WO2025101890A1 - Compositions and methods of treating disease - Google Patents

Abstract

The present disclosure generally relates to antibodies and antigen binding fragments thereof that specifically bind to Tmem273, and to compositions comprising such antibodies and antigen binding fragments thereof. The present disclosure further describes methods of treating diseases, such as those associated with Tmem273 expression, comprising use of such antibodies and antigen binding fragments thereof.

Description

COMPOSITIONS AND METHODS OF TREATING DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/598,018, filed November 10, 2023, the disclosure of which is hereby incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted herewith as an XML-formatted text file named “047162-7484WOl_sequence_listing.xml” created November 6, 2024, and having a size of 176,985 bytes is herein incorporated by reference in its entirety pursuant to 37 C.F.R. 1.52(e)(5)

BACKGROUND

Systemic sclerosis (SSc) has the highest mortality rate of the rheumatologic diseases. Current therapies to manage SSc progression are limited to nonspecific targeting of lymphocytes (using cyclophosphamide, methotrexate, or my cophenolate), targeting of B cells (using rituximab), targeting inflammation globally (through IL-6 pathway using tocilizumab), and autologous hematopoietic stem cell transplant (AHSCT), which approaches have proven unsatisfactory. While AHSCT appears to have some effects as a disease modifying therapy, it is an expensive and difficult option that is useful only early in disease for younger patients with aggressive disease and a poor prognosis. Furthermore, AHSCT carries a significant risk of treatment-associated death, serious infection, and cancer even in the setting of improved overall survival. In current practice immunosuppression is continued post-AHSCT to prevent SSc recurrence/progression. As such, AHSCT is a practicable intervention only for a small minority of SSc patients, and even then treatment with AHSCT comes with a number of severe risk factors and potential severe adverse events. Other biologies directed at T cells (abatacept, CTLA-4), B cells (belimumab. anti-BAFF), and cytokines (roiciguat IL-4/13 antagonist) have shown only modest benefits in small SSc clinical trials of similar magnitude as my cophenolate. Missing from the therapeutic armamentarium are interventions directed at unique pathophysiology of SSc.

Therefore, there remains a need in the field for disease modifying therapeutics targeting pathophysiology unique to SSc. BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

FIG. 1 presents sequences of the extracellular domains of human Tmem273 (SEQ ID NO: 1) and murine Tmem273 (SEQ ID NO: 2).

FIG. 2 presents the sequence of human Tmem273 extracellular domain fusion protein (hClO) (SEQ ID NO: 3) with annotations of engineered features. The engineered features include a human IL-2 signal peptide; added signal peptide amino acids; a stabilizing glycine (indicated by an arrow); an external domain of native human Tmem273; a linker (indicated by solid underline); and murine IgGl Fc.

FIG. 3A-FIG. 3C present sequences, schematics, and results related to production of human Tmem273 fusion protein human 293F cells. Human Tmem273 extracellular domain protein (hC 10) was produced by transient transfection of the expression vector (FIG. 3B) containing the KasI - EcoRV insert (FIG. 3A) (SEQ ID NO: 5) to produce the Fc fusion protein. KasI indicated by solid underline; EcoRV indicated by dashed underline. FIG. 3C presents an image taken of a 4-12% SDS-PAGE nonreducing gradient gel with purified hClO fusion protein. The predicted weight of the hClO homodimer is 56kd

FIG. 4 presents engineered features of the murine Tmem273 extracellular domain fusion protein (mClO) (SEQ ID NO: 6). Linker indicated by solid underline.

FIG. 5A-FIG. 5C present schematics and results related to production of murine Tmem273 fusion protein (mClO) in human 293F cells. Murine Tmem273 extracellular domain protein (mClO) was produced by transient transfection of the expression vector (FIG. 5B), containing the KasI - EcoRV insert (FIG. 5A) (SEQ ID NO: 7). to produce the Fc fusion protein. KasI indicated by solid underline; Ncoll indicated by dashed underline. FIG. 5C presents an image of a 4-12% SDS-PAGE nonreducing gradient gel with purified mClO fusion protein. The predicted weight of the hC 10 homodimer is 60kd

FIG. 6 presents a graphical representation of titers of hClO specific antibody at the time of final boost in the Balb/c mouse used for the hybridoma fusion.

FIG. 7 presents graphical representations of a PBMC flow cytometry screening for monoclonal antibody hC10-6Gl (right panel) and a control (left panel).

FIG. 8A-FIG. 8C presents graphical representations of results of a costimulation assay in which immobilization of hclO-6Gl with anti-CD3 and anti-CD28 selectively enhanced the relative production of IL-13 over IFN-y. and therefore the ratio of IL-13/IFN-y. * = p value <0.05; two-tailed Students T-test.

FIG. 9 presents an image of a gel confirming production of recombinant hC10-h6Gl human IgGl antibody.

FIG. 10 presents a graphical representation of relative binding of 6G1 and h6Gl to human (-hC10) and murine (-mC10) Tmem273 fusion proteins.

FIG. 11 A-FIG. 1 1C presents sequences, schematics, and results related to the binding of h6Gl to hClO (nominal antigen), mClO (murine homolog of nominal antigen), and hSlc24a3 (unrelated fusion protein sharing linker domain with hClO but not mClO). FIG. 11 A presents an alignment of human (SEQ ID NO: 8) and murine (SEQ ID NO: 9) Tmem273 extracellular and linker domains in addition to the hSlc24a3 Fc linker (SEQ ID NO: 10). FIG. 1 IB presents the results of a binding assay with serial dilution of high input h6GlmcAb to immobilized hClO (saturating), to demonstrate lower affinity cross-reactivity with mClO and no reactivity with hSlc24a3 (specificity) fusion proteins. FIG. 11C presents a gel image of hSlc24a3 fusion protein.

FIG. 12 presents a sequence alignment of extracellular domains of rat (SEQ ID NO: 11) and mouse (SEQ ID NO: 2) Tmem273.

FIG. 13 presents a graphical representation of results regarding rat anti -mouse Tmem273 extracellular domain (mClO) specific IgM monoclonal antibody 22G1 and control rat IgM monoclonal antibody specific for rat IgG Fc, demonstrating that mCl 0-22G1 did not specifically bind to rat IgG Fc. BSA = control well blocked with bovine serum albumin only.

FIG. 14 presents a graphical representation of results regarding costimulation of CD8yl3 T cell clone by 22G1 (anti-mClO) and 24H5 (control) hybridoma cells with anti- CD3 at two different concentrations (0.5 & 0.25 pg/ml). ** = p value < 0.01 two-tailed Students T-test.

FIG. 15 presents a gel image of recombinant mouse IgG2b monoclonal antibody mC10-m22Gl.

FIG. 16A-FIG. I6B present graphical representations of the binding of parent rat IgM monoclonal 22G1 (FIG. 16 A) and mouse-ized recombinant m22Gl (FIG. 16B) to mC10 (nominal antigen) and to hClO (human homolog of nominal antigen).

FIG. 17 presents a graphical representation of the weights of C. muridarum infected mice injected IP with 1 mg of isotype control recombinant antibody (squares) or anti-mouse Tmem273 mouse-ized recombinant IgG2b antibody m22Gl (circles). FIG. 18 presents Tmem273 mRNA signals analysis of different cell types from human tissues with signal > 20 nTPM in brain, adipose tissue, endothelial cells, and hematopoietic cells.

FIG. 19A-FIG. 19C present data and results related to flow cytometry staining of Tmem273 on peripheral blood T cells. FIG. 19A presents a representation of the gating strategy: Lymphocytes -^single cells- live cells- CD3 (30,000 events) CD4 vs CD8. FIG. 19B presents illustrative data showing Tmem273 pos frequency in CD4 and CD8 T cell subsets from a Healthy Control (HC and Early stage and Late stage SSc subjects. Gating separately on the CD4 and CD8 T cell subsets, Tmem273 was quantified with humanized monoclonal antibody hC10-6Gl conjugated to Alexa 546 (x-axis) plotted against side scatter (y-axis). FIG. 19C presents the absolute number of Tmem273 pos cells per 30,000 T cells calculated by FlowJo software in CD4 and CD8 subsets for the first 7 enrolled subjects (3 HC, 4 SSc) in an ongoing clinical investigation comparing healthy controls and rheumatoid arthritis subjects (disease control) to SSc subjects (20 subjects each group).

FIG. 20A-FIG. 20B present schematic representations and results related to a mouse study. FIG. 20A presents a schematic representation of the study. FIG. 20B presents a graphical representation of results related to analysis of the effects of an anti-murine Tmem273 antibody on Tmem positive CD4 and CD8 T cells in mice.

FIG. 21 presents images of a gross autopsy that was performed at the conclusion of a mouse model study described herein. Trichrome staining of lungs from mice that received either recombinant isotype control antibody (Con) or anti-Tmem273 antibody (m22Gl) on day 21, are ordered from lowest to highest number of recovered Tmem273 positive T cells. # Tmem273 positive CD4 and CD8 per 10,000 T cells; indicated for each inset here (CD4, CD8). Inset A) Con 1 (280, 290), Inset B) Con 2 (321, 267), Inset C) Con 3 (304, 201), Inset D) m22Gl-2 (467, 338), Inset E) m22Gl-3 (555, 435), Inset F) m22Gl-l (613, 513)

DETAILED DESCRIPTION

DEFINITIONS

Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry’, and nucleic acid hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e ., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

So that the disclosure may be more readily understood, select terms are defined below.

As used herein, the terms “a.” ’an.” or “the” are used to include one or more than one unless the context clearly dictates otherwise. By way of example, “an element” means one element or more than one element. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.”

In this disclosure, “comprises,” “comprising,” “containing,” and “having” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like; “consisting essentially of’ or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 and so forth, as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

The term "therapeutic" as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by any degree of suppression, remission, or eradication of a disease state.

