NGF analog fusion proteins for antigen specific immunotherapy and methods of use

Abstract

The present disclosure provides recombinantly manufactured fusion proteins comprising a nerve growth factor (NGF) protein fragment or an analog thereof linked to a canine Fc fragment. Embodiments include the administration of the fusion proteins to patients to reduce pain symptoms in dogs suffering from a pain-associated ailment such as osteoarthritis. Exemplary Fc fusion proteins and pharmaceutical formulations of exemplary Fc fusion proteins are provided, in addition to methods of use and preparation.

Description

ABC-052PCT / 59495-PCT3NGF ANALOG FUSION PROTEINS FOR ANTIGEN SPECIFIC IMMUNOTHERAPY AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 707,799, filed October 16, 2024, entitled “NGF ANALOG FUSION PROTEINS FOR ANTIGEN SPECIFIC IMMUNOTHERAPY AND METHODS OF USE”, incorporated by reference in its entirety herein, the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 707,803, filed October 16, 2024, entitled “NGF ANALOG FUSION PROTEINS FOR ANTIGEN SPECIFIC IMMUNOTHERAPY AND METHODS OF USE”, incorporated by reference in its entirety herein, and the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 707,813, filed October 16, 2024, entitled “NGF ANALOG FUSION PROTEINS FOR ANTIGEN SPECIFIC IMMUNOTHERAPY AND METHODS OF USE”, incorporated by reference in its entirety herein.SEQUENCE LISTING

[0002] The following application contains a sequence listing filed electronically as a Standard ST.26 compliant XML file entitled “ABC-052PCT.xml” created on October 10, 2025, as 23,249 bytes in size, the entire contents of which are incorporated by reference herein.TECHNICAL FIELD

[0003] The present technology relates to fusion proteins comprising an NGF protein or an analog thereof linked to Fc fragments and their use in the management of chronic pain in dogs.BACKGROUND

[0004] The following description of the background is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.Fc Fusion Proteins

[0005] Fc fusion proteins are comprised of a species-specific immunoglobin Fc domain that is linked to another peptide such as a protein or peptide with therapeutic potential. As used herein, the terms “fusion protein” and “Fc fusion protein” generally mean a protein comprising more than one part, for example from different sources (e.g., different proteins, polypeptides, cells, etc.), that are covalently linked through peptide bonds. Fc fusion proteins are preferablyABC-052PCT / 59495-PCT3 covalently linked by (i) connecting the genes that encode for each part into a single nucleic acid molecule and (ii) expressing in a host cell (e.g., HEK cell or CHO cell) the protein for which the nucleic acid molecule encodes. The fully recombinant synthesis approach is preferred over methods in which the therapeutic protein and Fc fragments are synthesized separately and then chemically conjugated. The chemical conjugation step and subsequent purification process increase the manufacturing complexity, reduce product yield, and increase cost.

[0006] The terms “Fc fragment,” “Fc region,” “Fc domain,” or “Fc polypeptide,” are used herein to generally mean a C-terminal region of an immunoglobulin heavy chain. The Fc fragment, region, domain, or polypeptide may be a native sequence Fc region or a variant / mutant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, they generally comprise some or all of the hinge region of the heavy chain, the CH2 region of the heavy chain, and the CH3 region of the heavy chain. The hinge region of a Fc fragment comprises amino acid sequences that connect the CHI domain of the heavy chain to the CH2 region of the heavy chain and contains one or more cysteines that form one or more interheavy chain disulfide bridges to form a homodimer of an Fc fusion protein from two identical but separate monomers of the Fc fusion protein. The hinge region may comprise all or part of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence.

[0007] The presence of the Fc domain increases the plasma half-life due to its interaction with the neonatal Fc-receptor (FcRn) in addition to slower renal clearance of the Fc fusion protein due to the large molecule size, resulting in in vivo recycling of the molecule achieving prolonged activity of the linked peptide and improved solubility and stability of the Fc fusion protein molecule. The Fc domain also enables Fc fusion proteins to interact with Fc receptors on immune cells. In some examples, the therapeutic protein or peptide is linked to the immunoglobin Fc domain via a linker. The therapeutic protein or peptide and linker replace the variable region of an antibody while keeping the Fc region intact.

[0008] An Fc receptor (FcR) generally means a receptor that binds to an Fc fragment or to the Fc region of an antibody. In examples, the FcR is a native sequence of a mammalian FcR, and the FcR is one which binds an Fc fragment or the Fc region of an IgG antibody (a gamma receptor) and includes without limitation, receptors of the Fc(gamma) receptor I, Fc(gamma) receptor la, Fc(gamma) receptor lib, and Fc(gamma) receptor III subclasses (and their speciesspecific equivalents, e.g., canine-specific equivalents), including allelic variants and alternatively spliced forms of these receptors. “FcR” also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG molecules to the fetus and is also responsible for the prolonged in vivo elimination half-lives of antibodies and Fc-fusion proteins in vivo. In examples, FcR of human origin are used in vitro (e.g., in an assay) to measure theABC-052PCT / 59495-PCT3 binding of Fc fusion proteins comprising Fc fragments of any mammalian origin so as to assess their FcR binding properties. Those skilled in the art will understand that mammalian FcR from one species (e.g., FcR of human origin) are sometimes capable of in vitro binding of Fc fragments from a second species (e.g., FcR of canine origin).Nerve Growth Factor (NGF)

[0009] Osteoarthritis, a pain-associated ailment, is the most common cause of chronic pain in humans and companion animals. Presently, one in four of the 77.2 million pet dogs in the United States are diagnosed with some form of arthritis (Clarke S (2015) Canine Osteoarthritis and treatments: A Review, Veterinary Science Development 5 10.4081 / vsd.2015.5931). Osteoarthritis is a chronic, non-curable, degenerative disease affecting moving joints, leading to motor disability. Medical management is complex and multimodal and focuses on functional and pharmacological treatments to slow its progression and alleviate pain. The presence of pain is often assumed because of motor claudication and improved function following treatment with anti-inflammatory drugs. In the absence of a cure, the primary therapeutic goal is to alleviate pain through pharmacological or immunological methods. Nonsteroidal anti-inflammatory drugs (NSAIDs) are only partially effective and do not provide complete pain relief in dogs with osteoarthritis. Moreover, ongoing treatments often have adverse effects, including serious gastro-intestinal and kidney toxicity. When NSAIDs are ineffective or poorly tolerated, adjunctive drugs such as corticosteroids or opioid analgesics may be indicated to relieve osteoarthritis-associated signs of moderate or severe pain. Unfortunately, the failure of conventional medications results in diminished quality of life, chronic pain, and suffering, which often leads to euthanasia.

[0010] Nerve growth factor (NGF) is a neuropeptide which was originally identified as a critical factor for the development and maintenance of sensory and sympathetic neurons in the developing nervous system, and later found to have a role in inflammatory hyperalgesia. NGF is the founding member of the neurotrophins, a family of secreted growth factors responsible for the growth, survival, and developmental plasticity of neuronal populations in the vertebrate peripheral and central nervous system. NGF has also been shown to play a key role in the generation of acute and chronic pain and in hyperalgesia in diverse pain states. NGF is expressed at high levels in damaged or inflamed tissues and facilitates pain transmission by nociceptive neurons through a variety of mechanisms.ABC-052PCT / 59495-PCT3SUMMARY OF THE PRESENT TECHNOLOGY

[0011] Described herein are fusion proteins, each comprising a respective Nerve Growth Factor (NGF) protein fragment and an Fc fragment, wherein the NGF fragment and the Fc fragment are connected by a peptide linker. In one or more embodiments, the NGF fragment comprises a NGF analog comprising a functional fragment, analog, or variant / mutant thereof. In one or more embodiments, the NGF analog comprises a NGF fragment of SEQ ID NO: 17, or a functional fragment, analog, or variant / mutant thereof.EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNIT VDPRLFK S SRLRSPRVLF STQPPREAADTQDLDFE VGGAAPFNRTHRS SRS S SH PIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETK CRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACV CVLSRKAVRRA (SEQ ID NO: 17).

[0012] In one or more embodiments, the NGF analog comprises a NGF fragment of SEQ ID NO: 19, or a functional fragment, analog, or variant / mutant thereof.EPHSESNVPAGHTIPQAHWTKLQHSLDTALRD ARSAP AAAIAARVAGQTRNIT VDPRLFK S SRLRSPRVLF STQPPREAADTQDLDFE VGGAAPFNRTHRS SRS S SH PIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETK CRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACV CVLSRKAVRDA (SEQ ID NO: 19).

[0013] In one or more embodiments, the Fc fragment comprises a sequence or functional fragment of SEQ ID NO: 2.DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFV DGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPS PIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNG QQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNH YTQESLSHSPG (SEQ ID NO: 2).

[0014] In one or more embodiments, the linker comprises the sequence GGGSGGGS (SEQID NO: 9).

[0015] In one or more embodiments, the linker comprises the sequence GGGGGSGGGSGGGGSGGS (SEQ ID NO: 21).

[0016] In one or more embodiments, the fusion protein comprises, consists essentially or even consists of a sequence of SEQ ID NO: 15.EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITV DPRLFKS SRLRSPRVLF STQPPREAADTQDLDFEVGGAAPFNRTHRS SRSS SHPIF HRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDABC-052PCT / 59495-PCT3PNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLS RKAVRRAGGGSGGGSDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVV DLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGK QFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFF PPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFIC AVMHEALHNHYTQESLSHSPG (SEQ ID NO: 15).

[0017] In one or more embodiments, the fusion protein comprises, consists essentially or even consists of a sequence of SEQ ID NO: 18.EPHSESNVPAGHTIPQAHWTKLQHSLDTALRD ARSAP AAAIAARVAGQTRNITV DPRLFKS SRLRSPRVLF STQPPREAADTQDLDFEVGGAAPFNRTHRS SRSS SHPIF HRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRD PNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLS RKAVRDAGGGSGGGSDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVV DLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGK QFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFF PPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFIC AVMHEALHNHYTQESLSHSPG (SEQ ID NO: 18).

[0018] In one or more embodiments, the fusion protein comprises, consists essentially or even consists of a sequence of SEQ ID NO: 20.EPHSESNVPAGHTIPQAHWTKLQHSLDTALRD ARSAP AAAIAARVAGQTRNITV DPRLFKS SRLRSPRVLF STQPPREAADTQDLDFEVGGAAPFNRTHRS SRSS SHPIF HRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRD PNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLS RKAVRDAGGGGGSGGGSGGGGSGGSDCPKCPAPEMLGGPSVFIFPPKPKDTLLI ARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVL PIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKN TVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDK SRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 20).

[0019] In one or more embodiments, the Fc fragment is glycosylated.

[0020] Also described herein are immunogenic compositions which comprise or consist essentially of a fusion protein(s) according to any embodiments or combinations of embodiments described herein and a pharmaceutically acceptable carrier. In one or more embodiments, the fusion protein is dispersed in the carrier. In one or more embodiments, the compositions further comprise an adjuvant. In one or more embodiments, the adjuvant is Montanide™ ISA-720. In one or more embodiments, the fusion protein is emulsified with theABC-052PCT / 59495-PCT3 adjuvant. In one or more embodiments, the emulsification is prepared onsite before administration. In one or more embodiments, the prepared emulsification is refrigerated (4°C) or room temperature stable for at least 8 hours, preferably up to 24 hours, preferably up to 48 hours or more. In one or more embodiments, the composition is an injectable formulation. In one or more embodiments, the composition is adapted for subcutaneous administration. In one or more embodiments, the composition is adapted for intramuscular administration. In one or more embodiments, the composition is adapted for therapeutic vaccination.

[0021] Also described herein are various methods for increasing antibody production in a patient against an antigenic agent, or alternatively or additionally methods of inducing an immune response in a patient against an NGF protein. The methods generally comprise administering a therapeutically effective amount of a fusion protein(s) or immunogenic composition(s) according to any embodiments or combinations of embodiments described herein to a patient. In one or more embodiments, the fusion protein or immunogenic composition is administered via injection. In one or more embodiments, the fusion protein or immunogenic composition is administered subcutaneously or intramuscularly. In one or more embodiments, the fusion protein or immunogenic composition is provided as a unit dosage form. In one or more embodiments, the fusion protein or immunogenic composition is co-administered with an adjuvant. In one or more embodiments, the methods further comprise preparing the fusion protein or immunogenic composition for administration, wherein the preparation comprises premixing the fusion protein or immunogenic composition with an adjuvant before administration. In one or more embodiments, pre-mixing comprises emulsifying the adjuvant and fusion protein to yield an emulsion and administering the emulsion to the patient.

[0022] Also described herein are methods of producing a fusion protein according to any embodiments or combinations of embodiments described herein. The methods generally comprising transiently transfecting a nucleic acid encoding for the fusion protein into a Chinese Hamster Ovary (CHO) cell, wherein the transfected CHO cell expresses the fusion protein. In one or more embodiments, the fusion protein is secreted by the cells into cell culture media, further comprising purifying or isolating the fusion protein from the media. Advantageously, the yield of the purified or isolated fusion protein is greater than 75 mg / L.

[0023] Also described herein are cells engineered to express a fusion protein according to any embodiments or combinations of embodiments described herein. In one or more embodiments, the cell is a CHO cell.

[0024] As described herein, the fusion protein(s) or immunogenic composition(s) according to any embodiments or combinations of embodiments described herein can be used in therapy and / or as a medicament.ABC-052PCT / 59495-PCT3

[0025] As described herein, the fusion protein(s) or immunogenic composition(s) according to any embodiments or combinations of embodiments described herein can be used for increasing antibody production in a patient.

[0026] As described herein, the fusion protein(s) or immunogenic composition(s) according to any embodiments or combinations of embodiments described herein can be used in treatment of pain associated with pain-associated ailments such as osteoarthritis.

[0027] As described herein, the fusion protein(s) or immunogenic composition(s) according to any embodiments or combinations of embodiments described herein can be used as a prophylactic, therapeutic and / or booster vaccine.

[0028] Particular embodiments concern the fusion protein consisting of SEQ ID NO: 15, or pharmaceutical compositions thereof for use in treatment to reduce the pain associated with inflammation and diseases such as osteoarthritis.

[0029] Particular embodiments concern the fusion protein consisting of SEQ ID NO: 18, or pharmaceutical compositions thereof for use in treatment to reduce the pain associated with inflammation and diseases such as osteoarthritis.

[0030] Particular embodiments concern the fusion protein consisting of SEQ ID NO: 20, or pharmaceutical compositions thereof for use in treatment to reduce the pain associated with inflammation and diseases such as osteoarthritis.

[0031] As described herein, the fusion protein(s) or immunogenic composition(s) according to any embodiments or combinations of embodiments described herein can be used in the manufacture of a medicament for the treatment of pain associated with inflammation and pain- associated ailments.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 shows a schematic representation of an NGF analog-Fc fusion protein homodimer. The glycosylation site shown is the conserved natural glycosylation site on the Fc fragment (glycosylation that may occur on the NGF analog polypeptide is not shown).

[0033] FIG. 2 illustrates a side-by-side sequence comparison of the human NGF-P of SEQ ID NO: 3 and the canine NGF-P of SEQ ID NO: 4.

[0034] FIG. 3 illustrates a side-by-side sequence comparison of the human IgGl immunoglobin Fc fragment of SEQ ID NO: 1 and the canine IgGB immunoglobin Fc fragment of SEQ ID NO: 2.

[0035] FIG. 4 illustrates a side-by-side sequence comparison of the NGF analog of SEQ ID NO: 6 and the shortened NGF analog of SEQ ID NO: 7.

[0036] FIG. 5 illustrates a side-by-side sequence comparison of the NGF analog of SEQ IDABC-052PCT / 59495-PCT3NO: 8 and the NGF analog of SEQ ID NO: 17.

[0037] FIG. 6 illustrates a side-by-side sequence comparison of the NGF analog of SEQ ID NO: 8 and the NGF analog of SEQ ID NO: 19.

[0038] FIG. 7A and FIG. 7B illustrate a side-by-side sequence comparison of the NGF analogs of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 17 and SEQ ID NO: 19.

[0039] FIG. 8A, FIG. 8B, FIG. 8C and FIG. 8D illustrate a side-by-side sequence comparison of the NGF analog-Fc fusion proteins of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18 and SEQ ID NO: 20.

[0040] FIG. 9 is a schematic diagram depicting example modes in which an antigen or an antigen-Fc fusion protein may interact with an antigen presenting cell, e.g., a dendritic cell.DETAILED DESCRIPTION

[0041] In the adult system, NGF has an important role in pro-nociception via the NGF- specific tyrosine kinase receptor (TrkA). In osteoarthritis, NGF is upregulated and secreted by inflammatory cells, fibroblasts, and synoviocytes. Activation of high-affinity NGF-specific TrkA receptors located in sensory fibers results in increased excitability and post-translational changes in the transient receptor potential vanilloid receptor 1 (TRPV1) cation channel. NGF released from inflamed tissues also activates infiltrating mast cells, which in turn further sensitize sensory neurons through secretion of various inflammatory factors. In patients with osteoarthritis whose tissues are inflamed, NGF levels tend to be higher than normal.

