fc+ with His tag (with mutations for half life extension) Amino acid sequence- SEQ ID NO:20 MDWTWILFLVAAATRVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEAPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGKGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEAPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGKGGGGSGG GGSQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLPGTAPKLLIYGNSNRPSGVPD RFSGSKSGTSASLAITGLQAEDETDYFCQSYDSSLSAWVFGGGTKVTVLGGGGSGGGGSGGGGS QVQLVQSGGGVVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEWVSAISANGGTTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANNGNYRGAFDIWGQGTMVTVSSGGGGSE VQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQK FKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTS RLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKSSHHHHHH G37-TRV-Fc DNA sequence- SEQ ID NO:21 atggactggacctggatcctgttcctggtggctgccgccacaagggtgcacagcgataagacccacacatgccctccctgtcctgcaccagaggcagc cggcggaccttccgtgttcctgtttccacccaagccaaaggacaccctgatgatcagcagaacccccgaggtgacatgcgtggtggtggacgtgtccca cgaggcacctgaggtgaagtttaactggtacgtggatggcgtggaggtgcacaatgccaagacaaagccctgcgaggagcagtacggctctacctata gatgcgtgagcgtgctgacagtgctgcaccaggattggctgaacggcaaggagtataagtgcgccgtgtctaataaggccctgcccgcccctatcgag aagaccatcagcaaggcaaagggacagccaagggagcctcaggtgtacacactgcctccatctagagaggagatgaccaagaaccaggtgagcctg acatgtctggtgaagggcttctatccatccgacatcgccgtggagtgggagtctaatggccagcccgagaacaattacaagaccacaccccctgtgctg gactccgatggctctttctttctgtatagcaagctgaccgtggataagtccaggtggcagcagggcaacgtgtttagctgctccgtgatgcacgaggccct gcacaatcactacacacagaagtctctgagcctgtcccctggcaagggcggcggcggcagcggcggcggcggcagcggcggcggcggcagcgg cggcggcggcagcggcggcggcggcagcggcggcggcggctccgacaagacccacacatgcccaccctgtccagcccctgaggcagccggcg gaccaagcgtgttcctgtttcctccaaagcctaaggataccctgatgatctcccggaccccagaagtcacctgcgtggtcgtggacgtgtctcacgaggc ccccgaggtgaagttcaactggtacgtggacggagtcgaagtgcacaatgccaagaccaagccatgtgaagagcagtacggctccacctatcgctgc gtgagcgtgctgacagtgctgcatcaggactggctgaatggcaaggaatataagtgcgccgtgagcaataaggccctgcctgccccaatcgagaagac catctctaaagccaaaggacagccaagggagccacaggtgtacacactgcccccttcccgcgaagaaatgactaaaaaccaggtgtctctgacctgcc tggtcaaaggcttctatcctagcgacatcgcagtggagtgggagtccaacggacagccagaaaataattacaagaccacaccacccgtgctggacagcDocket No.206193-0138-00WO gatggctccttctttctgtattctaaactgactgtggataagagcagatggcagcagggaaacgtgttttcttgtagcgtgatgcatgaggccctgcataacc actacacacagaagtccctgtctctgagtccaggcaagggcggcggcggcagcggcggcggcggctctcagagcgtgctgacacagcccccttccg tgtctggagcaccaggacagcgggtgaccatcagctgcacaggcagctcctctaacatcggcgccggctacgacgtgcactggtatcagcacctgcca ggaaccgcaccaaagctgctgatctacggcaactctaatagacctagcggcgtgccagatcggtttagcggatccaagtctggcaccagcgcctccctg gcaatcacaggactgcaggcagaggacgagacagattacttctgccagtcctatgatagctccctgtctgcctgggtgtttggcggcggaaccaaggtg accgtgctgggcggcggcggcagcggcggcggcggcagcggcggcggcggctcccaggtgcagctggtgcagagcggcggcggcgtggtgca gcctggcggctccctgagactgtcttgtgccgccagcggcttccctttttctagctacgcaatgagctgggtgcggcaggcaccaggcaagggactgga gtgggtgtccgccatctctgccaacggcggaaccacatactatgccgactccgtgaagggcaggttcaccatctccagagataactctaagaatacactg tacctgcagatgaacagcctgagggcagaggacaccgccgtgtactattgcgccaacaatggcaattataggggcgcctttgatatctggggccaggg caccatggtgacagtgtccagcggcggcggcggctcggaggtgcagctggtggagagcggcggcggcctggtgcagccaggcggcagcctgagg ctgtcctgtgcagcaagcggatactccttcaccggctatacaatgaattgggtgaggcaggcccctggcaagggcctggaatgggtggccctgatcaac ccctacaagggcgtgtccacctataatcagaagttcaaggaccgctttaccatctctgtggataagagcaagaacacagcctacctgcagatgaatagcc tgagagccgaggacacagccgtgtactattgcgcacggagcggatactatggcgactccgattggtattttgacgtgtggggccagggcaccctggtga cagtgagcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggctctgacatccagatgacccagtccccatctagcctgtctgcc agcgtcggcgacagggtgaccatcacatgtcgcgcctctcaggatatcaggaactacctgaattggtatcagcagaagcccggcaaggcccctaagct gctgatctactatacatccaggctggagtctggagtgccaagcaggttctccggatctggaagcggaaccgactacaccctgacaatctcctctctgcag