Anti-vasoactive intestinal peptide antibodies and uses thereof

Abstract

Disclosed herein are antibodies that specifically bind vasoactive intestinal peptide (anti-VIP antibodies). In certain embodiments, chimeric anti-VIP antibody sequences are useful in the treatment human patients with VIP associated disorders or conditions such as hematological or solid cancers. In certain embodiments, this disclosure relates to vectors and cells that express anti-VIP antibodies disclosed herein, kits, and pharmaceutical compositions comprising the same.

Description

[0001] ANTI- VASOACTIVE INTESTINAL PEPTIDE ANTIBODIES AND USES THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 729,731 filed December 9, 2024. The entirety of this application is hereby incorporated by reference for all purposes.

[0004] INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN XML FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM

[0005] The Sequence Listing associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is 25053PCT.xml. The XML file is 22,035 bytes, was created on December 9, 2025, and is being submitted electronically via the USPTO patent electronic filing system.

[0006] BACKGROUND

[0007] Vasoactive intestinal peptide (VIP) is a neuropeptide with immunosuppressive effects. The two receptors of VIP, vasoactive intestinal peptide receptors 1 and 2 (VPAC1 / VIPR1 and VPAC2 / VIPR2), are G protein-coupled receptors. VIP receptors are upregulated in some cancers and VIP antagonists were reported to inhibit cancer cell proliferation in certain cancers.

[0008] Moody et al. report a vasoactive intestinal peptide antagonist inhibits non-small cell lung cancer growth. PNAS, 1993, 90: 4345-4349.

[0009] Li et al. report VIPhyb, an antagonist of vasoactive intestinal peptide receptor, enhances cellular antiviral immunity in murine cytomegalovirus infected mice. PLoS One, 2013, 8(5), e63381.

[0010] Asano et al. report vasoactive intestinal peptide-VIPR2 signaling regulates tumor cell migration. Front Oncol, 2022, 12:852358.

[0011] Kittikulsuth et al. report inhibition of VIP signaling enhanced Ml macrophage polarization and macrophage phagocytic function resulting in tumor regression in a colon cancer model. Scientific Reports, 2023, 13:927.

[0012] Lester et al. report chemical modifications to enhance the drug properties of a VIP receptor antagonist (ANT) peptides. Int. I. Mol. Sci. 2024, 25, 4391. Sakamoto et al. report a VIPR2-selective antagonist KS-133 changes macrophage polarization and exerts potent anti-tumor effects as a single agent and in combination with an anti- PD-1 antibody. PLoS ONE, 2023, 18(7):e0286651.

[0013] See also WO2019209952, WO2018119380, US Pat. App. Pub. No. 2023 / 0174582, and WO2025213178.

[0014] References cited herein are not an admission of prior art.

[0015] SUMMARY

[0016] Disclosed herein are antibodies that specifically bind vasoactive intestinal peptide, antiVIP antibodies. In certain embodiments, chimeric recombinant humanized anti-VIP antibodies are useful in the treatment human patients with VIP associated disorders or conditions such as in the treatment of hematological or solid cancers. In certain embodiments, this disclosure relates to vectors and cells that express anti-VIP antibodies disclosed herein. In certain embodiments, this disclosure relates to kits, and pharmaceutical compositions comprising anti-VIP antibodies.

[0017] In certain embodiments, the anti-VIP antibodies comprise a heavy chain, wherein the heavy chain CDR1 is SYWIG (SEQ ID NO: 1), CDR2 is IIYPGDSDTRYSPSFQG (SEQ ID NO: 2), and a CDR3.

[0018] In certain embodiments, the CDR3 is VDYDGEP (SEQ ID NO: 3). In certain embodiments, the heavy chain comprises the amino acid sequence of GVHSEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPG DSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAVYYCARVDYDGEPFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALG (SEQ ID NO: 4).

[0019] In certain embodiments, the CDR3 is YSEYDGAYDI (SEQ ID NO: 5). In certain embodiments, the heavy chain comprises the amino acid sequence of GVHSEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPG DSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAVYYCARYSEYDGAYDIFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG (SEQ ID NO: 6).

[0020] In certain embodiments, the CDR3 is ASYGAGEP (SEQ ID NO: 7). In certain embodiments, the heavy chain comprises the amino acid sequence of GVHSEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPG DSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAVYYCARASYGAGEPFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG (SEQ ID NO: 9).

[0021] In certain embodiments, the antibody heavy chains comprise one or more of the following constant region mutations G236A, S239D, A330L, I332E, S267E, L328F, P238D, H268F, S324T, S228P, G236R, L328R, L234A, L235A, M252Y, S254T, T256E, M428L, and N434S.

[0022] In certain embodiments, the antibody comprises a light chain. In certain embodiment, the antibody comprises a light chain, wherein the light chain CDR1 is RASQSISSYLN (SEQ ID NO: 10), CDR2 is AASSLQS (SEQ ID NO: 11), and CDR3 is QQSYSTPLT (SEQ ID NO: 12). In certain embodiment, the antibody comprises a light chain having the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKRTV (SEQ ID NO: 13).

[0023] In certain embodiments, this disclosure relates to vectors or nucleic acids encoding antibodies as disclosed herein. In certain embodiment, this disclosure relates to a somatic cell comprising a vector or nucleic acid encoding antibodies as disclosed herein.

[0024] In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of an anti-VIP antibody or other agent as disclosed here to a subject in need thereof. In certain embodiments, the subject is a human patient. In certain embodiments, antibody is administered in combination with another chemotherapy agent. In certain embodiments, the chemotherapy agent is an immune checkpoint inhibitors, e.g., antibody or other agent that specifically binds, programmed cell death receptor 1 (PD-1), ligand (PD-L1), T-cell immunoglobulin and mucin domain-3 (TIM3), lymphocyte-associated gene 3 (LAG3), T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT), or cytotoxic T-lymphocyte- associated protein 4 (CTLA-4).

[0025] In certain embodiments, the cancer is a hematological cancer. In certain embodiments, the hematological cancer is acute myeloid leukemia (AML). In certain embodiments, the cancer is chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma, lung cancer, colon cancer, gliomas, thyroid cancer, head or neck cancers, pancreatic cancer, Kaposi sarcoma, breast cancer, prostate cancer, biliary cancer, or uveal melanoma. In certain embodiments, the cancer is a solid cancer or tumor, lung cancer, colon cancer, gliomas, thyroid cancer, head or neck cancers, pancreatic cancer, or uveal melanoma. In certain embodiments, the cancer is chronic lymphocytic leukemia, diffuse large B-cell lymphoma, Hodgkin lymphoma, Kaposi sarcoma, breast cancer, prostate cancer, or biliary cancer.

[0026] In certain embodiments, this disclosure relates to antibodies disclosed herein conjugated to an exogenous protein, albumin, fluorescent dye, or other label.

[0027] In certain embodiments, this disclosure relates to methods of labeling cells or a tissue comprising contacting or administering an effective amount of an anti-VIP antibody or other agent as disclosed here comprising a label to a subject and detecting, measuring, or quantifying the label on the cell or tissue. In certain embodiments, methods further comprise imaging the tissue or cells.

[0028] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0029] Figure 1A shows data using 10 ug / ml mAb concentration. Anti-VIP mAbs increased frequencies of CD69+ and 4-1BB+ CD4 T cells compared to anti- CD3 / CD28 / CD2 activation alone (ACT). ANT-308 is a peptide antagonist of the VIP receptor. ET5a is a control mAb.

[0030] Figure IB shows data using 10 ug / ml mAb concentration. Anti-VIP mAbs increased frequencies of CD69+ and 4-1BB+ CD8 T cells compared to anti- CD3 / CD28 / CD2 activation alone (ACT). ANT-308 is a peptide antagonist of the VIP receptor. ET5a = control mAb.

[0031] Figure 2 shows data indicating in vivo anti-tumor activity of anti-VIP mAbs.

[0032] Figure 3 shows data from an in-vitro test for VIP mAb T-cell activation anti-VIP mAb administration.

[0033] DETAILED DISCUSSION

[0034] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims or as amended during prosecution.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0036] An "embodiment" of this disclosure refers to an example, but not necessarily limited to such example. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0037] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

[0038] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

[0039] As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0040] The terms, “peptide” and “polypeptide” refer to a polymer of amino acids. Typically, the peptide has greater than 3 amino acids or 4 or more amino acids, or 5 or more amino acids, and may contain N or C terminal chemical modifications, alkyl esters, amides, etc. The amino acids can be natural occurring or non-naturally occurring amino acids. The term “comprising” in reference to a protein having a peptide sequence refers to a peptide that may contain additional 5’ (5’ terminal end) or 3’ (3’ terminal end) nucleotides or N- or C-terminal amino acids, i.e., the term is intended to include the peptide sequence within a larger peptide. "Consisting essentially of or "consists of or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein that exclude certain prior art elements to provide an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel character! stic(s) of the compositions or methods.