[0042] Blockade of NGF signaling by neutralization of extracellular NGF using specific antibodies has proven to be an effective antinociceptive approach. Recently, attention has been focused on monoclonal antibodies (mAb) that neutralize NGF activity to reduce hyperalgesia and behavioral indicators of pain in various animal models of inflammatory arthritis and patients with osteoarthritis. Administration of anti-NGF monoclonal antibodies (mAb) in passive immunization has been shown to be effective in reducing pain in animal models of arthritis pain. A canine-specific mAb, Librela, was approved by the US FDA in May 2023 for the control of pain associated with osteoarthritis in dogs. Evidence demonstrated that Librela is effective at controlling pain associated with osteoarthritis in dogs when at least two doses are given 28 days apart.

[0043] In human clinical studies, one anti-NGF monoclonal antibody, tanezumab, has been tested by the Food and Drug Administration as a treatment for moderate-to-severe osteoarthritis. In a phase III study of osteoarthritis patients receiving injections of tanezumab (Brown et alABC-052PCT / 59495-PCT32013 Arthritis and Rheumatism 65 (7) 1795-1803 it was found that tanezumab was superior to placebo for painful hip osteoarthritis. Additionally, a phase III study of tanezumab for treatment of lower back pain (Markman et al Pain. 2020 Sep l;161(9):2068-2078. doi: 10.10977j.pain.0000000000001928. PMID: 32453139; PMCID: PMC7431140.) demonstrated a statistically significant improvement in chronic low back pain. However, tanezumab does not have FDA or EMA approval due to adverse side effects.

[0044] There are drawbacks with monoclonal antibody therapy treatments. As shown with tanezumab, these treatments can have side effects affecting a significant proportion of the treated patients (which resulted in the withholding of FDA approval). In addition, a monoclonal antibody treatment will involve regular administrations (for example, Librela is a once a month treatment) and large doses, since antibodies are given exogenously and not produced by the patient’s immune system endogenously. Treatment must be given by injection which can be painful for the patient.

[0045] The required dosing regimen for a mAb NGF treatment incurs considerable monetary and manufacturing costs for producing the drug which get passed on to patients or pet owners, making the treatment unaffordable in many cases. Exogenously administered monoclonal antibodies may also become less efficacious over time if the body becomes immune to them.

[0046] Production of a therapeutic vaccine in the form of an NGF analog-Fc fusion protein for use as a treatment for NGF derived pain will not have these drawbacks. A vaccine approach to neutralize NGF in the body to reduce pain associated with osteoarthritis will require minimal intervention as the objective is to stimulate the body to manufacture its own endogenously- produced antibodies to neutralize the excess NGF. Each treatment will therefore be long-acting and only require a small dose to stimulate an immune reaction. Therefore, there is a considerable cost advantage as the scale of manufacturing of a low-dose therapeutic vaccine will be greatly reduced compared to a monoclonal antibody, thereby making the treatment accessible to more patients. For the therapeutic vaccine approach, an initial priming dose can be followed up with additional doses or booster injections spaced 6 months, 1 year, or even further apart to stimulate further immune reaction and antibody production. In addition, a vaccine that stimulates the body to produce endogenous antibodies is likely to create polyclonal antibodies, with the potential of a more robust response to neutralizing NFG in the body.

[0047] In diseases such as osteoarthritis, inflammation leads to increased levels of nerve growth factor (NGF) that stimulate further inflammation and increased sensitivity to pain. The up-regulation of NGF in inflamed tissues can regulate innervation and neuronal activity or peripheral neurons, inducing the release of immune-active neuropeptides and neurotransmitters.ABC-052PCT / 59495-PCT3It can also directly influence innate and adaptive immune responses. Expression of the TrkA and p75NTRNGF receptors is dynamically regulated in immune cells. While activating immune responses, NGF also activates pathways necessary to dampen the inflammatory response and limit tissue damage. Decreases in TrkA expression might prevent the activation by NGF of regulatory feedback mechanisms, contributing to the development and maintenance of chronic inflammation.

[0048] The present disclosure is directed to methods for making and using novel NGF based Fc fusion proteins (NGF analog-Fc fusion proteins) which allow for the cost-effective production of vaccines to cause a patient to produce antibodies against soluble NGF (e.g., a patient’s endogenously produced NGF) with the effect of reducing pain associated with inflammation and osteoarthritis. The present disclosure is specifically directed to methods for making and using NGF analog-Fc fusion proteins for use as a therapeutic vaccine which is efficacious for causing patients to create anti-NGF antibodies to the NGF protein, for example to decrease the levels of NGF or prevent NGF from binding to its receptor in order to reduce symptoms associated with osteoarthritis, including pain. The present disclosure is directed to the specification of amino acid sequences comprising an NGF analog or NGF analog fragment linked via a linker to an Fc fragment.

[0049] In an example, a pharmaceutical composition of a novel NGF analog-Fc fusion protein therapeutic vaccine is administered to patients requiring treatment for ameliorating pain associated with a disease. In examples, the novel NGF analog-Fc fusion protein has the effect of stimulating the patient to produce humoral immunity, for example generating increased levels of IgG and IgM antibodies compared to an untreated patient, where the antibodies bind NGF in the body. When the immune system develops a robust antibody response to NGF, excess levels of NGF are removed and may also be blocked from binding to cell-based NGF receptors, thereby alleviating pain and in some examples, reducing inflammation.

[0050] As previously noted, there are drawbacks with existing monoclonal antibody therapy treatments for chronic pain, including side effects and the necessity of frequent administrations by injection, which is inconvenient for owners and distressing for pets, leading to non- compliance with treatment regimes. The dose volume can be large and may vary depending on the body weight of the pet, requiring different Stock Keeping Units (SKUs) to treat a population of patients that have widely varying body weights, thereby further increasing manufacturing costs and costs for owners. A therapeutic vaccine-based approach to neutralize NGF in the body to reduce pain associated with osteoarthritis greatly simplifies the cost and burden on pet owners, as the treatment stimulates the body to manufacture its own antibodies against the administered NGF analog-Fc fusion protein, which can then bind and neutralize theABC-052PCT / 59495-PCT3 endogenously produced NGF. Because antibodies are produced endogenously, significantly fewer treatments are required. The body also may create polyclonal antibodies in response to the vaccine, with the potential of providing a more robust response to endogenously-produced NGF in the body.Equivalents and Definitions

[0051] As used herein, the articles “a” and “an” generally mean one or more than one, e.g., to at least one of the grammatical object of the article. The use of the words “a” or “an” when used in conjunction with the term “comprising” herein may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” As used herein, the phrase “and / or,” when used in a list of two or more items, generally means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and / or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0052] As used herein, “about” and “approximately” generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements.

[0053] As used herein, an amount of a molecule, compound, conjugate, or substance effective to treat a disorder (e.g., a disorder described herein), “therapeutically effective amount,” or “effective amount” generally means an amount of the molecule, compound, conjugate, or substance which is effective, upon single or multiple dose administration(s) to a patient, in treating a patient, or in curing, alleviating (e.g., alleviating associated symptoms such as pain), relieving or improving a patient with a condition or disorder (e.g., a disorder described herein) beyond that expected in the absence of such treatment.

[0054] As used herein, the term “analog” generally means a compound or conjugate (e.g., a compound or conjugate as described herein) having a chemical structure similar to that of another compound or conjugate but differing from it in at least one aspect.

[0055] As used herein, the term “antigen” or “antigenic agent” generally means any substance that causes a patient’s immune system to produce antibodies against it. An antigen may be a substance from the environment, such as chemicals, bacteria, viruses, or pollen, or an antigen may also be inside the body. In some cases, the antigen is endogenously-produced (e.g., a self-antigen). An example of a self-antigen is a NGF protein. An antigen (e.g., NGF) or an antigen analog (e.g., NGF analog, NGF antigen analog or NGF-analog) may also be covalently linked to another protein (e.g., an Fc fragment).ABC-052PCT / 59495-PCT3

[0056] As used herein, the term “antibody” or “antibody molecule” generally means an immunoglobulin molecule (Ig), or immunologically active portions of an immunoglobulin (Ig) molecule, i.e., a molecule that contains an antigen binding site that specifically binds, e.g., immunoreacts with, an antigen or a self-antigen. As used herein, the term “antibody domain” generally means a variable or constant region of an immunoglobulin. It is documented in the art that antibodies comprise several classes, for example IgA, IgM, or IgG in the case of mammals (e.g., dogs). Classes of mammalian IgG immunoglobulins can be further classified into different isotypes, such as IgGA, IgGB, IgGC and IgGD for dogs. Those skilled in the art will recognize that immunoglobulin isotypes of a given immunoglobulin class will comprise different amino acid sequences, structures, and functional properties from one another (e.g., different binding affinities to Fc(gamma) receptors or TrkA receptors). “Specifically binds” or “immunoreacts with” generally means that the antibody reacts with one or more antigenic determinants of the desired antigen and has a lower affinity for other polypeptides, e.g., does not react with other polypeptides.

[0057] As used herein, the term “NGF analog” generally means a protein comprising a peptide derived from or consisting of all or a portion of an NGF protein, which may have none, one or more than one amino acid deletions, mutations, or additions. For example, an NGF analog may be a native (wild-type) NGF sequence with no changes or mutations, a native NGF sequence comprising an additional peptide sequence combined with a native NGF protein, or an NGF protein with none, one or more than one amino acid deletions, mutations, or additions from a native NGF protein. In examples, an NGF analog may be a native ProNGF protein, which may have none, one or more than one amino acid deletions, mutations, or additions. In examples, an NGF analog may comprise a portion (e.g., a fragment) or truncated section of a native NGF or native ProNGF protein, which may have none, one or more than one amino acid deletions, mutations, or additions. In examples, an NGF analog may comprise a portion of an artificial sequence combined with all or a portion of a native NGF protein or native ProNGF protein, which may have none, one, or more than one amino acid deletions, mutations, or additions. As used herein the term NGF analog may comprise all or a portion of a ProNGF precursor protein, which may have none, one or more than one amino acid deletions, mutations, or additions. As used herein, “NGF analog” may refer to a polypeptide that contains a portion of an NGF protein sequence as given in SEQ ID NO: 8. In examples, an NGF analog may comprise a human NGF- P as given in SEQ ID NO: 3. In examples the NGF analog may comprise a canine NGF-P as given in SEQ ID NO: 4. In examples, the NGF analog may be linked to an Fc fragment or analog thereof, as illustrated in FIG. 1. In examples, the NGF analog may comprise an additional polypeptide sequence on the N-terminus of the NGF analog. In examples, an NGF analog mayABC-052PCT / 59495-PCT3 or may not have an additional polypeptide sequence on the N-terminus of the NGF analog and may or may not have one or more amino acid mutations as compared to a non-mutated NGF (e.g., an NGF sequence that is homologous to human NGF or canine NGF). An NGF analog can also be an NGF antigen analog.

[0058] As used herein, the term “dimer” generally means a protein or a fusion protein comprising two polypeptides linked covalently. In embodiments, two identical polypeptides are linked covalently (e.g., via disulfide bonds) forming a “homodimer” (diagrammatically represented in FIG. 1, which is an illustration of an NGF analog-Fc fusion protein homodimer). Referring to FIG. 1 in more detail, the NGF analog-Fc fusion protein polypeptide is connected via a linker to an Fc fragment. Disulfide bonds (the total number of disulfide bonds in actuality may be greater or less than the number shown in FIG. 1) create a homodimer from two identical Fc fusion proteins. The novel NGF analog-Fc fusion protein homodimer may be encoded by a single nucleic acid molecule, wherein the homodimer is made recombinantly inside a cell by first forming novel NGF analog-Fc fusion protein monomers and by then assembling two identical novel NGF analog-Fc fusion protein monomers into the homodimer upon further processing inside the cell.

[0059] As used herein, the terms “multimer,” “multimeric,” or “multimeric state” generally means non-covalent, associated forms of Fc fusion protein dimers that may be in equilibrium with Fc fusion protein dimers or may act as permanently aggregated versions of Fc fusion protein dimers (e.g., dimers of Fc fusion protein homodimers, trimers of Fc fusion protein homodimers, tetramers of Fc fusion protein homodimers, or higher order aggregates containing five or more Fc fusion protein homodimers). It may be expected that multimeric forms of Fc fusion proteins may have different physical, stability, or pharmacologic activities from that of fusion protein homodimers.

[0060] As used herein, an “NGF analog-Fc fusion protein” and a “novel NGF analog-Fc fusion protein” (which terms may be interchangeably used) generally mean an immunoglobin Fc domain that is linked to a NGF analog, which is useful in generating antibodies that specifically bind the NGF protein. For ease of reference, the term NGF, unless otherwise dictated by the context, encompasses protein residues consisting of fragments of the native NGF protein. As used herein, the general terms “fusion protein” and “Fc fusion protein” generally mean a protein comprising more than one part, for example from different sources (e.g., different proteins, polypeptides, cells, etc.), that are covalently linked through peptide bonds.

[0061] T lymphocytes expressing CD4+ are also known as helper T cells. This subset of T cells can be further subdivided into Thl cells and Th2 cells. Thl cells stimulate cellular immune response, participate in the inhibition of macrophage activation and stimulate B cells to produceABC-052PCT / 59495-PCT3IgM, IgGl . Th2 stimulates humoral immune response, promotes B cell proliferation and induces antibody production (IL-4).

[0062] As used herein, the term “activity,” “biological activity,” “potency,” “bioactive potency,” or “biological potency” generally means the extent to which an Fc fusion protein binds to or activates a cell receptor and / or exerts the production or reduction of native or foreign substances. As used herein, “in vitro activity” or “receptor activity” generally means the affinity with which an Fc fusion protein binds to a cell receptor and is typically measured by the concentration of an Fc fusion protein that causes the Fc fusion protein to reach half of its maximum binding (i.e., EC50 value).

[0063] As used herein, the term “biosynthesis,” “recombinant synthesis,” or “recombinantly made” generally means the process by which an Fc fusion protein is expressed within a host cell by transfecting the cell with a nucleic acid molecule (e.g., vector) encoding the Fc fusion protein (e.g., where the entire Fc fusion protein is encoded by a single nucleic acid molecule). Exemplary host cells include mammalian cells, e.g., CHO cells or HEK293 cells. The cells can be cultured using standard methods in the art and the expressed Fc fusion protein may be harvested and purified from the cell culture using standard methods in the art.

[0064] As used herein, the term “cell surface receptor” generally means a molecule such as a protein, generally found on the external surface of the membrane of a cell and which interacts with soluble molecules, e.g., molecules that circulate in the blood supply. In some embodiments, a cell surface receptor may include a host cell receptor (e.g., an TrkA receptor or p75(RTN) receptor) or an Fc receptor which binds to an Fc fragment or the Fc region of an antibody (e.g., an Fc(gamma) receptor, for example Fc(gamma) receptor I (or the equivalent canine Fc(gamma) receptor), or an Fc neonatal receptor, for example FcRn). As used herein, “in vitro activity” or “Fc(gamma) receptor activity” or “Fc(gamma) receptor binding” or “FcRn receptor activity” or “FcRn binding” generally means the affinity with which an Fc fusion protein binds to the Fc receptor (e.g. Fc(gamma) receptor or FcRn receptor) and is typically measured by the concentration of an Fc fusion protein that causes the Fc fusion protein to reach half of its maximum binding (i.e., EC50 value) as measured on an assay (e.g., an enzyme-linked immunosorbent assay (ELISA) assay) using OD 450 nm values as measured on a microplate reader.

[0065] As used herein, the term “immunogenic” or “immunogenicity” generally means the capacity for a given molecule or antigen (e.g., an Fc fusion protein of the present invention) to provoke the immune system of a target patient such that after administration of the molecule, the patient develops antibodies capable of binding all or specific portions of the molecule (i.e., anti-drug antibodies or ADA). In patients, (e.g., a canine) the antibody development may beABC-052PCT / 59495-PCT3 polyclonal (e.g., a mixture of antibodies capable of binding an Fc fusion protein). As used herein, the terms “neutralizing,” “neutralizing antibodies”, or “neutralizing anti-drug antibodies” generally mean the capacity for antibodies developed against an Fc fusion protein (e.g., an antigen or antigen analog-Fc fusion protein) to cross-react, bind and interfere with all or a portion of the self-antigen’s biological activity in the target patient. For example, in the case of a novel NGF analog-Fc fusion protein molecule (or a pharmaceutical composition thereof) administered to dogs, the immunogenicity generally means antibodies that bind to the NGF portion of the NGF analog-Fc fusion protein but then also cross-react, bind and interfere (e.g., neutralize) with the activity of endogenously produced NGF at a cell surface receptor (e.g., an NGF receptor). Likewise, antibodies generated by the administration of a novel NGF analog-Fc fusion protein molecule (or a pharmaceutical composition thereof) are neutralizing when those anti -NGF antibodies inhibit the binding between an endogenously produced protein (e.g., native NGF protein) in a patient and a patient’s host cells, which is directly related to the bioactivity of endogenously produced NGF in the patient.

[0066] As used herein, the term “immunogenic composition” generally means a pharmaceutical composition or mixture of substances comprising an immunogenic molecule, antigen or agent, that is suitable for administering to a patient. For example, an immunogenic composition may comprise an NGF analog-Fc fusion protein and a sterile aqueous solution or adjuvant or other carrier.

[0067] As used herein, the term “monomer” generally means a protein or a fusion protein comprising a single polypeptide. In embodiments, the “monomer” is a protein or a fusion protein, e.g., a single polypeptide, comprising a NGF analog polypeptide and an Fc fragment polypeptide, wherein the NGF fragment and Fc fragment polypeptides are joined by peptide bonds via a linker to form the single polypeptide. In embodiments, the monomer is encoded by a single nucleic acid molecule.

