Anti-CTLA4 monoclonal antibody and chimeric antigen receptor

Abstract

Compositions and methods are presented in which selected polypeptide compounds bind to CTLA-4. Most typically, binding is mediated by selected VH and / or VL domains, with preferred compounds being prepared as scFvs, IgGs, or CARs.

Description

Technical Field

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 038,111, filed on Jun. 11, 2020, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing The content of the ASCII text file of the sequence listing, which is 110 KB in size and has the file name 102719.0032PCT_REV005_ST25.txt, was created on Jun. 1, 2021, and was electronically submitted via EFS-Web together with this application, and is hereby incorporated by reference in its entirety.

[0003] Field of the Invention The field of the invention is compositions and methods of compounds that bind to cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), particularly antibodies, antibody fragments, scFvs, and fusion molecules comprising the VH and VL domains presented herein.

Background Art

[0004] The background description includes information that may be useful in understanding the present invention. It is not admitted that any of the information provided herein is prior art or relevant to the presently claimed invention, nor is it admitted that any publication specifically or implicitly referenced is prior art.

[0005] All publications and patent applications mentioned in this specification are hereby incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. If the definition or use of a term in an incorporated reference conflicts or contradicts the definition of that term provided herein, the definition of that term provided herein shall apply and the definition of that term in the incorporated reference shall not apply.

[0006] Immunotherapy is becoming a promising treatment strategy for various cancers. Among other options, cancer vaccine compositions, typically combined with immunostimulants, have shown significant therapeutic effects in at least some approaches. Unfortunately, the tumor microenvironment often has immunosuppressive effects on T cells and NK cells, frequently reducing the effectiveness of immunotherapy. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4, CD152) is a membrane glycoprotein expressed by activated effector T cells (T eff ) and is involved in the suppression of T cell proliferation, cell cycle progression, and cytokines (IL-2, IFN-γ). CTLA-4 is thought to exert its inhibitory function through multiple mechanisms, including competition with CD28-positive costimulation for binding to the common B7 ligands (CD80 / CD86) on antigen-presenting cells (APCs), and direct inhibitory effects via its cytoplasmic tail associated with signaling molecules.

[0007] To overcome immunosuppression by CTLA-4 activity, anti-CTLA-4 antibodies can be used as therapeutic effectors. Currently known anti-CTLA-4 antibodies include ipilimumab and tremelimumab, which are used alone or in combination with chemotherapy, cancer vaccines, or other antibodies (anti-PD-1 or anti-OX40) in the treatment of various cancers, including melanoma, non-small cell lung cancer (NSCLC), breast cancer, prostate cancer, pancreatic cancer, hepatocellular carcinoma, and mesothelioma. Ipilimumab increased long-term survival rates at a dose of 10 mg / kg in melanoma and had antitumor activity in patients with B cell lymphoma. However, ipilimumab was not effective in patients with metastatic castration-resistant prostate cancer. Although tremelimumab did not show the same advantages as ipilimumab, clinical trials of tremelimumab showed acceptable tolerance and clinically meaningful activity in patients with melanoma, refractory metastatic colorectal cancer, hepatocellular carcinoma, and malignant mesothelioma.

[0008] Unfortunately, ipilimumab and tremelimumab have significant adverse effects and are not always effective in reducing immunosuppression. Thus, despite the various systems and methods for interfering with CTLA-4 signaling known in the art, all or almost all of them have some drawbacks. Accordingly, there remains a need for compositions and methods for improved compounds that bind to CTLA-4. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0009] The subject matter of the present invention relates to various compositions and methods of polypeptides that bind to CTLA-4 with high specificity and affinity. In preferred embodiments, the polypeptide is constructed as an scFv, an antibody, or a part thereof, or as a chimeric molecule such as a CAR, N-803, or TxM derivative. In more preferred embodiments, the polypeptide comprises a VH and / or VL domain, has an amino acid sequence set forth in SEQ ID NOs: 1-59, or comprises a heavy or light chain of an antibody set forth in SEQ ID NOs: 60-71. As will be readily appreciated, these polypeptides can be used in a variety of diagnostic and therapeutic applications, particularly in reducing immunosuppression and treating cancer.

[0010] In one aspect of the subject matter of the present invention, the inventors contemplated a single-chain variable fragment (scFv) peptide comprising a first amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 36, 38, 40, 42, 44, 46, 48, 49, 51, 54, 56, and 58 H segment, and / or a V segment comprising a second amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 37, 39, 41, 43, 45, 47, 50, 53, 55, 57, and 59 L segment. Most typically, the V H segment and the V LThe segment is conjugated with a linker peptide (e.g., a glycine-rich peptide). Further, a single-chain variable fragment (scFv) peptide may further comprise at least two pairs of V H segments and V L segments, and the at least two pairs are linked to form a multivalent scFv. Optionally, the peptide may be present in a pharmaceutically acceptable carrier.