[0041] The term “consisting of’ in reference to a peptide having an amino acid sequence refers to a peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids expressly specified in the claim. In certain embodiments, the disclosure contemplates that the “N-terminus of a peptide consists of an amino acid sequence,” which refers to the N-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids specified in the claim; however, the C- terminus may be connected to additional amino acids, e.g., as part of a larger peptide. Similarly, the disclosure contemplates that the “C-terminus of a peptide consists of an amino acid sequence,” which refers to the C-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids specified in the claim; however; the N-terminus may be connected to additional amino acids, e g., as part of a larger peptide. In certain embodiments, the C-terminus may have 5, 10, 20, or 50 additional amino acids. In certain embodiments, the N-terminus may have 5, 10, 20, or 50 additional amino acids.

[0042] "Subject" refers to any animal, preferably a human patient, livestock, rodent, monkey or domestic pet. The term is used herein to encompasses apparently healthy, non-infected individuals or a patient who is known or suspected of being having a disease or condition. In certain embodiments, the subject is a human subject of advanced age or elderly e.g., more than 45, 55, 60, or 65 years old.

[0043] As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments of the present disclosure also contemplate treatment that merely reduces symptoms, and / or delays disease progression.

[0044] As used herein, the terms "prevent" and "preventing" include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.

[0045] A "label" refers to a detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. A label includes the incorporation of a radiolabeled amino acid or the covalent attachment of biotinyl moieties to a polypeptide that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as18F,35S, or131I) fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for labeled secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0046] A "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide and retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall (homologous) and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. With regard to any of the peptides disclosed herein, it is contemplated that such peptide may be variants contain one or more amino acid substitutions. In certain embodiments, the variant has greater than about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or similarity.

[0047] "Identity," as known in the art, is a relationship between two or more peptide sequences, as determined by comparing the sequences. In the art, "similarity" also refers to the degree of sequence relatedness between peptides as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods. Preferred methods to determine identity are designed to give the largest match between the sequences tested. In certain embodiments, sequence "identity" refers to the number of exactly matching amino acids (expressed as a percentage) in a sequence alignment between two sequences of the alignment calculated using the number of identical positions divided by the greater of the shortest sequence or the number of equivalent positions excluding overhangs wherein internal gaps are counted as an equivalent position. In certain embodiments, identity is determined by using the NCBI BLAST program using default values.

[0048] The term "nucleic acid" refers to a polymer of nucleotides, or a polynucleotide, e.g., RNA, DNA, or a combination thereof. The term is used to designate a single molecule, or a collection of molecules. Nucleic acids may be single stranded or double stranded and may include coding regions and regions of various control elements. In certain embodiments, this disclosure relates to nucleotide sequences or nucleic acids that encode the anti-VIP peptides as disclosed herein, or genetic constructs that include nucleotide sequences or nucleic acids and one or more elements. In certain embodiments, this disclosure relates to hosts or host cells (somatic cells) that contain such nucleotide sequences or nucleic acids, and / or that express (or are capable of expressing), the peptides disclosed herein.

[0049] In certain embodiments, nucleic acid sequences encode the anti-VIP antibodies disclosed herein. The nucleic acids can be DNA, RNA, mRNA or vectors encoding the same. Recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced. For example, typically the selectable marker gene confers resistance to drugs (anti-microbial agents), such as hygromycin or methotrexate in contact with a host cell into which the vector has been introduced. Standard molecular biology techniques can be used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the anti-VIP antibodies or cells coated with the anti-VIP antibodies from the culture medium. For example, the anti-VIP antibodies or cells can be isolated by affinity chromatography.

[0050] Anti-VIP antibodies

[0051] Vasoactive intestinal peptide (VIP) is a neuropeptide expressed in various tissues including the pancreas, intestines and central nervous system. The overexpression of VIP and its receptors is associated with increased growth and metastasis of breast, prostate, and lung malignancies. In addition, the interaction of VIP with its receptors on activated T cells results in immune suppression which further supports tumor growth. Furthermore, tumor supporting regulatory T cells promote by VIP-dependent mechanisms. Experiments indicate that blockade of VIP signaling inhibits tumor-mediated immune suppression and augments antitumor immune responses. Recent preclinical studies in acute myeloid leukemia and T lymphoblastic leukemia demonstrated that VIP receptor antagonists increase T cell-dependent anti-tumor responses. Peptides have a relatively short half-life; thus, long circulating antibodies that bind VIP and inhibit its immunosuppressive activities are needed.

[0052] Yeast display methods were used to screen a human scFv antibody library. Multiple rounds of FACS sorting using fluorochrome-bound VIP yielded candidate clones with scFv fragments having high VIP binding affinity; e.g., A6, C2, and D5. The scFv fragment VL and VH sequences were cloned into a plasmid and co-transfected into a human expression cell line to generate humanized monoclonal antibodies. Purified anti-VIP mAbs were tested for VIP binding using biotinylated VIP bound to streptavidin beads, using an anti-human FITC-conjugated secondary antibody in a flow-cytometric assay, and also using surface plasmon resonance (SPR) analysis. In vitro testing for increased T cell activation used a pool of MACS-purified T cells from three healthy donors that were incubated overnight with 50 lU / ml IL-2, followed by addition of the A6, C2, or D5 mAbs at 10, 50, or 100 pg / mL and activation with a sub-maximal concentration of anti- CD3 / CD28 / CD2. After 48 hours, T cells were analyzed for expression of CD69 and 4-1BB activation markers. In an initial in vivo test for anti -leukemia activity, groups of 10 DBA / 2 mice were inoculated s.c. with 1E5 P815 myeloid sarcoma cells, followed by treatment on day 7 using a single injection of 3 nmol of a pool of the 3 anti-VIP mAbs, control mAb, ANT-308, control peptide, or PBS. Mice were followed for tumor burden and survival.

[0053] Analysis of anti-VIP mAb binding on VIP-coated beads demonstrated near-equivalent VIP binding for A6, C2, and D5, with MFI 3.5-fold higher than control mAb. SPR analysis also showed VIP binding for all three mAbs, with better binding kinetics for the D5 mAb. In the in vitro activation assay, the anti-VIP mAbs increased CD69 and 4-1BB expression in CD4+ and CD8+ T cells compared to anti-CD3 / CD28 / CD2 activation alone. Of note, activation levels with anti-VIP antibodies were higher than those observed with ANT-308, a peptide antagonist of the VIP receptor. In the in vivo myeloid sarcoma model, all mice in the PBS, control peptide, and control antibody groups succumbed to tumor burden by 30 days post-inoculation. The single day 7, 3 nmol dose of combined anti-VIP mAbs resulted in improved survival equivalent to that achieved with 3 nmol ANT-308. Moreover, treatment of P815-bearing DBA / 2 mice with a single injection of the pool of anti-VIP antibodies led to initial regression of established s.c. P815 tumors, and sampling blood for cancer-specific T cells using a P815-specific tetramer showed 5-fold expansion of tetramer+ CD8+ T cells at 14 days following anti-VIP antibody administration.

[0054] Humanized anti-VIP antibodies are an attractive alternative to peptide-based VIP-receptor antagonists due to their improved pharmacokinetics. Anti-VIP Abs identified here bind Vasoactive Intestinal Peptide (VIP) HSDAVFTDNYTRLRKQMAVKKYLNSILN (SEQ ID NO: 8) in a beadbased flow cytometry assay, increase human T cell activation in vitro, and improve survival in a mouse mastocytoma model. Experiments indicate the mAbs and have the ability to improve the anti -tumor activity of T cells in pre-clinical AML models using repeated dosing strategies.

[0055] Identification, synthesis, and characterization of anti-VIP antibodies

[0056] A yeast display of a nonimmune human single-chain variable fragment (scFv) library was screened to identify VIP -binding scFvs. VIP binders were screened by several rounds of selection using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). The enriched binder population was cloned into single colonies of yeast cells by limited dilution. The binding affinities of VIP binding clones were evaluated via flow cytometry by titrating fluorescence-labeled VIP. Clones with high binding affinity (Kd < 500 nM) were selected for sequencing.

[0057] VIP binders were selected by MACS in four rounds. Selections include a brief incubation of yeast library and VIP -magnetic beads, then binders were pulled out using a magnet. Selected yeasts were expanded and induced by specific culture media before analyzed by flow cytometry. Flow cytometry experiments were performed as staining each round of yeast library by anti-c-myc antibody -FITC, VIP -biotin, and Streptavidin PerCP-Cyanine5.51MConjugate. Stained cells were analyzed by flow cytometer. Dot plots were created to show binding percentages of VIP+c-myc+ events from 4 rounds of enrichment.