cccgaggatttcgccacatactattgtcagcagggcaataccctgccttggacatttggccagggcaccaaggtggagatcaagagctccggcggcgg cggaagccagtccgtgctgacccagcccccttctgtgagcggagcaccaggacagagggtgaccatcagctgcacaggcagctcctctaacatcggc gccggctacgacgtgcactggtatcagcacctgccaggcacagcccccaagctgctgatctacggcaactctaatcggcctagcggcgtgccagatcg cttctccggctctaagagcggcacctccgcctctctggccatcacaggcctgcaggccgaggacgagaccgattacttctgccagagctatgacagctc cctgtccgcctgggtgtttggcggcggaaccaaggtgaccgtgctgggcggcggcggcagcggcggcggcggcagcggcggcggcggctctcag gtgcagctggtgcagagcggcggcggcgtggtgcagcctggcggctccctgaggctgtcttgtgcagcaagcggattccccttttctagctatgcaatgt cctgggtgcgccaggcacctggcaagggactggagtgggtgagcgccatctccgccaatggcggaaccacatactatgccgactctgtgaagggca ggttcaccatctccagagataactctaagaatacactgtacctgcagatgaactccctgagggccgaggacacagccgtgtactattgtgccaacaatgg caattatagaggcgcctttgatatctggggccagggcaccatggtgacagtgtcctcttgataa G37-TRV-Fc Amino acid sequence- SEQ ID NO:22 MDWTWILFLVAAATRVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEAPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEAPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGS GGGGSQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLPGTAPKLLIYGNSNRPSGV PDRFSGSKSGTSASLAITGLQAEDETDYFCQSYDSSLSAWVFGGGTKVTVLGGGGSGGGGSGGGDocket No.206193-0138-00WO GSQVQLVQSGGGVVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEWVSAISANGGTTYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANNGNYRGAFDIWGQGTMVTVSSGGGG SEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGG GGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYT SRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKSSGGGGSQSV LTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLPGTAPKLLIYGNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDETDYFCQSYDSSLSAWVFGGGTKVTVLGGGGSGGGGSGGGGSQVQLV QSGGGVVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEWVSAISANGGTTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCANNGNYRGAFDIWGQGTMVTVSS G37- TRV-Fc with His tag DNA sequence- SEQ ID NO:23 atggactggacctggatcctgttcctggtggctgccgccacaagggtgcacagcgataagacccacacatgccctccctgtcctgcaccagaggcagc cggcggaccttccgtgttcctgtttccacccaagccaaaggacaccctgatgatcagcagaacccccgaggtgacatgcgtggtggtggacgtgtccca cgaggcacctgaggtgaagtttaactggtacgtggatggcgtggaggtgcacaatgccaagacaaagccctgcgaggagcagtacggctctacctata gatgcgtgagcgtgctgacagtgctgcaccaggattggctgaacggcaaggagtataagtgcgccgtgtctaataaggccctgcccgcccctatcgag aagaccatcagcaaggcaaagggacagccaagggagcctcaggtgtacacactgcctccatctagagaggagatgaccaagaaccaggtgagcctg acatgtctggtgaagggcttctatccatccgacatcgccgtggagtgggagtctaatggccagcccgagaacaattacaagaccacaccccctgtgctg gactccgatggctctttctttctgtatagcaagctgaccgtggataagtccaggtggcagcagggcaacgtgtttagctgctccgtgatgcacgaggccct gcacaatcactacacacagaagtctctgagcctgtcccctggcaagggcggcggcggcagcggcggcggcggcagcggcggcggcggcagcgg cggcggcggcagcggcggcggcggcagcggcggcggcggctccgacaagacccacacatgcccaccctgtccagcccctgaggcagccggcg gaccaagcgtgttcctgtttcctccaaagcctaaggataccctgatgatctcccggaccccagaagtcacctgcgtggtcgtggacgtgtctcacgaggc ccccgaggtgaagttcaactggtacgtggacggagtcgaagtgcacaatgccaagaccaagccatgtgaagagcagtacggctccacctatcgctgc gtgagcgtgctgacagtgctgcatcaggactggctgaatggcaaggaatataagtgcgccgtgagcaataaggccctgcctgccccaatcgagaagac catctctaaagccaaaggacagccaagggagccacaggtgtacacactgcccccttcccgcgaagaaatgactaaaaaccaggtgtctctgacctgcc tggtcaaaggcttctatcctagcgacatcgcagtggagtgggagtccaacggacagccagaaaataattacaagaccacaccacccgtgctggacagc gatggctccttctttctgtattctaaactgactgtggataagagcagatggcagcagggaaacgtgttttcttgtagcgtgatgcatgaggccctgcataacc actacacacagaagtccctgtctctgagtccaggcaagggcggcggcggcagcggcggcggcggctctcagagcgtgctgacacagcccccttccg tgtctggagcaccaggacagcgggtgaccatcagctgcacaggcagctcctctaacatcggcgccggctacgacgtgcactggtatcagcacctgcca ggaaccgcaccaaagctgctgatctacggcaactctaatagacctagcggcgtgccagatcggtttagcggatccaagtctggcaccagcgcctccctg gcaatcacaggactgcaggcagaggacgagacagattacttctgccagtcctatgatagctccctgtctgcctgggtgtttggcggcggaaccaaggtg