[0011] In another aspect of the subject matter of the present invention, the inventors contemplate an antibody comprising a heavy chain comprising a first amino acid sequence selected from the group consisting of SEQ ID NOs: 60, 61, 62, 64, 65, 66, 68, and 70, and / or a light chain comprising a second amino acid sequence selected from the group consisting of SEQ ID NOs: 63, 67, 69, and 71. For example, suitable antibodies include an antibody having a heavy chain comprising the first amino acid sequence of SEQ ID NO: 68 and a light chain comprising the second amino acid sequence of SEQ ID NO: 69, or an antibody having a heavy chain comprising the first amino acid sequence of SEQ ID NO: 70 and a light chain comprising the second amino acid sequence of SEQ ID NO: 71.

[0012] In a further aspect of the subject matter of the present invention, the inventors contemplate a pharmaceutical composition for treating a patient having cancer, comprising a single-chain variable fragment (scFv) peptide or an antibody presented herein, and the single-chain variable fragment (scFv) peptide or antibody is present in a pharmaceutically acceptable carrier.

[0013] In yet another aspect of the subject matter of the present invention, the inventors contemplate a diagnostic composition comprising a single-chain variable fragment (scFv) peptide or an antibody presented herein, and the single-chain variable fragment (scFv) peptide or antibody further comprises a detectable label.

[0014] Furthermore, the inventors also contemplate a recombinant nucleic acid comprising a first nucleic acid segment encoding a first amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 36, 38, 40, 42, 44, 46, 48, 49, 51, 54, 56, and 58, and / or a second amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 37, 39, 41, 43, 45, 47, 50, 53, 55, 57, and 59. Preferably, although not necessarily, the first and second segments are in the same reading frame. Furthermore, the inventors also contemplate a recombinant nucleic acid comprising a first nucleic acid segment encoding a first amino acid sequence selected from the group consisting of SEQ ID NOs: 60, 61, 62, 64, 65, 66, 68, and 70, and / or a second amino acid sequence selected from the group consisting of SEQ ID NOs: 63, 67, 69, and 71. Preferably, although not necessarily, the first and second segments are in a bicistronic arrangement.

[0015] For example, a contemplated recombinant nucleic acid can comprise a first nucleic acid segment encoding the first amino acid sequence of SEQ ID NO: 68 and a second nucleic acid segment encoding the second amino acid sequence of SEQ ID NO: 69, or a first nucleic acid segment encoding the first amino acid sequence of SEQ ID NO: 70 and a second nucleic acid segment encoding the second amino acid sequence of SEQ ID NO: 71.

[0016] As will be readily appreciated, the recombinant nucleic acid may further comprise a third segment encoding a polypeptide that encodes at least a portion of a chimeric antigen receptor (CAR), and the CAR, when expressed intracellularly, has an antigen-binding extracellular domain comprising the first and / or second nucleic acid segments. Alternatively, the recombinant nucleic acid may further comprise a third segment encoding a polypeptide that encodes at least a portion of N-803 or TxM, and N-803 or TxM, when expressed intracellularly, has an antigen-binding domain comprising the first and / or second nucleic acid segments.

[0017] Accordingly, the inventors also contemplate recombinant cells that contain the recombinant nucleic acids described above (e.g., by transfection or genetic engineering). Among other suitable cell types, the recombinant cells can be recombinant T cells, recombinant NK cells, recombinant NKT cells, recombinant monocytes, recombinant macrophages, or recombinant dendritic cells. Alternatively, the recombinant cells can be production cells engineered to produce recombinant proteins such as CHO cells or EC7 cells.

[0018] In yet a further aspect contemplated, a method of reducing CTLA-4 mediated effects in a cell or tissue is presented, the method comprising contacting the cell or tissue with a single-chain variable fragment (scFv) peptide, which is an antibody presented herein, in an amount that reduces CTALA-4 mediated effects in the cell or tissue.

[0019] Thus, the inventors also contemplate a method of treating a patient having a tumor, the method comprising administering to the patient a pharmaceutical composition comprising a single-chain variable fragment (scFv) peptide or an antibody presented herein, or a recombinant cell presented herein. Most preferably, the pharmaceutical composition is administered to the patient in an effective dosage and schedule for treating the tumor.

[0020] Accordingly, from a different perspective, the inventors also contemplate a method of reducing immunosuppression in a patient having a tumor, the method comprising administering to the patient a pharmaceutical composition comprising a single-chain variable fragment (scFv) peptide or an antibody presented herein, or a recombinant cell presented herein. Most preferably, the pharmaceutical composition is administered to the patient in an effective dosage and schedule for reducing immunosuppression in the tumor microenvironment.

[0021] Accordingly, the use of the single-chain variable fragment (scFv) or antibody or recombinant cell presented herein is contemplated for use in medicine, particularly for treating cancer and / or for reducing immunosuppression in a patient having a tumor.

