Disclosed are compositions and methods related to anti-ACAT1 binding molecules that preferentially bind glycosylated ACAT1.

REFERENCE TO SEQUENCE LISTING

The sequence listing submitted on Apr. 17, 2023, as an .XML file entitled “10110-367WO1” created on Apr. 17, 2023, and having a file size of 19,512 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

Immuno-oncology today is sharply focused on T cells. However, B cells can present antigens and provide co-stimulation to T cells and coordinated T and B cell activation needs to occur to maximize immune protection. In the tumor microenvironment, T and B cells frequently interact, but cooperation between humoral and cellular responses remains incompletely understood. T follicular helper (Tfh) cell-driven B cell responses play a role in the effectiveness of checkpoint inhibitors. However, the drivers of Tfh conversion at tumor beds remain unknown. Some T:B cell intra-tumoral interactions take place in highly organized conglomerates that recapitulate the architecture of secondary lymph nodes, termed tertiary lymphoid structures (TLS). Similar to TLS in autoimmune conditions, some intra-tumoral TLS contain germinal centers with interdigitating follicular dendritic cells, plus an adjacent T cell zone composed by CD4+ and CD8+ lymphocytes and high endothelial venules. The presence of TLS is associated with positive outcomes in multiple human malignancies, including ovarian, pancreatic, colorectal, bladder and non-small cell lung cancer. Given the importance of B cells to a positive outcome and the association of the presence of TLS with positive outcomes, what are needed are new binding molecules that target tumors and facilitate TLS formation.

Disclosed are methods and compositions related to binding molecules of ACAT1.

In one aspect, disclosed herein are anti-anti-acetyl-CoA acetyltransferase 1 (ACAT1) binding molecules (such as, for example, antibodies, diabodies, nanobodies, scFv, sFv, and immunotoxins) that specifically binds glycosylated ACAT1, and wherein the anti-ACAT1 binding molecule comprising one or more variable heavy chain complementarity determining regions (CDRs) as set forth in SEQ ID Nos 1-3 (such as, for example the variable heavy chain as set forth in SEQ ID NO: 7) and one or more variable light chain complementarity determining regions (CDRs) as set forth in SEQ ID Nos 4-6 (such as, for example the variable light chain as set forth in SEQ ID NO: 9). In one aspect, the anti-ACAT1 binding molecule is an IgG, IgA, or IgM antibody.

Also disclosed herein are methods of treating, reducing, decreasing, inhibiting, ameliorating, and or preventing a cancer and/or metastasis (such as for example, ovarian, pancreatic, colorectal, bladder and non-small cell lung cancer) comprising administering to the subject the ACAT1 binding molecule of any preceding aspect. For example, disclosed herein are methods of treating, reducing, decreasing, inhibiting, ameliorating, and or preventing a cancer and/or metastasis (such as for example, ovarian cancer) in a subject in need thereof comprising administering to the subject an anti-acetyl-CoA acetyltransferase 1 (ACAT1) binding molecule (such as, for example, antibodies, diabodies, nanobodies, scFv, sFv, and immunotoxins) that specifically binds glycosylated ACAT1, and wherein the anti-ACAT1 binding molecule comprising one or more variable heavy chain complementarity determining regions (CDRs) as set forth in SEQ ID Nos 1-3 (such as, for example the variable heavy chain as set forth in SEQ ID NO: 7) and one or more variable light chain complementarity determining regions (CDRs) as set forth in SEQ ID Nos 4-6 (such as, for example the variable light chain as set forth in SEQ ID NO: 9). In one aspect, the anti-ACAT1 binding molecule is an IgG, IgA, or IgM antibody.

IV. DETAILED DESCRIPTION

An “increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.

A “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.

The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

“Biocompatible” generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.

A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be “positive” or “negative.”

“Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

A “pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

“Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms “carrier” or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents. As used herein, the term “carrier” encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

“Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.

“Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

“Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of type I diabetes. In some embodiments, a desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.