[0068] As used herein and as illustrated in FIG. 1, “N-terminus” generally means the start of a protein or polypeptide that is initiated by an amino acid containing a free amine group that is the alpha-amino group of the amino acid (e.g., the free amino that is covalently linked to one carbon atom that is located adjacent to a second carbon atom, wherein the second carbon atom is part of the carbonyl group of the amino acid). As used herein and as illustrated in FIG. 1, “C- terminus” generally means the end of a protein or polypeptide that is terminated by an amino acid containing a carboxylic acid group, wherein the carbon atom of the carboxylic acid group is located adjacent to the alpha-amino group of the amino acid.

[0069] As used herein, the term “carrier” is used herein to generally mean diluents, excipients, vehicles, and the like, in which the Fc fusion protein(s) may be dispersed, emulsified,ABC-052PCT / 59495-PCT3 or encapsulated for administration. Suitable carriers will be pharmaceutically acceptable. As used herein, the term “pharmaceutically acceptable” generally means not biologically or otherwise undesirable, in that it can be administered to a patient without excessive toxicity, irritation, or allergic response, and does not cause unacceptable biological effects or interact in a deleterious manner with any of the other components of the composition in which it is contained. A pharmaceutically acceptable carrier would naturally be selected to minimize any degradation of the compound or other agents and to minimize any adverse side effects in the patient, as would be well known to one of skill in the art. Pharmaceutically acceptable ingredients include those acceptable for veterinary use as well as human pharmaceutical use and will depend on the route of administration. Any carrier compatible with the excipient(s) and the Fc fusion protein(s) can be used. In example, and adjuvant may be considered one type or subclass of carrier.

[0070] As used herein, “pharmacodynamics” or “PD” generally means the biological effects of an Fc fusion protein in a patient. As an example, herein, the PD of a novel NGF analog-Fc fusion protein generally means the measure of the anti -NGF antibody titers over time in a patient after the administration of the novel NGF analog-Fc fusion protein.

[0071] As used herein, “pharmacokinetics” or “PK” generally means the characteristic interactions of an Fc fusion protein and the body of the patient in terms of its absorption, distribution, metabolism, and excretion. As an example, herein, the PK generally means the concentration of a novel NGF analog-Fc fusion protein in the blood or serum of a patient at a given time after the administration of the novel NGF analog-Fc fusion protein. As used herein, “half-life” generally means the time taken for the concentration of Fc fusion protein in the blood or serum of a patient to reach half of its original value as calculated from a first order exponential decay model for drug elimination. Fc fusion proteins with greater “half-life” values demonstrate greater duration of action in the target patient.

[0072] The terms “sequence identity,” “sequence homology,” “homology,” or “identical” in amino acid or nucleotide sequences as used herein generally describes that the same nucleotides or amino acid residues are found within the variant and reference sequences when a specified, contiguous segment of the nucleotide sequence or amino acid sequence of the variant is aligned and compared to the nucleotide sequence or amino acid sequence of the reference sequence. Methods for sequence alignment and for determining identity between sequences are known in the art, including the use of Clustal Omega, which organizes, aligns, and compares sequences for similarity, wherein the software highlights each sequence position and compares across all sequences at that position and assigns one of the following scores: an (asterisk) for sequence positions which have a single, fully conserved residue, a (colon) indicates conservationABC-052PCT / 59495-PCT3 between groups of strongly similar properties with scoring greater than 0.5 in the Gonnet PAM 250 matrix, and a (period) indicates conservation between groups of weakly similar properties with scoring less than or equal to 0.5 in the Gonnet PAM 250 matrix, a (dash) indicates a sequence gap, meaning that no local homology exists within a particular set of comparisons within a certain range of the sequences, and an empty space “ ” indicates little or no sequence homology for that particular position across the compared sequences.

[0073] With respect to optimal alignment of two nucleotide sequences, the contiguous segment of the variant nucleotide sequence may have additional nucleotides or deleted nucleotides with respect to the reference nucleotide sequence. Likewise, for purposes of optimal alignment of two amino acid sequences, the contiguous segment of the variant amino acid sequence may have additional amino acid residues or deleted amino acid residues with respect to the reference amino acid sequence. In some embodiments, the contiguous segment used for comparison to the reference nucleotide sequence or reference amino acid sequence will comprise at least 6, 10, 15, or 20 contiguous nucleotides, or amino acid residues, and may be 30, 40, 50, 100, or more nucleotides or amino acid residues. Corrections for increased sequence identity associated with inclusion of gaps in the variant’s nucleotide sequence or amino acid sequence can be made by assigning gap penalties. Methods of sequence alignment are known in the art.

[0074] In embodiments, the determination of percent identity or “homology” between two sequences is accomplished using a mathematical algorithm. For example, the percent identity of an amino acid sequence is determined using the Smith-Waterman homology search algorithm using an affine 6 gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix 62. In embodiments, the percent identity of a nucleotide sequence is determined using the Smith -Waterman homology search algorithm using a gap open penalty of 25 and a gap extension penalty of 5. Such a determination of sequence identity can be performed using, for example, the DeCypher Hardware Accelerator from TimeLogic.

[0075] As used herein, the term “homology” is generally used to compare two or more proteins by locating common structural characteristics and common spatial distribution of, for instance, beta strands, helices, and folds. Accordingly, homologous protein structures are defined by spatial analyses. Measuring structural homology involves computing the geometric- topological features of a space. One approach used to generate and analyze three-dimensional (3D) protein structures is homology modeling (also called comparative modeling or knowledgebased modeling) which works by finding similar sequences on the basis of the fact that 3D similarity reflects 2D similarity. Homologous structures do not imply sequence similarity as a necessary condition.ABC-052PCT / 59495-PCT3

[0076] As used herein, the terms “subject” and “patient” are generally intended to include mice, and canines. Exemplary canine subjects or patients include dogs having a disease or a disorder, e.g., osteoarthritis or another disease or disorder described herein, or normal subjects.

[0077] As used herein, the term “titer” or “yield” generally means the amount of a fusion protein product (e.g., an Fc fusion protein described herein) resulting from the biosynthesis (e.g., in a mammalian cell, e.g., in a HEK293 cell or CHO cell) per volume of the cell culture. The amount of product may be determined at any step of the production process (e.g., before or after purification), but the yield or titer is always stated per volume of the original cell culture. As used herein, the term “product yield” or “total protein yield” generally means the total amount of Fc fusion protein expressed by cells and purified via at least one affinity chromatography step (e.g., Protein A or Protein G) and includes monomers of Fc fusion protein, homodimers of Fc fusion protein, and higher-order molecular aggregates of homodimers of Fc fusion protein. As used herein, the term “percent homodimer” or “%homodimer” generally means the proportion of a fusion protein product (e.g., an Fc fusion protein described herein) that is the desired homodimer. As used herein, the term “homodimer titer” generally means the product of the %homodimer and the total protein yield after Protein A purification step reported per volume of the cell culture.

[0078] As used herein, the terms “treat” or “treating” or “treatment” of a patient having a disease or a disorder generally means an intervention performed with the intention of mitigating or preventing the symptoms (in particular, ameliorating or preventing pain) associated with the condition or disease, and / or reducing the duration of the symptoms associated with the condition or disease. Accordingly, “treatment” generally means both therapeutic treatment and prophylactic or preventative measures. Improvement after treatment may be manifested as a decrease or elimination of such symptoms, e.g., by a decrease or elimination of pain, and / or by a decrease in the duration of such symptoms. As an example, the compositions described herein are useful in treating pain due to osteoarthritis. Treating a patient having a condition or disease may refer to subjecting the patient with the condition or disease to a treatment regimen, for example the administration of a fusion protein such as an NGF analog-Fc fusion protein described herein, or a pharmaceutical composition of a fusion protein such as an NGF analog- Fc fusion protein described herein, such that the pain associated with the condition or disease is reduced, alleviated, relieved, altered, remedied, ameliorated, or improved. Treating includes administering an amount of a fusion protein such as an NGF analog-Fc fusion protein described herein, or a pharmaceutical composition of a fusion protein such as an NGF analog-Fc fusion protein described herein that is effective to reduce, alleviate, relieve, alter, remedy, ameliorate, improve, pain associated with the condition or disease. Treating includes administering anABC-052PCT / 59495-PCT3 amount effective to generate antibodies to an antigen (in this case NGF) in a patient that has a condition or disease that for which pain is a symptom with the intention of generating antibodies targeted to NGF (the antigen) to reduce the antigen and hence the severity or duration of the pain associated with the disease or disorder.

[0079] As used herein, a “therapeutic vaccine” generally means a treatment that introduces an antigen or antigen analog into a patient that has a condition or disorder associated with a selfantigen, with the goal that the patient’s immune system will create antibodies for the antigen or antigen analog, enabling the patient’s body to reduce the level of the self-antigen and have an ameliorating effect on symptoms of the condition or disorder that are associated with the selfantigen (for example, pain).

[0080] As used herein, a “prophylactic vaccine” or “prophylactic immunization” or “preventative vaccine” generally means the artificial establishment of specific immunity through the introduction of antigens into a patient that is not necessarily suffering with the symptoms of a condition or disorder, with the goal that the patient’s immune system will create antibodies against the antigen and thereby prevent any future suffering or increase in suffering with the symptoms of the disorder. The distinction between a prophylactic vaccine and a therapeutic vaccine, is that a therapeutic vaccine is typically administered to a patient that is already suffering from the symptoms that the immunity targets, and a prophylactic vaccine is typically administered to a patient in anticipation of the patient suffering from the symptoms that the immunity targets.

[0081] As used herein, “booster vaccine” generally means an extra administration of a vaccine after the patient has previously received an initial administration of a vaccine, or after a patient has acquired antibodies (i.e., has a measurable antibody titer) through having had a previous treatment which introduced antibodies to reduce the symptoms of the condition or disease, or having had previous exposure to the antigen. In some examples, an additional dose of a vaccine is beneficial to periodically to “boost” the immunity of a patient to an antigen, by increasing the patient’s antigen antibody titer, which in turn ameliorates the symptoms caused by the antigen.

[0082] As used herein, the phrase “effective amount” or “therapeutically effective amount” generally means a therapeutic or prophylactic amount of the NGF analog-Fc fusion protein of the present disclosure or pharmaceutical composition thereof, that elicit the desired therapeutic or prophylactic effect or response in stimulating the immune system of the patient to generate antibodies, as evidenced by the alleviation of some or all of such symptoms of the condition or disease, when administered in accordance with the desired treatment regimen. In examples, where the desired therapeutic or prophylactic effect or response is to alleviate symptoms of aABC-052PCT / 59495-PCT3 condition or disease, an amount of an NGF analog-Fc fusion protein of the present disclosure or pharmaceutical composition thereof may be considered therapeutically effective if symptoms and / or effects of the condition or disease are observably reduced in the patient after the treatment regime. The therapeutically effective dosage of an NGF analog-Fc fusion protein may vary depending on the size and species of the patient, and / or according to the mode of administration.

[0083] As used herein, when referring to an amino acid in some portion of an amino acid sequence, for example an NGF analog amino acid sequence, a cited amino acid position is referenced as the position of the amino acid counting from the beginning of the amino acid sequence itself. For example, consider the human ProNGF amino acid sequence of SEQ ID NO: 8.EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNIT VDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKRSSSH PIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETK CRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACV CVLSRKAVRRA (SEQ ID NO: 8).

[0084] A mutation of the first serine amino acid of this sequence would be described as a mutation of the 4thamino acid of the sequence. For example, if the first serine amino acid in SEQ ID NO: 8 were mutated to asparagine, this could be referred to as an S4N mutation of SEQ ID NO: 8.

[0085] As used herein, an “NGF fragment” generally means a portion of a novel NGF analog-Fc fusion protein that comprises some portion of the NGF protein given in SEQ ID NO: 8. In examples, the NGF fragment is linked to an Fc fragment or analog thereof, as illustrated in FIG. 1.

[0086] As used herein, a “pain-associated ailment” generally means a NGF-related disease or disorder that is associated with acute or chronic pain. Acute or chronic pain includes but is not limited to post-surgical pain, rheumatoid arthritis, pain associated with cancer and osteoarthritis, as well as pain related disorders and conditions which result in occurrence of pain. Non-limiting examples of pain-associated ailments (i.e., NGF-related diseases, disorders, or conditions) include general inflammation, surgical and post-surgical pain including pain from amputation, dental pain, pain from trauma, fracture pain, pain from abscess, neuropathic pain, hyperalgesia and allodynia, neuropathic pain, post herpetic neuralgia, diabetes including, but not limited to, diabetic neuropathy pain, stroke, thalamic pain syndrome, gout joint pain, osteoarthritis or rheumatoid arthritis pain, rheumatic diseases, lupus, psoriasis, sciatica, pain associated with musculoskeletal diseases including, but not limited to, chronic low back pain, fibromyalgia, sprains, trigeminal neuralgia, dysmenorrhea, endometriosis, ovarian cysts,ABC-052PCT / 59495-PCT3 visceral pain, prostatitis, cystitis, interstitial cystitis, erythromelalgia or pain caused by pancreatitis or kidney stones, general gastrointestinal disorders including, but not limited to, colitis, gastric ulceration and duodenal ulcers, gastroesophageal reflux, dyspepsia, inflammatory bowel disorders, irritable bowel syndrome, inflammatory bladder disorders, incisional pain, pain from burns and / or wounds, ankylosing spondylitis, periarticular pathologies, cancer pain including, but not limited to, pain from bone metastases and pain from cancer treatment. Other examples of pain-associated ailments (i.e., NGF-related diseases, conditions, or disorders) include malignant melanoma, rhinitis, bronchial disorders, and asthma, such as uncontrolled asthma with severe airway hyper-responsiveness, intractable cough; and pain from skin diseases or disorders with an inflammatory component such as, but not limited to, allergic skin reactions, dermatitis, and pruritis.Novel NGF Analog-Fc Fusion Proteins

[0087] Osteoarthritis is a prevalent disease in dogs leading to refractory pain and functional disability, frequently resulting in euthanasia. The present disclosure evaluates the beneficial effects of systemic active immunization against the nociceptive mediator nerve growth factor (NGF); for example, to mitigate osteoarthritis-associated pain, thus delaying functional deterioration and euthanasia in domesticated dogs. Active immunization against NGF may be used to complement conventional treatments currently offered to relieve refractory osteoarthritis-associated pain, thus improving dogs’ quality of life and sparing suffering dogs from euthanasia. The goal therefore is to create an Fc fusion protein comprising an NGF analog and a Fc fragment (e.g., human or canine) containing a site or residue with a tendency towards glycosylation in order to create a manufacturable conjugate that presents the antigen (NGF) in a novel manner to cause a patient to produce anti-NGF antibodies capable of cross-reacting and neutralizing endogenously-produced NGF. In neutralizing the endogenously produced NGF, the antibodies produced in response to the administered therapeutic vaccine are able to reduce the pain a subject experiences in response to a disease (e.g., osteoarthritis).

[0088] NGF is initially a complex of 3 proteins - Alpha-NGF (a.k.a. NGF-alpha, NGF-a or NGF-a), Beta-NGF (a.k.a. NGF-beta, NGF-b or NGF-0), and Gamma-NGF (a.k.a. NGF- gamma or NGF-y; 2: 1 :2 ratio) when expressed. This form of NGF is also referred to as ProNGF (e.g., an NGF precursor with a polypeptide located at the N-terminus of the NGF protein sequence). The gamma subunit of this complex acts as a serine protease and cleaves the N- terminal portion of the beta subunit, thereby activating the protein into functional NGF. The complete human ProNGF protein is shown below as SEQ ID NO: 8.EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITABC-052PCT / 59495-PCT3VDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKRSSSH PIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETK CRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACV CVLSRKAVRRA (SEQ ID NO: 8).

[0089] The term ‘nerve growth factor’ usually refers to the beta subunit of the protein, (Beta- NGF, NGF-Beta, NGF-b or NGF-P), the only component of the NGF complex that is biologically active (i.e., that acts as a signaling molecule). The human NGF-P protein is shown below as SEQ ID NO: 3.S S SHPIFHRGEF S VCD S VS VW VGDKTT ATDIKGKEVMVLGEVNINNS VFKQ YF FETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDT ACVCVLSRKAVRRA (SEQ ID NO: 3).

[0090] The canine NGF-P protein differs from human NGF-P protein by three amino acids. The canine NGF-P protein is shown below as SEQ ID NO: 4, where the three amino acid differences from human NGF-P are highlighted in bold. The changes in the canine NGF-P protein compared to the human NGF-P protein are the 6thamino acid in the human NGF-P protein is mutated from isoleucine to valine (I6V), the 62ndamino acid in the human NGF-P protein is mutated from asparagine to threonine (N62T), and the 117thamino acid in the human NGF-P protein is mutated from valine to glycine (VI 17G). The amino acid sequence of SEQ ID NO: 4 is shown below:S S SHP VFHRGEF S VCD S VS VWVGDKTT ATDIKGKEVMVLGEVNINNS VFKQ YF FETKCRDPTPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDT ACVCVLSRKAGRRA (SEQ ID NO: 4).