[0022] Various objects, features, aspects, and advantages of the subject matter of the present invention will become more apparent from the following detailed description of the preferred embodiments, taken in conjunction with the accompanying drawings in which like numerals represent like components.

Brief Description of the Drawings

[0023]

Figure 1

Figures 2A-D

Figure 2E

Figures 3A-D

Figure 3E

Modes for Carrying Out the Invention

[0024] The inventors have discovered various polypeptides that specifically bind to CTLA - 4, particularly to the VH and VL domains of antibodies, or fragments or fusion proteins containing the VH and / or VL domains (and the CDRs contained therein). Most advantageously, the VH and / or VL domains or the CDRs contained therein can be used to generate various therapeutic or diagnostic agents having high affinity and specificity for CTLA - 4.

[0025] Regarding the VH and VL domains discussed herein, it should be understood that the domains generally follow well-known nomenclature. Thus, the VH domain typically represents the variable fragment of the heavy chain of an antibody that binds to several constant domains (e.g., the VH domain that binds to the CH1, CH2, and CH3 domains of IgG). Similarly, the VL domain typically represents the variable fragment of the light chain of an antibody that binds to a constant domain (e.g., the VL domain that binds to CL in IgG). Therefore, as will be readily understood, each of the VH and VL domains typically has its respective framework regions (FRs) and complementarity-determining regions (CDRs) arranged consecutively as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Thus, based on the known framework regions of the VH and VL domains, one of ordinary skill in the art will readily recognize the respective CDRs of the VH or VL domain. The terms "domain" and "segment" are used interchangeably herein in conjunction with VH or VL.

[0026] Furthermore, as will be more readily appreciated, one of ordinary skill in the art can use the amino acid sequences of the VH segment and / or VL segment to generate recombinant isolated antibodies or fragments thereof. As used herein, the term "antibody" refers to an immunoglobulin molecule and the immunologically active portion of an immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that binds immunospecifically to an antigen. Thus, "antibody and its fragments" includes the entire immunoglobulin molecule (e.g., full size, whole IgG1, IgA, etc.) and antigen-binding fragments of the entire immunoglobulin molecule. Thus, fragments contemplated include scFv, Fab fragment, Fab' fragment, F(ab')2, disulfide-linked Fv (sdFv), Fv, and V H segment and / or V LAny fragment comprising either segment may be mentioned. When the antibody is an immunoglobulin, the immunoglobulin may comprise heavy or constant domains of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY) and any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2) to constitute different types of immunoglobulins. Further, “antibodies” may particularly include human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, and polyclonal antibodies. Thus, it should be understood that the VH and / or VL domains of SEQ ID NOs: 1-59 can be grafted onto any existing (typically human or humanized) antibody or antibody fragment using methods well known in the art.

[0027] The inventors further consider that the amino acid sequences of the VH segments and / or VL segments shown in SEQ ID NOs: 1-59 can be conjugated with a carrier protein to generate a hybrid protein having the VH segment and / or VL segment on its surface such that CTLA-4 can be captured or inhibited by the hybrid protein. Any suitable form of the carrier protein that stably holds the VH segment and / or VL segment and preferably provides access to the tumor microenvironment is considered. One particularly preferred carrier protein includes albumin, refolded albumin, and other proteins having an affinity for an antibody moiety (e.g., protein A, protein G, protein Z) conjugated with one or more VH segments and / or VL segments.

[0028] Typically, the VH segment and / or the VL segment is conjugated with an anchor molecule, whereby the VH segment and / or the VL segment can be conjugated with a carrier protein. For example, if the carrier protein is albumin, the anchor molecule can be a hydrophobic peptide or glycolipid of any suitable size that fits into one of the Sudlow sites I and II of albumin, or any other hydrophobic region of albumin. For example, the recombinant immunoglobulin protein against CTLA-4 described above can be conjugated with a carrier protein via its Fc domain. In other embodiments, the anchor molecule may comprise a hydrophobic peptide (e.g., having a length of at least 10 amino acids, 15 amino acids, 20 amino acids, 30 amino acids, etc.). In these embodiments, various configurations of the VH segment and / or the VL segment (e.g., in the form of scFv) and the hydrophobic peptide can be contemplated. For example, a monovalent scFv domain can be directly linked to the hydrophobic peptide, or a multivalent scFv can be directly linked to the hydrophobic peptide. Alternatively, one scFv domain can be directly linked to multiple hydrophobic peptides, or multiple scFv domains can be directly linked to multiple hydrophobic peptides.