accgtgctgggcggcggcggcagcggcggcggcggcagcggcggcggcggctcccaggtgcagctggtgcagagcggcggcggcgtggtgca gcctggcggctccctgagactgtcttgtgccgccagcggcttccctttttctagctacgcaatgagctgggtgcggcaggcaccaggcaagggactgga gtgggtgtccgccatctctgccaacggcggaaccacatactatgccgactccgtgaagggcaggttcaccatctccagagataactctaagaatacactg tacctgcagatgaacagcctgagggcagaggacaccgccgtgtactattgcgccaacaatggcaattataggggcgcctttgatatctggggccaggg caccatggtgacagtgtccagcggcggcggcggctcggaggtgcagctggtggagagcggcggcggcctggtgcagccaggcggcagcctgagg ctgtcctgtgcagcaagcggatactccttcaccggctatacaatgaattgggtgaggcaggcccctggcaagggcctggaatgggtggccctgatcaacDocket No.206193-0138-00WO ccctacaagggcgtgtccacctataatcagaagttcaaggaccgctttaccatctctgtggataagagcaagaacacagcctacctgcagatgaatagcc tgagagccgaggacacagccgtgtactattgcgcacggagcggatactatggcgactccgattggtattttgacgtgtggggccagggcaccctggtga cagtgagcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggctctgacatccagatgacccagtccccatctagcctgtctgcc agcgtcggcgacagggtgaccatcacatgtcgcgcctctcaggatatcaggaactacctgaattggtatcagcagaagcccggcaaggcccctaagct gctgatctactatacatccaggctggagtctggagtgccaagcaggttctccggatctggaagcggaaccgactacaccctgacaatctcctctctgcag cccgaggatttcgccacatactattgtcagcagggcaataccctgccttggacatttggccagggcaccaaggtggagatcaagagctccggcggcgg cggaagccagtccgtgctgacccagcccccttctgtgagcggagcaccaggacagagggtgaccatcagctgcacaggcagctcctctaacatcggc gccggctacgacgtgcactggtatcagcacctgccaggcacagcccccaagctgctgatctacggcaactctaatcggcctagcggcgtgccagatcg cttctccggctctaagagcggcacctccgcctctctggccatcacaggcctgcaggccgaggacgagaccgattacttctgccagagctatgacagctc cctgtccgcctgggtgtttggcggcggaaccaaggtgaccgtgctgggcggcggcggcagcggcggcggcggcagcggcggcggcggctctcag gtgcagctggtgcagagcggcggcggcgtggtgcagcctggcggctccctgaggctgtcttgtgcagcaagcggattccccttttctagctatgcaatgt cctgggtgcgccaggcacctggcaagggactggagtgggtgagcgccatctccgccaatggcggaaccacatactatgccgactctgtgaagggca ggttcaccatctccagagataactctaagaatacactgtacctgcagatgaactccctgagggccgaggacacagccgtgtactattgtgccaacaatgg caattatagaggcgcctttgatatctggggccagggcaccatggtgacagtgtcctctcaccaccaccaccaccactgataa G37- TRV-Fc with His tag Amino acid sequence- SEQ ID NO:24 MDWTWILFLVAAATRVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEAPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEAPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGS GGGGSQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLPGTAPKLLIYGNSNRPSGV PDRFSGSKSGTSASLAITGLQAEDETDYFCQSYDSSLSAWVFGGGTKVTVLGGGGSGGGGSGGG GSQVQLVQSGGGVVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEWVSAISANGGTTYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANNGNYRGAFDIWGQGTMVTVSSGGGG SEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGG GGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYT SRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKSSGGGGSQSV LTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLPGTAPKLLIYGNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDETDYFCQSYDSSLSAWVFGGGTKVTVLGGGGSGGGGSGGGGSQVQLVDocket No.206193-0138-00WO QSGGGVVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEWVSAISANGGTTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCANNGNYRGAFDIWGQGTMVTVSSHHHHHHDNA sequence- SEQ ID NO:25 atggactggacctggatcctgttcctggtggctgccgccacaagggtgcacagcgataagacccacacatgccctccctgtcctgcaccagaggcagc cggcggaccttccgtgttcctgtttccacccaagccaaaggacaccctgatgatcagcagaacccccgaggtgacatgcgtggtggtggacgtgtccca cgaggcacctgaggtgaagtttaactggtacgtggatggcgtggaggtgcacaatgccaagacaaagccctgcgaggagcagtacggctctacctata gatgcgtgagcgtgctgacagtgctgcaccaggattggctgaacggcaaggagtataagtgcgccgtgtctaataaggccctgcccgcccctatcgag aagaccatcagcaaggcaaagggacagccaagggagcctcaggtgtacacactgcctccatctagagaggagatgaccaagaaccaggtgagcctg acatgtctggtgaagggcttctatccatccgacatcgccgtggagtgggagtctaatggccagcccgagaacaattacaagaccacaccccctgtgctg gactccgatggctctttctttctgtatagcaagctgaccgtggataagtccaggtggcagcagggcaacgtgtttagctgctccgtgctgcacgaggccct