[0091] Previous work with insulin-Fc fusion proteins, such as is described in W02018107117A1 and W02020006529A1, has demonstrated that the choices of the protein sequence, the linker sequence, and the composition of the Fc domain can all potentially influence protein yields, purity, and bioactivity. For example, the application W02018107117A1 describes a combination of insulin analog, linker, and Fc domain that yield a non-bioactive insulin-Fc fusion protein. The application W02020006529A1 describes a combination of a different insulin analog, linker and Fc domain that yield a bioactive insulin-Fc fusion protein. Both the application W02018107117A1 and the application W02020006529A1 describe combinations of insulin analogs, linkers and Fc domains that have poor manufacturability of homogeneity.

[0092] In choosing the NGF analog for the novel NGF analog-Fc fusion protein it is conceivable that one could choose an NGF analog that includes some portion of a wild-type NGF. For example, the NGF analog for a novel NGF analog-Fc fusion protein may comprise allABC-052PCT / 59495-PCT3 or a portion of the human ProNGF of SEQ ID NO: 8, or may comprise all or a portion of the human NGF-P protein of SEQ ID NO: 3, or may comprise all or a portion of the canine NGF-P protein of SEQ ID NO: 4. In examples, the NGF analog may comprise additional amino acids or polypeptides, for example at the N-terminus of an NGF analog sequence (collectively then referred to as the NGF analog). In examples, one or more amino acids in the NGF analog of the novel NGF analog-Fc fusion protein may be deleted or mutated from their native state.

[0093] It is expected that different NGF analog-Fc fusion protein designs will result in different protein yields (see for example, Azadeh Beygmoradi, Ahmad Homaei, Roohullah Hemmati, Pedro Fernandes, Recombinant protein expression: Challenges in production and folding related matters, International Journal of Biological Macromolecules, Volume 233, 2023, 123407, ISSN 0141-8130, doi.org / 10.1016 / j.ijbiomac.2023. 123407, and Massimo Ste ani, Protein misfolding and aggregation: new examples in medicine and biology of the dark side of the protein world, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, Volume 1739, Issue 1, 2004, Pages 5-25, ISSN 0925-4439).

[0094] For example, larger or shorter NGF analog sequences in the NGF analog, when used to produce the NGF analog-Fc fusion protein, are expected to result in different protein yields. The resulting protein yield when the selected NGF analog is attached to an Fc fragment can be experimentally determined. The choice of the Fc fragment and the portion of the Fc fragment hinge region that is linked to the selected NGF analog impacts the manufacturability of the NGF analog-Fc fusion protein.

[0095] The novel NGF analog-Fc fusion protein may comprise a peptide linker. In examples, the therapeutic protein comprising the NGF analog is linked to the N-terminal side of the Fc fragment. In examples, the novel NGF analog-Fc fusion protein comprises domains in the following orientation from N- to C-termini: (N-terminus) — antigen — peptide linker — Fc fragment — (C-terminus) (e.g., (N-terminus) — NGF analog — peptide linker — Fc fragment — (C- terminus)). The length and composition of the linker connecting the NGF analog to the Fc fragment may impact the protein yield.

[0096] The design goal is to create an NGF analog-Fc fusion protein with a protein yield after production in transiently transfected CHO cells and protein A purification that may be greater than 5 mg / L, 10 mg / L, 20 mg / L, 50 mg / L or more preferably greater than 75 mg / L (e.g., greater than 80 mg / L, greater than 90 mg / L, greater than 100 mg / L).

[0097] As a first attempt in creating a novel NGF analog-Fc fusion protein for use in canines, a human NGF-P (SEQ ID NO: 3) was selected as the NGF analog. It was expected that using a human NGF will cause the NGF analog-Fc fusion protein to potentially be more immunogenic in animals (e.g., mice or dogs) than using a canine native sequence. The use of a human IgG FcABC-052PCT / 59495-PCT3 fragment may also increase immunogenicity of the NGF analog-Fc fusion protein further resulting in a greater immune reaction which will decrease levels of endogenous NGF in the patient through the greater production of antibodies against endogenous NGF. Human IgGl is known to be more immunogenic than other human IgG molecules, for example IgG2, IgG3 and IgG4.

[0098] In the human IgGl fragment of SEQ ID NO: 1, the C-terminus lysine on the native human IgGl fragment was eliminated. The C-terminal lysine that is found in native IgG isotype Fc fragment amino acid sequences (i.e., the lysine that represents the last amino acid of the Fc fragment sequence) is known to result in the accidental production of unwanted amino acid sequence variants during manufacturing (e.g., Fc fragments containing the C-terminal lysine becoming mixed with Fc fragments where the C-terminal lysine is omitted, which can occur during production of the desired protein within cells (Dick, LW., (2008) Biotechnol Bioeng. Aug 15; 100(6) ppll32-43 Therefore, it was expected that removing the C-terminal lysine would improve manufacturing yield and purity. In addition, the asparagine at the cNg site on the human IgGl fragment was conserved to preserve the glycan attachment during fusion protein production in host cells. It has been shown that IgG glycosylation may assist in increased immunogenicity through enhanced antigen presentation via binding to Fc(gamma)R receptors on antigen presenting immune cells present in a patient (e.g., a dog). Therefore, an NGF analog- Fc fusion protein comprising a glycosylated Fc fragment is expected to show increased immunogenicity over NGF analog-Fc fusion protein embodiments without glycosylation at the cNg site (Cobb BA. The history of IgG glycosylation and where we are now. Glycobiology. 2020 Mar 20;30(4):202-213. doi: 10.1093 / glycob / cwz065. PMID: 31504525; PMCID: PMC7 109348),

[0099] The human NGF-P of SEQ ID NO: 3 was linked using the peptide linker GGGSGGGS (SEQ ID NO: 9) to a human IgGl Fc fragment comprising the following sequence:DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG (SEQ ID NO: 1).

[0100] The resultant NGF analog-Fc fusion protein is given below as SEQ ID NO: 10.S S SHPIFHRGEF S VCD S VS VW VGDKTT ATDIKGKEVMVLGEVNINNS VFKQ YF FETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDT ACVCVLSRKAVRRAGGGSGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLABC-052PCT / 59495-PCT3MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 10).

[0101] The NGF analog-Fc fusion protein of SEQ ID NO: 10 was manufactured in CHO cells according to Example 1 and was purified according to Example 3. The Fc fusion protein structure of the NGF analog-Fc fusion protein of SEQ ID NO: 10 was confirmed according to Example 4, and sequence identification is performed according to Example 5. The resultant yield of the fusion protein produced was a titer of 7 mg / L, which was significantly below the design goal of 75 mg / L.

[0102] The use of non-native (i.e., non-canine) NGF analog in a dog may result in too much immunogenicity and have safety implications for the patient. In a second attempt to create an NGF analog-Fc fusion protein and reduce the risk of an adverse reaction in dogs, the human NGF analog was replaced by the canine NGF-P of SEQ ID NO: 4 as shown below:SSSHPVFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYF FETKCRDPTPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDT ACVCVLSRKAGRRA (SEQ ID NO: 4).

[0103] A sequence comparison of the human NGF-P of SEQ ID NO: 3 and the canine NGF- P of SEQ ID NO: 4 is shown in FIG. 2.

[0104] The use of a human Fc fragment in a dog may also result in too much immunogenicity and have safety implications for the patent. Therefore, the human IgG portion of the NGF analog-Fc fusion protein was replaced with a canine IgG. IgGB was chosen as it is known to have greater immunogenicity than other IgG in canines (IgGA, IgGC and IgGD). As with the human IgGl Fc fragment, the C-terminal lysine that is found in native canine or IgG isotype Fc fragment amino acid sequences (i.e., the lysine that represents the last amino acid of the Fc fragment sequence) was omitted to prevent the accidental production of unwanted amino acid sequence variants during manufacturing, e.g., Fc fragments containing the C-terminal lysine becoming mixed with Fc fragments where the C-terminal lysine is omitted, which can occur during production of the desired protein within cells (Dick, LW., (2008) Biotechnol Bioeng. Aug 15;100(6) pp!132-43). The canine IgGB Fc fragment sequence lacking a C- terminal lysine is given in SEQ ID NO: 2:DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFV DGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPS PIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNG QQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHABC-052PCT / 59495-PCT3YTQESLSHSPG (SEQ ID NO: 2).

[0105] A sequence comparison of the human IgGl of SEQ ID NO: 1 and the canine IgGB of SEQ ID NO: 2 is shown in FIG. 3.

[0106] The NGF analog of SEQ ID NO: 4 was linked to the Fc fragment of SEQ ID NO: 2 via the linker GGGSGGGS (SEQ ID NO: 9). The resulting NGF analog-Fc fusion protein of SEQ ID NO: 11 is shown below:SSSHPVFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYF FETKCRDPTPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDT ACVCVLSRKAGRRAGGGSGGGSDCPKCPAPEMLGGPSVFIFPPKPKDTLLIART PEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIG HQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNT VSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDK SRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 11).

[0107] The NGF analog-Fc fusion protein of SEQ ID NO: 11 was manufactured in CHO cells according to Example 1 and was purified according to Example 3. The Fc fusion protein structures of the NGF analog-Fc fusion protein of SEQ ID NO: 11 was confirmed according to Example 4, and sequence identification is performed according to Example 5. This produced a protein yield titer of 10 mg / L, which was below the preferred design goal of a titer exceeding 75 mg / L.

[0108] In a further attempt to produce a novel NGF analog-Fc fusion protein with acceptable manufacturing yield, the canine NGF-P protein (SEQ ID NO: 4) was extended to incorporate a part of the human ProNGF precursor protein. The addition of a fragment of the human ProNGF to the canine NGF-P sequence is intended to render the resulting NGF analog-Fc fusion protein biologically inactive at the endogenous NGF receptor and decrease the probability that the NGF analog-Fc will cause an increase in pain when administered to a patient. This ProNGF fragment comprised an octapeptide which was linked to the N-terminus of the canine NGF-P sequence. Additionally, the octapeptide was mutated at amino acid number 8 from arginine (R) to glycine (G), i.e., R8G resulting in the sequence NRTHRSKG (SEQ ID NO: 16). This mutation was intended to prevent the octapeptide from cleaving off of the NGF analog-Fc fusion protein, in order to allow the resulting NGF analog-Fc fusion protein to generate immunogenicity but to prevent the NGF-P from significantly activating the cell receptor and causing additional pain when administered to the patient. The resulting NGF analog of SEQ ID NO: 5 is shown below:NRTHRSKGSSSHPVFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNI NNSVFKQYFFETKCRDPTPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQ AAWRFIRIDTACVCVLSRKAGRRA (SEQ ID NO: 5).ABC-052PCT / 59495-PCT3

[0109] The NGF analog of SEQ ID NO: 5 was linked via the peptide linker GGGSGGGS (SEQ ID NO: 9) to the canine IgGB Fc fragment of SEQ ID NO: 2 comprising the following sequence:DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFV DGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPS PIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNG QQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNH YTQESLSHSPG (SEQ ID NO: 2).

[0110] The resultant novel NGF analog-Fc fusion protein is given below (SEQ ID NO: 12): NRTHRSKGSSSHPVFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNI NNSVFKQYFFETKCRDPTPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQ AAWRFIRIDTACVCVLSRKAGRRAGGGSGGGSDCPKCPAPEMLGGPSVFIFPP KPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNG TYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVL PPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSY FLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 12).[OHl] The novel NGF analog-Fc fusion protein of SEQ ID NO: 12 was manufactured in CHO cells according to Example 1 and was purified according to Example 3. The Fc fusion protein structure of the novel NGF analog-Fc fusion protein of SEQ ID NO: 12 was confirmed according to Example 4, and sequence identification is performed according to Example 5. The addition of the mutated human octapeptide ProNGF precursor (SEQ ID NO: 16) resulted in a protein titer of 57 mg / L, which was a significant improvement over the NGF analog-Fc fusion protein of SEQ ID NO: 10 and the NGF analog-Fc fusion protein of SEQ ID NO: 11. However, this was still below the preferred design target of 75 mg / L.

[0112] In a further attempt to produce an NGF analog-Fc fusion protein with acceptable manufacturing yield, the NGF analog of the NGF analog-Fc fusion protein was replaced with a mutated canine NGF-P protein. The canine NGF-P protein was mutated at the 4thamino acid with a histidine (H) to aspartic acid (D) substitution (H4D). This mutation is expected to allow the generation of antibodies to the canine NGF-P but to prevent the NGF protein sequence from binding to the cell receptors and generating pain. The resulting canine NGF analog is SEQ ID NO: 6, with the H4D mutation shown in bold below:S S SDP VFHRGEF S VCD S VS VWVGDKTT AT D I KGK E VM VLGE VN I N N S VFKQ YF FETKCRDPTPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDT ACVCVLSRKAGRRA (SEQ ID NO: 6).ABC-052PCT / 59495-PCT3

[0113] The mutated NGF analog of SEQ ID NO: 6 was linked via the peptide linker GGGSGGGS (SEQ ID NO: 9) to the canine IgGB Fc fragment of SEQ ID NO: 2 in order to create the NGF analog-Fc protein SEQ ID NO: 13 as shown below:SSSDPVFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYF FETKCRDPTPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDT ACVCVLSRKAGRRAGGGSGGGSDCPKCPAPEMLGGPSVFIFPPKPKDTLLIART PEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIG HQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNT VSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDK SRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 13).

[0114] The NGF analog-Fc fusion protein of SEQ ID NO: 13 was manufactured in CHO cells according to Example 1 and was purified according to Example 3. The Fc fusion protein structure of the novel NGF analog-Fc fusion proteins of SEQ ID NO: 13 was confirmed according to Example 4, and sequence identification is performed according to Example 5. This produced a protein yield titer of 39 mg / L which was still below the preferred design target of 75 mg / L.

[0115] In a further attempt to create an NGF analog-Fc fusion protein with acceptable manufacturing yield, the canine NGF analog with the H4D mutation (SEQ ID NO: 6) was truncated from the C-terminus end to yield a shorter sequence (SEQ ID NO: 7). The intention of the truncation was to avoid unpaired cysteine sites whose free thiols could lead to improper folding and aggregation of the NGF analog-Fc fusion protein. As with SEQ ID NO: 6, the inclusion of the H4D mutation in the NGF analog was expected to allow the generation of antibodies to the NGF-P but to minimize the NGF analog from binding to nerve cell receptors and contributing to pain. The resulting canine NGF analog is SEQ ID NO: 7, with the H4D mutation shown in bold below:S S SDP VFHRGEF S VCD S VS VWVGDKTT AT D I KGK E VM VLGE VN I N N S VF (SEQ ID NO: 7).

[0116] A sequence comparison of the NGF analog of SEQ ID NO: 6 and the NGF analog of SEQ ID NO: 7 is shown in FIG. 4. The NGF analog of SEQ ID NO: 7 was linked via the peptide linker GGGSGGGS (SEQ ID NO: 9) to the canine IgGB Fc fragment (SEQ ID NO: 2). The resultant NGF analog-Fc fusion protein was SEQ ID NO: 14, shown below:SSSDPVFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFGGGS GGGSDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQI SWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNK ALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWABC-052PCT / 59495-PCT3QSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEAL HNHYTQESLSHSPG (SEQ ID NO: 14).

[0117] The NGF analog-Fc fusion protein of SEQ ID NO: 14 was manufactured in CHO cells according to Example 1 and was purified according to Example 3. The Fc fusion protein structure of the novel NGF analog-Fc fusion protein of SEQ ID NO: 14 was confirmed according to Example 4, and sequence identification is performed according to Example 5. This produced a protein yield titer of 4 mg / L which was still below the preferred design target of 75 mg / L.

[0118] In a further attempt to create an NGF analog-Fc fusion protein that met the manufacturing target titer yield, the NGF analog of the fusion protein was replaced with a portion of the human ProNGF sequence of SEQ ID NO: 8. As stated previously, the ProNGF sequence is expected to be less biologically inactive at the NGF receptor, so the combination of the ProNGF with the Fc fragment was expected to stimulate the desired immune effect of generating NGF antibodies without further pain stimulation. Additionally, using the human ProNGF sequence was expected to potentially generate a greater immune reaction in dogs than using a native canine ProNGF sequence in dogs. However, it was believed that the longer human ProNGF sequence of SEQ ID NO: 8 would not be manufacturable at acceptable protein yield titers due to undesirable aggregation or improper protein folding, or that it may be subject to proteolytic cleavage. Mutations in the Pro portion of a ProNGF to remove lysine-arginine (KR) and arginine-arginine (RR) proteolytic cleavage sites may prevent proteolytic digestion (cleaving) of the ProNGF during cell culture. Cleaving of the ProNGF has the potential to create a mixture of different compound mixtures that may result in more onerous separation and purification, thereby decreasing manufacturing yield. Thus, incorporating mutations capable of reducing cleaving will improve manufacturing yield. Mutations were therefore made to the human ProNGF of SEQ ID NO: 8. A first mutation was made at position 61 of the human ProNGF of SEQ ID NO: 8, where the lysine was mutated to serine (K61S). A further mutation was made at position 62 of the human ProNGF of SEQ ID NO: 8, where arginine was mutated to serine (R62S). These mutations were made in an attempt to decrease proteolytic degradation by eliminating a lysine-arginine (KR) highly basic charged proteolytic cleavage site and replacing the lysine and arginine amino acids with neutral serine amino acids (SS) at positions 61 and 62. A third mutation made in an attempt to decrease proteolytic degradation was made at position 102 of the human ProNGF of SEQ ID NO: 8, where lysine was mutated to serine (K102S) to eliminate a further lysine-arginine (KR) proteolytic cleavage site at positions 102 and 103 and replace it with a serine-arginine (SR) motif. A fourth mutation was made at position 32 of the human ProNGF of SEQ ID NO: 8 where the arginine was mutated to aspartic acid (R32D) to eliminate the arginine-arginine (RR) motif at positions 31 and 32. A fifth mutationABC-052PCT / 59495-PCT3 was made at position 222 of the human ProNGF of SEQ ID NO: 8 where the arginine was mutated to aspartic acid (R222D) to eliminate the arginine-arginine (RR) motif at positions 221 and 222. These fourth and fifth mutations were made in an attempt to decrease proteolytic degradation by eliminating the arginine-arginine (RR) highly basic charged proteolytic cleavage sites and replacing them with a negatively charged arginine-aspartic acid (RD) motif. The resulting NGF analog was SEQ ID NO: 19, shown below with the mutations shown in bold type:EPHSESNVPAGHTIPQAHWTKLQHSLDTALRD ARSAP AAAIAARVAGQTRNITV DPRLFKSSRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSSRSSSHPIF HRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRD PNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLS RKAVRDA (SEQ ID NO: 19).