[0029] As an alternative or in addition, one or more VH segments and / or VL segments may be conjugated with an intermediate molecule having an anchor portion that binds to a carrier protein. In a preferred embodiment, the inventors contemplate that the intermediate molecule provides multiple binding sites for the VH segment and / or VL segment such that multiple target recognition domains can be carried via a single binding site on the carrier protein. Suitable intermediate molecules may include any protein, glycolipid, organic molecule, or inorganic molecule that does not provide any significant toxicity to naive tissue. For example, suitable intermediate molecules may include nanoparticles (e.g., quantum dots, gold nanoparticles, magnetic nanoparticles, nanotubes, polymeric nanoparticles, dendrimers, etc.), or beads (e.g., polystyrene beads, latex beads, Dynabeads, etc.). Preferably, the nanoparticles and / or beads have dimensions of less than 1 μm, preferably less than 100 nm. The nanoparticles may be cross-linked to or partially coated with a hydrophobic tail that provides an anchor to a carrier protein (e.g., albumin). One or more VH segments and / or VL segments may also be cross-linked to or partially coat the nanoparticles (e.g., via an additional tail domain linked to the target recognition domain for cross-linking, etc.).

[0030] In another example, suitable intermediate molecules may include beads (e.g., polystyrene beads, latex beads, Dynabeads, etc.) conjugated with an antibody to a carrier protein. Thus, if the carrier protein is albumin, the beads may be conjugated with an α-albumin antibody (e.g., by cross-linking, coating, etc.) such that the beads can bind to the carrier protein with high affinity and specificity. One or more VH segments and / or VL segments may also be cross-linked to or partially coat the beads (e.g., via an additional tail domain linked to the target recognition domain for cross-linking, thiol-mediated cross-linking, etc.).

[0031] In some embodiments, the scFv peptide can form a recombinant immunoglobulin protein complex that comprises or mimics the ALT-803 (IL-15 superagonist complex, see, e.g., Blood 2015 126:1957) or TxM (targeted ALT-803-based scaffold platform, see, e.g., URL, altorbioscience.com / our-science / il-15-protein-superagonist-and-scaffold-technology / ) structure. Preferably, the inventors contemplate that when the immunoglobulin protein complex mimics the TxM structure, the scFv peptide can be directly (or indirectly via a linker) conjugated to one or more interleukin-15 (IL-15) binding motifs and / or one or more ligands for the IL-15 binding motif (e.g., IL-15, IL-15N72D, etc.). Thus, when the recombinant immunoglobulin protein complex mimics the TxM IgG1 structure, the recombinant immunoglobulin protein complex may comprise 1-4 scFv peptides against CTLA-4.

[0032] Furthermore, a recombinant immunoglobulin protein complex that mimics the TxM structure and has one or more scFv peptides conjugated to an IL-15 binding motif or its ligand may also comprise a binding domain for a tumor-specific antigen or a patient-specific and tumor-specific neoepitope (e.g., an scFv peptide against a neoepitope, etc.). For example, the recombinant immunoglobulin protein complex may comprise two scFv peptides against CTLA-4 conjugated to two IL-15 binding motifs and two scFv peptides against a neoepitope conjugated to two IL-15 binding motif ligands. Preferably, the neoepitope is patient-specific and tumor-specific and is identified, for example, by omics analysis of sequence data as disclosed in U.S. Patent Application Publication Nos. 2012 / 0059670A1 and 2012 / 0066001A1.

[0033] In some embodiments, the scFv or a fragment thereof can be generated using one sequence encoding a VH segment among SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 36, 38, 40, 42, 44, 46, 48, 49, 51, 54, 56, and 58. In other embodiments, the scFv, recombinant antibody or a fragment thereof can be generated using one sequence encoding a VL segment among SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 37, 39, 41, 43, 45, 47, 50, 53, 55, 57, and 59. In still other embodiments, the scFv, recombinant antibody or a fragment thereof can be generated using one sequence encoding a VH segment among SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 36, 38, 40, 42, 44, 46, 48, 49, 51, 54, 56, and 58, and one sequence encoding a VL segment among SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 37, 39, 41, 43, 45, 47, 50, 53, 55, 57, and 59. In these embodiments, it is preferred to pair the sequences encoding the VH segment and the sequences encoding the VL segment having a common designator in the sequence listing using distinct VH and VL identifiers (e.g., 73-2VH and 73-2VL shown in SEQ ID NOs: 1 and 2, respectively).However, as shown in more detail below, it is also contemplated that any pair of VH and VL segments can be generated to form a CTLA-4 binding molecule by selecting one VH segment from SEQ ID NO: xx and one VL segment from SEQ ID NO: yy, where xx is any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 36, 38, 40, 42, 44, 46, 48, 49, 51, 54, 56, and 58, and yy is any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 37, 39, 41, 43, 45, 47, 50, 53, 55, 57, and 59.