gcactcccactacacacagaagtctctgagcctgtcccctggcaagggcggcggcggcagcggcggcggcggcagcggcggcggcggcagcgg cggcggcggcagcggcggcggcggcagcggcggcggcggctccgacaagacccacacatgcccaccctgtccagcccctgaggcagccggcg gaccaagcgtgttcctgtttcctccaaagcctaaggataccctgatgatctcccggaccccagaagtcacctgcgtggtcgtggacgtgtctcacgaggc ccccgaggtgaagttcaactggtacgtggacggagtcgaagtgcacaatgccaagaccaagccatgtgaagagcagtacggctccacctatcgctgc gtgagcgtgctgacagtgctgcatcaggactggctgaatggcaaggaatataagtgcgccgtgagcaataaggccctgcctgccccaatcgagaagac catctctaaagccaaaggacagccaagggagccacaggtgtacacactgcccccttcccgcgaagaaatgactaaaaaccaggtgtctctgacctgcc tggtcaaaggcttctatcctagcgacatcgcagtggagtgggagtccaacggacagccagaaaataattacaagaccacaccacccgtgctggacagc gatggctccttctttctgtattctaaactgactgtggataagagcagatggcagcagggaaacgtgttttcttgtagcgtgctgcacgaggccctgcactcc cactacacacagaagtccctgtctctgagtccaggcaagggcggcggcggcagcggcggcggcggctctcagagcgtgctgacacagcccccttcc gtgtctggagcaccaggacagcgggtgaccatcagctgcacaggcagctcctctaacatcggcgccggctacgacgtgcactggtatcagcacctgc caggaaccgcaccaaagctgctgatctacggcaactctaatagacctagcggcgtgccagatcggtttagcggatccaagtctggcaccagcgcctcc ctggcaatcacaggactgcaggcagaggacgagacagattacttctgccagtcctatgatagctccctgtctgcctgggtgtttggcggcggaaccaag gtgaccgtgctgggcggcggcggcagcggcggcggcggcagcggcggcggcggctcccaggtgcagctggtgcagagcggcggcggcgtggt gcagcctggcggctccctgagactgtcttgtgccgccagcggcttccctttttctagctacgcaatgagctgggtgcggcaggcaccaggcaagggact ggagtgggtgtccgccatctctgccaacggcggaaccacatactatgccgactccgtgaagggcaggttcaccatctccagagataactctaagaatac actgtacctgcagatgaacagcctgagggcagaggacaccgccgtgtactattgcgccaacaatggcaattataggggcgcctttgatatctggggcca gggcaccatggtgacagtgtccagcggcggcggcggctcggaggtgcagctggtggagagcggcggcggcctggtgcagccaggcggcagcctg aggctgtcctgtgcagcaagcggatactccttcaccggctatacaatgaattgggtgaggcaggcccctggcaagggcctggaatgggtggccctgat caacccctacaagggcgtgtccacctataatcagaagttcaaggaccgctttaccatctctgtggataagagcaagaacacagcctacctgcagatgaat agcctgagagccgaggacacagccgtgtactattgcgcacggagcggatactatggcgactccgattggtattttgacgtgtggggccagggcaccct ggtgacagtgagcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggctctgacatccagatgacccagtccccatctagcctg tctgccagcgtcggcgacagggtgaccatcacatgtcgcgcctctcaggatatcaggaactacctgaattggtatcagcagaagcccggcaaggcccc taagctgctgatctactatacatccaggctggagtctggagtgccaagcaggttctccggatctggaagcggaaccgactacaccctgacaatctcctctc tgcagcccgaggatttcgccacatactattgtcagcagggcaataccctgccttggacatttggccagggcaccaaggtggagatcaagagctccggcg gcggcggaagccagtccgtgctgacccagcccccttctgtgagcggagcaccaggacagagggtgaccatcagctgcacaggcagctcctctaacat cggcgccggctacgacgtgcactggtatcagcacctgccaggcacagcccccaagctgctgatctacggcaactctaatcggcctagcggcgtgccaDocket No.206193-0138-00WO gatcgcttctccggctctaagagcggcacctccgcctctctggccatcacaggcctgcaggccgaggacgagaccgattacttctgccagagctatgac agctccctgtccgcctgggtgtttggcggcggaaccaaggtgaccgtgctgggcggcggcggcagcggcggcggcggcagcggcggcggcggct ctcaggtgcagctggtgcagagcggcggcggcgtggtgcagcctggcggctccctgaggctgtcttgtgcagcaagcggattccccttttctagctatg caatgtcctgggtgcgccaggcacctggcaagggactggagtgggtgagcgccatctccgccaatggcggaaccacatactatgccgactctgtgaa gggcaggttcaccatctccagagataactctaagaatacactgtacctgcagatgaactccctgagggccgaggacacagccgtgtactattgtgccaac aatggcaattatagaggcgcctttgatatctggggccagggcaccatggtgacagtgtcctcttgataa G37- TRV-Fc+ (with mutations for half life extension) Amino acid sequence- SEQ ID NO:26 MDWTWILFLVAAATRVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEAPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGKGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEAPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGKGGGGSGG GGSQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLPGTAPKLLIYGNSNRPSGVPD RFSGSKSGTSASLAITGLQAEDETDYFCQSYDSSLSAWVFGGGTKVTVLGGGGSGGGGSGGGGS QVQLVQSGGGVVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEWVSAISANGGTTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANNGNYRGAFDIWGQGTMVTVSSGGGGSE VQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQK FKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTS RLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKSSGGGGSQSV LTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLPGTAPKLLIYGNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDETDYFCQSYDSSLSAWVFGGGTKVTVLGGGGSGGGGSGGGGSQVQLV QSGGGVVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEWVSAISANGGTTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCANNGNYRGAFDIWGQGTMVTVSS G37- TRV-Fc+ with His tag (with mutations for half life extension) DNA sequence- SEQ ID NO:27 atggactggacctggatcctgttcctggtggctgccgccacaagggtgcacagcgataagacccacacatgccctccctgtcctgcaccagaggcagc cggcggaccttccgtgttcctgtttccacccaagccaaaggacaccctgatgatcagcagaacccccgaggtgacatgcgtggtggtggacgtgtccca cgaggcacctgaggtgaagtttaactggtacgtggatggcgtggaggtgcacaatgccaagacaaagccctgcgaggagcagtacggctctacctata gatgcgtgagcgtgctgacagtgctgcaccaggattggctgaacggcaaggagtataagtgcgccgtgtctaataaggccctgcccgcccctatcgag aagaccatcagcaaggcaaagggacagccaagggagcctcaggtgtacacactgcctccatctagagaggagatgaccaagaaccaggtgagcctg acatgtctggtgaagggcttctatccatccgacatcgccgtggagtgggagtctaatggccagcccgagaacaattacaagaccacaccccctgtgctgDocket No.206193-0138-00WO gactccgatggctctttctttctgtatagcaagctgaccgtggataagtccaggtggcagcagggcaacgtgtttagctgctccgtgctgcacgaggccct gcactcccactacacacagaagtctctgagcctgtcccctggcaagggcggcggcggcagcggcggcggcggcagcggcggcggcggcagcgg cggcggcggcagcggcggcggcggcagcggcggcggcggctccgacaagacccacacatgcccaccctgtccagcccctgaggcagccggcg gaccaagcgtgttcctgtttcctccaaagcctaaggataccctgatgatctcccggaccccagaagtcacctgcgtggtcgtggacgtgtctcacgaggc ccccgaggtgaagttcaactggtacgtggacggagtcgaagtgcacaatgccaagaccaagccatgtgaagagcagtacggctccacctatcgctgc gtgagcgtgctgacagtgctgcatcaggactggctgaatggcaaggaatataagtgcgccgtgagcaataaggccctgcctgccccaatcgagaaga ccatctctaaagccaaaggacagccaagggagccacaggtgtacacactgcccccttcccgcgaagaaatgactaaaaaccaggtgtctctgacctgc ctggtcaaaggcttctatcctagcgacatcgcagtggagtgggagtccaacggacagccagaaaataattacaagaccacaccacccgtgctggacag cgatggctccttctttctgtattctaaactgactgtggataagagcagatggcagcagggaaacgtgttttcttgtagcgtgctgcacgaggccctgcactc ccactacacacagaagtccctgtctctgagtccaggcaagggcggcggcggcagcggcggcggcggctctcagagcgtgctgacacagcccccttc cgtgtctggagcaccaggacagcgggtgaccatcagctgcacaggcagctcctctaacatcggcgccggctacgacgtgcactggtatcagcacctg ccaggaaccgcaccaaagctgctgatctacggcaactctaatagacctagcggcgtgccagatcggtttagcggatccaagtctggcaccagcgcctc cctggcaatcacaggactgcaggcagaggacgagacagattacttctgccagtcctatgatagctccctgtctgcctgggtgtttggcggcggaaccaa ggtgaccgtgctgggcggcggcggcagcggcggcggcggcagcggcggcggcggctcccaggtgcagctggtgcagagcggcggcggcgtgg tgcagcctggcggctccctgagactgtcttgtgccgccagcggcttccctttttctagctacgcaatgagctgggtgcggcaggcaccaggcaagggac tggagtgggtgtccgccatctctgccaacggcggaaccacatactatgccgactccgtgaagggcaggttcaccatctccagagataactctaagaata cactgtacctgcagatgaacagcctgagggcagaggacaccgccgtgtactattgcgccaacaatggcaattataggggcgcctttgatatctggggcc agggcaccatggtgacagtgtccagcggcggcggcggctcggaggtgcagctggtggagagcggcggcggcctggtgcagccaggcggcagcct gaggctgtcctgtgcagcaagcggatactccttcaccggctatacaatgaattgggtgaggcaggcccctggcaagggcctggaatgggtggccctga tcaacccctacaagggcgtgtccacctataatcagaagttcaaggaccgctttaccatctctgtggataagagcaagaacacagcctacctgcagatgaa tagcctgagagccgaggacacagccgtgtactattgcgcacggagcggatactatggcgactccgattggtattttgacgtgtggggccagggcaccct ggtgacagtgagcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggctctgacatccagatgacccagtccccatctagcctg tctgccagcgtcggcgacagggtgaccatcacatgtcgcgcctctcaggatatcaggaactacctgaattggtatcagcagaagcccggcaaggcccc taagctgctgatctactatacatccaggctggagtctggagtgccaagcaggttctccggatctggaagcggaaccgactacaccctgacaatctcctctc tgcagcccgaggatttcgccacatactattgtcagcagggcaataccctgccttggacatttggccagggcaccaaggtggagatcaagagctccggc ggcggcggaagccagtccgtgctgacccagcccccttctgtgagcggagcaccaggacagagggtgaccatcagctgcacaggcagctcctctaac atcggcgccggctacgacgtgcactggtatcagcacctgccaggcacagcccccaagctgctgatctacggcaactctaatcggcctagcggcgtgcc