In one aspect, disclosed herein are anti-anti-acetyl-CoA acetyltransferase 1 (ACAT1) binding molecules (such as, for example, antibodies, diabodies, nanobodies, scFv, sFv, and immunotoxins) that specifically binds glycosylated ACAT1, and wherein the anti-ACAT1 binding molecule comprising one or more variable heavy chain complementarity determining regions (CDRs) as set forth in SEQ ID Nos 1-3 (such as, for example the variable heavy chain as set forth in SEQ ID NO: 7) and one or more variable light chain complementarity determining regions (CDRs) as set forth in SEQ ID Nos 4-6 (such as, for example the variable light chain as set forth in SEQ ID NO: 9). In one aspect, the anti-ACAT1 binding molecule is an IgG, IgA, or IgM antibody.

The term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with glycosylated ACAT1. The antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.

The disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Pat. No. 5,804,440 to Burton et al. and U.S. Pat. No. 6,096,441 to Barbas et al.

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.

As used herein, the term “antibody or fragments thereof” encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab′)2, Fab′, Fab, Fv, sFv, scFv, and the like, including hybrid fragments. Thus, fragments of the antibodies that retain the ability to bind their specific antigens are provided. For example, fragments of antibodies which maintain ACAT1 binding activity are included within the meaning of the term “antibody or fragment thereof.” Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).

Also included within the meaning of “antibody or fragments thereof” are conjugates of antibody fragments and antigen binding proteins (single chain antibodies).

The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, M.J. Curr. Opin. Biotechnol. 3:348-354, 1992).

As used herein, the term “antibody” or “antibodies” can also refer to a human antibody and/or a humanized antibody. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.

(2) Human Antibodies

The disclosed human antibodies can be prepared using any technique. The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)). Specifically, the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. Antibodies having the desired activity are selected using Env-CD4-co-receptor complexes as described herein.

Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Accordingly, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or antibody chain (or a fragment thereof, such as an sFv, Fv, Fab, Fab′, F(ab′)2, or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody.

To generate a humanized antibody, residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen). In some instances, Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

(4) Administration of Antibodies

Administration of the antibodies can be done as disclosed herein. Nucleic acid approaches for antibody delivery also exist. The broadly neutralizing anti ACAT1 antibodies and antibody fragments can also be administered to patients or subjects as a nucleic acid preparation (e.g., DNA or RNA) that encodes the antibody or antibody fragment, such that the patient's or subject's own cells take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment. The delivery of the nucleic acid can be by any means, as disclosed herein, for example.

It is understood that as discussed herein the use of the terms homology and identity mean the same thing as similarity. Thus, for example, if the use of the word homology is used between two non-natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking at the similarity or relatedness between their nucleic acid sequences. Many of the methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity regardless of whether they are evolutionarily related or not.

In general, it is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein, is through defining the variants and derivatives in terms of homology to specific known sequences. This identity of particular sequences disclosed herein is also discussed elsewhere herein. In general, variants of genes and proteins herein disclosed typically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

It is understood that any of the methods typically can be used and that in certain instances the results of these various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein.

For example, as used herein, a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above. For example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods. As another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods. As yet another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated homology percentages).

3. Pharmaceutical Carriers/Delivery of Pharmaceutical Products

The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, “topical intranasal administration” means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.

Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

b) Therapeutic Uses

Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of the antibody used alone might range from about 1 μg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.

4. Method of Treating Cancer

The disclosed anti-CAT1 binding molecules can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphomas such as B cell lymphoma and T cell lymphoma; mycosis fungoides; Hodgkin's Disease; myeloid leukemia (including, but not limited to acute myeloid leukemia (AML) and/or chronic myeloid leukemia (CML)); bladder cancer; brain cancer; nervous system cancer; head and neck cancer; squamous cell carcinoma of head and neck; renal cancer; lung cancers such as small cell lung cancer, non-small cell lung carcinoma (NSCLC), lung squamous cell carcinoma (LUSC), and Lung Adenocarcinomas (LUAD); neuroblastoma/glioblastoma; ovarian cancer; pancreatic cancer; prostate cancer; skin cancer; hepatic cancer; melanoma; squamous cell carcinomas of the mouth, throat, larynx, and lung; cervical cancer; cervical carcinoma; breast cancer including, but not limited to triple negative breast cancer; genitourinary cancer; pulmonary cancer; esophageal carcinoma; head and neck carcinoma; large bowel cancer; hematopoietic cancers; testicular cancer; and colon and rectal cancers.