[0119] A sequence comparison of the NGF analog of SEQ ID NO: 8 and the NGF analog of SEQ ID NO: 19 is shown in FIG. 6.

[0120] The NGF analog of SEQ ID NO: 19 was linked to the canine IgGB sequence of SEQ ID NO: 2 via the linker GGGSGGGS (SEQ ID NO: 9). This created the NGF analog-Fc fusion protein of SEQ ID NO: 18 as shown below:EPHSESNVPAGHTIPQAHWTKLQHSLDTALRD ARSAP AAAIAARVAGQTRNITV DPRLFKS SRLRSPRVLF STQPPREAADTQDLDFEVGGAAPFNRTHRS SRSS SHPIF HRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRD PNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLS RKAVRDAGGGSGGGSDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVV DLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGK QFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFF PPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFIC AVMHEALHNHYTQESLSHSPG (SEQ ID NO: 18).

[0121] The NGF analog-Fc fusion protein of SEQ ID NO: 18 was manufactured in CHO cells according to Example 1 and was purified according to Example 3. The Fc fusion protein structure of the novel NGF analog-Fc fusion protein of SEQ ID NO: 18 was confirmed according to Example 4, and sequence identification is performed according to Example 5. Unexpectedly the protein yield titer increased to a level of 248 mg / L. This sequence exceeded the design goal of an NGF analog-Fc fusion protein with a titer of over 75 mg / L.

[0122] In a further attempt to create an NGF analog-Fc fusion protein that met the manufacturing target titer yield and that increased the titer yield, the NGF analog of SEQ ID NO: 19 was linked to the canine IgGB sequence of SEQ ID NO: 2 via the linker GGGGGSGGGSGGGGSGGS (SEQ ID NO: 21). The longer linker of SEQ ID NO: 21 (i.e.,ABC-052PCT / 59495-PCT3 compared to the linker of SEQ ID NO: 9) was used to address potential folding challenges with the resulting NGF analog-Fc fusion protein. This created the NGF analog-Fc fusion protein of SEQ ID NO: 20 as shown below:EPHSESNVPAGHTIPQAHWTKLQHSLDTALRD ARSAP AAAIAARVAGQTRNITV DPRLFKS SRLRSPRVLF STQPPREAADTQDLDFEVGGAAPFNRTHRS SRSS SHPIF HRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRD PNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLS RKAVRDAGGGGGSGGGSGGGGSGGSDCPKCPAPEMLGGPSVFIFPPKPKDTLLI ARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVL PIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKN TVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDK SRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 20).

[0123] The NGF analog-Fc fusion protein of SEQ ID NO: 20 was manufactured in CHO cells according to Example 1 and was purified according to Example 3. The Fc fusion protein structure of the novel NGF analog-Fc fusion protein of SEQ ID NO: 20 was confirmed according to Example 4, and sequence identification is performed according to Example 5. The protein yield titer of SEQ ID NO: 20 was measured as 245 mg / L and remained approximately constant relative to the protein yield titer of SEQ ID NO: 18 (248 mg / L). SEQ ID NO: 20 exceeded the design goal of an NGF analog-Fc fusion protein with a titer of over 75 mg / L.

[0124] In a further attempt to create an NGF analog-Fc fusion protein that met the manufacturing target titer yield and that increased the titer yield, the fourth (R32D) and fifth (R222D) mutations of the NGF analog of SEQ ID NO: 19 were reverted in an attempt to push the isoelectric point of the molecule to a higher value and increase solubility and minimize the potential for aggregation.

[0125] The resulting NGF analog was SEQ ID NO: 17, shown below with the mutations shown in bold type:EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAP AAAIAARVAGQTRNITV DPRLFKSSRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSSRSSSHPIF HRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRD PNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLS RKAVRRA (SEQ ID NO: 17).

[0126] A sequence comparison of the NGF analog of SEQ ID NO: 8 and the NGF analog of SEQ ID NO: 17 is shown in FIG. 5. A sequence comparison of the NGF analogs of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 17 and SEQ ID NO: 19 is shown in FIG. 7A and FIG. 7B.ABC-052PCT / 59495-PCT3

[0127] The NGF analog of SEQ ID NO: 17 was linked to the canine IgGB sequence of SEQ ID NO: 2 via the linker GGGSGGGS (SEQ ID NO: 9). This created the NGF analog-Fc fusion protein of SEQ ID NO: 15 as shown below:EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITV DPRLFKS SRLRSPRVLF STQPPREAADTQDLDFEVGGAAPFNRTHRS SRSS SHPIF HRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRD PNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLS RKAVRRAGGGSGGGSDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVV DLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGK QFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFF PPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFIC AVMHEALHNHYTQESLSHSPG (SEQ ID NO: 15).

[0128] The NGF analog-Fc fusion protein of SEQ ID NO: 15 was manufactured in CHO cells according to Example 1 and was purified according to Example 3. The Fc fusion protein structure of the novel NGF analog-Fc fusion protein of SEQ ID NO: 15 was confirmed according to Example 4, and sequence identification is performed according to Example 5. Unexpectedly the protein yield titer increased to 300 mg / L. This sequence exceeded the design goal of an NGF analog-Fc fusion protein with a titer of over 75 mg / L.

[0129] The protein yields of the NGF analog-Fc fusion proteins of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18 and SEQ ID NO: 20 are shown below in Table 1. A sequence comparison of the NGF analog- Fc fusion proteins of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18 and SEQ ID NO: 20 is shown in FIG. 8A, FIG. 8B, FIG. 8C and FIG. 8D.ABC-052PCT / 59495-PCT3NGF Analog-Fc Fusion Proteins for Use as a Therapeutic Vaccine

[0130] An NGF analog-Fc fusion protein may be used as a vaccine. In one or more embodiments, the NGF analog-Fc fusion protein is provided in a pharmaceutical composition. Injection of any protein into a patient (e.g., a dog) may induce an immune response, the magnitude and type of which is highly dependent on the “status” of the respective immune system. For example, injection of a foreign antigen (Ag) relative to a self Ag may induce a greater immune response in an immune system that maintains central and peripheral tolerance mechanisms. Moreover, foreign Ag administration to an immune system that has been primed to previous exposure to the respective Ag (e.g., a viral infection) will lodge a more rapid and elevated immune response relative to that of an Ag-naive system. The immunological basis of this priming is two-fold: 1) an Ag-naive immune system has naive B and T lymphocytes that have a much higher threshold of activation than do the Ag-primed “memory” cells of a Ag- primed immune system, such that the antigen-presenting cells (APCs) that present Ag require much less Ag to activate primed memory T cells, and 2) due to expansion of memory T cells during the Ag priming exposure, there are inherently greater numbers of such cells upon reexposure to an injected Ag. Note that dominant APCs are dendritic cells (DCs) and macrophages that present Ag in complex with Major Histocompatibility Complex (MHC) molecules on theirABC-052PCT / 59495-PCT3 surface to T cell Ag receptors. FIG. 9 is a schematic diagram depicting example modes in which an antigen may interact with an antigen presenting cell, e.g., a dendritic cell.

[0131] In another example, injection of a self Ag (or self Ag-Fc fusion protein) is designed to induce an immune response against a self Ag (e.g., a patient’ s endogenously-produced antigen or endogenously-produced protein, e.g., endogenously-produced NGF), but needs to overcome immune system central and peripheral tolerance mechanisms. To do so, the antigen is exposed to the immune system to cause an immune response (e.g., generate antibodies against the self Ag) in a manner that overcomes these tolerance mechanisms. In one embodiment, this is done through the use of a carrier (e.g., an adjuvant). In another embodiment, this can be done through linking of the self Ag or a self Ag analog to an Fc fragment in order to increase the antigen presentation of the self Ag via Fc fragment-Fc(gamma)R binding as depicted in FIG. 9. In another embodiment, this is done through linking of the self Ag or self Ag analog to an Fc fragment and using a carrier (e.g., an adjuvant).

[0132] Generation of an immune response against a self Ag may be done through a priming approach. The immunological basis of this priming is two-fold: 1) an Ag-naive immune system has naive B and T lymphocytes that have a much higher threshold of activation than do the Ag- primed “memory” cells of a Ag-primed immune system, such that the antigen-presenting cells (APCs) that present Ag require much less Ag to activate primed memory T cells, and 2) due to expansion of memory T cells during the Ag priming exposure, there are inherently greater numbers of such cells upon re-exposure to an injected self Ag or self Ag analog. Note that dominant APCs are dendritic cells (DCs) and macrophages that present Ag in complex with Major Histocompatibility Complex (MHC) molecules on their surface to T cell Ag receptors. FIG. 9 is a schematic diagram depicting example modes in which an antigen may interact with a presenting cell, e.g., a dendritic cell.

[0133] Further enhancement of an immune response against a self Ag may be done through a boosting approach. The immunological basis of this boosting is to allow for an expansion of memory T cells during the Ag priming exposure, so as to enhance the numbers of such cells upon re-exposure to an injected self Ag or self Ag analog. Boosting can be done through one or more follow-on injections after a priming injection. Boosting (e.g., booster injections) may contain a carrier (e.g., an adjuvant) or no carrier (no adjuvant). Boosting may increase the level of antibodies against a self Ag, create higher affinity antibodies against a self Ag, or both increase the level and the affinity of antibodies against a self Ag.

[0134] Antigen presenting cells (APCs) can influence both the “magnitude” and “type” of response to an Ag. B cells participate in the immune response directly by humoral immunity (antibody production) and also participate in the T cell immune response as specific APCs thatABC-052PCT / 59495-PCT3 selectively capture and present antigens to T cells. Both of these B cell functions are achieved through activation of the surface B cell receptor (BCR), which is essentially a membrane bound antibody that binds specifically to a particular antigen. Multivalent soluble antigens such as the Fc-fusion homodimer containing the specific antigen can be recognized by BCRs and activate them. Thus, the NGF analog-Fc fusion protein homodimers can i) activate B cells through antigen-specific BCR activation leading to an increase in antibody production, and ii) through B cell mediated APC activity, increase T cell recognition and reactivity directed specifically against the NGF epitopes. Thus, these fusion proteins can activate either humoral immunity, cellular immunity or a combination of humoral and cellular immunity after administration.Adjuvants

[0135] In some examples, APC activation is the conceptual basis of many immune enhancing substances called adjuvants. Dominant APCs are dendritic cells (DCs) and macrophages that present Ag in complex with Major Histocompatibility Complex (MHC) molecules on their surface to T cell Ag receptors. Some adjuvants are designed to trick the immune system into reacting to the inj ected vaccine Ag as if it were part of an ongoing infection (i.e., infectious agents provide such natural viral or bacterial adjuvant substances). Therefore, adjuvants activate APCs for greater Ag-presentation capabilities necessary to overcome the high activation threshold of naive T cells, in addition to shaping their development into the Thl immune system response, Th2 immune system response or a mixture of Thl and Th2 immune system responses. Some T cells provide critical help to B cells that specifically bind the respective Ag to produce Ag-specific antibody (Ab) titers.

[0136] The novel NGF analog-Fc fusion protein used as a vaccine may be co-administered with an adjuvant to enhance or otherwise alter the immune response in the target patient. In examples, known adjuvants may be used in a pharmaceutical composition of the NGF analog- Fc fusion protein to enhance the induction of anti -NGF antibodies.

[0137] Examples of adjuvants that may be employed in the pharmaceutical compositions disclosed herein include but are not limited to oil-in-water, amorphous aluminum hydroxyphosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate (Alum), Freund’s adjuvant (complete and / or incomplete), squalene, AS02, AS03, AS04, MF59, AS01B, QS-21, CpG 1018, ISCOMS, Montanide™ ISA-51, Montanide™ ISA-720, Montanide™ GEL 01 PR, Montanide™ GEL 02 PR, Sepivac SWE, polylactide coglycolide (PLG), monophosphoryl lipid A (MPL), Detox, AGP [RC-529], DC Chol, OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), saponin-based adjuvants (e.g. Quil-A®, QS 21, or other adjuvants made fromABC-052PCT / 59495-PCT3Quillaja saponaria bark extracted saponins), modified LT and CT, hGM-CSF, hIL-12, Immudaptin, inert vehicles, such as gold particles as well as various experimental adjuvants from sources such as Advax (Australia) such as AddaVax (Invivogen) or other Advax -based vaccine adjuvants.

[0138] In some examples, the selected adjuvant may be MF59 (Novartis) and AS-03 (GlaxoSmithKline). A custom formulation of MF59 (Novartis) or an equivalent such as AddaVax (Invivogen) or other Advax-based vaccine adjuvants from Vaxine Pvt Ltd. (Australia) may be used in a pharmaceutical composition of the NGF analog-Fc fusion protein. In examples, the NGF analog-Fc fusion protein is co-administered with the Montanide™ ISA-720 adjuvant to enhance or otherwise alter the immune response in the target patient. In other examples, the NGF analog-Fc fusion protein is co-administered with a saponin-based adjuvant to enhance or otherwise alter the immune response in the target patient. In still other examples, the NGF analog-Fc fusion protein is co-administered with Quil-A® adjuvant to enhance or otherwise alter the immune response in the target patient.

[0139] In one or more embodiments, the NGF analog-Fc fusion protein formulation is prepared onsite for administration. In one aspect, the NGF analog-Fc fusion protein is mixed with an adjuvant onsite under sterile mixing conditions. In one aspect, the NGF analog-Fc fusion protein and adjuvant are thoroughly mixed and / or emulsified to prepare a homogenous emulsion for administration to the patient. The adjuvanted formulation of the NGF analog-Fc fusion protein or a pharmaceutical composition thereof is administered to a patient by subcutaneous (s.c.) injection or intramuscular (i.m.) injection, as the s.c. or i.m. injection sites are more likely to induce a strong antibody response due to there being more dendritic cells (DCs) in the subcutaneous and intramuscular spaces.

[0140] As described above, in some cases, it may be advantageous to use an adjuvant in the pharmaceutical composition in order to increase the quantity of anti-NGF antibody titers as measured according to Example 8. The use of an adjuvant may be especially advantageous in older patients who experience altered immune competence with increasing age, so-called immunosenescence, which is the result of changes at multiple levels of the immune system over time. Once a patient has measurable antibodies, upon re-challenge with NGF or an NGF analog- Fc fusion protein, the patient will exhibit rapid development of anti-NGF antibodies.Primary NGF Analog-Fc Fusion Protein Vaccines Evaluated in Mice

[0141] The efficacy of an exemplary NGF analog-Fc fusion proteins of this disclosure or pharmaceutical compositions thereof may be initially evaluated in mice immunization studies for their capacity to induce anti-NGF protein IgG titers when administered according to theABC-052PCT / 59495-PCT3 procedure in Example 9. BALB / c mice are a relevant animal model that has been extensively used for preclinical immunogenicity assessment of vaccines. This strain generates robust Ab responses when immunized with adjuvanted and non-adjuvanted vaccine candidates. Moreover, mouse-specific reagents are widely available for evaluating the kinetics and characteristics of a variety of immune responses to vaccination, including relevant Ab isotypes and T cell responses (e.g., Thl vs. Th2 responses). Therefore, the BALB / c mouse model is selected to evaluate the immunogenicity of NGF analog-Fc fusion protein vaccines with respect to dose, potentiation by adjuvants, routes of administration, and dosing frequency required to achieve optimal Ab responses.

[0142] Briefly, target mice (e.g., BALB / c mice) are injected three times at predetermined intervals (e.g., on Day 0, Day 21 and Day 42) with an exemplary NGF analog-Fc fusion protein (with or without Montanide™ ISA 720 adjuvant) or pharmaceutical composition thereof, and serum is collected at regular intervals (e.g., every 7-14 days beginning at Day 14).

[0143] After administering one or more than one treatment of the NGF analog-Fc fusion protein of SEQ ID NO: 15, the NGF analog-Fc fusion protein of SEQ ID NO: 18 or the NGF analog-Fc fusion protein of SEQ ID NO: 20 to N=5 BALB / c mice according to Example 9, anti- NGF IgG antibody titers were measured according to Example 8.