To “treat"’ a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

As used herein, to “alleviate” a disease means reducing the severity of one or more symptoms of the disease.

The terms “patient”, “subject”, and “individual” are used interchangeably and are intended to include living organisms that may be subjected to treatment for a given disease, e.g, mammals. A “subject”, “patient”, or “individual”, as used herein, can be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals, as well as simian and non- human primate mammals. Preferably, the subject is human.

As used herein, the term “complementary” generally refers to the ability of a single strand of a polynucleotide (or portion thereof) to hybridize to an anti-parallel polynucleotide strand (or portion thereof) by contiguous base-pairing between the nucleotides (that is not interrupted by any unpaired nucleotides) of the anti-parallel polynucleotide single strands, thereby forming a double-stranded polynucleotide between the complementary strands. A first polynucleotide is said to be “completely complementary” to a second polynucleotide strand if each and every nucleotide of the first polynucleotide forms base-paring with nucleotides within the complementary region of the second polynucleotide. A first polynucleotide is not completely complementary (i.e.. partially complementary) to the second polynucleotide if at least one nucleotide in the first polynucleotide does not base pair with the corresponding nucleotide in the second polynucleotide. The degree of complementarity between polynucleotide strands has significant effects on the efficiency and strength of annealing or hybridization between polynucleotide strands. This is of particular importance in amplification reactions, which depend upon binding between polynucleotide strands. It is well-known in the art that sequences need not be completely complementary in order for hybridization to occur.

An oligonucleotide primer is “complementary” to a target polynucleotide if at least 50% (preferably. 60%. more preferably 70%, 80%, still more preferably 90% or more) nucleotides of the primer form base-pairs with nucleotides on the target polynucleotide.

As used herein, “hybridization” means the pairing of complementary oligomeric compounds (e.g.. a single strand of a polynucleotide pairing with an anti -parallel polynucleotide strand). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases). For example, the natural base adenine is nucleobase complementary to the natural nucleobases thymidine and uracil which pair through the formation of hydrogen bonds. The natural base guanine is nucleobase complementary to the natural bases cytosine and 5-methyl cytosine. Hybridization can occur under varying circumstances.

As used herein, the term “specifically hybridizes” refers to the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hy bridizes to another nucleic acid site.

By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such crossspecies reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity' does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope '‘A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

As used herein, the term “substantially the same” amino acid sequence is defined as a sequence with at least 70%, preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least 99% homology with another amino acid sequence, as determined by the FASTA search method in accordance with Pearson & Lipman, 1988, Proc. Natl. Inst. Acad. Sci. USA 85:2444-48.

As used herein, the terms “effective amount” and “therapeutically effective amount” are used interchangeably and refer to the amount required to reduce or improve at least one symptom or change in a clinical marker of a disease relative to an untreated patient. The effective amount of the treatment used for therapeutic treatment of the disease varies depending upon the manner of the specific disorder, condition or disease, extent of the disorder, condition or disease, and administration of the cells, as well as the age, body weight, and general health of the subject. The effective amount is capable of achieving a particular desired biological result and/or provides a therapeutic or prophylactic benefit.

As used herein, the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (z.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

As used herein, the term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

As used herein, the term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g, naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

As used herein, the term “identity” refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

As used herein, the term “conservative sequence modifications” is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g, lysine, arginine, histidine), acidic side chains (e.g, aspartic acid, glutamic acid), uncharged polar side chains (e.g, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for the ability' to bind antigens using the functional assays described herein.

As used herein, the term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a polypeptide naturally present in a living animal is not “isolated ’ but the same nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

An “isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, z.e., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids that have been substantially purified from other components which naturally accompany the nucleic acid, i.e.. RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzy me digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

The term “recombinant polypeptide” as used herein is defined as a polypeptide produced by using recombinant DNA methods, such as for, example, a recombinant antibody or antigen binding fragment thereof.

The term “recombinant DNA” as used herein is defined as DNA produced by joining pieces of DNA from different sources.

“Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination. A variant of a nucleic acid or peptide may be a naturally occurring such as an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.

By the term "modi Tied” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

As used herein, the term “oligonucleotide” typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (z.e., A, T, C, G), this also includes an RNA sequence (z.e., A, U, C, G) in which “U” replaces “T.” Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

“Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v ), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, “nucleic acid” and “polynucleotide” as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides” and which comprise one or more “nucleotide sequence(s)”. The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences (z.e., “nucleotide sequences”) which are obtained by any means available in the art, including, without limitation, recombinant means, z.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

The term “immune response” as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and/or activate lymphocytes to remove the antigen.

“Instructional material.” as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of the composition and/or compound of the invention in a kit. The instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the invention or be shipped together with a container that contains the compound and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. Delivery of the instructional material may be, for example, by physical delivery of the publication or other medium of expression communicating the usefulness of the kit, or may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website.

The term “antigen” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

The term “epitope” as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and/or T cell responses. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e.. they are multivalent. In general, an epitope is roughly about 10 amino acids and/or sugars in size. In some instances, the epitope is about 4-18 amino acids, about 5-16 amino acids, about 6-14 amino acids, about 7-12, or about 8-10 amino acids. One skilled in the art understands that generally the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity and therefore distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.

As used herein, “antibody” or “Ab” refers to an immunoglobulin molecule capable of recognizing and binding to a specific target or antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, and so forth, through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. The term can encompass any ty pe of antibody, including but not limited to monoclonal antibodies, polyclonal antibodies, “antigen-binding fragments" (or portion), such as Fab, Fab', F(ab')2, Fd, Fv, Fc, and so forth, of intact antibodies that retain the ability to specifically bind to a given antigen, an isolated complementarity determining region (CDR), bispecific antibodies, heteroconjugate antibodies, mutants thereof, fusion proteins having an antibody, or antigen-binding fragment thereof, (e.g., a domain antibody), single chain (ScFv) and single domain antibodies (e g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies. triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Holliger and Hudson, 2005, Nature Biotechnology 23(9): 1126-1136), humanized antibodies, chimeric antibodies and any other modified configuration of the immunoglobulin molecule that includes an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibodies may be murine, rat, human, or any other origin (including chimeric or humanized antibodies).

Native or naturally occurring antibodies and native immunoglobulins are typically heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains (LC) and two identical heavy chains (HC). Each heavy chain has a variable domain (VH) followed by a number of constant domains or regions (e.g. hinge, CHI, CH2 or CH3), referred to as “CH domains’". Each light chain has a variable domain (VL) and a constant domain, referred to as “CL domain”. The term “constant region” or “constant domain” of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as Fc receptor (FcR) binding, participation of the antibody in antibody-dependent cellular toxicity (ADCC), opsonization, initiation of complement dependent cytotoxicity, and mast cell degranulation. The constant regions of the antibodies may be derived from constant regions of any one of IgA, IgD, IgE, IgG, IgM, any isotypes thereof (e.g., IgGl, IgG2, IgG3, or IgG4 isotypes of IgG), as well as subclasses and mutated versions thereof.

CHI domain includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain that extends, e.g. from about positions 1 18-215 according to the EU index of Kabat. The CHI domain is adjacent to the VH domain and amino terminal to the hinge region of an immunoglobulin heavy chain molecule, and does not form a part of the Fc region of an immunoglobulin heavy chain.

The hinge region includes the portion of a heavy chain molecule that joins the CHI domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antgen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains.

CH2 domain includes the portion of a heavy chain immunoglobulin molecule that extends, e.g. from about positions 231-340 according to the EU index of Kabat. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. In some aspects, the antibody (or fragment thereof) of the invention comprises a CH2 domain derived from an IgG molecule, such as IgGl, IgG2, IgG3, or IgG4. In some aspects, the IgG is a human IgG.

CH3 domain includes the portion of a heavy chain immunoglobulin molecule that extends approximately 1 10 residues from N-terminus of the CH2 domain, e.g. from about positions 341-447 according to the EU index of Kabat. The CH3 domain typically forms the C -terminal portion of the antibody. In some immunoglobulins, however, additional domains may extend from CH3 domain to form the C-terminal portion of the molecule (e.g. the CH4 domain in the p chain of IgM and the E chain of IgE). In some aspects, the antibody (or fragment thereof) of the invention comprises a CH3 domain derived from an IgG molecule, such as IgGl, IgG2, IgG3, or IgG4. In some aspects, the IgG is a human IgG.

CL domain includes the constant region domain of an immunoglobulin light chain that extends, e.g. from about positions 108-214 according to the EU index of Kabat. The CL domain is adjacent to the VL domain. In some aspects, the antibody (or fragment thereof) of the invention comprises a kappa light chain constant domain (CLK). In some aspects, the antibody (or fragment thereof) comprises a lambda light chain constant domain (CLX). CLK has known polymorphic loci CLK-V/A45 and CLK-LA/83 (using Kabat numbering) thus allowing for polymorphisms Km(l): CLK-V45/L83; Km(l,2): CLK-A45/L83; and Km(3): CLK-A45/V83. Polypeptides and antibodies of the invention may have antibody components with any of these light chain constant regions.

The Fc region generally comprises a CH2 domain and a CH3 domain. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230 (according to the EU index of Kabat), to the carboxyl-terminus thereof. A Fc region may be a native sequence Fc region or a variant Fc region. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

A “variable region"’ of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As known in the art. the variable regions of the heavy (VH) and light (VL) chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.

A CDR of a variable domain may be identified in accordance with the definitions of the Kabat (Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), Chothia (Chothia et al., Nature 342:877-883, (1989)), the accumulation of both Kabat and Chothia, AbM definition (derived using Oxford Molecular’s AbM antibody modeling software (now Accelrys®)), contact definition (based on observed antigen contacts, set forth in MacCallum et al., J. Mol. Biol., 262:732-745, (1996)), and/or conformational definition (Makabe et al., Journal of Biological Chemistry, 283: 1156-1166, 2008) or any method of CDR determination well known in the art. As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. For the present invention, the CDRs set forth in Table 2 below were derived using Kabat and Chothia definitions. The anti-EDB antibodies, or antigenbinding fragment thereof, of the present invention include one or more CDR(s) (such as one. two, three, four, five, or all six CDRs).