[0058] Sequences of the isolated scFv revealed that a unique section of complementarity- determining region 3 (CDR3) of the heavy chain which has a dominant role in VIP binding. Multiple clones with similar but distinct CDR3 sequences produced a useful range of binding affinities for further development. Yeast display is an effective technology for identifying human scFvs that bind to the immunosuppressive neuropeptide, VIP. CDR3 of scFv heavy chains were influential in VIP recognition. In certain embodiments, this disclosure relates to human monoclonal antibodies that binds to the VIP peptide as represented by amino acid sequences and neutralizes its immunosuppressive properties on T cells and monocytes.

[0059] In certain embodiments, the DNA sequence of the monoclonal antibody heavy chain is of scFv_clone_A6_Heavy having following sequence wherein Agel (start of variable domain, bold) Sall (bold, end of variable domain / start of hlgGl constant domain) and stop codon (bold TGA): ACCGGTGTACATTCCGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCC CGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGG ATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTAT CCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAG CCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGAC ACGGCCGTGTATTACTGTGCGAGGGTTGATTACGATGGTGAACCATTTGATTACTGGG GCCAAGGTACCCTGGTCACTGTCTCCAGTGCGTCGACCAAGGGCCCATCGGTCTTCC CCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGG TCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGACCA GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA GCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGA ATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGAC AAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTC TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC ACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTA CGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACA ACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATG GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAA ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC ATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGC TCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC ATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAT GA (SEQ ID NO: 14). In certain embodiments, the DNA sequence of the monoclonal antibody heavy chain is of scFv_clone_C2_Heavy having following sequence wherein Agel (start of variable domain, bold) Sall (bold, end of variable domain / start of hlgGl constant domain) and stop codon (bold TGA): ACCGGTGTACATTCCGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCC CGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGG ATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTAT CCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAG CCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGAC ACGGCCGTGTATTACTGTGCGAGGTACTCTGAATACGATGGTGCATACGATATCTTTGA TTACTGGGGCCAAGGTACCCTGGTCACTGTCTCCAGTGCGTCGACCAAGGGCCCATC GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGG CTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGC CCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTC CCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTG CAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAAT CTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGAC CGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCC TGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATC GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT GCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA ACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACA GCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCG GGTAAATGA (SEQ ID NO: 15)

[0060] In certain embodiments, the DNA sequence of the monoclonal antibody heavy chain is of scFv_clone_D5_Heavy having following sequence wherein Agel (start of variable domain, bold) Sall (bold, end of variable domain / start of hlgGl constant domain) and stop codon (bold TGA): ACCGGTGTACATTCCGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCC CGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGG ATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTAT CCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAG CCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGAC ACGGCCGTGTATTACTGTGCGAGGGCATCTTACGGTGCAGGTGAACCATTTGATTACT GGGGCCAAGGTACCCTGGTCACTGTCTCCAGTGCGTCGACCAAGGGCCCATCGGTCT TCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCCCTGA CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCA GCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTC AGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGA GGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG TACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGA GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC CCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAG GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG

[0061] TAAATGA (SEQ ID NO: 16).

[0062] In certain embodiments, the DNA sequence of the monoclonal antibody light chain is of scFv_clone_C2_Light(Kappa) having following sequence wherein Agel (start of variable domain, bold), BsiWI (bold, end of variable domain / start of hlgGl constant domain) and stop codon (bold TGA):

[0063] ACCGGTGTACATTCCGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGT AGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAA TTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGT TTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACT CTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTT ACAGTACCC'CGCTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAA ACG'TXCfzGTG GCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTG CCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAA GGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACA GCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTAC GAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTC ACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 17). It is contemplated that the monoclonal antibodies

[0064] In certain embodiments, this disclosure relates to the treatment of patients with cancer by administering a human monoclonal antibody protein in which the final concentration of antibody is between 1 nanomolar and one micromolar.

[0065] In certain embodiments, the human monoclonal antibody is administered to patients with cancer in combination with a monoclonal antibody to PD1, Tim3, LAG3, or TIGIT.

[0066] In certain embodiments, the type of cancer is from the following list: acute myeloid leukemia, chronic lymphocytic leukemia, lung cancer, colon cancer, gliomas, thyroid cancer, head and neck cancers, pancreatic cancer, uveal melanoma.

[0067] In certain embodiments, the anti-VIP antibodies comprising a heaving chain CDR3 which is VDYDGEP (SEQ ID NO: 3). In certain embodiments, the anti-VIP antibodies comprise a heavy chain, wherein the heavy chain CDR1 is SYWIG (SEQ ID NO: 1), CDR2 is IIYPGDSDTRYSPSFQG (SEQ ID NO: 2), and a CDR3 which is VDYDGEP (SEQ ID NO: 3). In certain embodiments, the heavy chain comprises the amino acid sequence of GVHSEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPG DSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAVYYCARVDYDGEPFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALG (SEQ ID NO: 4).

[0068] In certain embodiments, CDR1 is SYWIG (SEQ ID NO: 1), CDR2 is IIYPGDSDTRYSPSFQG (SEQ ID NO: 2), and CDR3 is YSEYDGAYDI (SEQ ID NO: 5). In certain embodiments, the heavy chain comprises the amino acid sequence of GVHSEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPG DSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAVYYCARYSEYDGAYDIFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG (SEQ ID NO: 6).

[0069] In certain embodiments, CDR1 is SYWIG (SEQ ID NO: 1), CDR2 is IIYPGDSDTRYSPSFQG (SEQ ID NO: 2), and CDR3 is ASYGAGEP (SEQ ID NO: 7). In certain embodiments, the heavy chain comprises the amino acid sequence of GVHSEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPG DSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAVYYCARASYGAGEPFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG (SEQ ID NO: 9).

[0070] In certain embodiments, the antibody heavy chains comprise one or more of the following constant region mutations G236A, S239D, A330L, I332E, S267E, L328F, P238D, H268F, S324T, S228P, G236R, L328R, L234A, L235A, M252Y, S254T, T256E, M428L, N434S.

[0071] In certain embodiments, the antibody comprises a light chain. In certain embodiment, the antibody comprises a light chain, wherein the light chain CDR1 is RASQSISSYLN (SEQ ID NO: 10), CDR2 is AASSLQS (SEQ ID NO: 11), and CDR3 is QQSYSTPLT (SEQ ID NO: 12). In certain embodiment, the antibody comprises a light chain having the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKRTV (SEQ ID NO: 13).

[0072] In certain contexts, an “antibody” refers to a protein-based molecule that is naturally produced by animals in response to the presence of a protein or other molecule or that is not recognized by the animal’s immune system to be a “self’ molecule, i.e., recognized by the animal to be a foreign molecule, i.e., an antigen to the antibody. The immune system of the animal will create an antibody to specifically bind the antigen (or any cell or organism attached to the antigen) and thereby targeting the antigen for degradation or elimination. It is well recognized by skilled artisans that the molecular structure of a natural antibody can be synthesized and altered by laboratory techniques. Recombinant engineering can be used to generate fully synthetic antibodies or fragments thereof providing control over variations of the amino acid sequences of the antibody. Thus, the term “antibody” is intended to include natural antibodies, monoclonal antibody, or non- naturally produced synthetic antibodies, such as specific binding single chain antibodies, bispecific antibodies, or fragments thereof. These antibodies may have chemical modifications. The term "monoclonal antibodies" refers to a collection of antibodies encoded by the same nucleic acid molecule that are optionally produced by a single hybridoma (or clone thereof) or other cell line, or by a transgenic mammal such that each monoclonal antibody will typically recognize the same antigen. The term "monoclonal" is not limited to any particular method for making the antibody, nor is the term limited to antibodies produced in a particular species, e.g., mouse, rat, etc.

[0073] In humans, from a structural standpoint, an antibody is a combination of proteins: two heavy chain proteins and two light chain proteins. Alternatively, other animals produce antibodies from nucleic acids that encode a single protein. In humans, the heavy chains are longer than the light chains. The two heavy chains typically have the same amino acid sequence. Similarly, the two light chains typically have the same amino acid sequence. Each of the heavy and light chains contain a variable segment that contains amino acid sequences which participate in binding to the antigen. The variable segments of the heavy chain do not have the same amino acid sequences as the light chains. The variable segments are often referred to as the antigen binding domains. The antigen and the variable regions of the antibody may physically interact with each other at specific smaller segments of an antigen often referred to as the "epitope." Epitopes usually consist of surface groupings of molecules, for example, amino acids or carbohydrates. The terms “variable region,” "antigen binding domain," and "antigen binding region" refer to that portion of the antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. Small binding regions within the antigenbinding domain that typically interact with the epitope are also commonly alternatively referred to as the "complementarity-determining regions, or CDRs."