[0144] As previously discussed, adjuvants activate APCs for greater Ag-processing and Ag- presentation capabilities which are necessary to overcome the high activation threshold of naive T cells. It is expected that when the NGF analog-Fc fusion protein of SEQ ID NO: 15, the NGF analog-Fc fusion protein of SEQ ID NO: 18 or the NGF analog-Fc fusion protein of SEQ ID NO: 20 is combined with Montanide™ ISA 720 adjuvant (30% / 70% v / v), the anti-NGF protein antibody titers at approximately 35 days after the first injection on Day 0 will be greater compared to the NGF analog-Fc fusion protein without adjuvant.

[0145] The kinetic response, that is the duration of response, to dose levels varying from 1 pg to 100 pg after 1, 2, and 3 doses is expected to demonstrate increasing anti-NGF protein antibody titers at all dose levels up to at least 56 days post vaccination.NGF Analog-Fc Fusion Proteins for Use as a Booster Vaccine

[0146] In examples, an exemplary NGF analog-Fc fusion protein of this disclosure, for example the NGF analog-Fc fusion protein of SEQ ID NO: 15, the NGF analog-Fc fusion protein of SEQ ID NO: 18 or the NGF analog-Fc fusion protein of SEQ ID NO: 20, may be used as a booster vaccine. Administration of the NGF analog-Fc fusion protein to patients that already have low but measurable antibody levels to the NGF self-antigen (e.g., after a primary vaccination with an NGF-analog Fc fusion protein) to amplify their antibody titers and increaseABC-052PCT / 59495-PCT3 their neutralization against the endogenously produced NGF. NGF analogs are synthesized to maximize antigenicity and overall manufacturability, while the Fc region prolongs antigen residence time and / or binding to Fc(gamma)R receptors present on APCs. Without wishing to be bound to any particular theory of mechanism, it is believed that during the longer in vivo residence time, the naturally glycosylated Fc fragment will help bind Fc(gamma) receptors on antigen-presenting cells (APCs), which will in turn cause greater presentation of the NGF analog antigen to T-cells and / or B-cells, which is expected to produce a strong immune response (e.g. antibody titers) to the NGF antigen present on the NGF analog-Fc fusion protein, and that these antibody titers will be able to bind and neutralize the endogenously produced target protein (e.g. NGF-P). Specifically, the APCs internalize the NGF antigen via Fc(gamma) receptors, and then process and present NGF fragments to CD4+ Th cells that in turn promote (“help”) B cell activation and anti -NGF IgG (i.e., Ab) production.

[0147] Antigen-presenting cells may be, for example, dendritic cells (DCs), monocytes or macrophages that can internalize the molecules of the NGF analog-Fc fusion protein of SEQ ID NO: 15, the molecules of the NGF analog-Fc fusion protein of SEQ ID NO: 18 or the molecules of the NGF analog-Fc fusion protein of SEQ ID NO: 20 via Fc-receptor mediated phagocytosis (e.g., through the Fc region of the NGF analog-Fc fusion protein binding to the Fc(gamma) receptors in immune cells). Fc-mediated uptake of the NGF analog-Fc fusion protein by, for example, a subset of DCs (e.g., cDC2s) promotes the development of anti-NGF T helper 2 (Th2) cells through secretion of IL-10 and IL-33. Anti-NGF Th2 cells activate anti-NGF B-cells, for example by cross linking their antigen receptors to allow the B-cells to attract the Th2 cells. B- cell antigen receptor (BCR) mediated uptake binds the NGF of the NGF analog-Fc fusion protein molecules, then delivers the NGF antigen to intracellular sites where it is degraded and returned to the B-cell surface as peptides bound to MHC class II molecules. The peptide MCH class II complex can be recognized by the NGF-specific helper T cells simulating them to make proteins that in turn cause the B-cell to proliferate and its progeny to differentiate into B cells that secrete anti-NGF antibodies. The NGF analog-Fc fusion protein of SEQ ID NO: 15, the NGF analog-Fc fusion protein of SEQ ID NO: 18 or the NGF analog-Fc fusion protein of SEQ ID NO: 20 may increase exposure of the NGF analog fragment to antigen producing cells over a protracted period of time due to the presence of the Fc fragment and the Fc-FcRn receptor interactions that enable the NGF analog-Fc fusion protein to have a prolonged in vivo pharmacokinetic half-life. Furthermore, and as previously described, the glycosylated Fc fragment in the NGF analog-Fc fusion protein of SEQ ID NO: 15, the glycosylated Fc fragment in the NGF analog-Fc fusion protein of SEQ ID NO: 18 and the glycosylated Fc fragment in the NGF analog-Fc fusion protein of SEQ ID NO: 20 are expected to help induce a strong immuneABC-052PCT / 59495-PCT3 response directed to the therapeutic or antigen portion of the fusion protein through binding of the Fc to Fc(gamma)R receptors on immune cells, thereby increasing NGF-analog uptake and processing in a manner described in FIG. 9. These properties in combination are expected to significantly increase the amount of anti-NGF antibodies while also decreasing the amount of antigen necessary to produce the required immune response.

[0148] In examples, a therapy comprising treatment of a patient with the NGF analog-Fc fusion protein of SEQ ID NO: 15, the NGF analog-Fc fusion protein of SEQ ID NO: 18, the NGF analog-Fc fusion protein of SEQ ID NO: 20 or a pharmaceutical composition of any of these NGF analog-Fc fusion proteins, may consist of a booster vaccine administered to patients that are already antibody-positive to NGF (e.g., patients that may already have received an initial dose of NGF analog-Fc fusion protein vaccine), as a means to amplify their antibody titers and affinity. Furthermore, a therapy comprising a treatment of a patient with the NGF analog-Fc fusion protein of SEQ ID NO: 15, the NGF analog-Fc fusion protein of SEQ ID NO: 18, the NGF analog-Fc fusion protein of SEQ ID NO: 20 or a pharmaceutical composition of any of these NGF analog-Fc fusion proteins, may be administered as a booster vaccine to patients that have been previously immunized with a vaccine against NGF as a means to amplify their antibody titers and affinity specifically against NGF. Such a therapy is important in cases where priming vaccines are not 100% effective and / or where the induced antibody titers wane over time. In examples, the NGF analog-Fc fusion protein of SEQ ID NO: 15, the NGF analog-Fc fusion protein of SEQ ID NO: 18, the NGF analog-Fc fusion protein of SEQ ID NO: 20 or a pharmaceutical composition of any of these NGF analog-Fc fusion proteins may be administered to a patient by subcutaneous injection (s.c.) or intramuscularly (i.m.), as the s.c. or i.m. injection sites are more likely to induce a strong antibody response due to there being more dendritic cells (DCs) in the subcutaneous and intramuscular spaces.Fc Fusion Protein Production

[0149] In embodiments, a fusion protein can be expressed by a cell as described in more detail in the Examples section.Expression and Purification

[0150] An NGF analog-Fc fusion protein can be expressed recombinantly, e.g., in a eukaryotic cell, e.g., mammalian cell or non-mammalian cell. Exemplary mammalian cells used for expression include CHO cells or HEK293 cells. CHO cells can be subdivided into various strains or subclasses, (e.g., CHO DG44, CHO-M, CHO-SE™ and CHO-K1), and some of these cell strains may be genetically engineered for optimal use with a particular type of nucleic acidABC-052PCT / 59495-PCT3 molecule (e.g., a vector comprising DNA) or a particular cell growth media composition as described in the Examples section. Cells may be transfected with a nucleic acid molecule (e.g., vector) encoding the NGF analog-Fc fusion protein (e.g., where the entire NGF analog-Fc fusion protein is encoded by a single nucleic acid molecule). CHO cells may be transfected with a vector that encodes for the NGF analog-Fc fusion protein, but this process only results in temporary expression of the NGF analog-Fc fusion protein for a period of time (e.g., 3 days, 4 days, 5, days, 7 days, 10 days, 12 days, 14 days, or more) before the host cell stops expressing appreciable levels of the NGF analog-Fc fusion protein (i.e., transient transfection). CHO cells that are transiently transfected with nucleic acid sequences encoding for NGF analog-Fc fusion proteins often allow for more rapid production of recombinant proteins which facilitates making and screening multiple NGF analog-Fc fusion protein candidates. CHO cells may be transfected with a vector that is permanently incorporated into the host cell DNA and leads to consistent and permanent expression (i.e., stable transfection) of the NGF analog-Fc fusion protein as long as the cells are cultured appropriately. CHO cells and CHO cell lines that are stably transfected with nucleic acids encoding for NGF analog-Fc fusion proteins often take longer to develop, but they often produce higher protein yields and are more amenable to manufacturing low-cost products (e.g., products for use in the veterinary pharmaceutical market). Cells and cell lines can be cultured using standard methods known in the art. Synthesis and methods of making an NGF analog-Fc fusion protein in transiently transfected CHO cells is described in Example 1, and synthesis and methods of making an NGF analog-Fc fusion protein in stably transfected CHO cells is described in Example 2.

[0151] In examples, the NGF analog-Fc fusion protein may be purified or isolated from the cells (e.g., by lysis of the cells). The NGF analog-Fc fusion protein is secreted by the cells and may be purified or isolated from the cell culture media in which the cells were grown. Purification of the NGF analog-Fc fusion protein can include using column chromatography (e.g., affinity chromatography) or using other separation methods based on differences in size, charge, and / or affinity for certain molecules. Purification of the NGF analog-Fc fusion protein involves selecting or enriching for proteins containing an Fc fragment, e.g., by using Protein A beads or a Protein A column that cause proteins containing an Fc fragment to become bound with affinity at neutral solution pH to the Protein A covalently conjugated to the Protein A beads. The bound NGF analog-Fc fusion protein may then be eluted from the Protein A beads by a change in a solution variable (e.g., a decrease in the solution pH). Other separation methods such as ion exchange chromatography and / or gel filtration chromatography can also be employed alternatively or additionally. Purification of the NGF analog-Fc fusion protein may further comprise filtering or centrifuging the protein preparation, diafiltration, ultrafiltration,ABC-052PCT / 59495-PCT3 and filtration through porous membranes of various sizes, as well as final formulation with excipients. Purification of an NGF analog-Fc fusion protein is described in Example 3.

[0152] The purified NGF analog-Fc fusion protein can be characterized, e.g., for purity, protein yield, structure, and / or activity, using a variety of methods, e.g., absorbance at 280 nm (e.g., to determine protein yield), size exclusion or capillary electrophoresis (e.g., to determine the molecular weight, percent aggregation, and / or purity), mass spectrometry (MS) and / or liquid chromatography (LC-MS) (e.g., to determine purity and / or glycosylation), and / or ELISA (e.g., to determine extent of binding, e.g., affinity, to a NGF antibody or a cell receptor such as a TrkA or p75(NTR) cell receptor). Exemplary methods of characterization are also described in Example 5 and Example 6.

[0153] The protein yield of an NGF analog-Fc fusion protein after production in transiently transfected CHO cells and protein A purification may be greater than 5 mg / L, 10 mg / L, or 20 mg / L, or more preferably greater than 50 mg / L (e.g., greater than 60 mg / L, greater than 70 mg / L, greater than 80 mg / L, greater than 90 mg / L, greater than 100 mg / L).Pharmaceutical Compositions and Routes of Administration

[0154] The amount and concentration of the NGF analog-Fc fusion protein in the pharmaceutical compositions, as well as the quantity of the pharmaceutical composition administered to a patient, can be selected based on clinically relevant factors, such as medically relevant characteristics of the patient (e.g., age, weight, gender, other medical conditions, and the like), the solubility of compounds in the pharmaceutical compositions, the potency and activity of the compounds, and the manner of administration of the pharmaceutical compositions.

[0155] Formulations of the present disclosure include those suitable for parenteral administration. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by intravenous, intramuscular, or subcutaneous injection.

[0156] Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical compositions of the disclosure include water, saline, ethanol, salts, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate, buffering agents, such as potassium and / or sodium phosphates, pH buffers, such as hydrochloric acid and / or sodium hydroxide, and the like. Proper fluidity can be maintained, for example, by the use of coating or emulsifier materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants, e.g., Tween-like surfactants.ABC-052PCT / 59495-PCT3In some examples, the pharmaceutical composition (e.g., as described herein) comprises a Tween-like surfactant, e.g., polysorbate-20, Tween-20 or Tween-80. In some examples, the pharmaceutical composition (e.g., as described herein) comprises a Tween-like surfactant, e.g., Tween-80, at a concentration between about 0.001% and about 2%, or between about 0.005% and about 0.1%, or between about 0.01% and about 0.5%. In other examples, aqueous and nonaqueous carriers comprise an adjuvant. In still other examples, the aqueous and non-aqueous carriers comprise Quil-A®.

[0157] The NGF analog-Fc fusion protein may be administered as a bolus, infusion, or an intravenous push, or administered through syringe injection, pump, pen, needle, or indwelling catheter. The NGF analog-Fc fusion protein may be administered by a subcutaneous bolus injection. In examples, the NGF analog-Fc fusion protein or a pharmaceutical composition thereof is administered to a patient by subcutaneous injection (s.c.) or intramuscularly (i.m.), as the s.c. or i.m. injection sites are more likely to induce a strong antibody response due to there being more dendritic cells (DCs) in the subcutaneous and intramuscular spaces. Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow- release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non- degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site. Additional pharmaceutically acceptable ingredients for use in the compositions include buffering agents, salts, stabilizing agents, diluents, preservatives, antibiotics, isotonic agents, and the like.Dosages

[0158] In use, a therapeutically effective amount of the NGF analog-Fc fusion protein is administered to a patient in need thereof. Administration of the NGF analog-Fc fusion protein elicits an immune response in the patient, and more specifically an immune response against NGF, relieving chronic pain symptoms caused e.g., by osteoarthritis. The immune response is demonstrated by a lack of observable clinical symptoms, or reduction of clinical symptoms normally displayed by an afflicted patient. In another embodiment, a method of activating an immune cell at a site of infection or disease is provided comprising administering a therapeutically effective amount of the NGF analog-Fc fusion protein to a patient. In another aspect, a method of increasing antibody production in a patient is provided comprising administering a therapeutically effective amount of the NGF analog-Fc fusion protein to a patient.ABC-052PCT / 59495-PCT3

[0159] Actual dosage levels of the NGF analog-Fc fusion protein can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient. The selected dosage level will depend upon a variety of factors including the activity of the particular fusion protein employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular fusion protein employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. In general, a suitable dose of an NGF analog-Fc fusion protein will be the amount that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0160] The immunogenic formulation is provided, in various aspects, in unit dosage form for ease of administration and uniformity of dosage. “Unit dosage form” as used herein generally means physically discrete units suited as unitary dosages for the patient to be treated, each unit containing a predetermined quantity of the NGF analog-Fc fusion protein calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms is dictated by and are directly dependent on the unique characteristics of the excipient(s) and therapeutic agent(s) and the particular biological effect to be achieved. In one or more embodiments, the formulation is provided in a kit of components for administration of the NGF analog-Fc fusion protein to the patient. In one or more embodiments, a pharmaceutical composition comprising the NGF analog-Fc fusion protein dispersed in a suitable carrier is provided in a unit dosage form (e.g., vial). In one or more embodiments, the kit further comprises a discrete unit dosage form (e.g., vial) containing an adjuvant and / or other carrier system for onsite mixing of the NGF analog-Fc fusion protein for administration. In one or more embodiments, the kit comprises one or more emulsifying needles and syringes for onsite mixing of the immunogenic formulation for administration. In one or more embodiments, the kit comprises one or more dosing syringes for administering the prepared immunological composition to the patient. In one or more embodiments, the kit further comprises instructions for preparing the immunogenic composition and / or administering the immunogenic composition.

[0161] The present disclosure contemplates formulation of an NGF analog-Fc fusion protein in any of the aforementioned pharmaceutical compositions and preparations. Furthermore, the present disclosure contemplates administration via any of the foregoing routes of administration. One of skill in the art can select the appropriate formulation, dose level and route of administration based on the condition being treated and the overall health, age, and size of theABC-052PCT / 59495-PCT3 patient being treated.EXAMPLES

[0162] The present technology is further illustrated by the following Examples. It is to be understood, however, that these examples are provided by way of illustration, and nothing therein should be taken as a limitation upon the overall scope of the technology.General Examples for Synthesis, Purification and Validation of NGF Analog-Fc Fusion ProteinsExample 1: Synthesis and Methods of Makins an NGF Analog-Fc Fusion Protein in Transiently Transfected CHO Cells.

[0163] NGF analog-Fc fusion proteins were synthesized as follows. A gene sequence of interest was constructed using proprietary software (Curia, Belmont, CA) and was cloned into a high expression mammalian vector. CHO cells were seeded in a shake flask 24 hours before transfection and were grown using serum-free chemically defined media. A DNA expression construct that encoded the NGF analog-Fc fusion protein of interest was transiently transfected into a suspension of CHO cells using the (Curia, Belmont, CA) standard operating procedure for transient transfection. After 20 hours, the cells were counted to determine the viability and viable cell count, and the titer was measured by ForteBio® Octet® (Pall ForteBio LLC, Fremont, CA). Additional readings were taken throughout the transient transfection production run. The culture was harvested on or after Day 14.Example 2: Synthesis and Methods of Making an NGF Analog-Fc Fusion Protein in CHO Cells.