[0034] The inventors also contemplate that an scFv, recombinant antibody or fragment thereof can be generated using an amino acid sequence encoding a VH segment and / or VL segment that is at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical to any one of SEQ ID NO: xx or yy. Most typically, when the identity is not 100%, the different amino acids are located in one or more of the framework regions of the sequence and do not affect the CDR regions of a given sequence. In a further embodiment, the binding affinity of the scFv peptide, recombinant antibody or fragment thereof is preferably 100 nM or less, or 50 nM or less, or 20 nM or less, or 10 nM or less, or 5 nM or less, or 2 nM or less, or 1 nM or less. For example, any scFv having a particular VH and / or VL domain presented herein can be selected and then subjected to affinity maturation via random mutagenesis in the CDR regions of VH and VL. In such an example, typically, it is contemplated that 4 or fewer, or 3 or fewer, or 2 or fewer amino acids are changed across all CDR regions of a given sequence. Thus, as will be appreciated, multiple binders can be isolated from an affinity maturation process in which the binding properties are improved.

[0035] Most typically, the VH and VL segments in an scFv peptide or similar construct are conjugated via a linker or spacer, which are typically 5 to 40 amino acids, preferably 10 to 30 amino acids, more preferably 20 to 30 amino acids. In some embodiments, the linker can conjugate to the N-terminus of the VH segment and the C-terminus of the VL segment. In other embodiments, the linker can conjugate to the N-terminus of the VL segment and the C-terminus of the VH segment. The inventors further contemplate that a glycine-rich sequence for the linker (e.g., (G4S)n, where n is from 1 to 5, etc.) is preferred to provide structural flexibility between the VH and VL segments. Inclusion of one or more serine or threonine residues in the linker is also contemplated to enhance the solubility of the scFv peptide, recombinant antibody or fragment thereof. There are numerous methods known in the art for making linkers and scFvs, and all such known methods are considered suitable for use herein.

[0036] Furthermore, the scFv peptide can include multiple VH and VL segments to form a bivalent or multivalent scFv. In some embodiments, the multiple VH and / or VL segments may have the same amino acid sequence (e.g., a multivalent scFv having three VH segments and three VL segments, where all VH segments have the same VH and all VL segments have the same VL domain). In other embodiments, at least two of the VH and / or VL segments may have different amino acid sequences (e.g., a multivalent scFv having three VH segments and three VL segments, where two of the VH segments are the same VH domain, one VH segment is different, two of the VL segments are the same VL domain, and one VL segment is different).

[0037] Preferably, the binding affinity (Kd) of the scFv, recombinant antibody or fragment thereof for CTLA-4 (at least one of the 72-mer and 77-mer IL-8) is at least 1×10 -7 M or less, preferably 1×10 -8 M or less, more preferably 1×10 -9 M or less. The inventors considered that even if an scFv, recombinant antibody, or fragment thereof is produced using the same amino acid sequence of the V H segment and / or V L segment, the binding affinity of the scFv, recombinant antibody, or fragment thereof for CTLA-4 may be different due to differences in structure. For example, Tables 1 to 4 provide different affinities (measured by KD values) of different scFv clones, and Table 5 shows different affinities (measured by KD values) of various recombinant antibody (IgG1) clones.

[0038] It is further considered that the scFv peptide (or the VH and VL segments of the antibody or fragments thereof) can be encoded by a single recombinant nucleic acid. In such an embodiment, the recombinant nucleic acid includes at least two nucleic acid segments: a first nucleic acid segment encoding the VH segment of the subject of the present invention and a second nucleic acid segment encoding the VL segment. However, it is also considered that the first and second nucleic acid segments encoding the VH and VL segments, respectively, are at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical to any one of SEQ ID NOs: 1 to 59. Most preferably, the two nucleic acid segments are in the same reading frame such that the two nucleic acid segments can be translated into a single protein having two peptide segments.

[0039] Furthermore, the recombinant nucleic acid may include a third nucleic acid segment between the first and second nucleic acid segments that encodes a linker peptide (preferably a G-rich or otherwise flexible linker peptide), which is typically 5 to 40 amino acids, preferably 10 to 30 amino acids, more preferably 20 to 30 amino acids. In this embodiment, it is particularly preferred that the three nucleic acid segments are in the same reading frame so that the three nucleic acid segments can be translated into a single protein having three peptide segments.

[0040] On the other hand, when the CTLA-4 binding molecule is an antibody (e.g., IgG), the VH and VL segments presented herein can be encoded on a single recombinant bicistronic construct, or these segments can be encoded on two separate constructs. Most typically, the nucleic acid encoding the VH domain also encodes one or more CH domains (e.g., CH1-CH2-CH3 in the case of IgG) in frame. Similarly, the nucleic acid encoding the VL domain also encodes one CH domain (e.g., CL in the case of IgG) in frame. Most typically, the constant domains of the heavy and light chains are humanized, although other mammalian sequences are also considered suitable. Furthermore, it should be recognized that the recombinant nucleic acid preferably (although not necessarily) has a codon usage frequency optimized for the cells that express the antibody.