agatcgcttctccggctctaagagcggcacctccgcctctctggccatcacaggcctgcaggccgaggacgagaccgattacttctgccagagctatga cagctccctgtccgcctgggtgtttggcggcggaaccaaggtgaccgtgctgggcggcggcggcagcggcggcggcggcagcggcggcggcgg ctctcaggtgcagctggtgcagagcggcggcggcgtggtgcagcctggcggctccctgaggctgtcttgtgcagcaagcggattccccttttctagctat gcaatgtcctgggtgcgccaggcacctggcaagggactggagtgggtgagcgccatctccgccaatggcggaaccacatactatgccgactctgtga agggcaggttcaccatctccagagataactctaagaatacactgtacctgcagatgaactccctgagggccgaggacacagccgtgtactattgtgcca acaatggcaattatagaggcgcctttgatatctggggccagggcaccatggtgacagtgtcctctcaccaccaccaccaccactgataa G37- TRV-Fc+ with His tag (with mutations for half life extension) Amino acid sequence- SEQ ID NO:28 MDWTWILFLVAAATRVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEAPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSNKALDocket No.206193-0138-00WO PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGKGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEAPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGKGGGGSGG GGSQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLPGTAPKLLIYGNSNRPSGVPD RFSGSKSGTSASLAITGLQAEDETDYFCQSYDSSLSAWVFGGGTKVTVLGGGGSGGGGSGGGGS QVQLVQSGGGVVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEWVSAISANGGTTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANNGNYRGAFDIWGQGTMVTVSSGGGGSE VQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQK FKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTS RLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKSSGGGGSQSV LTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLPGTAPKLLIYGNSNRPSGVPDRFSGSK SGTSASLAITGLQAEDETDYFCQSYDSSLSAWVFGGGTKVTVLGGGGSGGGGSGGGGSQVQLV QSGGGVVQPGGSLRLSCAASGFPFSSYAMSWVRQAPGKGLEWVSAISANGGTTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCANNGNYRGAFDIWGQGTMVTVSSHHHHHH linked Fc Domain Amino acid sequence- SEQ ID NO:29 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVKFNWYVDGVEV HNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVKFNWYVDG VEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK linked Fc Domain (with mutations for half-life extension) Amino acid sequence- SEQ ID NO:30 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVKFNWYVDGVEV HNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVKFNWYVDG VEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPRDocket No.206193-0138-00WO EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK linked Fc Domain DNA sequence- SEQ ID NO:31 gataagacccacacatgccctccctgtcctgcaccagaggcagccggcggaccttccgtgttcctgtttccacccaagccaaaggacaccctgatgatc agcagaacccccgaggtgacatgcgtggtggtggacgtgtcccacgaggcacctgaggtgaagtttaactggtacgtggatggcgtggaggtgcaca atgccaagacaaagccctgcgaggagcagtacggctctacctatagatgcgtgagcgtgctgacagtgctgcaccaggattggctgaacggcaagga gtataagtgcgccgtgtctaataaggccctgcccgcccctatcgagaagaccatcagcaaggcaaagggacagccaagggagcctcaggtgtacaca ctgcctccatctagagaggagatgaccaagaaccaggtgagcctgacatgtctggtgaagggcttctatccatccgacatcgccgtggagtgggagtct aatggccagcccgagaacaattacaagaccacaccccctgtgctggactccgatggctctttctttctgtatagcaagctgaccgtggataagtccaggtg gcagcagggcaacgtgtttagctgctccgtgatgcacgaggccctgcacaatcactacacacagaagtctctgagcctgtcccctggcaagggcggcg gcggcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggctccgacaag acccacacatgcccaccctgtccagcccctgaggcagccggcggaccaagcgtgttcctgtttcctccaaagcctaaggataccctgatgatctcccgg accccagaagtcacctgcgtggtcgtggacgtgtctcacgaggcccccgaggtgaagttcaactggtacgtggacggagtcgaagtgcacaatgccaa gaccaagccatgtgaagagcagtacggctccacctatcgctgcgtgagcgtgctgacagtgctgcatcaggactggctgaatggcaaggaatataagt gcgccgtgagcaataaggccctgcctgccccaatcgagaagaccatctctaaagccaaaggacagccaagggagccacaggtgtacacactgcccc cttcccgcgaagaaatgactaaaaaccaggtgtctctgacctgcctggtcaaaggcttctatcctagcgacatcgcagtggagtgggagtccaacggac agccagaaaataattacaagaccacaccacccgtgctggacagcgatggctccttctttctgtattctaaactgactgtggataagagcagatggcagca