[0074] "Single chain antibodies" refer to a single peptide containing naturally or non-naturally occurring sequences, including synthetically modified peptide sequences, derived from an antibody variable region that specifically binds an antigen of interest. Single chain antibodies are sometimes fragments or variants of naturally occurring mammalian antibodies. Such antibodies are sometimes referred to as single-domain antibodies (sdAbsor or VHHs), or camelid singledomain antibodies, e.g., when derived from an animal of Camelidae family, e.g., lamas, camels.

[0075] In certain contexts, an “antibody” refers to a protein-based molecule that is naturally produced by animals in response to the presence of a protein or other molecule or that is not recognized by the animal’s immune system to be a “self’ molecule, i.e., recognized by the animal to be a foreign molecule, i.e., an antigen to the antibody. The immune system of the animal will create an antibody to specifically bind the antigen, and thereby targeting the antigen for degradation or elimination, or any cell or organism attached to the antigen. It is well recognized by skilled artisans that the molecular structure of a natural antibody can be synthesized and altered by laboratory techniques. Recombinant engineering can be used to generate fully synthetic antibodies or fragments thereof providing control over variations of the amino acid sequences of the antibody. Thus, the term “antibody” is intended to include natural antibodies, monoclonal antibody, or non-naturally produced synthetic antibodies, such as specific binding single chain antibodies, bispecific antibodies, or fragments thereof. These antibodies may have chemical modifications. The term "monoclonal antibodies" refers to a collection of antibodies encoded by the same nucleic acid molecule that are optionally produced by a single hybridoma (or clone thereof) or other cell line, or by a transgenic mammal such that each monoclonal antibody will typically recognize the same antigen. The term "monoclonal" is not limited to any particular method for making the antibody, nor is the term limited to antibodies produced in a particular species, e.g., mouse, rat, etc.

[0076] From a structural standpoint, a human antibody is a combination of proteins: two heavy chain proteins and two light chain proteins. The heavy chains are longer than the light chains. The two heavy chains typically have the same amino acid sequence. Similarly, the two light chains typically have the same amino acid sequence. Each of the heavy and light chains contain a variable segment that contains amino acid sequences which participate in binding to the antigen. The variable segments of the heavy chain do not have the same amino acid sequences as the light chains. The variable segments are often referred to as the antigen binding domains. The antigen and the variable regions of the antibody may physically interact with each other at specific smaller segments of an antigen often referred to as the "epitope." Epitopes usually consist of surface groupings of molecules, for example, chains of amino acids or carbohydrates. The terms “variable region,” "antigen binding domain," and "antigen binding region" refer to that portion of the antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. Small binding regions within the antigenbinding domain that typically interact with the epitope are also commonly alternatively referred to as the "complementarity-determining regions, or CDRs."

[0077] With regard to variable chain immunoglobulins, the location of binding complementarity- determining regions (CDRs) sometimes varies depending on the specific sequence context and animal. The CDRs can be determined through epitope studies and sequence alignment comparisons of the constant and framework regions for the specific animal. As is well-known in the art that there are multiple conventions to define and describe the CDRs of a VH or VHH fragment, such as the Kabat definition (which is based on sequence variability) and the Chothia definition (which is based on the location of the structural loop regions).

[0078] In general, identifying CDRs can be accomplished using Kabat or Chothia antibody sequence criteria. Note that there are examples where these constant features do not occur; however, the Cys residues are the most common conserved features.

[0079] For CDR-L1, the start residue is approximately 24 to 30 after the first amino acid and typically after a Cys. The residue after is typically a Trp such as Trp-Tyr-Gln, Trp-Leu-Gln, Trp- Phe-Gln, or Trp-Tyr-Leu. Length is typically 10 to 17 residues. CDR-L2 starts about 16 residues after the end of LI . The residues before are typically Ile-Tyr, Val-Tyr, Ile-Lys, Ile-Phe, with a length of about 4-7 residues. CDR-L3 starts at about 33 residues after end of L2 before a Cys residue with a length of about 7 to 11 residues typically ending before a Phe-Gly.

[0080] For CDR-H1, the start residue is approximately 26 to 30 after the first amino acid and typically 4 amino acids after a Cys and typically ends with Trp, e.g., Trp-Val, but also, Trp-Ile, Trp-Ala. The length is typically about 6 to 12 residues. CDR-H2 typically starts at about 4-15 residues after the end of CDR-H1. Residues before the start are typically Trp-Ile-Gly but can be a number of variations, and residues after typical ends with Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala- Thr / Ser / Ile / Ala. The length can vary from about 8 to 20 amino acids; CDR-H3 is typically about 30-33 residues after the end of CDR-H2, and often identified 3 amino acids after a Cys, such as in the example Cys-Ala-Arg. The end is sometimes identified before residues such as Trp-Gly. The length can vary widely, e.g., 4-25 or more depending on the animal.

[0081] A "chimeric antibody" is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such that the entire molecule is not naturally occurring. Examples of chimeric antibodies include those having a variable region derived from a non-human antibody and a human immunoglobulin constant region. The term is also intended to include antibodies having a variable region derived from one human antibody grafted to an immunoglobulin constant region of a predetermined sequences or the constant region from another human for which there are allotypic differences residing in the constant regions of any naturally occurring antibody having the variable regions, e.g., CDRs 1, 2, and 3 of the light and heavy chain. Human heavy chain genes exhibit structural polymorphism (allotypes) that are inherited as a haplotype. The serologically defined allotypes differ within and between population groups. See Jefferis et al. mAb, 1 (2009), pp. 332-338.

[0082] In certain embodiments, the antibody, antigen binding fragment, the light chain, or the heavy chain as disclosed herein comprises a non-naturally occurring chimeric amino acid sequence such that there is at least one mutation that is not present in naturally occurring antibodies comprising one or all of the six CDRs.

[0083] The term "antibody fragment" refers to an antibody which comprises less than a complete, intact antibody. Complete human antibodies comprise two functionally independent parts or fragments: an antigen binding fragment known as "Fab," and a carboxy terminal crystallizable fragment known as the "Fc" fragment. The Fab fragment includes the first constant domain from both the heavy and light chain (CHI and CL1) together with the variable regions from both the heavy and light chains that bind the specific antigen. Each of the heavy and light chain variable regions includes three complementarity determining regions (CDRs) and framework amino acid residues which separate the individual CDRs. The Fc region comprises the second and third heavy chain constant regions (CH2 and CH3) and is involved in effector functions such as complement activation and attack by phagocytic cells. In some antibodies, the Fc and Fab regions are separated by an antibody "hinge region," and depending on how the full-length antibody is proteolytically cleaved, the hinge region may be associated with either the Fab or Fc fragment. For example, cleavage of an antibody with the protease papain results in the hinge region being associated with the resulting Fc fragment, while cleavage with the protease pepsin provides a fragment wherein the hinge is associated with both Fab fragments simultaneously. Because the two Fab fragments are covalently linked following pepsin cleavage, the resulting fragment is termed the F(ab')2 fragment.

[0084] The term, "humanized" refers to an antibody containing one or more amino acid mutations so that immunogenicity upon administration in human patients, e.g., due to "pre-existing antibodies", is reduced, made highly unlikely, or nonexistent. Anaphylaxis is a severe allergic reaction to an allergen, e.g., polypeptide. Non-human proteins contain amino acid residues that may be immunogenic when targeted by preexisting antibodies circulating in a human patient. Thus, it is desirable to mutate residues within a therapeutic antibody so that the peptide sequences are similar to peptide sequences that commonly occurs in human antibodies / proteins, provided that the desirable therapeutic properties are retained, thereby reducing the risk of undesirable immune reactions. In antibodies, this is typically accomplished by transfer of complementarity-determining regions (CDRs) of a non-human antibody to a human framework sequence, yielding a human like antibody with reduced immunogenicity. Another method entails comparing sequences, preferably framework sequences, and identifying amino acid substitutions providing "humanized" sequences frequently found within human antibody sequence repertoire. These humanized sequences reduce the risk of undesirable immune reactions providing an antibody that is substantially non- immunogenic in humans and retain the affinity and activity of the original polypeptide. In one example humanization, e g., framework region humanization, one screens the available human consensus sequences for existing known sequences that are most similar to the original sequence.