[0041] The inventors further contemplate that scFv peptides, antibodies or fragments thereof can be formulated as pharmaceutical compositions for administration to a patient having a tumor so as to reduce or inhibit signal transduction by CTLA-4 and the attendant immunosuppression. Thus, it is contemplated that the scFv peptides, antibodies or fragments thereof can be formulated in any pharmaceutically acceptable carrier (e.g., as a sterile injectable composition) in an amount of at least 1 ml, preferably at least 5 ml, more preferably at least 20 ml per dosage unit for a therapeutic formulation. However, alternative formulations are also considered suitable for use herein, and all known routes and modes of administration are considered herein. As used herein, the term "administering" refers to both direct and indirect administration of the compounds and compositions considered herein, where direct administration is typically performed by a healthcare provider (e.g., a physician, nurse, etc.), while indirect administration typically includes the step of providing or making available the compounds and compositions to a medical professional for direct administration. In some embodiments, the pharmaceutical formulation is administered by systemic injection, including subcutaneous injection, subdermal injection, or intravenous injection. In other embodiments, it is contemplated that the formulation is administered via intratumoral injection, as shown in more detail below.

[0042] One exemplary method and use of a pharmaceutical composition comprising an scFv peptide, an antibody or a fragment thereof, comprising the VH and VL segments described above, is to reduce CTLA-4 mediated effects in a target tissue or target cell. As used herein, CTLA-4 mediated effects refer to any biological consequence directly or indirectly induced by the activity of CTLA-4 in a tissue or in the microenvironment of a tissue. Most typically, this effect can be observed as a decrease in T cell activity (and in particular cytotoxic cell death by T cells and NK cells), which can be enhanced or observed in the tumor microenvironment. From another perspective, a decrease in IL-8 mediated effects in vivo can also be observed by physiological phenomena. For example, a decrease in CTLA-4 signaling can be observed through a reduction or inhibition of tumor growth by tumor shrinkage, apoptosis of tumor cells, or necrosis. Similarly, a decrease in CTLA-4 signaling in a patient, particularly in the tumor microenvironment, reduces the tendency of the tumor to cause local or distant metastases.

[0043] Regarding the dosage and schedule of administration of the pharmaceutical composition to a patient, it is contemplated that the dosage and / or schedule may vary depending on the type of peptide (e.g., scFv, antibody, antibody fragment, any combination of two of these, all combinations, etc.), the type and prognosis of the disease (e.g., tumor type, size, location), and the health status of the patient (including age, gender, etc.). Although it may vary, the dosage and schedule are selected and adjusted such that the formulation does not cause any significant toxic effects on the host's normal cells, yet the effect of CTLA-4 in the tumor microenvironment is reduced by at least 20%, preferably at least 30%, more preferably at least 40%, and most preferably at least 50% within 3 hours, 6 hours, 12 hours, 24 hours, 72 hours, or 1 week. For example, if the CTLA-4 binder is an antibody, a suitable dosage of the antibody can be 0.1 - 1.0 mg, or 1.0 - 5.0 mg, or 5.0 - 10.0 mg, or 10 - 50 mg, or 50 - 100 mg per single administration.

[0044] Furthermore, the inventors contemplate that the effect of an scFv, antibody or fragment thereof against CTLA-4 can be enhanced by co-administration of one or more cancer drug therapies. Examples of cancer drug therapies include fulvestrant, aldoxorubicin, docetaxel, tumor necrosis therapeutics (e.g., 131 I-chTNT-3, etc.), avelumab (a human monoclonal IgG1 antibody that inhibits the interaction between PD-L1 and its receptor), a bracurial-targeted vaccine (e.g., ETBX-051 (Ad5[E1-, E2b-]-bracurial)), a Her2-targeted vaccine (e.g., ETBX-021, etc.), a MUC-1-targeted vaccine (e.g., ETBX-061 (Ad5[E1-, E2b-]-MUC1)), and yeast vaccines (e.g., GI-4000 (GI-4014, GI-4015, GI-4016, GI-4020), GI-6207, GI-6301), and adenovirus- or yeast-based vaccines against tumor-specific neoepitopes, but are not limited thereto. Details of examples of these cancer drug therapies are described in International Publication No. PCT / US17 / 40297, which is hereby incorporated by reference in its entirety.

[0045] If desired, the CTLA-4 binder can also be co-administered with cell-based therapies (simultaneously or at intervals of one or more days), and particularly preferred cell-based therapies include the transfusion of NK cells and / or T cells. Of course, it should be understood that such cells can be genetically modified to express one or more recombinant molecules to increase target-specific tumor cell death.