An “isolated antibody”, as used herein, refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds EDB is substantially free of antibodies that specifically bind antigens other than EDB). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. It is also understood that by reading this definition, for example, an antibody (or moiety' or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target.

The term “compete”, as used herein with regard to an antibody, means that a first antibody, or an antigen-binding fragment thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antibody, or an antigen-binding fragment thereof, such that the result of binding of the first antibody w ith its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. How ever, where each antibody detectably inhibits the binding of the other antibody with its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to “cross-compete” with each other for binding of their respective epitope(s). Both competing and cross-competing antibodies are encompassed by the present invention. Regardless of the mechanism by which such competition or cross -competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope, or portion thereof), the skilled artisan would appreciate, based upon the teachings provided herein, that such competing and/or cross-competing antibodies are encompassed and can be useful for the methods disclosed herein.

The term “monoclonal antibody” or “mAb” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

In some instances, a monovalent antibody can have more than one antigen binding sites, but the binding sites are from different antigens. In some aspects of the invention, the antibody, or antigen-binding fragment thereof, may include a “bivalent antibody”, i.e.. having two antigen binding sites per molecule (e g., IgG). In some instances, the two binding sites have the same antigen specificities. Alternatively, bivalent antibodies may be bispecific. A “bispecific,” “dual-specific” or “bifunctional” antibody is a hybrid antibody having two different antigen binding sites. The two antigen binding sites of a bispecific antibody bind to two different epitopes, which may reside on the same or different protein targets.

The term “chimeric antibody” is intended to refer to antibodies in which part or all of the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

As used herein, “humanized” or “CDR grafted” antibody refers to forms of nonhuman (e.g. murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab. Fab', F(ab')2 or other antigen binding subsequences of antibodies) that contain minimal sequence derived from a non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from one or more CDRs of the recipient are replaced by residues from one or more CDRs of a non-human species (donor antibody) such as mouse, rat. or rabbit having the desired specificity, affinity, and capacity. Antibodies of the invention can be produced using techniques well known in the art, e.g., recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art (see, for example, Jayasena, S. D., Clin. Chem., 45: 1628-50 (1999) and Fellouse, F. A., et al, J. Mol. Biol., 373(4):924-40 (2007)). Additional guidance may be found in Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor. N.Y. (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003).

Nucleic acids encoding the heavy and light chains of the antibodies used to prepare the antibodies or antigen binding fragments thereof can be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest may be maintained in vector in a host cell and the host cell can then be expanded and frozen for future use. Production of recombinant monoclonal antibodies in cell culture can be carried out through cloning of antibody genes from B cells by means known in the art. See, e g. Tiller et al., J. Immunol. Methods 329: 112-124, 2008; U.S. Patent No. 7,314,622.

For all heavy chain constant region amino acid positions discussed in the present invention, numbering is according to the Eu index first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1 ):78-85, describing the amino acid sequence of myeloma protein Eu, which is the first human IgGl sequenced. The Eu index of Edelman et al. is also set forth in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service. National Institutes of Health, Bethesda. Thus, the “EU index as set forth in Kabaf’ or “EU index of Kabat” refers to the residue numbering system based on the human IgGl Eu antibody of Edelman et al. as set forth in Kabat 1991.

As used herein, the term “host cell” includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of this invention.

As used herein, the term “sample” generally refers to any material or substance that may contain an analyte, such as an analyte of interest. In some aspects, the sample comprises more than one analyte, e.g., more than one analyte of interest.

ANTI-TMEM273 ANTIBODIES AND ANTIGEN BINDING FRAGMENTS

THEREOF

Systemic sclerosis (SSc) is an autoimmune disease directed at unknown antigen(s) with unique pathology based on chronic endotheliitis causing wide ranging ischemia, tissue injury, and progressive scarring. Biopsies of affected sites show perivascular lymphocytic infiltrates and apoptosis of endothelial cells. In milder cases the ischemic injury is limited to skin, limited cutaneous SSc (IcSSc, scleroderma). In more severe disease, diffuse cutaneous SSc, ischemic injury can be systemic affecting skin, mucosa, and critical organs including lungs, kidneys, gastrointestinal tract, and heart. There remains a need in the field for disease modifying therapeutics targeting pathophysiology unique to SSc.

Furthermore, fibrosis is a major contributor to scarring and infertility caused by Chlamydia infection, as well as the disease manifestation of systemic sclerosis (SSc). A major pathway for scarring involves TNF-alpha and IL-13 induction of TGF-beta that drives scarring biology (Fichtner -Feigl et al. 2006, Nature Medicine 12: 99-106). It is conventionally known that T cells, including CD8 T cells, in affected tissue produce cytokines that drive the synthesis of extracellular matrix proteins by fibroblasts resulting in excess fibrosis. Research in the mouse model for Chlamydia genital tract infections has shown unambiguously that scarring and infertility are mediated by CD8 T cells. Perhaps more specifically, it was previously shown in the mouse model that the CD8 T cell response to Chlamydia genital tract infections is atypical and includes antigen-specific CD8 T cells that produce interferon gamma, IL-13 and tumor necrosis factor alpha (TNF-alpha) (Johnson et al, 2014. Immunology 142: 248-257). Similarly, in SSc, progressive scarring and fibrosis have been attributed to CD8IL-13 T cells (Fushiotti et al, 2009, Arthritis Rheum 60: 1119- 28). In early stage SSc skin lesions CD8 T cells predominate, in late stage SSc predominate, with IL-13 present in all stages (Fuschiotti et al, 2013, Arthritis & Rheumatism 65: 236-246). Additionally, activated CD8 T cells from SSc patients cause human dermal fibroblasts to express collagen and fibrinogen. The SSc CD8 T cell-driven dermal fibroblast product of scarring proteins is blocked by neutralization of IL- 13, thereby directly tying the CD8IL-13 T cell subset and IL-13 to the immunopathology' underlying SSc (Fuschiotti et al, 2013, Arthritis Rheum 65: 236-46). In the murine chlamydia model CD4 and CD8 T cells producing interferon gamma, IL-13, and TNF-alpha (referred to as CD4_y13 and CD8_y13) uniquely express Tmem273 (Johnson et al, 2018, Infect Immun e00614-17, supplemental material; Johnson et al 2020. J Infect Dis 11: 1895-1906). In theory, monoclonal antibodies that deplete CD4_TB and CD8713 T cells may diminish inflammatory cytokines including TNF-alpha and IL-13 that are known to drive scarring pathophysiology relevant to SSc.

As such, the present disclosure generally relates to an isolated or recombinant antibody or antigen binding fragment thereof which specifically binds to transmembrane protein 273 (Tmem273), to compositions comprising such antibodies or antigen binding fragments thereof, and to methods of using such antibodies and antigen binding fragments thereof. In one aspect, the present disclosure generally relates to an isolated or recombinant antibody or antigen binding fragment thereof which specifically binds to transmembrane protein 273 (Tmem273), wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL). wherein: the Vu comprises: (a) a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12; a CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 14; (b) a CDR1 comprising the amino acid sequence of SEQ ID NO: 58; a CDR2 comprising the amino acid sequence of SEQ ID NO: 59; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 60; (c) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 71; (d) a CDR1 comprising the amino acid sequence of SEQ ID NO: 80: a CDR2 comprising the amino acid sequence of SEQ ID NO: 81; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 82; (e) a CDR1 comprising the amino acid sequence of SEQ ID NO: 91; a CDR2 comprising the amino acid sequence of SEQ ID NO: 92; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 93; or (f) a CDR1 comprising the amino acid sequence of SEQ ID NO: 102; a CDR2 comprising the amino acid sequence of SEQ ID NO: 103; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 104; the VL comprises: (g) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17; a CDR2 comprising the amino acid sequence of KVS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19; (h) a CDR1 comprising the amino acid sequence of SEQ ID NO: 64: a CDR2 comprising the amino acid sequence of YTS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 65; (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 75; a CDR2 comprising the amino acid sequence of YTS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 76; (j) a CDR1 comprising the amino acid sequence of SEQ ID NO: 86; a CDR2 comprising the amino acid sequence of KVS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 87; (k) a CDR1 comprising the amino acid sequence of SEQ ID NO: 97; a CDR2 comprising the amino acid sequence of FAS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 98; or (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 108; a CDR2 comprising the amino acid sequence of LVS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 109. . Moreover, in one aspect, the present disclosure generally relates to an isolated or recombinant antibody or antigen binding fragment thereof which specifically binds to transmembrane protein 273 (Tmem273). wherein the antibody or antigen binding fragment thereof comprises a heavy chain comprising a variable region (VH) chain comprising VH complementarity determining regions (CDR) CDR1, CDR2 and CDR3 respectively of the amino acid sequences of SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30; and further comprises a light chain comprising a variable region (VL) comprising VL complementarity determining regions (CDR) CDR1, CDR2 and CDR3 respectively of the amino acid sequences of SEQ ID NO: 33, LAS, and SEQ ID NO: 35.

In some aspects, the antibody or antigen binding fragment thereof comprises: (a) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 12; the CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 17; the CDR2 comprising the amino acid sequence of KVS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 19; (b) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 58; the CDR2 comprising the amino acid sequence of SEQ ID NO: 59; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 60; and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 64; the CDR2 comprising the amino acid sequence of YTS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 65; (c) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 69; the CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 71; and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 75; the CDR2 comprising the amino acid sequence of YTS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 76; (d) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 80; the CDR2 comprising the amino acid sequence of SEQ ID NO: 81; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 82: and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 86; the CDR2 comprising the amino acid sequence of KVS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 87; (e) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 91; the CDR2 comprising the amino acid sequence of SEQ ID NO: 92; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 93; and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 97; the CDR2 comprising the amino acid sequence of FAS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 98; or (f) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 102; the CDR2 comprising the amino acid sequence of SEQ ID NO: 103; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 104; and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 108; the CDR2 comprising the amino acid sequence of LVS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 109.