In one aspect, the treatment of the cancer can include any of the ACAT1 binding molecules disclosed herein. Thus, disclosed herein are methods of treating, reducing, decreasing, inhibiting, ameliorating, and or preventing a cancer and/or metastasis (such as for example, ovarian, pancreatic, colorectal, bladder and non-small cell lung cancer) comprising administering to the subject any of the ACAT1 binding molecules disclosed herein. For example, disclosed herein are methods of treating, reducing, decreasing, inhibiting, ameliorating, and or preventing a cancer and/or metastasis (such as for example, ovarian, pancreatic, colorectal, bladder and non-small cell lung cancer) in a subject in need thereof comprising administering to the subject an anti-acetyl-CoA acetyltransferase 1 (ACAT1) binding molecule (such as, for example, antibodies, diabodies, nanobodies, scFv, sFv, and immunotoxins) that specifically binds glycosylated ACAT1, and wherein the anti-ACAT1 binding molecule comprising one or more variable heavy chain complementarity determining regions (CDRs) as set forth in SEQ ID Nos 1-3 (such as, for example the variable heavy chain as set forth in SEQ ID NO: 7) and one or more variable light chain complementarity determining regions (CDRs) as set forth in SEQ ID Nos 4-6 (such as, for example the variable light chain as set forth in SEQ ID NO: 9). In one aspect, the anti-ACAT1 binding molecule is an IgG, IgA, or IgM antibody.

It is understood and herein contemplated that the disclosed treatment regimens can used alone or in combination with any anti-cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel

We wanted to examine the composition and density of B cell and T cells infiltrates in ovarian cancer to determine the difference between cancer with and without TLS. Using immunofluorescence we were able to characterize the CD3 positive cells (T cells) and CD19 positive cells (B cells) in ovarian cancer cells stained with DAPI (FIG. 1A). Using nearest neighbor analysis we observed that in TLS positive cancers a denser population of T and B cells (FIG. 1B). Looking at the survival of subjects with TLS compared to those without, we observed that Human ovarian cancer (HGSOC) tertiary lymphoid structures (TLSs) are associated with improved outcome (FIG. 1C). Looking at the number of B cells, CD4 T cells, and CD8T cells, we observed an increased number of B cells, CD4 T cells, and CD8T cells in TLS positive cancers compared to TLS negative cancers (FIG. 1D). Measuring the interaction strength and as shown by a CIRCOS plot, TLS positive cancers had a stronger interaction of B cells, CD4 T cells, and CD8T cells when compared with TLS negative cancers (FIG. 1E). We next examined the TLS from different human ovarian cancers (HGSOC) using laser microdissection (LCM) and adaptive immunosequencing (FIG. 2). Immunosequencing revealed that variable heavy chain (VH) sequences are highly clonal revealing 10 related sequences CARGRSSSSYYMDVW (SEQ ID NO: 11), CARASVPAPITGTIIWFDPW (SEQ ID NO: 12), CARVESYPSEYYYFGMDVW (SEQ ID NO: 13), CATTYSSSSGFDCW (SEQ ID NO:14), CARASVPAPITGTIIWFDPW (SEQ ID NO: 15), CARGRSSSSYYMDVW (SEQ ID NO: 16), CTHDWRNW (SEQ ID NO: 17), CVKSAGRTSASPSYYYMDVW (SEQ ID NO:18), CARGRGRDCRSSNCYYMDVW (SEQ ID NO: 19), and CARGRSSSSYYMDVW (SEQ ID NO: 20). Results showed a strong oligoclonal B cell response in the TLS areas, with a broader repertoire in whole tumor.

As shown in FIG. 2, the most dominant VH sequence (74.8% of clonotypes in that TLS). As shown in FIG. 3, we know that the most dominant antibody in TLS is an IgA. We then used multiplex PCR and sequencing to identify the two most dominant variable light chains (VL) sequences (one kappa and one lambda) (FIG. 4). We then generated dimeric IgA by combining the heavy chain in IgA backbone, with kappa and lambda light chains independently along with J-chain. To make these recombinant antibodies, the VH is cloned in PBMN-I GFP (retroviral transduction and GFP sort). Next the J-chain is cloned in pcDNA 3 (Lipofectamine transfection and G418/Geneticin selection). Finally, the light chain is cloned in pVITRO1 (Lipofectamine transfection and Hygromycin selection).