[0046] In particularly preferred embodiments, such modified NK cells or T cells can be engineered to express a chimeric antigen receptor (CAR). In some embodiments, the CAR of the NK cell or T cell can target an antigen specific to one or more cancers or tumors, but in embodiments of particular interest, the CAR expressed by the NK cell or T cell targets CTLA-4. Generally, a suitable CAR is known to include a single-chain variable fragment (scFv) linked to at least one intracellular signaling domain. The scFv recognizes and binds to an antigen (e.g., an antigen or CTLA-4) on the target cell, causing activation of the effector cell. The signaling domain will most often include an immunoreceptor tyrosine-based activation domain (ITAM) important for intracellular signaling by the receptor. Suitable CARs include first-generation, second-generation, and third-generation CARs that can be expressed in a variety of cells, particularly T cells and NK cells.

[0047] First-generation CARs typically include one cytoplasmic signaling domain. For example, one type of first-generation CAR for T cells can include a signaling domain from the Fc epsilon receptor gamma (FcεRIγ) that includes one ITAM, and another type can include a signaling domain from CD3ζ that includes three ITAMs. Second-generation CARs typically use CD28 as the most common intracellular co-stimulatory domain alongside CD3ζ. Alternatively, second-generation CARs can also include the 4-1BB intracellular signaling domain together with CD3ζ. Third-generation CARs typically include CD3ζ, CD28, and the 4-1BB intracellular signaling domain. See also WO 2016 / 201304 and WO 2018 / 076391 for further third-generation CARs.

Examples

[0048] The affinity of the selected scFv molecules for human CTLA-4 was performed via surface plasmon resonance (SPR). More specifically, using a Pioneer FE instrument, the binding affinity between the scFv and recombinant CTLA-4 protein was measured at 25°C. Briefly, a biotinylated anti-FLAG M2 antibody was immobilized on a PCH biosensor (Molecular Devices / ForteBio) coated with neutravidin. The 3×FLAG-tagged scFv was captured by anti-FLAG M2 on the sensor, and the binding affinity was measured using a one-step injection with recombinant human CTLA-4 protein. Exemplary results for the selected scFvs are shown in Tables 1 and 2 below. In the display of the clones in Tables 1-4 below, it should be noted that the scFv clones contain both the VH and VL domains with the indicated SEQ ID NOs.

[0049]

Table 1

[0050]

Table 2

[0051] Similarly, the affinity of the selected scFv molecules for mouse CTLA-4 was measured by surface plasmon resonance (SPR). More specifically, using a Pioneer FE instrument, the binding affinity between the scFv and recombinant CTLA-4 protein was measured at 25°C. Briefly, a biotinylated anti-FLAG M2 antibody was immobilized on a PCH biosensor (Molecular Devices / ForteBio) coated with neutravidin. The 3×FLAG-tagged scFv was captured by anti-FLAG M2 on the sensor, and the binding affinity was measured using a one-step injection with recombinant mouse CTLA-4 protein. Exemplary results for the selected scFvs are shown in Tables 3 and 4 below.

[0052]

Table 3

[0053]

Table 4

[0054] In further experiments, the selected VH and VL domains were grafted onto a human IgG scaffold, and K was measured by SPR (Pioneer FE) or Octet (Red96e) at 25 °C or 37 °C. D All values shown in Table 5 are ×10 -9 M. Antibodies were captured on the chip surface using an anti-human Fc antibody (SPR) or an AHC sensor (Octet). As shown in Table 5, human or mouse CTLA-4 was used as the analyte.

[0055]

Table 5

[0056] The inventors further examined an antibody (IgG1) that blocks the CD80 / CTLA4 complex by BLI using human CTLA-4 / human CD80 or mouse CTLA-4 / mouse CD80. Here, the Octet Red96e instrument was used to test the ability of an αCTLA-4 antibody to block the binding of CD80 to CTLA-4. Briefly, biotinylated CTLA-4 was immobilized on an SA biosensor (Molecular Devices / ForteBio). Next, 10 μg / mL (67 nM) of αCTLA-4 was used to load the CTLA-4-coated biosensor. Finally, 250 nM of CD80 was used to test the binding to CTLA-4 in the presence of the αCTLA-4 antibody, and exemplary results are shown in Figure 1.

[0057] Following the in vitro tests described above, the inventors then proceeded to conduct various in vivo experiments to examine the anti-tumor activity of the selected IgG constructs. Specifically, the inventors used yeast lysate (pTK170, yeast expressing a tumor-specific neoepitope), anti-OX40 antibody, and the selected anti-CTLA-4 antibodies shown to test intratumoral immunization in a B16F10 mouse tumor model. Four groups of animals were used: 1. PBS; 2. pTK170 + aOX40 + a-CTLA4-7 (SEQ ID NO: 68, SEQ ID NO: 69); 3. pTK170 + aOX40 + a-CTLA4-8 (SEQ ID NO: 70, SEQ ID NO: 71); and 4. pTK170 + aOX40 + a-CTLA4-9H10. The dose of pTK170 yeast was 5 YU / injection, and anti-OX40 and anti-CTLA4 were each administered at 100 μg per injection.