In some aspects, the antibody or antigen binding fragment specifically binds to the extracellular domain of Tmem273. In some aspects, the antibody specifically binds to mouse (SEQ ID NO: 2) and/or human (SEQ ID NO: 1) Tmem273.

In some aspects, the antibody or antigen binding fragment thereof comprises a VH comprising an amino acid sequence at least 70%, at least 75%, at least 80%, at least, 90%, at least 91%, a least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 15; SEQ ID NO: 61; SEQ ID NO: 72; SEQ ID NO: 83; SEQ ID NO: 94; and SEQ ID NO: 105. In some aspects, the antibody or antigen binding fragment thereof comprises a VH comprising an amino acid sequence at least 90%. at least 95%. or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 15; SEQ ID NO: 61; SEQ ID NO: 72; SEQ ID NO: 83; SEQ ID NO: 94; and SEQ ID NO: 105. In some aspects, the antibody or antigen binding fragment thereof comprises a VH comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 15; SEQ ID NO: 61; SEQ ID NO: 72; SEQ ID NO: 83; SEQ ID NO: 94; and SEQ ID NO: 105. In some aspects, the antibody or antigen binding fragment thereof comprises a VH comprising an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 15; SEQ ID NO: 61; SEQ ID NO: 72; SEQ ID NO: 83; SEQ ID NO: 94; and SEQ ID NO: 105. In some aspects, the antibody or antigen binding fragment thereof comprises a VH comprising an amino acid sequence at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 15; SEQ ID NO: 61; SEQ ID NO: 72; SEQ ID NO: 83; SEQ ID NO: 94; and SEQ ID NO: 105. In some aspects, the antibody or antigen binding fragment thereof comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15; SEQ ID NO: 61; SEQ ID NO: 72; SEQ ID NO: 83; SEQ ID NO: 94; and SEQ ID NO: 105

In some aspects, the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence at least 70%, at least 75%, at least 80%, at least, 90%, at least 91%, a least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20; SEQ ID NO 66: SEQ ID NO: 77; SEQ ID NO: 88; SEQ ID NO: 99; and SEQ ID NO: 110. In some aspects, the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20; SEQ ID NO 66: SEQ ID NO: 77; SEQ ID NO: 88; SEQ ID NO: 99; and SEQ ID NO: 110. In some aspects, the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20; SEQ ID NO 66: SEQ ID NO: 77; SEQ ID NO: 88; SEQ ID NO: 99; and SEQ ID NO: 110. In some aspects, the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20; SEQ ID NO 66: SEQ ID NO: 77; SEQ ID NO: 88; SEQ ID NO: 99; and SEQ ID NO: 110. In some aspects, the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20; SEQ ID NO 66: SEQ ID NO: 77; SEQ ID NO: 88; SEQ ID NO: 99; and SEQ ID NO: 110. In some aspects, the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20; SEQ ID NO 66: SEQ ID NO: 77; SEQ ID NO: 88; SEQ ID NO: 99; and SEQ ID NO: 110.

In some aspects, the antibody or antigen binding fragment thereof comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 20; (b) a VH comprising the amino acid sequence of SEQ ID NO: 61 and a VL comprising the ammo acid sequence of SEQ ID NO: 66; (c) a VH comprising the amino acid sequence of SEQ ID NO: 72 and a VL comprising the amino acid sequence of SEQ ID NO: 77; (d) a VH comprising the amino acid sequence of SEQ ID NO: 83 and a VL comprising the amino acid sequence of SEQ ID NO: 88; (e) a VH comprising the amino acid sequence of SEQ ID NO: 94 and a VL comprising the amino acid sequence of SEQ ID NO: 99; or (f) a VH comprising the amino acid sequence of SEQ ID NO: 105 and a VL comprising the amino acid sequence of SEQ ID NO: 110.

In some aspects, the antibody or antigen binding fragment thereof is a humanized antibody or antigen binding fragment thereof. In some aspects, the antibody or antigen binding fragment thereof is a chimeric antibody or antigen binding fragment thereof.

In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain comprising an amino acid sequence at least 70%, at least 75%, at least 80%, at least, 90%. at least 91%. a least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 22. In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 22. In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 22. In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 22. In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 22. In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 22.

In some aspects, the antibody or antigen binding fragment thereof comprises a light chain comprising an amino acid sequence at least 70%, at least 75%, at least 80%, at least, 90%, at least 91%, a least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 25. In some aspects, the antibody or antigen binding fragment thereof comprises a light chain comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 25. In some aspects, the antibody or antigen binding fragment thereof comprises a light chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 25. In some aspects, the antibody or antigen binding fragment thereof comprises a light chain comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 25. In some aspects, the antibody or antigen binding fragment thereof comprises a light chain comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 25. In some aspects, the antibody or antigen binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 25. In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 22 and a light chain comprising the amino acid sequence of SEQ ID NO: 25.

In some aspects, the antigen or antigen binding fragment thereof comprises a heavy chain and/or the light chain comprising a signal peptide. In some aspects, the heavy chain comprises the amino acid sequence of SEQ ID NO: 23 and/or the light chain comprises the amino acid sequence of SEQ ID NO: 26. In some aspects, the antibody or antigen binding fragment thereof comprises an IgGl, IgG2, IgG3 or IgG4 constant region or fragment thereof. In some aspects, the antibody or antigen binding fragment thereof comprises an IgGl constant region or fragment thereof. In some aspects, the antibody or antigen binding fragment thereof is directly or indirectly attached to a detectable label or a therapeutic agent. In some aspects, the antibody or antigen binding fragment thereof is directly or indirectly- attached to a therapeutic agent. In some aspects, the therapeutic agent comprises a cytotoxic drug. In some aspects, the therapeutic agent comprises an anti-CD3 antibody or antigen binding fragment thereof. In some aspects, the antigen binding fragment is an Fab fragment, an Fab' fragment, or an F(ab’)2 fragment.

In some aspects, the antibody specifically binds to mouse Tmem273 (SEQ ID NO: 2). In some aspects, the antibody or antigen binding fragment thereof comprises a Vn comprising an amino acid sequence at least 70%, at least 75%, at least 80%, at least, 90%, at least 91%, a least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 31. In some aspects, the antibody or antigen binding fragment thereof comprises a Vn comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 31. In some aspects, the antibody or antigen binding fragment thereof comprises a VH comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 31. In some aspects, the antibody or antigen binding fragment thereof comprises a VH comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 31. In some aspects, the antibody or antigen binding fragment thereof comprises a VH comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 31. In some aspects, the antibody or antigen binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 31.

In some aspects, the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence at least 70%, at least 75%, at least 80%, at least, 90%, at least 91%, a least 92%, at least 93%, at least 94%. at least 95%. at least 96%. at least 97%. at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 36. In some aspects, the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence at least 90%. at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 36. In some aspects, the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 36. In some aspects, the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 36. In some aspects, the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 36. In some aspects, the antibody or antigen binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 36. In some aspects, the antibody or antigen binding fragment thereof comprises a VH of the amino acid sequence of SEQ ID NO: 31 and a VL of the amino acid sequence of SEQ ID NO: 36.

In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain comprising an amino acid sequence at least 70%, at least 75%, at least 80%, at least, 90%, at least 91%, a least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%. or at least 99% identical to the amino acid sequence of SEQ ID NO: 38. In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 38. In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 38. In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 38. In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 38. In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38.

In some aspects, the antibody or antigen binding fragment thereof comprises a light chain comprising an amino acid sequence at least 70%. at least 75%, at least 80%, at least. 90%, at least 91%, a least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 41. In some aspects, the antibody or antigen binding fragment thereof comprises a light chain comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 41. In some aspects, the antibody or antigen binding fragment thereof comprises a light chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 41. In some aspects, the antibody or antigen binding fragment thereof comprises a light chain comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 41. In some aspects, the antibody or antigen binding fragment thereof comprises a light chain comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 41. In some aspects, the antibody or antigen binding fragment thereof comprises a light chain comprises the amino acid sequence of SEQ ID NO: 41 . In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 41.

In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain and/or the light chain comprises a signal peptide. In some aspects, the heavy chain comprises the amino acid sequence of SEQ ID NO: 23 and/or the light chain comprises the amino acid sequence of SEQ ID NO: 26. In some aspects, the antibody or antigen binding fragment thereof is directly or indirectly attached to a detectable label or a therapeutic agent. In some aspects, the antibody or antigen binding fragment thereof is directly or indirectly attached to a therapeutic agent. In some aspects, the therapeutic agent comprises a cytotoxic drug. In some aspects, the therapeutic agent comprises an anti-CD3 antibody or antigen binding fragment thereof. In some aspects, the antigen binding fragment is an Fab fragment, an Fab' fragment, or an F(ab^?)2 fragment.

In some aspects, the antibody or antigen binding fragment thereof comprises a mouse constant region. In some aspects, the constant region is IgGl, IgG2a, IgG2b, or IgG3, or a fragment thereof. In some aspects, the constant region is IgG2b.

Polynucleotides and Vectors

In some aspects, an antibody or antigen binding fragment thereof as described herein is encoded by a polynucleotide. In some aspects, the polynucleotide comprises recombinant or isolated or synthetic DNA. In some aspects, the polynucleotide comprises recombinant or isolated or synthetic RNA. In some aspects, the polynucleotide encodes a recombinant or synthetic polypeptide comprising the antibody or antigen binding fragment thereof. In some aspects, a vector encodes the antibody or antigen binding fragment thereof. In some aspects, the vector is an expression vector.