gggaaacgtgttttcttgtagcgtgatgcatgaggccctgcataaccactacacacagaagtccctgtctctgagtccaggcaag linked Fc domain with mutations for half-life extension DNA sequence- SEQ ID NO:32 gataagacccacacatgccctccctgtcctgcaccagaggcagccggcggaccttccgtgttcctgtttccacccaagccaaaggacaccctgatgatc agcagaacccccgaggtgacatgcgtggtggtggacgtgtcccacgaggcacctgaggtgaagtttaactggtacgtggatggcgtggaggtgcaca atgccaagacaaagccctgcgaggagcagtacggctctacctatagatgcgtgagcgtgctgacagtgctgcaccaggattggctgaacggcaagga gtataagtgcgccgtgtctaataaggccctgcccgcccctatcgagaagaccatcagcaaggcaaagggacagccaagggagcctcaggtgtacaca ctgcctccatctagagaggagatgaccaagaaccaggtgagcctgacatgtctggtgaagggcttctatccatccgacatcgccgtggagtgggagtct aatggccagcccgagaacaattacaagaccacaccccctgtgctggactccgatggctctttctttctgtatagcaagctgaccgtggataagtccaggtg gcagcagggcaacgtgtttagctgctccgtgctgcacgaggccctgcactcccactacacacagaagtctctgagcctgtcccctggcaagggcggcg gcggcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggctccgacaag acccacacatgcccaccctgtccagcccctgaggcagccggcggaccaagcgtgttcctgtttcctccaaagcctaaggataccctgatgatctcccgg accccagaagtcacctgcgtggtcgtggacgtgtctcacgaggcccccgaggtgaagttcaactggtacgtggacggagtcgaagtgcacaatgccaa gaccaagccatgtgaagagcagtacggctccacctatcgctgcgtgagcgtgctgacagtgctgcatcaggactggctgaatggcaaggaatataagt gcgccgtgagcaataaggccctgcctgccccaatcgagaagaccatctctaaagccaaaggacagccaagggagccacaggtgtacacactgcccc cttcccgcgaagaaatgactaaaaaccaggtgtctctgacctgcctggtcaaaggcttctatcctagcgacatcgcagtggagtgggagtccaacggacDocket No.206193-0138-00WO agccagaaaataattacaagaccacaccacccgtgctggacagcgatggctccttctttctgtattctaaactgactgtggataagagcagatggcagca gggaaacgtgttttcttgtagcgtgctgcacgaggccctgcactcccactacacacagaagtccctgtctctgagtccaggcaag Fc domain Amino acid sequence- SEQ ID NO:33 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVKFNWYVDGVEV HNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fc domain with mutations for half-life extension Amino acid sequence- SEQ ID NO:34 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVK FNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCAVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALH SHYTQKSLSLSPGK Table 1: Sequence Information Sequence Identifier  Sequence Type  Description SEQ ID NO:1  Nucleotide  CA9 VL SEQ ID NO:2  Amino Acid  CA9 VL SEQ ID NO:3  Nucleotide  CA9 VH SEQ ID NO:4  Amino Acid  CA9 VH SEQ ID NO:5  Nucleotide  CD3 VH SEQ ID NO:6  Amino Acid  CD3 VH SEQ ID NO:7  Nucleotide  CD3 VL SEQ ID NO:8  Amino Acid  CD3 VL SEQ ID NO:9  Nucleotide  G37_DBiTE (BTE) SEQ ID NO:10  Amino Acid  G37_DBiTE (BTE) SEQ ID NO:11  Nucleotide  G37_DBiTE with His tag (BTE) SEQ ID NO:12  Amino Acid  G37_DBiTE with His tag (BTE) Docket No.206193-0138-00WO SEQ ID NO:13  Nucleotide  G37‐DBiTE‐Fc (PBTE) SEQ ID NO:14  Amino Acid  G37‐DBiTE‐Fc (PBTE)  SEQ ID NO:15  Nucleotide  G37‐DBiTE‐Fc with His tag (PBTE) SEQ ID NO:16  Amino Acid  G37‐DBiTE‐Fc with His tag (PBTE) SEQ ID NO:17  Nucleotide  G37‐DBiTE‐Fc+ (PBTE) SEQ ID NO:18  Amino Acid  G37‐DBiTE‐Fc+ (PBTE) SEQ ID NO:19  Nucleotide  G37‐DBiTE‐Fc+ with His tag (PBTE) SEQ ID NO:20  Amino Acid  G37‐DBiTE‐Fc+ with His tag (PBTE) SEQ ID NO:21  Nucleotide  G37‐TRV‐Fc (PMTE) SEQ ID NO:22  Amino Acid  G37‐TRV‐Fc (PMTE) SEQ ID NO:23  Nucleotide  G37‐TRV‐Fc with His tag (PMTE) SEQ ID NO:24  Amino Acid  G37‐TRV‐Fc with His tag (PMTE) SEQ ID NO:25  Nucleotide  G37‐TRV‐Fc+ (PMTE) SEQ ID NO:26  Amino Acid  G37‐TRV‐Fc+ (PMTE) SEQ ID NO:27  Nucleotide  G37‐TRV‐Fc+ with His tag (PMTE) SEQ ID NO:28  Amino Acid  G37‐TRV‐Fc+ with His tag (PMTE) SEQ ID NO:29  Amino Acid  Linked Fc Domain  SEQ ID NO:30   Amino Acid  Linked Fc Domain with mutations for half‐life extension  SEQ ID NO:31   Nucleotide   Linked Fc Domain  SEQ ID NO:32  Nucleotide   Linked Fc Domain with mutations for half‐life extension  SEQ ID NO:33  Amino Acid  Fc domain  SEQ ID NO:34   Amino Acid  Fc domain with mutations for half‐life extension