[0164] A CHO cell line is originally derived from CHO-K1 (Curia, Belmont, CA), and the endogenous glutamine synthetase (GS) genes are knocked out by recombinant technology using methods known in the art. Stable expression DNA vectors are designed and optimized for CHO expression and GS selection and incorporated into a high expression mammalian vector (Curia, Belmont, CA). The sequence of each completed construct is confirmed prior to initiating scale- up experiments. The suspension-adapted CHO cells are cultured in a humidified 5% CO2 incubator at 37°C in a chemically defined media (CD OptiCHO; Invitrogen, Carlsbad, CA). No serum or other animal-derived products are used in culturing the CHO cells.

[0165] Approximately 80 million suspension-adapted CHO cells, growing in CD OptiCHO media during the exponential growth phase, are transfected by electroporation using MaxCyte® STX® system (MaxCyte, Inc., Gaithersburg, MD) with 80 pg DNA to create a stable CHO cellABC-052PCT / 59495-PCT3 line for each NGF analog-Fc fusion protein (DNA construct contains the full-length sequence of the NGF analog-Fc fusion protein). After twenty-four hours, the transfected cells are counted and placed under selection for stable integration of the NGF analog-Fc fusion genes. The transfected cells are seeded into CD OptiCHO selection media containing between 0-100 pM methionine sulfoximine (MSX) at a cell density of 0.5* 106cells / mL in a shaker flask and incubated at 37°C with 5% CO2. During a selection process, the cells are spun down and resuspended in fresh selection media every 2-3 days until the CHO stable pool recovers its growth rate and viability. The cell culture is monitored for growth and titer.

[0166] The cells are grown to 2.5* 106cells per mL. At the time of harvest for cell banking, the viability is expected to be above 95%. The cells are then centrifuged, and the cell pellet is resuspended in the CD OptiCHO media with 7.5% dimethyl sulfoxide (DMSO) to a cell count of 15x 106cells per mL per vial. Vials are cryopreserved for storage in liquid nitrogen.

[0167] A small scale-up production is performed using the CHO cells as follows. The cells are scaled up for production in CD OptiCHO growth medium containing 100 pM MSX at 37°C and fed every 2-4 days as needed, with CD OptiCHO growth medium supplemented with glucose and additional amino acids as necessary for approximately 14-21 days. The conditioned media supernatant harvested from the stable pool production run is clarified by centrifuge spinning. The protein is run over a Protein A (Mab Select, GE Healthcare, Little Chalfont, United Kingdom) column pre-equilibrated with binding buffer. Washing buffer is then passed through the column until the OD280 value (NanoDrop, Thermo Fisher Scientific) is measured to be at or near background levels. The NGF analog-Fc fusion protein is eluted using a low pH buffer, elution fractions are collected, and the OD280 value of each fraction is recorded. Fractions containing the target NGF analog-Fc fusion protein are pooled and optionally further filtered using a 0.2 pm membrane filter.

[0168] The cell line is optionally further subcloned to monoclonality and optionally further selected for high titer NGF analog-Fc-fusion protein-expressing clones using the method of limiting dilution, a method known to those skilled in the art. After obtaining a high titer, monoclonal NGF analog-Fc fusion protein-expressing cell line, production of the NGF analog- Fc fusion protein is accomplished as described above in growth medium without MSX, or optionally in growth medium containing MSX, to obtain a cell culture supernatant containing the recombinant, CHO-made, NGF analog-Fc fusion protein. The MSX concentration is optionally increased over time to exert additional selectivity for clones capable of yielding higher product titers.Example 3: Purification of an NGF Analog-Fc Fusion Protein Manufactured in CHO Cells.ABC-052PCT / 59495-PCT3

[0169] Purification of an NGF analog-Fc fusion protein was performed as follows. Conditioned media supernatants containing the secreted NGF analog-Fc fusion protein were harvested from the CHO production runs and were clarified by centrifugation. The supernatant containing the desired NGF analog-Fc fusion protein was run over a Protein A column, washed and eluted using a low pH gradient. Afterwards, the eluted fractions containing the desired protein were pooled and buffer exchanged into 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer. A final filtration step was performed using a 0.2 pm membrane filter. The final protein concentration was calculated from the solution optical density at 280 nm. Further optional purification by ion-exchange chromatography (e.g., using an anion exchange bead resin or a cation exchange bead resin), gel filtration chromatography, or other methods was performed as necessary.Example 4: NGF Analog-Fc Fusion Protein Structure Confirmation by Non-Reducing and Reducing SDS-PAGE.

[0170] An NGF analog-Fc fusion protein sample for analysis was prepared in loading buffer (± reductant; e.g., beta-mercaptoethanol) and denatured at 70°C for 10 min before loaded into the NuPAGE™ Gel system (Thermo Fisher Scientific). After electrophoresis, the gel was stained with SimplyBlue™ SafeStain. Under non-reducing conditions, the sample was run against known molecular weight (MW) protein standards, and the eluting peak represents the ‘apparent’ MW of the fusion protein homodimer.

[0171] Under reducing conditions (e.g., using beta-mercaptoethanol to break disulfide bonds of the NGF analog-Fc fusion protein homodimer), the apparent MW of the resulting NGF analog-Fc fusion protein monomer was compared against half the molecular weight of the NGF analog-Fc fusion protein homodimer as a way of determining that the structural purity of the NGF analog-Fc fusion protein was likely to be correct.Example 5: NGF Analog-Fc Fusion Protein Sequence Identification by LC-MS with Glycan Removal.

[0172] To obtain an accurate estimate of the NGF analog-Fc fusion protein mass via mass spectroscopy (MS), the sample is first treated to remove naturally occurring glycan that might interfere with the MS analysis. 100 pL of an NGF analog-Fc fusion protein dissolved in 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer solution is first buffer exchanged into 0.1 M Tris, pH 8.0 buffer containing 5 mM EDTA using a Zeba desalting column (Pierce, Thermo Fisher Scientific, Waltham, MA). 2 *x 1.67 pL of PNGase F enzyme (Prozyme N-ABC-052PCT / 59495-PCT3 glycanase) is added to this solution to remove N-linked glycan present in the fusion protein (e.g., glycan linked to the side chain of the asparagine located at the cNg-N site), and the mixture is incubated at 37°C overnight in an incubator. The sample is then analyzed via LC-MS (NovaBioassays, Woburn, MA) resulting in a molecular mass of the molecule which corresponds to the desired homodimer without the glycan. This mass is then further corrected since the enzymatic process used to cleave the glycan from the cNg-asparagine also deaminates the asparagine side chain to form an aspartic acid, and in doing so the enzymatically treated homodimer gains 2 Da overall, corresponding to a mass of 1 Da for each chain present in the homodimer. Therefore, the actual molecular mass is the measured mass minus 2 Da to correct for each of the enzymatic modifications of the NGF analog-Fc fusion protein structure in the analytical sample.Example 6: %Homodimer by Size-Exclusion Chromatography for an NGF Analog-Fc Fusion Protein.

[0173] Size-exclusion chromatography (SEC-HPLC) of NGF analog-Fc fusion proteins is carried out using an Agilent HPLC (Santa Clara, CA) or Waters 2795HT HPLC (Waters Corporation, Milford, MA) connected to a Photodiode array at a wavelength of 280 nm. 100 pL or less of a sample containing a NGF analog-Fc fusion protein of interest is injected into a MAbPac SEC-1, 5 pm, 4 x 300 mm column (Thermo Fisher Scientific, Waltham, MA) operating at a flow rate of 0.2 mL / min and with a mobile phase comprising 50 mM sodium phosphate, 300 mM NaCl, and 0.05% w / v sodium azide, pH 6.2. The MAbPac SEC-1 column operates on the principle of molecular size separation. Therefore, larger soluble NGF analog-Fc aggregates (e.g., multimers of NGF analog-Fc fusion protein homodimers) elute at earlier retention times, and the non-aggregated homodimers elute at later retention times. In separating the mixture of homodimers from aggregated multimeric homodimers via analytical SEC-HPLC, the purity of the NGF analog-Fc fusion protein solution in terms of the percentage of nonaggregated homodimer is ascertained.Example 7: In vitro Fc (Gamma) and FcRn Receptor Binding Affinity for an NGF Analog-Fc Fusion Protein.

[0174] The binding of an NGF analog-Fc fusion protein to Fc(gamma) receptors at pH 7.4 is conducted using an ELISA assay as follows. Human Fc(gamma) receptors I, Ila, lib, III and the FcRn receptor are used as mammalian receptors. An NGF analog-Fc fusion protein is diluted to 10 pg / mL in sodium bicarbonate buffer at pH 9.6 and coated on Maxisorp (Nunc) microtiterABC-052PCT / 59495-PCT3 plates overnight at 4°C, after which the microplate strips are washed 5 times with PBST (PBS / 0.05% Tween-20) buffer and blocked with Superblock blocking reagent (Thermo Fisher Scientific). Serial dilutions of biotinylated rhFc(gamma) receptors (recombinant human Fc(gamma)RI, Fc(gamma)RIIa, Fc(gamma)RIIb, Fc(gamma)RIII, FcRn; R&D Systems) are prepared in PBST / 10% Superblock buffer from 6000 ng / mL to 8.2 ng / mL and loaded at 100 pL / well onto the microplate strips coated with the NGF analog-Fc fusion protein. The microtiter plate is incubated for 1 hour at room temperature after which the microplate strips are washed 5 times with PBST and then loaded with 100 pL / well of streptavidin-HRP diluted 1 :10000 in PBST / 10% Superblock buffer. After incubating for 45 min, the microplate strips are washed again 5 times with PBST. Trimethylbenzidine (TMB) is added to reveal the bound Fc(gamma) or FcRn receptor proteins and stopped with ELISA stop reagent (Boston Bioproducts). The plate is read in an ELISA plate reader at 450 nm, and the OD values (proportional to the binding of each rhFc(gamma) or FcRn receptor to the NGF analog-Fc fusion protein) are plotted against log concentrations of each rhFc(gamma) receptor or FcRn receptor added to each well to generate binding curves using GraphPad Prism software.Example 8: hi vitro ELISA Assay for Evaluating Anti-NGF Antibody Levels in Serum.

[0175] Serum anti-NGF antibody titers were determined by enzyme-linked immunosorbent assay (ELISA). Serum samples were diluted in sample dilution buffer in Sample Dilution Buffer (SDB; contained a mixture of 10% Superblock (Thermo Fisher Scientific) in PBS / 0.05% Tween 20) at 1 : 100-1 :500 and then added to microtiter plate wells that were coated with human NGF- beta (Millipore Sigma, Catalog# N1408-.1MG) protein in carbonate buffer, blocked with SuperBlock™ (Thermo Fisher Scientific) and then incubated with diluted serum test samples for one hour. It should be noted that human NGF-beta has a known sequence and structure that is similar to that from other species (e.g., dogs). Mouse IgG antibody (non-specific, Jackson Immunoresearch) in carbonate buffer was directly coated to microplate wells with the mouse IgG antibody pre-diluted via serial dilutions to create a standard curve that allowed for quantitation of antibody (Ab) titers in the serum test samples. After washing the plate with wash buffer (PBS / 0.05% Tween 20; PBST) to remove all unbound serum proteins, the bound NGF specific mouse IgG Abs bound to the plate (precoated mouse IgG standards or mouse anti-NGF Abs bound to the NGF-coated microplate) were detected by incubating microplate wells with goat anti-mouse-specific IgG Fc conjugated to HRP enzyme diluted between 1 :4000 and 1 : 30000 (SouthernBiotech, Catalog# 1013-05) in SDB for 1 hour. Following washes with PBST buffer, trimethylbenzidine (TMB) reagent was added to each well that was catalyzed by the HRPABC-052PCT / 59495-PCT3 enzyme and incubated for 10-20 minutes. This caused a colorimetric change that was proportional to the amount of bound HRP-antibody conjugate. The enzyme substrate reaction was then stopped by the addition of Stop Reagent (1% H2SO4) and the color intensity (optical density, OD) of each well was measured using a spectrophotometric microplate reader at 450 nm wavelength. The OD450 values were further converted to antibody titers (e.g., levels) using the mouse IgG standards, using a 4-parameter regression curve fit algorithm in SoftMax Pro software (Molecular Devices). Responders to the administered therapy in each test article group were defined as animals having an antibody titer on a given day that was more than twice the level of that measured on Day 0 for each individual animal.Example 9: In vivo General Preclinical Evaluation of the Effectiveness of NGF Analog-Fc Formulations in Inducing an Anti-NGF I f} Ab Titer in Mice.

[0176] Groups of female BALB / c mice (N=5; (Jackson Laboratories, Bar Harbor, ME)) at 6-8 weeks of age were injected s.c. on Days 0 and 21 with the respective dose levels of test compounds made via Example 1 and purified via Example 3. Mice were administered up to three subcutaneous doses of between 15 - 100 pg of the NGF analog-Fc fusion protein of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14. SEQ ID NO: 15, SEQ ID NO: 18 and SEQ ID NO: 20 with Montanide™ ISA 720 (30% / 70% v / v). Additional dosing was performed on Day 42 and an additional blood collection was performed on Day 56.

[0177] All mice were non-terminally bled via submandibular venipuncture before dosing and 14 days after each injection. Blood samples were allowed to clot and were centrifuged to obtain serum samples for anti-NGF antibody titer assessment by ELISA according to the methods described in Example 8.

[0178] The anti-NGF antibody titer values of the novel NGF analog-Fc fusion proteins of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18 and SEQ ID NO: 20 are shown in Table 2.ABC-052PCT / 59495-PCT3*LLOQ: Lower Limit of Quantification.Example 10: In vivo General Preclinical Evaluation of the Effectiveness of NGF Analog-FcFormulations in Inducing an Anti-NGF IgG Ab Titer in Dogs.

[0179] Groups of dogs of mixed ages are injected either subcutaneously (s.c.) or intramuscularly (i.m.) on Days 0 and 14 with adjuvant, and again on Day 21 without adjuvant, with the respective dose levels of test compounds made via Example 1 and purified via Example 3. Dogs are administered up to three s.c. or i.m. doses of 90 pg of the NGF analog-Fc fusionABC-052PCT / 59495-PCT3 protein of SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 20 with or without adjuvants (Montanide™ ISA 720 (30% / 70% v / v), Montanide™ GEL-02, or Advac / CpG mixture).

[0180] All dogs are non-terminally bled via submandibular venipuncture before dosing (Day 0) and 14 days after each injection (Day 14, Day 28, Day 42). Additional blood collections are performed on Day 56 and additional booster dosing may be performed on Day 56. Blood samples are allowed to clot and are centrifuged to obtain serum samples for anti-NGF antibody titer assessment by ELISA according to the methods described in Example 11.Example 11: In vitro ELISA Assay for Evaluating Anti-NGF Antibody Levels in Canine Serum.

[0181] Serum anti-NGF antibody titers are determined by enzyme-linked immunosorbent assay (ELISA). Serum samples are diluted in Sample Dilution Buffer (SDB; contains a mixture of 10% Superblock (Thermo Fisher Scientific) in PBS / 0.1% Tween 20 and 10% horse serum) at 1 :100-1 :500 and are then added to microtiter plate wells that are coated with human NGF- beta (Millipore Sigma, Catalog# N1408-.1MG) protein in carbonate buffer, blocked with SuperBlock™ (Thermo Fisher Scientific) and are then incubated with diluted serum test samples for one hour at 22° C. Human NGF-beta has a known sequence and structure that is similar to that from other species, including dogs. Dog IgG antibody (Rockland Inc., Catalog# 004-0102- 0005) in carbonate buffer is directly coated to microplate wells with the dog IgG antibody prediluted via serial dilutions to create a standard curve that allows for quantitation of antibody (Ab) titers in the serum test samples. After washing the plate with wash buffer (PBS / 0.05% Tween 20) to remove all unbound serum proteins, the NGF specific dog IgG Abs bound to the plate (precoated dog IgG standards or dog anti-NGF Abs bound to the NGF-coated microplate) are detected by incubating microplate wells with goat anti-cat-IgG F(ab’)2 conjugated to HRP enzyme diluted between 1 :4000 and 1 :30000 (Jackson ImmunoResearch, Catalog# 102-035- 006) in PBS / 0.1% Tween-20 / 10% Superblock buffer for 1 hour at 22° C. Following washes with wash buffer and purified water, trimethylbenzidine (TMB) reagent is added to each well that is catalyzed by the HRP enzyme and is incubated for 5-20 minutes. This causes a colorimetric change that is proportional to the amount of bound HRP-antibody conjugate. The enzyme substrate reaction is then stopped by the addition of Stop Reagent (1% H2SO4) and the color intensity (optical density, OD) of each well is measured using a spectrophotometric microplate reader at 450 nm wavelength. The OD450 values are further converted to antibody titers (e.g., levels) using the dog IgG standards, using a 4-parameter regression curve fit algorithm in SoftMax Pro software (Molecular Devices). Responders to the administered therapy in each test article group are defined as animals having an antibody titer on a given day that is more thanABC-052PCT / 59495-PCT3 twice the level of that measured on Day 0 for each individual animal.Example 12: In vivo General Preclinical FAduaiion of the Effectiveness of EGF FiaffpEc Formulations in Indticing Anti-NGF IgG Ab Titers in Dogs.

[0182] Groups of dogs (N=l or more per group) of mixed ages (8 to 10 years of age) are injected intramuscularly (i.m.) on Days 0, 14, and 28 with adjuvant mixed with NGF analog-Fc fusion protein made via Example 1 and purified via Example 3. Dogs are administered up to three i.m. doses of 10-90 pg of the NGF analog-Fc fusion protein homodimer of SEQ ID NO: 15 with adjuvants (either Quil-A® [Saponin-based adjuvant] (1 : 1 v / v) or ISA-720 (1 :1 v / v) or SEPIVAC SWE™ (1 : 1 v / v)).