[0085] In certain embodiments, the antibody heavy chain is an IgGl, IgG2, IgG3, and / or IgG4 constant domain. In certain embodiments, the antibody, antigen binding fragment, or heavy chain, comprises a human constant domain from an immunoglobulin constant region (Fc) having one, two, three, four, five, six, or more of the following mutations G236A, S239D, A33OL, I332E, S267E, L328F, P238D, H268F, S324T, S228P, G236R, L328R, L234A, L235A, M252Y, S254T, T256E, M428L, N434S. In certain embodiments, the antibody heavy chain is an IgGl, IgG2, IgG3, and / or IgG4 constant domain. With regard to the Fc mutations reported herein, the sequences are in reference to following amino acid sequence starting at amino acid 119:

[0086] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL Q S SGLYSL S SWT VP S S SLGTQT YICNVNHKPSNTK VDKK VEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 18).

[0087] In certain embodiments, the antibody heavy chains comprise one or more of the following constant region mutations P329G, D265A, N297A, N297G, N297Q, F243L, R292P, F296Y, Y300L, V305I, P396L, S298A, E333A, K334A, L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E, K326W, E333S, E345R, E430G, S440Y, L235E, and / or N325S.

[0088] In certain embodiments, the heavy chain is an IgG4 comprising one or more of the following mutations, S228P, F234A, L235A, K196Q, F296Y, E365K, R409K, H435R, L445P, G446 (deletion), K447 (deletion), M428L, N434S, K447 (deletion), E233P, F234V, D2654A, and / or L235E. Often, the numbering of amino acid residues is performed by the method described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of VH CDR2 and inserted residues (for instance residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence. Alternatively, the numbering of amino acid residues may be performed by the EU-index or EU numbering system (also described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Afurther numbering system of amino acid residues in antibodies is the IMGT numbering system (Lefranc, M.-P. et al., Dev. Comp. Immunol, 29, 185- 203 (2005)). Alternatively, the numbering of amino acid residues is performed by the EU-index also described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0089] In certain embodiments, the heavy chain is an IgG4 comprising one or more of the following mutations, S228P, S217P, G220T, P224H, P225T, and / or K196P.

[0090] Table 1. A representative list of mutations in an IgG4 molecule. In certain embodiments, this disclosure contemplates antibodies with a heavy chain (Fc domain) and known mutations. The antibody can be a single heavy chain antibody or also contain a light chain. As used herein, a “mutation,” “mutant,” or the like of an antibody heavy chain sequence refers to the expression of a variant amino acid(s) within a heavy chain antibody defined by positions compared to base amino acids within the constant regions of the heavy chain (Fc). The mutants may be constructed by building peptide sequences synthetically or, more typically, constructed using recombinant nucleic acid techniques, e.g., expression of the heavy chain in a cell from a template nucleic acid. Due to three codon translation of amino acids from nucleic acid, several three nucleotide codons may express the same amino acid variant. Sometimes the variant is due to a single nucleotide change, and sometimes the variant is due to more than one nucleotide change. Thus, reference to a “mutation,” “mutant,” or the like of a heavy chain antibody sequence are not necessarily limited to solely single nucleotide changes.

[0091] A "heterologous" nucleic acid sequence or peptide sequence refers to a nucleic acid sequence or a peptide sequence that does not naturally occur, e.g., because the whole sequence contains a segment from other plants, bacteria, viruses, other organisms, or joinder of two sequences that occur the same organism but are joined together in a manner that does not naturally occur in the same organism or any natural state.

[0092] The term "recombinant" when made in reference to a nucleic acid molecule refers to a nucleic acid molecule which is comprised of segments of nucleic acid joined together by means of molecular biological techniques provided that the entire nucleic acid sequence does not occurring in nature, i.e., there is at least one mutation in the overall sequence such that the entire sequence is not naturally occurring even though separately segments may occur in nature. The segments may be joined in an altered arrangement such that the entire nucleic acid sequence from start to finish does not naturally occur. The term "recombinant" when made in reference to a protein or a peptide refers to a protein molecule that is expressed using a recombinant nucleic acid molecule.

[0093] The terms "vector" or " expression vector " refer to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free expression system. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

[0094] In certain embodiments, this disclosure contemplates a vector encoding an antibody / protein(s) containing the same as disclosed herein in operable combination with a heterologous promoter or other transcription initiation element, translation initiation site, element that allows for translation initiation, translational enhancers, cis-acting nucleic acid DNA / RNA regions that promote internal initiation of protein synthesis. In certain embodiments, the promoter is a positive inducible promoter, negative inducible promoter, chemically inducible promoter, temperature inducible promoter, or light inducible promoter.

[0095] In certain embodiments, the disclosure relates to recombinant antibodies / proteins comprising sequences disclosed herein or variants or fusions thereof wherein the amino terminal end, or the carbon terminal end of the amino acid sequence are optionally attached to a heterologous amino acid sequence, label, or reporter molecule.

[0096] In certain embodiments, the disclosure relates to the recombinant vectors comprising a nucleic acid encoding an antibodies / recombinant protein as disclosed herein. In certain embodiments, the recombinant vector optionally comprises a mammalian, human, insect, viral, bacterial, bacterial plasmid, yeast associated origin of replication or gene such as a gene or retroviral gene or lentiviral LTR, TAR, RRE, PE, SLIP, CRS, and INS nucleotide segment or gene selected from tat, rev, nef, via, vpr, vpu, and vpx or structural genes selected from gag, pol, and env. In certain embodiments, the recombinant vector optionally comprises a gene vector element (nucleic acid) such as a selectable marker region, lac operon, a CMV promoter, a hybrid chicken B-actin / CMV enhancer (CAG) promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, internal ribosome entry site (IRES) sequence, cis-acting woodchuck post regulatory element (WPRE), scaffold-attachment region (SAR), inverted terminal repeats (ITR), c-myc tag coding region, metal affinity tag coding region, streptavidin binding peptide tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, SV40 polyadenylation signal, SV40 origin of replication, Col El origin of replication, fl origin, pBR322 origin, or pUC origin, TEV protease recognition site, loxP sites, Cre recombinase coding region, or a multiple cloning site such as having 5, 6, or 7 or more restriction sites within a continuous segment of less than 50 or 60 nucleotides or having 3 or 4 or more restriction sites with a continuous segment of less than 20 or 30 nucleotides.

[0097] As used herein, the term “conjugated” refers to linking molecular entities directly through covalent bonds, or by other specific binding interactions, such as indirectly due to hydrogen bonding and other Van der Waals forces, e.g., hydrogen bonding and other Van der Waals forces are involved in non-covalent binding interaction that occur at room temperature between a protein and a protein receptor. The force to break a covalent bond is high, e.g., about 1500 pN for a carbon to carbon bond. The force to break a combination of strong protein non-covalent interactions is typically a magnitude less, e.g., biotin to streptavidin is about 150 pN. Thus, a skilled artisan would understand that conjugation must be strong enough to bind molecular entities in order to implement the intended results.

[0098] The term "specific binding agent" refers to a molecule, such as a proteinaceous molecule, which binds a target molecule with a greater affinity than other random molecules or proteins. Examples of specific binding agents include antibodies that bind an epitope of an antigen or a receptor which binds a ligand. "Specifically binds" refers to the ability of a specific binding agent (such as an ligand, receptor, enzyme, antibody or binding region / fragment thereof) to recognize and bind a target molecule or polypeptide, such that its affinity (as determined by, e.g., affinity ELISA or other assays) is at least 10 times greater, but optionally 50 times, 100, 250 or 500 times greater, or even at least 1000 times greater than the affinity of the same for any other or other random molecule or polypeptide.

[0099] A "linking group" refers to any variety of molecular arrangements that can be used to bridge to molecular moieties together, e.g., a peptide bridge. An example formula are chemical chains of -Rn- wherein R is selected individually and independently at each occurrence as:

[0100] -CRnRn-, -CHRn-, -CH-, -C-, -CH2-, -C(OH)Rn, -C(OH)(OH)-, -C(OH)H, -C(Hal)Rn-, -C(Hal)(Hal)-, -C(Hal)H-, -C(CN)Rn-, -C(CN)(CN)-, -C(CN)H-, -O-, -S-, -N-, -NH-, -NRn-, -(C=O)-, -(C=NH)-, -(C=S)-, -(C=CH)-, which may contain single, double, or triple bonds individually and independently between the R groups. If an R is branched with an Rnit may be terminated with a group such as -CH3, -H, -CH=CH2, -CCH, -OH, -SH, -NH2, -N3, -CN, or -Hal, or two branched Rs may form an aromatic or non-aromatic cyclic structure. It is contemplated that in certain instances, the total Rs or “n” may be less than 100 or 50 or 25 or 10. It is contemplated that in certain instances, each Rnmay be individually and independently an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15, 17, 18, 19, 20, 21, 22, 23, 24, and 25. Examples of linking groups include bridging alkyl, amide, and alkoxyalkyl groups.

[0101] Methods of Use

[0102] In certain embodiments, this disclosure relates to methods of using antibodies, fragments and agents disclosed herein in the treatment or prevention of associated diseases and conditions. In certain embodiments, the pharmaceutical composition is administered either systemically or locally, i.e., parenteral, subcutaneous, intravenous, intramuscular, intranasal, intratumorally, or any other path of administration.