CDR regions are underlined, Chothia prediction

M  D  W  T  W  R  F  L  F  V  V  A  A  A  T  G  V  Q  S  Q

V  Q  L  V  Q  S  G  A  E  V  K  K  P  G  S  S  V  K  V  S

C  K  A  S  G  G  T  F  S  S  Y  A  I  S  W  V  R  Q  A  P

G  Q  G  L  E  W  M  G  G  I  I  P  I  F  G  T  A  N  Y  A

Q  K  F  Q  G  R  V  T  I  T  A  D  E  S  T  S  T  A  Y  M

E  L  T  G  L  T  S  Q  D  T  A  V  Y  Y  C  A  R  E  D  I

L  M  I  Y  S  Y  F  G  M  D  V  W  G  Q  G  T  T  V  T  V

M  D  W  T  W  R  I  L  F  L  V  A  A  A  T  G  A  H  S  E

V  Q  L  V  E  S  G  G  G  L  V  Q  P  G  G  S  L  R  L  S

C  A  A  S  G  F  N  I  K  D  T  Y  I  H  W  V  R  Q  A  P

G  K  G  L  E  W  V  A  R  I  Y  P  T  N  G  Y  T  R  Y  A

D  S  V  K  G  R  F  T  I  S  A  D  T  S  K  N  T  A  Y  L

Q  M  N  S  L  R  A  E  D  T  A  V  Y  Y  C  S  R  W  G  G

D  G  F  Y  A  M  D  Y  W  G  Q  G  T  L  V  T

Next, we examined the reactivity of the kappa and lambda antibodies. The recombinant antibody with the kappa light chain sequence was able to identity a target in tumor lysates and other tumors. By contrast, the antibody with a lambda light chain did not bind any targets (FIG. 5).

To determine the effect of the TLS-derived antibodies on cancer cells, we implanted OVCAR3 cancer cells or primary cancer cells from an ovarian tumor in mice. After 10 days, Recombinant antibodies comprising a Kappa light chain (TLS-κ), Lambda light chain (TLS-λ), or a control IgA were administered via intratumoral injection every 3-4 days and tumor growth was measured. As shown in FIG. 6, TLS-κ derived antibody abrogates the growth of human ovarian cancer. Tumor volume, weight, and images of OVCAR3 (FIG. 6A) and primary cancer 81908 (FIG. 6B) tumors treated with IgA kappa antibody, IgA lambda antibody, irrelevant antibody, or vehicle controls. While the TLS-λ antibody showed no improvement over an irrelevant antibody, TLS-κ antibodies showed marked improvement in reducing tumor volume and weight in both cancers.

We then assayed the binding of the TLS-κ and TLS-λ antibodies to determine their target. As shown in FIG. 7A, the TLS-k antibodies target ACAT1, wherein the TLS-λ antibodies had no specific target. We then measured the antibodies against peptides of ACAT1 to identify specific peptide targets (FIG. 7B) and binding intensity (FIG. 7C). To see if the targets were glycosylated or not, we compared the binding to TLS-κ in both OVCAR3 and primary cancer cells (PCL) that had been glycosylated or deglycosylated. Staining showed that TLS-K strongly associated with glycosylated targets on at best only weakly associated with deglycosylated targets (FIG. 7D). Checking the specificity of the TLS-k antibody we observed binding only in cancers with ACAT1 FIG. 8).

anti-ACAT1 variable heavy chain amino acid

sequence

anti-ACAT1 variable heavy chain nucleic acid

sequence

anti-ACAT1 variable light chain amino acid

sequence

anti-ACAT1 variable light chain nucleic acid

sequence

Variable Heavy Chain CDR3 amino acid sequence

Variable Heavy Chain CDR3 amino acid sequence

Variable Heavy Chain CDR3 amino acid sequence

SEQ ID NO:14

Variable Heavy Chain CDR3 amino acid sequence

Variable Heavy Chain CDR3 amino acid sequence

Variable Heavy Chain CDR3 amino acid sequence

Variable Heavy Chain CDR3 amino acid sequence

Variable Heavy Chain CDR3 amino acid sequence

Variable Heavy Chain CDR3 amino acid sequence

Variable Heavy Chain CDR3 amino acid sequence