In some aspects, the polynucleotide encoding the antibody or antigen binding fragment thereof is operably linked to a transcriptional control element, e.g., a promoter, and enhancer, and so forth. Suitable promoter and enhancer elements are known to those of skill in the art. In some aspects, the polynucleotide encoding the antibody or antigen binding fragment thereof is operably linked to a promoter. For expression in a bacterial cell, suitable promoters include, but are not limited to, lacl, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, suitable promoters include, but are not limited to, light and/or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, and so forth, is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to. alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), and so forth), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, and so forth), steroid regulated promoters (e g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, and so forth), metal regulated promoters (e.g., metallothionein promoter systems, and so forth), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, and so forth), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, and so forth), light regulated promoters, synthetic inducible promoters, and the like.

For expression in a yeast cell, a suitable promoter is a constitutive promoter such as an ADH1 promoter, a PGK1 promoter, an ENO promoter, a PYK.1 promoter and the like; or a regulatable promoter such as a GALI promoter, a GAL10 promoter, an ADH2 promoter, a PHOS promoter, a CUP1 promoter, a GALT promoter, a MET25 promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1 promoter, and A0X1 (e.g., for use in Pichia). Selection of the appropriate vector and promoter is well within the level of ordinary' skill in the art. Suitable promoters for use in prokaryotic host cells include, but are not limited to, a bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; in vivo regulated promoters, such as an ssaG promoter or a related promoter (see, e.g., U.S. Patent Publication No. 20040131637), a pagC promoter (Pulkkinen and Miller, J. Bacteriol. (1991) 173(1): 86-93; Alpuche-Aranda et al., Proc. Natl. Acad. Sci. USA (1992) 89(21): 10079-83), a nirB promoter (Harbome et al. Mol. Micro. (1992) 6:2805-2813), and the like (see, e.g., Dunstan et al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al.. Vaccine (2004) 22:3243-3255; and Chatfield et al., Biotechnol. (1992) 10:888-892); a sigma70 promoter, e.g., a consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); a stationary phase promoter, e.g., a dps promoter, an spv promoter, and the like; a promoter derived from the pathogenicity' island SPI-2 (see, e.g.. WO96/17951 ); an actA promoter (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70: 1087-1096); an rpsM promoter (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996). 22:367); a tet promoter (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology', Protein— Nucleic Acid Interaction. Macmillan, London. UK, Vol. 10. pp. 143-162); an SP6 promoter (see, e.g., Melton et al., Nucl. Acids Res. (1984) 12:7035); and the like. Suitable strong promoters for use in prokaryotes such as Escherichia coli include, but are not limited to Trc, Tac, T5, T7, and PLambda. Non-limiting examples of operators for use in bacterial host cells include a lactose promoter operator (LacI repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator), a tryptophan promoter operator (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of try ptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and a tac promoter operator (see, e.g., deBoer et al., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:21-25).

Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Other constitutive promoter sequences may also be used, including, but not limited to a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, the EF-1 alpha promoter, as well as human gene promoters such as, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

In some aspects, the polynucleotide or vector containing the suitable promoter is irreversibly switched through the induction of an inducible system. Suitable systems for induction of an irreversible switch are well known in the art, e.g., induction of an irreversible switch may make use of a Cre-lox-mediated recombination (see, e.g., Fuhrmann-Benzakein, et al.. Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinase, endonuclease, ligase, recombination sites, and so forth known to the art may be used in generating an irreversibly switchable promoter. Methods, mechanisms, and requirements for performing site-specific recombination, described elsewhere herein, find use in generating irreversibly switched promoters and are well known in the art, see, e.g.. Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), the disclosures of which are incorporated herein by reference.

A polynucleotide encoding the antibody or antigen binding fragment thereof of the present disclosure can be present within an expression vector and/or a cloning vector. An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and/or maintenance of the vector. Suitable expression vectors include, e.g., plasmids, viral vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. The following vectors are provided by way of example and should not be construed in anyway as limiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala. Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44. PXR1. pST-Ki, pST-KiT, pSG (Stratagene) pSVK.3, pBPV, pMSG and pSVL (Pharmacia). Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia vims; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al.. H. Gene Ther. (1999) 5: 1088-1097; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated vims (see, e.g., Ah et al., Hum. Gene Ther. (1998) 9: 81- 86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al.. Gene Ther. (1997) 4:683 690, Rolling et al.. Hum. Gene Ther. (1999) 10: 641-648; Ali et al.. Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al.. Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see. e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al.. J. Virol. (1999) 73: 7812-7816); a retroviral vector (e.g.. Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Vims, Harvey' Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma vims, and mammary tumor virus); and the like.

Additional expression vectors suitable for use are, e.g., without limitation, a lentivirus vector, a gamma retrovirus vector, a foamy virus vector, an adeno-associated virus vector, an adenovirus vector, a pox virus vector, a herpes virus vector, an engineered hy brid virus vector, a transposon mediated vector, and the like. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology⁷ manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.

In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Patent No. 6,326,193).

In some aspects, an expression vector can be used to introduce the antibody or antigen binding fragment thereof into a cell. Accordingly, an expression vector (e.g., a lentiviral vector) of the present invention may comprise a nucleic acid encoding for the antibody or antigen binding fragment thereof. In some aspects, the expression vector will comprise additional elements that will aid in the functional expression of the antibody or antigen binding fragment thereof encoded therein. In some aspects, an expression vector comprising a nucleic acid encoding for the antibody or antigen binding fragment thereof further comprises a mammalian promoter. In one aspect, the vector further comprises an elongation-factor-1 - alpha promoter (EF-la promoter). Use of an EF-la promoter may increase the efficiency in expression of downstream transgenes. Physiologic promoters (e.g.. an EF-la promoter) may be less likely to induce integration mediated genotoxicity, and may abrogate the ability of the retroviral vector to transform stem cells. Other physiological promoters suitable for use in a vector are known to those of skill in the art and may be incorporated into a vector of the present invention. In some aspects, the vector further comprises a non-requisite cis acting sequence that may improve titers and gene expression. One non-limiting example of a nonrequisite cis acting sequence is the central polypurine tract and central termination sequence (cPPT/CTS) which is important for efficient reverse transcription and nuclear import. Other non-requisite cis acting sequences are known to those of skill in the art and may be incorporated into a vector (e.g., lenti viral vector) of the present invention. In some aspects, the vector further comprises a posttranscriptional regulatory element. Posttranscriptional regulatory elements may improve RNA translation, improve transgene expression and stabilize RNA transcripts. One example of a posttranscriptional regulatory element is the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Accordingly, in some aspects a vector for the present invention further comprises a WPRE sequence. Various posttranscriptional regulator elements are known to those of skill in the art and may be incorporated into a vector of the present invention. A vector of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, packaging sequences, and 5’ and 3’ long terminal repeats (LTRs). The term ‘'long terminal repeat” or “LTR” refers to domains of base pairs located at the ends of retroviral DNAs which comprise U3, R and U5 regions. LTRs generally provide functions required for the expression of retroviral genes (e.g., promotion, initiation and polyadenylation of gene transcripts) and to viral replication. In one aspect, a vector of the present invention includes a 3’ U3 deleted LTR. Accordingly, a vector of the present invention may comprise any combination of the elements described herein to enhance the efficiency of functional expression of transgenes.

In some aspects, a polynucleotide encoding the antibody or antigen binding fragment thereof can be RNA, e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those of skill in the art; any known method can be used to synthesize RNA comprising a sequence encoding the antibody or antigen binding fragment thereof of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a nucleotide sequence encoding the antibody or antigen binding fragment thereof of the present disclosure into a host cell can be carried out in vitro, ex vivo or in vivo. For example, a host cell can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding an antibody or antigen binding fragment thereof of the present disclosure.

In order to assess the expression of a polypeptide or portions thereof, the expression vector to be introduced into a cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In some aspects, the selectable marker may be carried on a separate piece of DNA and used in a cotransfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, without limitation, antibiotic-resistance genes.

Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory⁷ sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assessed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include, without limitation, genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

In one aspect, the present disclosure generally relates to an isolated polynucleotide comprising a nucleotide sequence encoding an antibody or antigen binding fragment thereof as described herein. In one aspect, the present disclosure generally relates to a vector comprising a polynucleotide as described herein. In one aspect, the present disclosure generally relates to an isolated host cell that recombinantly produces the antibody or antigen binding fragment thereof as described herein.

PHARMACEUTICAL COMPOSITIONS

Also provided herein are compositions comprising an antibody or antigen binding fragment thereof as described herein, such as compositions comprising a polynucleotide encoding the antibody or antigen binding fragment thereof and/or compositions comprising the isolated or recombinant antibody or antigen binding fragment thereof. Among the compositions are pharmaceutical compositions and formulations for administration, such as for treating SSc, chlamydia, asthma, and/or fibrosis. Also provided are therapeutic methods for administering the antibody or antigen binding fragment thereof and compositions to subjects, e.g., patients. In one aspect, the present disclosure generally relates to a pharmaceutical composition comprising an antibody or antigen binding fragment as described herein and at least one pharmaceutically acceptable carrier.

Also provided are compositions for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the amount of the antibody or antigen binding fragment thereof for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some aspects, the composition includes at least one additional therapeutic agent. In some aspects, the additional therapeutic agent comprises a cytotoxic drug and/or an anti-CD3 antibody or antigen binding fragment thereof.

The term “pharmaceutical formulation” or “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride: hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, buty l or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).

Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary- methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some aspects, the pharmaceutical composition further includes other pharmaceutically active agents or drugs. The pharmaceutical composition in some aspects contains the antibody or antigen binding fragment thereof in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some aspects is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single administration of the antibody or antigen binding fragment thereof, by multiple administrations of the antibody or antigen binding fragment thereof, or by continuous administration of the antibody or antigen binding fragment thereof.

Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some aspects, the antibody or antigen binding fragment thereof is administered parenterally. The term “parenteral/’ as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some aspects, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. Compositions in some aspects are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods wi th specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary' substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.

Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

The formulations to be used for in vivo administration are generally sterile. Sterility' may be readily accomplished, e.g., by filtration through sterile filtration membranes.

METHODS

In one aspect, the present disclosure generally relates to a method of treating, ameliorating, and/or preventing a disease or condition associated with Tmem273 expression. wherein the method comprises contacting a cell that is capable of expressing Tmem273 with an antibody or antigen binding fragment thereof as described herein. In some aspects, the cell is a T cell. In some aspects, the T cell is a CD8 T cell. In some aspects, the T cell is a CD8yl3 T cell. In some aspects, the cell expresses at least one of TNFa, IL-13, IFN- y, and combinations thereof.

In one aspect, the present disclosure generally relates to a method of treating, ameliorating, and/or preventing systemic sclerosis (SSc) in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of an antibody or antigen binding fragment as described herein. Furthermore, in one aspect, the present disclosure generally relates to a method of treating, ameliorating, and/or preventing chlamydia in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of an antibody or antigen binding fragment as described herein. Moreover, in one aspect, the present disclosure generally relates to a method of treating, ameliorating, and/or preventing asthma in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of an antibody or antigen binding fragment as described herein. In some aspects, the asthma is chronic asthma. Furthermore, in one aspect, the present disclosure is generally related to a method of treating, ameliorating, and/or preventing fibrosis in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of an antibody or antigen binding fragment as described herein.

SEQUENCE TABLE

EXAMPLES

The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the instant specification should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Example 1: Tmem272 Fusion Proteins and Anti-Tmem273 Antibodies

Unique to systemic sclerosis among the autoimmune diseases is IL-13, and its source. In SSc significant producers of IL- 13 are effector CDS T cells polarized to coproduce interferon gamma (IFN-y) and IL-13. This unusual phenotype was characterized as a dysregulated CD8 T cell population. Investigation of this anomalous CD8 T cell subset found it was present in elevated levels in peripheral blood of SSc patients, that supernatants from activated SSc CD8 T cells induced dermal fibroblasts to produce collagen (scar), and that fibroblast collagen production could be blocked by antibodies that neutralized IL- 13, by reducing TGFp. Previous work had shown that the combination of TNFa and IL- 13 triggered stromal cells to produce TGFp, the master regulator of scarring and an important mechanism in SSc fibrosis. The same pathway underpins scarring in chronic asthma fibrosis (see, for example. Miyahara, N., et al., J Immunol 2004; 172:2549-58; and Dakhama. A., et al.. Allergy 2013; 68:666-73, each of which is hereby incorporated by reference in its entirety herein).

In the same window, parallel chlamydia pathogenesis research found that CD8 T cells coproducing IFN-y and IL- 13 (CD8yl3) were generated during murine immune responses to Chlamydia muridarum genital tract infections (Johnson, R.M., et al., Immunology 2014; 142:248-57), and that a chlamydia-specific CD8yl3 T cell clone protected mice from C. muridarum genital tract immunopathology (scarring) (Johnson, R.M., et al., J Infect Dis 2020). The latter finding brought into question whether CD8y 13 T cells were dysregulated or a minority CD8 T cell subset generated during mucosal immune responses. Because the authors of these studies had conventional CD8 and CD8yl3 murine T cell clones, they did comparative gene expression micro array analysis to identify biomarkers unique to CD8yl3 T cells and determined whether they were relevant to CD8 T cells circulating in the peripheral blood of SSc patients. The murine T cell clone expression micro-array comparison was functionally single cell RNAseq before that technology was generally available. Comparison of the murine CD8 T cell clones identified 1810011H1 IRik, the murine homolog of human C10orfl28, a hypothetical cell surface protein, as a biomarker specific to CD8yl3 T cells. Supporting the position that CD8yl3 T cells may be a normal component of mucosal immune responses, an RNAseq study of T cell subsets resident in the spleen and genital tracts of chlamydia immune mice found Tmem273 transcripts markedly enriched in genital tract resident CD8 T cells versus splenic CD8 T cells (Johnson, R.M., et al, J Infect Dis 2022; 225:2033-42). In the context of SSc, CD8yl3 T cells are likely a mucosal T cell response continuously activated by an autoantigen, causing a normal healing response to crossover to pathologic progressive scarring. Translational research utilizing a custom polyclonal antibody specific for the extracellular domain of human C10orfl28 for flow sorting and comparative downstream micro array analysis of purified CD8 T cells from an SSc patient with severe disease versus a healthy control confirmed that C10orfl28, now know n as Tmem273, was a biomarker for CD8y 13 T cells in the SSc patient, and identified other likely SSc biomarkers including Amtl, Cep85L IL-5. Alox5, Clc. and Amelx. That investigation established an association of C10orfl28 with IL-13 in SSc CD8 T cells, and was incorporated into US patent 9,683,262 B2. Tmem273 is also uniquely expressed by the majority' of CD4 T cells polarized to produce interferon gamma, TNF-alpha, and IL-13 (Johnson et al, 2018, Infect Immun 86: e00614-17 supplemental material).

To advance Tmem273 research fusion proteins linking the extracellular domains of human (SEQ ID NO: 1) and murine (SEQ ID NO: 2) Tmem273 (FIG. 1) to mouse IgGl and rat IgG2b Fc domains were engineered for immunization and monoclonal antibody generation in mice and rats respectively. The open reading frame for the human Tmem273 extracellular domain was engineered with the human IL-2 signal sequence and included an additional stabilizing glycine residue added to the mature n-terminus (FIG. 2) (SEQ ID NO: 3). The human Tmem273 fusion protein w as produced in human 293F cells (FIG. 3A-FIG. 3C). Human Tmem273 extracellular domain protein (hClO) was produced by transient transfection of the expression vector (FIG. 3B) containing the KasI - EcoRV insert (FIG. 3A) (SEQ ID NO: 5) to produce the Fc fusion protein. FIG. 3C presents an image taken of a 4- 12% SDS-PAGE nonreducing gradient gel with purified hClO fusion protein. The predicted weight of the hClO homodimer is 56kd.

The mouse Tmem273 fusion protein was engineered with the human Tmem273 signal peptide to generate a fusion protein with the matured native murine Tmem273 n-terminus QVLAT... (SignalP-4.1 server) when expressed in human 293F cells (FIG. 4) (SEQ ID NO: 6).

The murine Tmem273 fusion protein (mClO) was produced in human 293F cells (FIG. 5A-FIG. 5C). Murine Tmem273 extracellular domain protein (mClO) was produced by transient transfection of the expression vector (FIG. 5B), containing the KasI - EcoRV insert (FIG. 5A) (SEQ ID NO: 7), to produce the Fc fusion protein. FIG. 5C presents an image of a 4-12% SDS-PAGE nonreducing gradient gel with purified mClO fusion protein. The predicted weight of the hClO homodimer is 60kd.

For the human Tmem273 protein, Balb/c mice were immunized subcutaneously with 12.5 pg of hClO fusion protein in Sigma Adjuvant twice at a two-week interv al, then boosted unadjuvanted 12.5 pg dose injected retro-orbitally 12 weeks later with an SP2/0 fusion three days post retro-orbital boost. The mouse used for the hybridoma fusion had high-specific titers to hClO at the time of boosting (FIG. 6).

The murine Tmem273 fusion protein (mClO) was shipped to Green Mountain Antibodies, Inc. to immunize rats and generate rat anti-mouse Tm273 extracellular domain monoclonal antibodies.

The mouse anti-human Tmem273 hybridoma fusion yielded -forty hClO-specific mouse IgM and IgG monoclonal antibodies. After additional investigation monoclonal antibody hC10-6Gl was selected for further development based on specific staining of human T cells in PBMC populations and specific-enhancement of IL- 13 production when coimmobilized with anti-CD3 and -CD28 monoclonal antibodies.

Example 2: Anti-Tmem273 Antibody Analysis

For flow cytometry screening, PBMCs from a deidentified healthy control subject were Fc blocked with donkey IgG (Jackson ImmunoResearch cat# 017-000-003) then separately stained with 25 anti-hC lO monoclonal antibodies, washed, stained with anti-mouse IgG (Alexa 647, minimal cross-reactivity⁷; Biolegend cat# 405322), washed, and finally stained with anti-CD3 (APC/Cy7, Biolegend cat# 300318). Stained PBMC were gated on lymphocytes using forward/side scatter, then CD3 positivity and analyzed for the percentage of Tmem273 positive cells compared to “2nd step only” (no Tmem273 monoclonal antibody) negative control.

Anti-hClO monoclonal antibodies of the IgM subclass universally did not stain T cells (data not shown). Tmem273 has a predicted 17 amino acid (aa) extracellular surface domain while mouse IgM exists as a 900kd pentamer, suggesting steric hindrance may account for lack of IgM monoclonal antibody binding to human T cells. Seven hClO IgG monoclonal antibodies (15Okd) showed binding to a population of T cells. -10% of total, not present in the '‘2nd step only’’ control. The PBMC flow cytometry screen for monoclonal antibody hC10-6Gl is shown in FIG. 7.