[0103] In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of an anti-VIP antibody and / or other agent as disclosed here to a subject in need thereof. In certain embodiments, the subject is a human patient. In certain embodiments, antibody is administered in combination with another chemotherapy agent. In certain embodiments, the chemotherapy agent is an immune checkpoint inhibitors, e.g., antibody or other agent that specifically binds, programmed cell death receptor 1 (PD-1), ligand (PD-L1), T-cell immunoglobulin and mucin domain-3 (TIM3), lymphocyte-associated gene 3 (LAG3), T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT), or cytotoxic T-lymphocyte- associated protein 4 (CTLA-4). In certain embodiments, the chemotherapy agent is a therapeutic antibody or other agent that specifically binds PD1, Tim3, LAG3, or TIGIT.

[0104] In certain embodiments, the cancer is a hematological cancer. In certain embodiments, hematological cancer is acute myeloid leukemia or chronic lymphocytic leukemia. In certain embodiments, the cancer is a solid cancer or tumor, lung cancer, colon cancer, gliomas, thyroid cancer, head or neck cancers, pancreatic cancer, or uveal melanoma.

[0105] "Cancer" refers any of various cellular diseases with malignant neoplasms characterized by the proliferation of cells. It is not intended that the diseased cells must actually invade surrounding tissue and metastasize to new body sites. Cancer can involve any tissue of the body and have many different forms in each body area. Within the context of certain embodiments, whether "cancer is reduced" may be identified by a variety of diagnostic manners known to one skill in the art including, but not limited to, observation the reduction in size or number of tumor masses or if an increase of apoptosis of cancer cells observed, e.g., if more than a 5 % increase in apoptosis of cancer cells is observed for a sample compound compared to a control without the compound. It may also be identified by a change in relevant biomarker or gene expression profile, such as PSA for prostate cancer, HER2 for breast cancer, or others.

[0106] The cancer to be treated in the context of the present disclosure may be any type of cancer or tumor. These tumors or cancer include, and are not limited to, tumors of the hematopoietic and lymphoid tissues or hematopoietic and lymphoid malignancies, tumors that affect the blood, bone marrow, lymph, and lymphatic system. Hematological malignancies may derive from either of the two major blood cell lineages: myeloid and lymphoid cell lines. The myeloid cell line normally produces granulocytes, erythrocytes, thrombocytes, macrophages and mast cells; the lymphoid cell line produces B, T, NK and plasma cells. Lymphomas, lymphocytic leukemias, and myeloma are from the lymphoid line, while acute and chronic myelogenous leukemia, myelodysplastic syndromes and myeloproliferative diseases are myeloid in origin.

[0107] Also contemplated are malignancies located in the colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, hypophysis, testicles, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, genitals or urinary apparatus and, more particularly, childhood acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult (primary) hepatocellular cancer, adult (primary) liver cancer, adult acute lymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin's disease, adult Hodgkin's lymphoma, adult lymphocytic leukemia, adult non-Hodgkin's lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignant tumors, anal cancer, astrocytoma, cancer of the biliary tract, cancer of the bladder, bone cancer, brain stem glioma, brain tumors, breast cancer, cancer of the renal pelvis and ureter, primary central nervous system lymphoma, central nervous system lymphoma, cerebellar astrocytoma, brain astrocytoma, cancer of the cervix, childhood (primary) hepatocellular cancer, childhood (primary) liver cancer, childhood acute lymphoblastic leukemia, childhood acute myeloid leukemia, childhood brain stem glioma, childhood cerebellar astrocytoma, childhood brain astrocytoma, childhood extracranial germ cell tumors, childhood Hodgkin's disease, childhood Hodgkin's lymphoma, childhood visual pathway and hypothalamic glioma, childhood lymphoblastic leukemia, childhood medulloblastoma, childhood non-Hodgkin's lymphoma, childhood supratentorial primitive neuroectodermal and pineal tumors, childhood primary liver cancer, childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhood visual pathway and hypothalamic glioma, chronic lymphocytic leukemia, chronic myeloid leukemia, cancer of the colon, cutaneous T-cell lymphoma, endocrine pancreatic islet cells carcinoma, endometrial cancer, ependymoma, epithelial cancer, cancer of the esophagus, Ewing's sarcoma and related tumors, cancer of the exocrine pancreas, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic biliary tract cancer, cancer of the eye, breast cancer in women, Gaucher's disease, cancer of the gallbladder, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal tumors, germ cell tumors, gestational trophoblastic tumor, tricoleucemia, head and neck cancer, hepatocellular cancer, Hodgkin's disease, Hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancers, intraocular melanoma, islet cell carcinoma, islet cell pancreatic cancer, Kaposi sarcoma, cancer of kidney, cancer of the larynx, cancer of the lip and mouth, cancer of the liver, cancer of the lung, lymphoproliferative disorders, macroglobulinemia, breast cancer in men, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, occult primary metastatic squamous neck cancer, primary metastatic squamous neck cancer, metastatic squamous neck cancer, multiple myeloma, multiple myeloma / plasmatic cell neoplasia, myelodysplastic syndrome, myelogenous leukemia, myeloid leukemia, myeloproliferative disorders, paranasal sinus and nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma during pregnancy, nonmelanoma skin cancer, non-small cell lung cancer, metastatic squamous neck cancer with occult primary, buccopharyngeal cancer, malignant fibrous histiocytoma, malignant fibrous osteosarcoma / histiocytoma of the bone, epithelial ovarian cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, paraproteinemias, purpura, parathyroid cancer, cancer of the penis, phaeochromocytoma, hypophysis tumor, neoplasia of plasmatic cells / multiple myeloma, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell cancer, cancer of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, cancer of the salivary glands, sarcoidosis, sarcomas, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous neck cancer, stomach cancer, pineal and supratentorial primitive neuroectodermal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, transitional renal pelvis and ureter cancer, trophoblastic tumors, cell cancer of the renal pelvis and ureter, cancer of the urethra, cancer of the uterus, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic glioma, cancer of the vulva, Waldenstrom's macroglobulinemia, Wilms' tumor and any other hyperproliferative disease, as well as neoplasia, located in the system of a previously mentioned organ.

[0108] A “chemotherapy agent,” “chemotherapeutic,” “anti-cancer agent,” or the like, refer to molecules that are recognized to aid in the treatment of a cancer. Contemplated examples include the following molecules or derivatives such as abemaciclib, abiraterone acetate, methotrexate, paclitaxel, adriamycin, acalabrutinib, brentuximab vedotin, ado-trastuzumab emtansine, aflibercept, afatinib, netupitant, palonosetron, imiquimod, aldesleukin, alectinib, alemtuzumab, pemetrexed disodium, copanlisib, melphalan, brigatinib, chlorambucil, amifostine, aminolevulinic acid, anastrozole, apalutamide, aprepitant, pamidronate disodium, exemestane, nelarabine, arsenic trioxide, ofatumumab, atezolizumab, bevacizumab, avelumab, axicabtagene ciloleucel, axitinib, azacitidine, carmustine, belinostat, bendamustine, inotuzumab ozogamicin, bevacizumab, bexarotene, bicalutamide, bleomycin, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, brigatinib, busulfan, irinotecan, capecitabine, fluorouracil, carboplatin, carfilzomib, ceritinib, daunorubicin, cetuximab, cisplatin, cladribine, cyclophosphamide, clofarabine, cobimetinib, cabozantinib-S-malate, dactinomycin, crizotinib, ifosfamide, ramucirumab, cytarabine, dabrafenib, dacarbazine, decitabine, daratumumab, dasatinib, defibrotide, degarelix, denileukin diftitox, denosumab, dexamethasone, dexrazoxane, dinutuximab, docetaxel, doxorubicin, durvalumab, rasburicase, epirubicin, elotuzumab, oxaliplatin, eltrombopag olamine, enasidenib, enzalutamide, eribulin, vismodegib, erlotinib, etoposide, everolimus, raloxifene, toremifene, panobinostat, fulvestrant, letrozole, filgrastim, fludarabine, flutamide, pralatrexate, obinutuzumab, gefitinib, gemcitabine, gemtuzumab ozogamicin, glucarpidase, goserelin, propranolol, trastuzumab, topotecan, palbociclib, ibritumomab tiuxetan, ibrutinib, ponatinib, idarubicin, idelalisib, imatinib, talimogene laherparepvec, ipilimumab, romidepsin, ixabepilone, ixazomib, ruxolitinib, cabazitaxel, palifermin, pembrolizumab, ribociclib, tisagenlecleucel, lanreotide, lapatinib, olaratumab, lenalidomide, lenvatinib, leucovorin, leuprolide, lomustine, trifluridine, olaparib, vincristine, procarbazine, mechlorethamine, megestrol, trametinib, temozolomide, methylnaltrexone bromide, midostaurin, mitomycin C, mitoxantrone, plerixafor, vinorelbine, necitumumab, neratinib, sorafenib, nilutamide, nilotinib, niraparib, nivolumab, tamoxifen, romiplostim, sonidegib, omacetaxine, pegaspargase, ondansetron, osimertinib, panitumumab, pazopanib, interferon alfa-2b, pertuzumab, pomalidomide, mercaptopurine, regorafenib, rituximab, rolapitant, rucaparib, siltuximab, sunitinib, thioguanine, temsirolimus, thalidomide, thiotepa, trabectedin, valrubicin, vandetanib, vinblastine, vemurafenib, vorinostat, zoledronic acid, or combinations thereof such as cyclophosphamide, methotrexate, 5 -fluorouracil (CMF); doxorubicin, cyclophosphamide (AC); mustine, vincristine, procarbazine, prednisolone (MOPP); adriamycin, bleomycin, vinblastine, dacarbazine (ABVD); cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP); bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin, 5- fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX); methotrexate, vincristine, doxorubicin, cisplatin (MVAC).