[0058] Exemplary results of tumor volumes are shown in FIGS. 2A-2D, and the Kaplan-Meier plots of the animals are shown in FIG. 2E. As can be readily seen, all anti-CTLA4 groups (FIGS. 2B-2D) significantly decreased tumor growth of both treated and untreated tumors compared to PBS (FIG. 2A). There is still no statistically significant difference among clones 7, 8, and 9H10 at a dose of 100 μg. The non-injected tumor volumes are shown in FIGS. 3A-3D, where FIG. 3A is the PBS control and FIGS. 3B-3D show treatment with anti-CTLA4 compounds. The corresponding Kaplan-Meier plots of the animals are shown in FIG. 3E.

[0059] In some embodiments, it should be understood that the numbers representing properties such as components, concentrations, and amounts such as reaction conditions, which are used to describe and claim specific embodiments of the present invention, may in some cases be modified by the term "about". Accordingly, in some embodiments, the numerical parameters set forth in the written description and the appended claims are approximations that may vary depending upon the desired properties sought to be obtained by a particular embodiment. The description of a range of values herein is merely intended to serve as a simple method of referring individually to each value within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein.

[0060] As used herein, the term "administering" a pharmaceutical composition or drug refers to both direct and indirect administration of the pharmaceutical composition or drug, where direct administration of the pharmaceutical composition or drug is typically performed by a medical professional (e.g., a physician, nurse, etc.), and indirect administration includes the step of making the pharmaceutical composition or drug available to a medical professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.). It should be further noted that the terms "predicting" or "forecasting" a condition, susceptibility to the onset of a disease, or response to an intended treatment mean including the act or prediction of predicting a condition, susceptibility, and / or response, including but not limited to the progression, improvement, and / or duration of a condition in a subject (however, treatment or diagnosis is not included).

[0061] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples or exemplary language (e.g., "such as") provided with respect to specific embodiments herein is merely intended to make the present invention more readily understood and is not intended to limit the scope of the present invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.

[0062] Throughout the description of this specification and the following claims, the meanings of "a", "an", and "the" include references to the plural unless the context clearly dictates otherwise. As used in the description of this specification, unless the context clearly dictates otherwise, the meaning of "in" includes "in" and "on". Also, in the usage of this specification, unless the context clearly dictates otherwise, the term "coupled to" is intended to include both direct coupling (where two elements coupled to each other are in contact with each other) and indirect coupling (where at least one additional element is positioned between the two elements). Accordingly, the terms "coupled to" and "coupled with" are used synonymously.

[0063] It will be apparent to those skilled in the art that many more modifications other than those already described can be made without departing from the inventive concept of this specification. Accordingly, the subject matter of this invention is not limited except as by the appended claims. Further, in interpreting both this specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted in a non-exclusive manner to refer to elements, components, or steps, indicating that the referenced elements, components, or steps may be present, utilized, or combined with other elements, components, or steps not explicitly referenced. When the claims of this specification refer to at least one selected from the group consisting of A, B, C,.... and N, the text should be interpreted as requiring only one element from the group, not A + N, or B + N, etc.

Claims

1. An antibody or a fusion protein containing the same, comprising a variable heavy chain (VH) segment and a variable light chain (VL) segment each consisting of SEQ ID NO: 68 and SEQ ID NO: 69, respectively.

2. An antibody comprising a heavy chain containing the first amino acid sequence of SEQ ID NO: 68 and a light chain containing the second amino acid sequence of SEQ ID NO:

69.

3. A pharmaceutical composition for treating a patient having cancer, comprising the antibody according to claim 2, wherein the antibody is present in a pharmaceutically acceptable carrier.

4. A diagnostic composition comprising the antibody according to claim 2, wherein the antibody further comprises a detectable label.

5. A recombinant nucleic acid comprising a first nucleic acid segment encoding the first amino acid sequence of SEQ ID NO: 68 and a second nucleic acid segment encoding the second amino acid sequence of SEQ ID NO:

69.

6. The recombinant nucleic acid according to claim 5, further comprising a third nucleic acid segment encoding a polypeptide containing N-803.

7. A recombinant cell comprising the recombinant nucleic acid according to claim 5 or 6.

8. The recombinant cell according to claim 7, wherein the cell is a T cell, NK cell, NKT cell, monocyte, macrophage, or dendritic cell.

9. The recombinant cell according to claim 7, wherein the cell is a CHO cell or an EC7 cell.

10. A method for reducing the CTLA-4 mediated effect in a cell or tissue, comprising contacting the cell or tissue in vitro with an antibody or fusion protein according to claim 1 or 2 in an amount that reduces the CTLA-4 mediated effect in the cell or tissue.

11. Use of the antibody or fusion protein according to claim 1 or 2 for the manufacture of a medicament.

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