For the costimulatory investigation, Nunc maxisorp plates (cat# 44-2404-21) were coated overnight at 4°C with 50 pl of PBS containing Ipg/ml of anti-CD3 (Biolegend cat# 300438). anti-CD28 (Biolegend cat# 302934). without and with 1 pg/ml of hC10-6Gl (all azide-free). Wells were washed with 150pl of RPMI medium supplemented with 10% FBS (characterized, Gibco), 5xl0‘⁵M 2-ME, 1% Glutamax I, 12.5 pg/ml gentamicin, then 75k human PBMC from a healthy subject added to each well (media, 3/28, 3/28/6G1) in RPMI medium containing recombinant human IL-7 (R&D systems cat# 207IL005) at a final concentration of 2 r|g/ml. Supernatants collected at 72h were analyzed for IFNy (Invitrogen cat#s 14-7318-81 & 13-7319-81) and IL-13 (Invitrogen cat#s 14-7139-81 & 13-7138-81) levels by ELISA using recombinant human IFN-y and IL-13 (Biolegend cat#s 570202 & 571102) as standards and streptavidin-HRP (BD Pharmingen cat# 554066) with TMB substrate for detection.

Referring now to FIG. 8A-FIG. 8C, addition of hC10-6Gl to immobilized anti- CD3/28 resulted in a -50% increase in IFN-y production (FIG. 8A) compared with a 300% increase in IL- 13 (FIG. 8B), accounting for the increased IL-13/IFN-y ratio (FIG. 8C). The selective enhancement of IL- 13 production by hC10-6Gl engagement of Tmem273 directly linked Tmem273 to IL-13 production in human T cells. Tmem273 served as a mechanismbased biomarker for CD8 T cells that coproduce IFN-y and IL-13.

The complementarit -determining regions (CDR) mouse IgGl monoclonal hC10-6Gl were determined by sequencing, with the sequences presented below.

Heavy Chain

DNA Sequence (SEQ ID NO: 16) gaggtgaagcttctcgagtctggaggtggcctggtgcagcctggaggatccctgaaactctcctgtgcagcctcaggattcgattttagt agatactggatgagttgggtccggcaggctccagggaaagggctagaatggattggagaaattaatccagatagcagtacgataaac tatacgccatctctaaaggataaattcatcatctccagagacaacgccaaaaatacgctgtacctgcaaatgagcaaagtgagatctga ggacacagccctttattactgtgcaagactcagctgggggggggctat ggactactggggtcaaggaacctcagtcaccgtctcctcag

Predicted Protein Sequence (SEQ ID NO: 15)

Complementarity determining regions (CDRs) are underlined.

EVKLLESGGGLVOPGGSLKLSCAASGFDFSRYWMSWVROAPGKGLEWIGEINPDSS TINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCARLSWGGAMDYWGOGT SVTVSS

Light Chain

DNA Sequence (SEQ ID NO: 21) gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagagcattgtacat agtaatggaaacacgtattttgaatggtacctgcagaaaccaggccagtctccaaagctcctgatctacaaagtttccaaccgattttctg gggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagttt attactgctttcaaggttcacatgttccattcacgttcggctcggggacaaagttggaaataaaac

Predicted Protein Sequence (SEQ ID NO: 20)

Complementarity determining regions (CDRs) are underlined.

DVLMTOTPLSLPVSLGDQASISCRSSQSIVHSNGNTYFEWYLQKPGOSPKLLIYKVSN RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK

The hC10-6Gl antibody heavy and light chain sequences were sent to Absolute

Antibody, LTD to engineer a humanized recombinant hC10-6Gl monoclonal antibody of the IgGl subclass (depleting) using their proprietary Prometheus™ software platform. These sequences are presented below. hC10-h6Gl-Human - Heavy chain

DNA (SEQ ID NO: 24)

ATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCGCTAGCTGAAGTGA

AACTGCTTGAGAGCGGCGGGGGCCTAGTTCAACCCGGCGGCAGCCTGAAGCTGA

GCTGCGCCGCAAGCGGCTTCGACTTCAGCAGATACTGGATGAGCTGGGTGAGAC

AAGCCCCCGGCAAGGGCCTGGAGTGGATCGGCGAGATCAACCCCGACAGCAGCA

CCATCAACTACACCCCTAGCCTGAAGGACAAGTTCATCATCAGCAGAGACAACG

CCAAGAACACCCTGTACCTGCAGATGAGCAAGGTGAGAAGCGAGGACACCGCCC

TGTACTACTGCGCTAGACTGAGCTGGGGCGGGGCCATGGACTACTGGGGCCAAG

GCACAAGCGTGACCGTCTCGAGCGCCAGCACCAAAGGTCCATCCGTGTTTCCGCT

CGCCCCGTCCTCAAAGTCGACCTCCGGAGGCACTGCCGCCCTGGGCTGCCTTGTC

AAGGACTATTTCCCCGAACCTGTCACGGTGTCCTGGAACAGCGGCGCCCTGACTT

CCGGAGTGCACACCTTCCCCGCCGTCCTGCAATCCAGCGGCCTGTACTCACTGTC

ATCCGTTGTGACTGTCCCGTCGTCCAGCCTGGGAACCCAAACCTACATTTGCAAC

GTGAATCACAAACCATCGAATACCAAGGTCGATAAGAAAGTCGAGCCGAAGTCA

TGCGACAAGACTCACACCTGTCCGCCTTGCCCAGCCCCAGAACTGCTCGGCGGCC CTTCGGTGTTTTTGTTTCCGCCGAAGCCGAAGGACACTCTGATGATCTCACGCAC TCCAGAGGTGACTTGCGTGGTGGTCGATGTTTCGCACGAGGACCCGGAAGTGAA

ATTCAACTGGTATGTCGACGGGGTGGAAGTGCATAATGCCAAGACGAAGCCGAG

GGAGGAACAGTACAACTCCACCTACAGAGTGGTTTCAGTCCTTACCGTCCTCCAT

CAAGATTGGCTGAACGGAAAGGAGTACAAATGTAAGGTGTCGAACAAAGCGTTG

CCGGCCCCTATCGAAAAGACTATCAGCAAGGCCAAAGGACAGCCGCGGGAGCCG

CAAGTGTACACCCTCCCGCCTTCGCGGGACGAGCTGACCAAGAATCAGGTGTCC

CTTACTTGCCTGGTGAAGGGATTCTACCCCTCGGATATCGCAGTCGAATGGGAAT

CGAATGGACAGCCAGAAAACAACTACAAGACCACTCCCCCGGTGCTCGACTCCG

ACGGTTCCTTCTTCCTGTACTCGAAGCTGACCGTGGACAAATCACGCTGGCAGCA

GGGAAACGTGTTTAGCTGCAGCGTGATGCATGAGGCGCTGCATAATCACTACAC

CCAGAAGTCACTCTCGCTCAGCCCAGGGAAG

Protein (signal peptide in bold) (SEQ ID NO: 23)

MPLLLLLPLLWAGALAEVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVR

QAPGKGLEWIGEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYC

ARLSWGGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP

EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT

KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS

HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC

KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV

EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

YTQKSLSLSPGK hC10-h6Gl-Human - Kappa

DNA- gene (SEQ ID NO: 27)

ATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCGCTAGCTGACGTGC

TGATGACACAGACCCCCCTGAGCCTGCCCGTGAGCCTGGGCGACCAAGCAAGCA

TCAGCTGCAGAAGCTCTCAGAGCATCGTGCACAGCAACGGCAACACCTACTTCG

AGTGGTACCTGCAGAAGCCTGGGCAGAGCCCCAAGCTGCTGATCTACAAGGTGA

GCAATCGGTTCAGTGGGGTGCCTGACAGATTCAGTGGTAGCGGTAGCGGCACCG

ACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGGACCTGGGCGTGTACTACT

GCTTCCAAGGCAGCCACGTGCCCTTCACCTTCGGCAGCGGCACCAAACTCGAGAT

CAAGAGAACTGTGGCCGCGCCGTCAGTGTTTATCTTCCCTCCATCGGATGAACAG

CTTAAGTCCGGCACGGCGTCTGTGGTCTGCCTGCTCAATAACTTTTACCCTAGGG

AAGCTAAAGTCCAATGGAAAGTGGATAACGCCCTGCAGTCAGGAAACAGCCAGG

AATCGGTTACCGAACAGGACAGCAAGGACAGCACTTACTCCTTGTCGTCGACTCT TACTCTGAGCAAGGCCGATTACGAGAAGCACAAGGTCTACGCCTGCGAGGTCAC CCATCAGGGACTCTCGTCCCCGGTGACCAAATCCTTCAATAGAGGCGAATGC

Protein (signal peptide in bold) (SEQ ID NO: 26)

MPLLLLLPLLWAGALADVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYFEW YLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSH VPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

The antibody engineering work was successful based on production of the recombinant hC10-h6Gl human IgGl antibody (FIG. 9) and retention of antigen specificity.

To confirm humanized hC10-6Gl (h6Gl) recombinant monoclonal antibody retained antigen specificity, it was compared to the parent mouse antibody 6G1 for binding to hClO and mClO fusion proteins. Nunc maxisorp plates with coated with hClO and mClO (2 pg/ml in PBS, 50 pl/well = 100 r|g/well, overnight at 4°C). Wells were blocked with PBS containing 1.5% BSA and 0.05% Tween 20, then incubated with serial dilutions of parent and humanized monoclonal antibodies from saturating (1.5

0.55 r|g mcAb /well) to sub saturating (0.019 -> 0.002 pg/well). Detection of the original mouse antibody 6G1 was with goat anti-mouse light chain-HRP (minimal cross reactivity , Jackson ImmunoResearch cat# 115-035-174). The humanized h6Gl recombinant antibody was detected with goat antihuman Ig Fc (minimal cross reactivity, Jackson ImmunoResearch cat# 109-036-098). Second step HRP -conjugated antibodies were diluted 1 :6000 in blocking buffer. Detection was with TMB substrate (O.D. 450 nm).

Referring now to FIG. 10, the original mouse hC10-6Gl monoclonal antibody had high affinity for hClO and limited but detectable cross-reactivity with mClO, while humanized h6Gl retained high affinity for hClO and had increased affinity for mClO (compare h6Gl-mC10 to 6Gl-mC10 in FIG. 10).