[0109] In certain embodiments, the chemotherapy agent is an anti-PD-1, anti-PD-Ll anti-CTLA4 antibody or combinations thereof, such as an anti-CTLA4 (e.g., ipilimumab, tremelimumab) and anti-PDl (e.g., nivolumab, pembrolizumab, cemiplimab) and anti-PD-Ll (e.g., atezolizumab, avelumab, durvalumab).

[0110] In certain embodiments, the method of administration is in a subject with a lymphodepleted environment due to prior or concurrent administration of lymphodepleting agents. In certain embodiments, lymphodepleting agents (e.g., cyclophosphamide and fludarabine).

[0111] In certain embodiments, this disclosure relates to methods of labeling cells or a tissue comprising contacting or administering an effective amount of an anti-VIP antibody or other agent as disclosed here comprising a label to a subject in need thereof and detecting, measuring, or quantifying the label in a solution or on the cell or tissue. In certain embodiments, methods further comprise imaging the solution, cell, or tissue.

[0112] In certain embodiments, methods further comprise the step of detecting and / or measuring the emissions and creating an image indicating or highlighting the location of the anti-VIP antibody containing the label bound to a VIP peptide or VIP peptide bound to a VIP peptide receptor in the subject, e.g., from an organ or area of the subject. In certain embodiments, methods further comprise the step of detecting the emissions and creating an image indicating or highlighting the location of the labeled antibody in the subject.

[0113] In certain embodiments, this disclosure relates to methods of testing compounds for the ability to bind VIP comprising contacting, mixing, or administering an effective amount of a labeled anti-VIP antibody as reported herein with the test compound and determining whether the test compound reduces the ability of the anti-VIP antibody to bind VIP in a solution, cell, growth media, or tissue. A “test compound” can be any variety of organic compounds such as small molecules, proteins, antibodies, nucleobases, nucleobase polymers, and known therapeutic agents or therapeutic candidates.

[0114] In certain embodiments, methods further comprise the step of quantifying the label emission providing an emission quantity and recording the emission quantity on a non-transitory computer readable media. In certain embodiments, methods further comprise correlating the emission quantity to concentration of VIP or VIP bound to a VIP receptor in the tissue, cancer, tumor, brain, nerve, neuron cell, or cerebrospinal fluid. In certain embodiments, methods further comprise reporting the emission quantity to the subject or a medical professional.

[0115] Pharmaceutical compositions and kits

[0116] In certain embodiments, this disclosure relates to pharmaceutical compositions comprising an anti-VIP antibody, nucleic acid, vector or cell as reported herein. The pharmaceutical compositions provided herein may generally include one or more pharmaceutically acceptable and / or approved carriers, additives, antibiotics, anti-inflammatory agents, preservatives, diluents and / or stabilizers. Such auxiliary substances can be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, or the like. Suitable carriers are typically large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like.

[0117] In certain embodiments, the nucleic acid or recombinant vector encodes an anti-VIP antibody or fragments disclosed herein having at least one open reading frame that can be translated by a cell or an organism provided with the nucleic acid, DNA, RNA, or mRNA. If more than one peptide, e.g., heavy and / or light chain, of an anti-VIP antibody is translated, the peptides can be expressed in one vector / nucleic acid or in multiple (a plurality of) separate nucleic acids / vectors wherein the product of translation is an anti-VIP antibody or fragments disclosed herein disclosed herein. The product may also be a fusion protein composed of more than one anti- VIP antibody or fragments disclosed herein, e.g., a fusion protein that has two or more anti-VIP antibody or fragments disclosed herein, wherein an anti-VIP antibody or fragments disclosed herein are optionally linked by self-cleaving linker sequences.

[0118] In certain embodiments, nucleic acid, DNA, RNA, or mRNA may be designed to have two (bicistronic) or more (multi ci str onic) open reading frames (ORF). An open reading frame in this context is a sequence including a start codon that can be used as a location to start translation of the encoded nucleic acid into a recombinant protein herein. Translation of such nucleic acid(s) yields two (bicistronic) or more (multicistronic) identical or distinct translation products / proteins (provided the ORFs are not identical). For expression such nucleic acids may comprise an internal ribosomal entry site (IRES) sequence which allows for expression of two or more anti-VIP antibodies or fragments disclosed herein on a single nucleic acid molecule.

[0119] In certain embodiments, the anti-VIP antibodies or fragment disclosed herein, nucleic acid, or recombinant vector may be administered naked without being associated with any further vehicle, e.g., mRNA or DNA.

[0120] In certain embodiments, the anti-VIP antibody or fragments disclosed herein, nucleic acid, recombinant vector or cell may be administered in a pharmaceutical composition having a pharmaceutically acceptable excipient selected from lactose, sucrose, mannitol, triethyl citrate, dextrose, cellulose, methyl cellulose, ethyl cellulose, hydroxyl propyl cellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, croscarmellose sodium, polyvinyl N-pyrrolidone, crospovidone, ethyl cellulose, povidone, methyl and ethyl acrylate copolymer, polyethylene glycol, fatty acid esters of sorbitol, lauryl sulfate, gelatin, glycerin, glyceryl monooleate, silicon dioxide, titanium dioxide, talc, corn starch, carnauba wax, stearic acid, sorbic acid, magnesium stearate, calcium stearate, castor oil, mineral oil, calcium phosphate, starch, carboxymethyl ether of starch, iron oxide, triacetin, acacia gum, esters, or salts thereof.

[0121] In certain embodiments, the pharmaceutical composition is in the form of a sterilized pH buffered aqueous salt solution or a saline phosphate buffer between a pH of 6 to 8, optionally comprising a saccharide or polysaccharide.

[0122] In certain embodiment, the pharmaceutically acceptable excipient is a cationic or polycationic compound and / or with a polymeric carrier. In certain embodiments, the anti-VIP antibody or fragments disclosed herein, nucleic acid, or recombinant vector are in a pharmaceutical composition associated with or complexed with a cationic or polycationic compound or a polymeric carrier. In certain embodiments, the cationic or polycationic compound is protamine, spermine, spermidine, poly-L-lysine (PLL), poly-histidine, or poly-arginine, cationic polysaccharides, such as chitosan, polybrene, cationic polymers, polyethyleneimine (PEI), homo- and co-polymers of lactic acid and glycolic acid, or polymethylmethacrylate. Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, albumin, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, citric acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, hydrophilic polymers such as polyvinyl pyrrolidone, cellulose based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, gelatin, polyethylene polyoxypropylene block polymers, polyethylene glycol and antioxidants including ascorbic acid and methionine; preservatives; low molecular weight (less than about 10 residues) polypeptides; proteins; and amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine. In certain embodiments, the excipient may be one or more selected from the list consisting of NaCl, trehalose, sucrose, mannitol, and / or glycine.

[0123] The disclosure also encompasses products obtainable by further processing of a liquid formulation, such as a frozen, lyophilized or spray-dried product. Upon reconstitution, these solid products can become liquid formulations as described herein (but are not limited thereto). In its broadest sense, therefore, the term "formulation" encompasses both liquid and solid formulations. However, solid formulations are understood as derivable from the liquid formulations (e.g. by freezing, freeze-drying or spray-drying).

[0124] In certain embodiments, the formulations are isotonic in relation to human blood. Isotonic solutions possess the same osmotic pressure as blood plasma, and so can be intravenously infused into a subject without changing the osmotic pressure of the blood plasma.