[0183] All dogs are non -terminally bled via venipuncture before dosing (Day -1) and 14 days after each injection (Day 14, Day 28, Day 42). Additional blood collections are performed intermittently between Day 56 and Day 120. Blood samples are allowed to clot and are centrifuged to obtain serum samples for anti-NGF antibody titer assessment by ELISA according to the methods described in Example 11.

[0184] It is expected that the mean anti-NGF antibody titer values treated with the NGF analog-Fc fusion protein homodimer of SEQ ID NO: 15 plus Quil-A® adjuvant in dogs will be higher than the anti-NGF antibody titers exhibited by the NGF analog-Fc fusion protein homodimer of SEQ ID NO: 15 plus ISA-720 or SEPIVAC SWE™ adjuvant.Example 13: In vivo General Preclinical Evaluation of the Effectiveness of NGF Analog-Fc Formulations in Inducing Anti-NGF IgG Ab Titers in Dogs.

[0185] Groups of dogs (N=l or more per group) of mixed ages (8 to 10 years of age) are injected intramuscularly (i.m.) on Days 0, 14, and 28 with adjuvant mixed with NGF analog-Fc fusion protein made via Example 1 and purified via Example 3. Dogs are administered up to three i.m. doses of 10-90 pg of the NGF analog-Fc fusion protein homodimer of SEQ ID NO: 18 with adjuvants (either Quil-A® [Saponin-based adjuvant] (1 : 1 v / v) or ISA-720 (1 :1 v / v) or SEPIVAC SWE™ (1 : 1 v / v)).

[0186] All dogs are non -terminally bled via venipuncture before dosing (Day -1) and 14 days after each injection (Day 14, Day 28, Day 42). Additional blood collections are performed intermittently between Day 56 and Day 120. Blood samples are allowed to clot and are centrifuged to obtain serum samples for anti-NGF antibody titer assessment by ELISA according to the methods described in Example 11.

[0187] It is expected that the mean anti-NGF antibody titer values treated with the NGFABC-052PCT / 59495-PCT3 analog-Fc fusion protein homodimer of SEQ ID NO: 18 plus Quil-A® adjuvant in dogs will be higher than the anti-NGF antibody titers exhibited by the NGF analog-Fc fusion protein homodimer of SEQ ID NO: 18 plus ISA-720 or SEPIVAC SWE™ adjuvant.Example 14: In vivo General Preclinical Evaluation of the Effectiveness of NGF Analo -Fc Formulations in Inducing Anti-NGF I gG Ah Tilers in Dogs.

[0188] Groups of dogs (N=l or more per group) of mixed ages (8 to 10 years of age) are injected intramuscularly (i.m.) on Days 0, 14, and 28 with adjuvant mixed with NGF analog-Fc fusion protein made via Example 1 and purified via Example 3. Dogs are administered up to three i.m. doses of 10-90 pg of the NGF analog-Fc fusion protein homodimer of SEQ ID NO: 20 with adjuvants (either Quil-A® [Saponin-based adjuvant] (1 : 1 v / v) or ISA-720 (1 :1 v / v) or SEPIVAC SWE™ (1 : 1 v / v)).

[0189] All dogs are non -terminally bled via venipuncture before dosing (Day -1) and 14 days after each injection (Day 14, Day 28, Day 42). Additional blood collections are performed intermittently between Day 56 and Day 120. Blood samples are allowed to clot and are centrifuged to obtain serum samples for anti-NGF antibody titer assessment by ELISA according to the methods described in Example 11.

[0190] It is expected that the mean anti-NGF antibody titer values treated with the NGF analog-Fc fusion protein homodimer of SEQ ID NO: 18 plus Quil-A® adjuvant in dogs will be higher than the anti-NGF antibody titers exhibited by the NGF analog-Fc fusion protein homodimer of SEQ ID NO: 20 plus ISA-720 or SEPIVAC SWE™ adjuvant.Example 15: In vivo General Preclinical Evaluation of the Effectiveness of NGF Analog-Fc Formulations for Pain Mitigation in Dogs with Naturally Occurring Osteoarthritis.

[0191] Groups of dogs (N = 5 per group) of mixed ages are injected intramuscularly (i.m.) on Days 0, 14, and 28 with adjuvant, with NGF analog-Fc fusion protein homodimer prepared according to Example 1 and purified according to Example 3. The dogs are injected with up to three i.m. doses of 90 pg of the NGF analog-Fc fusion protein homodimer formulated with adjuvant (Quil-A® [saponin adjuvant] 1 :1 v / v). As a control, an anti-NGF monoclonal antibody treatment (Librela®, bedinvetmab, Zoetis Animal Health, Parsippany, NJ) is administered on Days 0 and 28 at 0.5 mg / kg subcutaneously (s.c.) according to the manufacturer’s instructions, and serum is collected as described in this Example.

[0192] All dogs are non-terminally bled via venipuncture before dosing (Day -1) and 14 days after each injection (Day 13, Day 27, Day 42). Additional blood collections are performedABC-052PCT / 59495-PCT3 on Day 56. Blood samples are allowed to clot and centrifuged to obtain serum, which are analyzed for anti-NGF antibody titers by ELISA according to the methods described in Example 11.

[0193] The dogs are assessed for pain-related functional performance using a modified Canine Brief Pain Inventory (mCBPI) questionnaire. Assessments are conducted over four consecutive days at baseline (Days -4 to -1) and during the treatment phase on Days 10-13, 23-26, and 52-56, at approximately the same time each day. The mCBPI is a laboratory-adapted version of the validated Canine Brief Pain Inventory (CBPI) clinical questionnaire, modified to account for differences between owner-based evaluations and laboratory technician assessments of canine pain (Brown DC, Bell M, Rhodes L. Power of treatment success definitions when the Canine Brief Pain Inventory is used to evaluate carprofen treatment for the control of pain and inflammation in dogs with osteoarthritis. Amer J Vet Rsch 2013; 74( 12): 1467-1473). Lower mCBPI total pain scores and lower mCBPI mobility scores are desirable and correlate with lower pain and more mobility, respectively.

[0194] Specifically, the ability of each dog to walk, trot, gallop, rear, jump over a low obstacle, climb and descend stairs, jump down from a perch, and general activity is evaluated in parallel with a subjective pain score assigned for each observed behavior. All assessments are performed according to standard operating procedures by the same technician for the duration of the study to minimize inter-observer variability; if the technician is unavailable, sessions are recorded and evaluated via video analysis. Staff conducting the mCBPI assessments are blinded from which therapies are given to the treatment groups. Comparisons of mean scores within groups for various timepoints compared to baseline levels are performed using a paired student’ s t-test, with p < 0.05 representing statistical significance at the 95% confidence level. Means and standard error of the means were calculated and shown in the table below. It is expected that treatment of dogs with the homodimer of SEQ ID NO: 15 plus Quil-A® adjuvant will demonstrate similar reductions in the mCBPI total pain and functionality scores as bedinvetmab.Example 16: CE-SDS Characterization of NGF-analog Fc fusion proteins.

[0195] CE-SDS analysis was performed in a LabChip® GXII (Perkin Elmer, Waltham, MA) on a solution of a purified NGF antigen analog Fc fusion protein homodimer dissolved in 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer, and the resulting electropherogram was plotted. Under non-reducing conditions, the sample was run against known molecular weight (MW) protein standards, and the eluting peak represented the ‘apparent’ MW of the NGF antigen analog-Fc fusion protein homodimer.

[0196] Under reducing conditions (e.g. using beta-mercaptoethanol to break disulfide bondsABC-052PCT / 59495-PCT3 of the NGF antigen analog-Fc fusion protein homodimer), the apparent MW of the resulting the NGF antigen analog-Fc fusion protein monomer is compared against half the molecular weight of the NGF antigen analog-Fc fusion protein homodimer as a way of determining that the structural purity of the NGF antigen analog-Fc fusion protein homodimer is likely to be correct.

[0197] The non-reducing and reducing main peaks found via CE-SDS analysis for the NGF antigen analog-Fc fusion protein homodimer synthesized in CHO cells are shown in Table 3, and 2* the apparent MW of the resulting NGF antigen analog-Fc fusion protein monomer under reducing conditions was compared to the molecular weight of the NGF antigen analog-Fc fusion protein homodimer under non-reducing conditions. The results in Table 3 illustrate that the structural conformations of the NGF antigen analog-Fc fusion protein homodimer are likely to be correct.EQUIVALENTS

[0198] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0199] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in anyABC-052PCT / 59495-PCT3 other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the disclosure, or aspects of the disclosure, is / are referred to as comprising particular elements and / or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprise(s),” “comprising,” “contain(s),” and “containing” are intended to be open and the use thereof permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0200] Additional advantages of the various embodiments of the technology will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments but is not necessarily included. Thus, the present invention encompasses a variety of combinations and / or integrations of the specific embodiments described herein.

[0201] The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting "greater than about 10" (with no upper bounds) and a claim reciting "less than about 100" (with no lower bounds).

Claims

ABC-052PCT / 59495-PCT3We claim:

1. A fusion protein comprising a NGF analog and an Fc fragment, wherein the NGF analog and the Fc fragment are connected by a peptide linker, wherein the NGF analog comprises the following sequence:EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTR NITVDPRLFKSSRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSS RSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFK QYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAW RFIRIDTACVCVLSRKAVRRA (SEQ ID NO: 17).

2. The fusion protein of claim 1, wherein the Fc fragment comprises the following sequence:DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQIS WFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVN NKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDID VEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICA VMHEALHNHYTQESLSHSPG (SEQ ID NO: 2).

3. The fusion protein of claim 1 or 2, wherein the linker comprises the following sequence:GGGSGGGS (SEQ ID NO: 9).

4. The fusion protein of any of claims 1-3, wherein the fusion protein comprises the following sequence:EPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTR NITVDPRLFKSSRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSS RSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFK QYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAW RFIRIDTACVCVLSRKAVRRAGGGSGGGSDCPKCPAPEMLGGPSVFIFPPKP KDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFN GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSV YVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQL DEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 15).

5. The fusion protein of any of claims 1-4, wherein the Fc fragment is glycosylated.

6. An immunogenic composition comprising a fusion protein according to any of claims 1-ABC-052PCT / 59495-PCT35 and a pharmaceutically acceptable carrier.

7. The immunogenic composition of claim 6, further comprising an adjuvant.

8. A method for increasing antibody production in a subject against an antigenic agent, the method comprising administering a therapeutically effective amount of a fusion protein according to any of claims 1-5 to said subject.

9. The method of claim 8, wherein the subject is antibody naive prior to administration of the fusion protein.

10. The method of claim 8 or 9, wherein the subject has a measurable antibody titer against said antigenic agent prior to administration of the fusion protein.

11. The method of any of claims 8-10, wherein the fusion protein is administered via injection.

12. The method of any of claims 8-11, wherein the fusion protein is administered subcutaneously or intramuscularly.

13. The method of any of claims 8-12, wherein the fusion protein is co-administered with an adjuvant.

14. A method of producing a fusion protein of any of claims 1-5, said method comprising transiently transfecting a nucleic acid encoding for the fusion protein into a CHO cell, wherein the transfected CHO cell expresses the fusion protein, and wherein the yield of the purified or isolated fusion protein is greater than 75 mg / L in any of the foregoing expression systems.

15. A cell engineered to express a fusion protein of any of claims 1-5.

16. A cDNA encoding a fusion protein of any of claims 1-5.

17. A fusion protein comprising the sequence of SEQ ID NO: 15 or pharmaceutical composition thereof for use in treatment of pain associated with a pain-associated ailment.

18. A fusion protein comprising the sequence of SEQ ID NO: 15 or pharmaceutical composition thereof for use in treatment of pain associated with osteoarthritis.

19. A fusion protein comprising a NGF analog and an Fc fragment, wherein the NGF analog and the Fc fragment are connected by a peptide linker, wherein the NGF analog comprises the following sequence:EPHSESNVPAGHTIPQAHWTKLQHSLDTALRD ARSAP AAAIAARVAGQTRABC-052PCT / 59495-PCT3NITVDPRLFKSSRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSS RSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFK QYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAW RFIRIDTACVCVLSRKAVRDA (SEQ ID NO: 19).

20. The fusion protein of claim 19, wherein the Fc fragment comprises the following sequence:DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQIS WFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVN NKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDID VEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICA VMHEALHNHYTQESLSHSPG (SEQ ID NO: 2).

21. The fusion protein of claim 19 or 20, wherein the linker comprises the following sequence:GGGSGGGS (SEQ ID NO: 9).

22. The fusion protein of any of claims 19-21, wherein the fusion protein comprises the following sequence:EPHSESNVPAGHTIPQAHWTKLQHSLDTALRD ARSAP AAAIAARVAGQTR NITVDPRLFKSSRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSS RSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFK QYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAW RFIRIDTACVCVLSRKAVRDAGGGSGGGSDCPKCPAPEMLGGPSVFIFPPKP KDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFN GTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSV YVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQL DEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 18).

23. The fusion protein of any of claims 19-22, wherein the Fc fragment is glycosylated.

24. An immunogenic composition comprising a fusion protein according to any of claims 19-23 and a pharmaceutically acceptable carrier.

25. The immunogenic composition of claim 24, further comprising an adjuvant.

26. A method for increasing antibody production in a subject against an antigenic agent, the method comprising administering a therapeutically effective amount of a fusion proteinABC-052PCT / 59495-PCT3 according to any of claims 19-23 to said subject.

27. The method of claim 26, wherein the subject is antibody naive prior to administration of the fusion protein.

28. The method of claim 26 or 27, wherein the subject has a measurable antibody titer against said antigenic agent prior to administration of the fusion protein.

29. The method of any of claims 26-28, wherein the fusion protein is administered via injection.

30. The method of any of claims 26-29, wherein the fusion protein is administered subcutaneously or intramuscularly.

31. The method of any of claims 26-30, wherein the fusion protein is co-administered with an adjuvant.

32. A method of producing a fusion protein of any of claims 19-23, said method comprising transiently transfecting a nucleic acid encoding for the fusion protein into a CHO cell, wherein the transfected CHO cell expresses the fusion protein, and wherein the yield of the purified or isolated fusion protein is greater than 75 mg / L in any of the foregoing expression systems.

33. A cell engineered to express a fusion protein of any of claims 19-23.

34. A cDNA encoding a fusion protein of any of claims 19-23.

35. A fusion protein comprising the sequence of SEQ ID NO: 18 or pharmaceutical composition thereof for use in treatment of pain associated with a pain-associated ailment.

36. A fusion protein comprising the sequence of SEQ ID NO: 18 or pharmaceutical composition thereof for use in treatment of pain associated with osteoarthritis.

37. The fusion protein of claim 19 or 20, wherein the linker comprises the following sequence:GGGGGSGGGSGGGGSGGS (SEQ ID NO: 21).

38. The fusion protein of any of claims 19, 20 or 37, wherein the fusion protein comprises the following sequence:EPHSESNVPAGHTIPQAHWTKLQHSLDTALRD ARSAP AAAIAARVAGQTR NITVDPRLFKSSRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSS RSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKABC-052PCT / 59495-PCT3QYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAW RFIRIDTACVCVLSRKAVRDAGGGGGSGGGSGGGGSGGSDCPKCPAPEML GGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQT AKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTIS KARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQE PESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHY TQESLSHSPG (SEQ ID NO: 20).

39. The fusion protein of any of claims 19, 20, 37 or 38, wherein the Fc fragment is glycosylated.

40. An immunogenic composition comprising a fusion protein according to any of claims 19, 20, 37, 38 or 39 and a pharmaceutically acceptable carrier.

41. The immunogenic composition of claim 40, further comprising an adjuvant.

42. A method for increasing antibody production in a subject against an antigenic agent, the method comprising administering a therapeutically effective amount of a fusion protein according to any of claims 19, 20, 37, 38 or 39 to said subject.

43. The method of claim 42, wherein the subject is antibody naive prior to administration of the fusion protein.

44. The method of claim 42 or 43, wherein the subject has a measurable antibody titer against said antigenic agent prior to administration of the fusion protein.

45. The method of any of claims 42-44, wherein the fusion protein is administered via injection.

46. The method of any of claims 42-45, wherein the fusion protein is administered subcutaneously or intramuscularly.

47. The method of any of claims 42-46, wherein the fusion protein is co-administered with an adjuvant.

48. A method of producing a fusion protein of any of claims 19, 20, 37, 38 or 39, said method comprising transiently transfecting a nucleic acid encoding for the fusion protein into a CHO cell, wherein the transfected CHO cell expresses the fusion protein, and wherein the yield of the purified or isolated fusion protein is greater than 75 mg / L in any of the foregoing expression systems.

49. A cell engineered to express a fusion protein of any of claims 19, 20, 37, 38 or 39.ABC-052PCT / 59495-PCT350. A cDNA encoding a fusion protein of any of claims 19, 20, 37, 38 or 39.

51. A fusion protein comprising the sequence of SEQ ID NO: 20 or pharmaceutical composition thereof for use in treatment of pain associated with a pain-associated ailment.

52. A fusion protein comprising the sequence of SEQ ID NO: 20 or pharmaceutical composition thereof for use in treatment of pain associated with osteoarthritis.

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