[0213] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents.Docket No.206193-0138-00WO

[0214] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.

Claims

Docket No.206193-0138-00WO CLAIMS What is claimed is:

1. A method of treating or preventing renal cell carcinoma in a subject, comprising adminsitering to the subject a composition comprising one or more synthetic binding molecule, or a binding fragment thereof, or a nucleic acid molecule encoding comprising one or more synthetic binding molecule, or a binding fragment thereof, wherein the one or more synthetic binding molecule comprises at least one antigen binding domain specific for binding to carbonic anhydrase 9 (CA9), and at least one immune cell engaging domain.

2. The method 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.

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

4. The method of claim 3, wherein the immune cell engaging domain targets CD3.

5. The method of claim 1, wherein the synthetic binding molecule comprises: a) a CA9 heavy chain amino acid sequence of SEQ ID NO: 4, or a fragment thereof comprising at least the CDRs; b) a CA9 light chain amino acid sequence of SEQ ID NO: 2, or a fragment thereof comprising at least the CDRs; c) a CD3 heavy chain amino acid sequence of SEQ ID NO: 6, or a fragment thereof comprising at least the CDRs; andDocket No.206193-0138-00WO d) a CD3 light chain amino acid sequence of SEQ ID NO: 8, or a fragment thereof comprising at least the CDRs.

6. The method of claim 5, wherein the synthetic binding molecule further comprises: e) an Fc domain selected from SEQ ID NO: 29 or SEQ ID NO:

30.

7. The method of claim 6, wherein the synthetic binding molecule further comprises: f) a second CA9 heavy chain amino acid sequence of SEQ ID NO: 4, or a fragment thereof comprising at least the CDRs; and g) a second CA9 light chain amino acid sequence of SEQ ID NO: 2, or a fragment thereof comprising at least the CDRs.

8. The method of claim 5, wherein the synthetic binding molecule comprises SEQ ID NO:10 or SEQ ID NO:

12.

9. The method of claim 6, wherein the synthetic binding molecule comprises SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 or SEQ ID NO:

20.

10. The method of claim 7, wherein the synthetic binding molecule comprises SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO:

28.

11. The method of claim 1, wherein the method comprises administering a nucleic acid molecule comprising: a) the nucleotide sequence of SEQ ID NO:3 encoding the CA9 heavy chain, or a fragment thereof encoding at least the CDRs;Docket No.206193-0138-00WO b) the nucleotide sequence of SEQ ID NO:1 encoding the CA9 light chain, or a fragment thereof encoding at least the CDRs; c) the nucleotide sequence of SEQ ID NO:5 encoding the CD3 heavy chain, or a fragment thereof encoding at least the CDRs; and d) the nucleotide sequence of SEQ ID NO:7 encoding the CD3 light chain, or a fragment thereof encoding at least the CDRs.

12. The method of claim 11, wherein the nucleic acid molecule further comprises: e) a nucleotide sequence selected from SEQ ID NO: 31 and SEQ ID NO:32 encoding the Fc domain.

13. The method of claim 12, wherein the nucleic acid molecule further comprises: f) the nucleotide sequence of SEQ ID NO:3 encoding a second CA9 heavy chain, or a fragment thereof encoding at least the CDRs; g) the nucleotide sequence of SEQ ID NO:1 encoding a second CA9 light chain, or a fragment thereof encoding at least the CDRs.

14. The method of claim 11, wherein the nucleic acid molecule comprises SEQ ID NO:9 or SEQ ID NO:

11.

15. The method of claim 12, wherein the nucleic acid molecule comprises SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 or SEQ ID NO:

19.

16. The method of claim 13, wherein the nucleic acid molecule comprises SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27.Docket No.206193-0138-00WO 17. The method of any one of claims 11-16, wherein the nucleotide sequence is operably linked to a nucleic acid sequence encoding an IgE leader sequence.

18. The method of any one of claims 11-17, wherein the nucleic acid molecule comprises an expression vector.

19. The method of any of claims 1-18, wherein the composition comprises a pharmaceutically acceptable excipient.

20. The method of any of claims 1-19, wherein the renal cell carcinoma is metastatic.

21. The method of any of claims 1-20, wherein the subject has a von Hippel- Lindau (VHL) mutation.

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