[0125] In certain embodiments, this disclosure relates to kits containing materials useful for the treatment, or prevention of diseases or conditions as described herein. In certain embodiments, the kit comprises a container, a product label and a package insert. Suitable containers include, for example, bottles, vials, syringes, and boxes containing the same. The containers may be of a variety of materials, e.g., glass, plastic, or cardboard. The container holds the composition which is effective in treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-VIP antibody or fragments disclosed herein, nucleic acid, recombinant vector, or cells as disclosed herein. The product label on, or associated with, the container indicates that the composition is used for treating the condition of choice. In certain embodiments, the kit may further comprise a second container comprising a pharmaceutically acceptable buffer, such as a phosphate buffer saline or a citrate buffered saline. It may further include other materials desirable from a user or commercial standpoint, including other buffers, diluents, filters, needles, and syringes. In certain embodiments, a dosage unit form can be, e.g., in the format of a prefilled syringe, an ampoule, cartridge or a vial.

[0126] In certain embodiments, this disclosure relates to kits or articles of manufacture, comprising an anti-VIP antibody or fragments disclosed herein, nucleic acid, recombinant vector or cell as disclosed herein and instructions for use by, e.g., a healthcare professional. The kits or articles of manufacture may include a container, vial, or a syringe containing the formulation as described herein. Preferably, the container, vial, or syringe is composed of glass, plastic, or a polymeric material. The syringe, ampoule, cartridge, or vial can be manufactured of any suitable material, such as glass or plastic and may include rubber materials, such as rubber stoppers for vials and rubber plungers and rubber seals for syringes and cartridges.

[0127] Development of Humanized antibodies against VIP for increased T cell anti-leukemia activity

[0128] Acute myeloid leukemia (AML) is a prevalent hematological malignancy in adults, with a low 5-year survival rate. Current treatments, such as allogeneic bone marrow transplantation, have limited applicability due to high morbidity and treatment-associated mortality, and current immune checkpoint therapies using anti-PDl and anti-PDLl antibodies are typically ineffective in AML patients.

[0129] Vasoactive intestinal peptide (VIP) is a conserved peptide hormone with immunosuppressive properties. VIP is frequently upregulated in AML and expressed by activated T cells as a co-inhibitory ligand, downregulating T cell anti-leukemia activity. Peptide antagonist of VIP and VIP receptor signaling were developed such as ANT-308, and these agents demonstrated increased T cell anti-tumor activity in murine leukemia and pancreatic cancer models. Disclosed herein are humanized anti-VIP monoclonal antibodies as an alternative therapeutic to treat AML.

[0130] Yeast display methods were used to screen a human scFv antibody library. Multiple rounds of FACS sorting using fluorochrome-bound VIP yielded three top candidate clones with scFv fragments having high VIP binding affinity: A6, C2, and D5. The scFv fragment VL and VH sequences were cloned into a plasmid and co-transfected into a human expression cell line to generate humanized monoclonal antibodies.

[0131] Humanized anti-VIP mAbs are an attractive alternative to peptide-based VIP-receptor antagonists due to their improved pharmacokinetics. Yeast-display single-chain antibody technology, selective sorting, and expression in a producer cell line was used to identify and generate anti-VIP human mAbs. Experiments indicate the anti-VIP mAbs increase human T cell activation in vitro, and improve survival in a mouse mastocytoma model.

[0132] A6, C2 and D5 Anti-VIP mAbs Specifically Bind to VIP on Beads

[0133] Purified anti-VIP mAbs were tested for VIP binding using biotinylated VIP bound to streptavidin beads, using an anti -human FITC-conjugated secondary antibody in a flow-cytometric assay. Anti-VIP mAbs increased T cell activation. Pooled MACS-purified T cells from 3 healthy donors were incubated overnight with 50 lU / ml IL-2, followed by addition of the A6, C2, or D5 mAbs at 10, 50, or 100 ug / mL and activation with a sub-maximal concentration of anti- CD3 / CD28 / CD2. After 48 hours, T cells were analyzed for expression of CD69 and 4-1BB activation markers. Experiments were performed using 10 pg / mL mAb concentration. Anti-VIP mAbs increased frequencies of CD69+ and 4-1BB+ CD4 and CD8 T cells compared to anti- CD3 / CD28 / CD2 activation alone (ACT). Of note, frequencies of activated T cells with anti-VIP mAbs were generally higher than those observed with ANT-308, a peptide antagonist of the VIP receptor. A PE-conjugated anti-VIP commercial mouse antibody showed specific binding on the bead, indicating effective coating of the streptavidin beads with biotinylated VIP. The three novel human anti-VIP mAbs, A6, C2 and D5, all show binding to the VIP-coated beads with about 3.5 higher MFI compared to a negative control human antibody (anti-flu mAb).

[0134] Injection of anti-VIP mAbs improved survival in P815 myeloid sarcoma bearing mice

[0135] In an in vivo test for anti-leukemia activity, groups of 10 DBA / 2 mice were inoculated s.c. with 1E5 P815 myeloid sarcoma cells, followed by treatment on day 7 using a single injection of 3 nmol of a pool of the 3 anti-VIP mAbs A6, C2 and D5, control mAb, ANT-308, control scrambled peptide, or PBS. Mice were followed for tumor burden and survival. Mice in control groups succumbed to tumor burden by 30 days post tumor inoculation. The single day 7, 3 nmol dose of anti-VTP mAbs resulted in improved survival time in this group, equivalent to that achieved with 3 nmol ANT-308.

[0136] Humanized anti-VIP mAbs are an attractive alternative to peptide-based VIP-receptor antagonists due to their improved pharmacokinetics. Yeast-display single-chain antibody technology, selective sorting, and expression in a producer cell line was used to identify and generate anti-VIP human mAbs. Experiments indicate the anti-VIP mAbs increase human T cell activation in vitro, and improve survival in a mouse mastocytoma model.

Claims

CLAIMS1. An antibody that specifically binds vasoactive intestinal peptide wherein the antibody comprises a heavy chain, wherein the heavy chain CDR1 is SYWIG (SEQ ID NO: 1), CDR2 is IIYPGDSDTRYSPSFQG (SEQ ID NO: 2), and a CDR3.

2. The antibody of claim 1, wherein the CDR3 is VDYDGEP (SEQ ID NO: 3).

3. The antibody of claim 2, wherein the heavy chain comprises the amino acid sequence of GVHSEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGHYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAVYYCARVDYDGEPFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG (SEQ ID NO: 4).

4. The antibody of claim 1, wherein the CDR3 is YSEYDGAYDI (SEQ ID NO: 5).

5. The antibody of claim 4, wherein the heavy chain comprises the amino acid sequence of GVHSEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPG DSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAVYYCARYSEYDGAYDIFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG (SEQ ID NO: 6).

6. The antibody of claim 1, wherein the CDR3 is ASYGAGEP (SEQ ID NO: 7).

7. The antibody of claim 6 wherein the heavy chain comprises the amino acid sequence of GVHSEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPG DSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAVYYCARASYGAGEPFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG (SEQ ID NO: 9).

8. The antibody of any of claims 1-7, wherein the heavy chain comprises one or more of the following constant region mutations G236A, S239D, A330L, I332E, S267E, L328F, P238D, H268F, S324T, S228P, G236R, L328R, L234A, L235A, M252Y, S254T, T256E, M428L, and N434S.

9. The antibody of any of claims 1-7, wherein the antibody comprises a light chain, wherein the light chain CDR1 is RASQSISSYLN (SEQ ID NO: 10), CDR2 is AASSLQS (SEQ ID NO: 11), and CDR3 is QQSYSTPLT (SEQ ID NO: 12).

10. The antibody of claim 9, wherein the antibody comprises a light chain having the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKRTV (SEQ ID NO: 13).

11. A vector or nucleic acid encoding the antibody as in any of claims 1-10.

12. A somatic cell comprising a vector or nucleic acid of claim 11.

13. The antibody as in any of claims 1-10 conjugated to an exogenous peptide, albumin, fluorescent dye, or other label.

14. A method of treating cancer comprising administering an effective amount of an antibody as in any of claims 1-10 to a subject in need thereof.

15. The method of claim 14, wherein the subject is a human patient.

16. The method of claim 14, wherein the antibody is administered in combination with another chemotherapy agent.

17. The method of claim 16, wherein the chemotherapy agent is a therapeutic antibody or other agent that specifically binds PD1, Tim3, LAG3, or TIGIT.

18. The method of claim 14, wherein the cancer is a hematological cancer.

19. The method of claim 18, wherein the hematological cancer is acute myeloid leukemia (AML).

20. The method of claim 14, wherein the cancer is chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma, lung cancer, colon cancer, gliomas, thyroid cancer, head or neck cancers, pancreatic cancer, Kaposi sarcoma, breast cancer, prostate cancer, biliary cancer, or uveal melanoma.

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