To confirm binding specificity of h6Gl for human and murine Tmem273 extracellular domains rather than spurious non-specific or protein linker binding, additional studies were performed incorporating fusion protein, Slc24a3-mIgGl, containing the 1^st extracellular domain of human Slc24a3 in the same mouse IgGl Fc fusion construct as hClO with anearly identical linker domain. Nunc maxisorp plates were coated ON at 4°C with 50 pl PBS containing 2 pg/ml of each fusion protein (hClO, mClO, hSlc24a3; 100 T)g/well). Binding of h6Gl to the fusion proteins was tested in the saturating range (1.5 -> 0.055 T|g/well) relevant to cross reactivity with mClO (FIG. 11A-FIG. 11C).

Referring now to FIG. 11 A-FIG. 11 C binding of h6Gl to hC 10 and mC 10 was highly specific (FIG. 1 IB) and represented cross reactivity between highly homologous human (SEQ ID NO: 1) and murine (SEQ ID NO: 2) Tmem273 extracellular domains (FIG. 11A). Not wishing to be bound by theory, the only plausible shared epitope between mClO and hClO is the highly homologous extracellular domain highlighted by the grey bar of FIG. 11A. Lack of binding to hSlc24a3 at high concentrations of h6Gl ruled out spurious non-specific binding or binding to linker domain sequences. FIG. 11C presents a gel image of hSlc24a3 fusion protein.

Additional mouse monoclonal antibodies against the extracellular domain of human Tmem273 were produced and are described below. These antibodies included 5C12. 5A9, 3E8, 3B8, and 1F3.

5C12

Heavy Chain

DNA sequence (SEQ ID NO: 63) caggtccagctgcagcagtctggacctgaactggtgaagcctggggcctcagtgaagatttcctgcaaagcttctggctacgcattca gtaactcttggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacggatttatcctggagatggagatactaac tacaatgggagattcaaggacaaggccgcactgactgcagacaaatcctccaacacagcctacatgcagctcagcagcctgacctct gtggactctgcggtctatttctgtgcaagccctattactacggtaataactaacgaggctttggactactggggtcaaggaacctcagtca ccgtctcctcag

Predicted Protein Sequence (SEQ ID NO: 61)

Complementarity determining regions (CDRs) are underlined.

OVOLOQSGPELVKPGASVKISCKASGYAFSNSWMNWVKORPGOGLEWIGRIYPGDG DTNYNGRFKDKAALTADKSSNTAYMOLSSLTSVDSAVYFCASPITTVITNEALDYW GQGTSVTVSS

Light Chain

DNA sequence (SEQ ID NO: 68) gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcagggcattag caattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctattacacatcaagtttacactcaggagtcccatcaa ggttcagtggcagtgggtctgggacagattattctctcaccatcaccaacctggaacctgaagatattgccacttactattgtcagcagta tattaagcttccgtggacgttcggtggaggcaccaagctggaaatcaaac

Predicted Protein Sequence (SEQ ID NO: 66)

Complementarity) determining regions (CDRs) are underlined DIOMTOTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGV PSRFSGSGSGTDYSLTITNLEPEDIATYYCOQYIKLPWTFGGGTKLEIK

5A9

Heavy Chain

DNA sequence (SEQ ID NO: 74) caggtccagctgcagcagtctggacctgaactggtgaagcctggggcctcagtgaagattcctgcaaagcttctggctacgcattca gtggctcttggatgaactgggtaaagcagaggcctggacagggtcttgagtggattggacggatttatcctggagatggagatactaa ctacaatgggaagttcaggggcaaggccacactgactgcagacaaatcctccaacacagcctacatgcagctcagcagcctgacctc tgtggactctgcggtctatttctgtgcaagccctattactacggtagtatctaacgaggctttggactactggggtcaaggaacctctgtca ccgtctcctcag

Predicted Protein Sequence (SEQ ID NO: 72)

Complementarity determining regions (CDRs) are underlined

OVOLOQSGPELVKPGASVKISCKASGYAFSGSWMNWVKORPGOGLEWIGRIYPGDG DTNYNGKFRGKATLTADKSSNTAYMQLSSLTSVDSAVYFCASPITTVVSNEALDYW GQGTSVTVSS

Light Chain

DNA sequence (SEQ ID NO: 79) gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcaggacattag caattatttaaactggtatcagcagaaaccagatggaactgtaaactcctgatctatacacatcaagtttacactcaggagtcccatcaa ggttcagtggcagtgggtctgggacagatttttctctcaccatcaccaacctggaacctgaagatatgccacttactattgtcagcagtat attaagcttccgtggacgttcggtggaggcaccaagctggaaatcaaac

Predicted Protein Sequence (SEQ ID NO: 77)

Complementarity determining regions (CDRs) are underlined

DIOMTOTTSSLSASLGDRVTISCSASODISNYLNWYOOKPDGTVKLLIYYTSSLHSGV PSRFSGSGSGTDFSLTITNLEPEDIATYYCOOYIKLPWTFGGGTKLEIK

3E8

Heavy Chain

DNA sequence (SEQ ID NO: 85) caagttactctaaaagagtctggccctgggatattgaagccctcacagaccctcagtctgacttgttctttctctgggttttcactgagcact tctggtatgggtgtaggctggattcgtcagccttcagggaagggtctggagtggctggcacacattggtgggatgatgataagtactat aacccatccctgaagagccagctcacaatctccaaggatacctccagaaaccaggtatcctcaagatcaccagtgtggacactgcag atactgccacttactactgtgctcggtactatactatgctatggactactggggtcaaggaacctcagtcaccgtctcctcag Predicted Protein Sequence (SEQ ID NO: 83)

Complementarity determining regions (CDRs) are underlined

OVTLKESGPGILKPSOTLSLTCSFSGFSLSTSGMGVGWIROPSGKGLEWLAHIWWDD DKYYNPSLKSOLTISKDTSRNOVFLKITSVDTADTATYYCARYYYYAMDYWGOGTS VTVSS

Light Chain

DNA sequence (SEQ ID NO: 90) gatgtttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagagcattgtacat agtaatggaaacacctattagaatggtacctgcagaaaccaggccagtctccaaagctcctgatctacaaagtttccaaccgattttctg gggtcccagacaggtcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtt atactgcttcaaggtcacatgtccgtacacgtcggaggggggaccaagctggaaataaaac

Predicted Protein Sequence (SEQ ID NO: 88)

Complementarity determining regions (CDRs) are underlined

DVLMTOTPLSLPVSLGDOASISCRSSOSIVHSNGNTYLEWYLQKPGOSPKLLIYKVSN RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK

3B8

Heavy Chain

DNA sequence (SEQ ID NO: 96) caggtccaactgcagcagcctggggctgagctggtgaggcctggggctcagtgaagctgtcctgcaaggctctggctacacctca ccagctactggatgaactgggtgaagcagaggcctggacaaggcctgaatggattggtatgattgatcctcagacagtgaaactca ctacaatcaaatgtcaaggacaaggccacattgactgtagacaaatcctccaccacagcctacatgcatctcagcagcctgacatctg aggactctgcggtctattactgtgcaagagaccccctttatgctatggactactggggtcaaggaacctcagtcaccgtctcttcag

Predicted Protein Sequence (SEQ ID NO: 94)

Complementarity^ determining regions (CDRs) are underlined

OVOLOQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKORPGOGLEWIGMIDPSD SETHYNOMFKDKATLTVDKSSTTAYMHLSSLTSEDSAVYYCARDPLYAMDYWGOG TSVTVSS

Light Chain

DNA sequence (SEQ ID NO: 101) gacattgtgatgacacagtctccctcctccctggctatgtcagtaggacagaaggtcactatgagctgcaagtccagtcagagcctttta aatagtggcaatcaaaagaactttttggcctggtaccagcaaaaaccaggacagtctcctaaacttctagtatacttgcatccactaggg atctggggtccctgatcgctcataggcagtggatctgggacagattcactcttaccatcagcagtgtgcagtctgaagacctggcag atacttctgtcagcaacattatagcactccgtacacgttcggaggggggaccaagctggaaataaaac Predicted Protein Sequence (SEQ ID NO: 99)

Complementarity determining regions (CDRs) are underlined

DIVMTOSPSSLAMSVGOKVTMSCKSSOSLLNSGNOKNFLAWYOQKPGOSPKLLVYF ASTRDSGVPDRFIGSGSGTDFTLTISSVOSEDLADYFCOOHYSTPYTFGGGTKLEIK

1F3

Heavy Chain

DNA sequence (SEQ ID NO: 107) gatgtgcagcttcaggagtcgggacctggcctggtgaaaccttctcagtctctgtccctcacctgcactgtcactggctactcaatcacc agtgattatgcctggaactggatccggcagtttccaggaaacaaactggagtggatgggctacataagctacagtggtagcactagct acaacccatctctcaaaagtcgaatctctatcactcgagacacatccaagaaccagttcttcctgcagttgaattctgtgactactgagga cacagccacatattactgtacatccctaggggcttactggggccaagggactctggtcactgtctctgcag

Predicted Protein Sequence (SEQ ID NO: 105)

Complementarity determining regions (CDRs) are underlined

DVOLQESGPGLVKPSOSLSLTCTVTGYSITSDYAWNWIROFPGNKLEWMGYISYSGS ISYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCISLGAYWGQGTLVTVSA

Light Chain

DNA sequence (SEQ ID NO: 112) gatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttag atagtgatggaaagacatatttgaattggttgttacagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactc tggagtccctgacaggttcactggcagtggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtt tattattgctggcaaggtacacattttccgtacacgttcggaggggggaccaagctggaaataaaac

Predicted Protein Sequence (SEQ ID NO: 110)

Complementarity determining regions (CDRs) are underlined

DVVMTOTPLTLSVTIGOPASISCKSSOSLLDSDGKTYLNWLLORPGOSPKRLIYLVSK LDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWOGTHFPYTFGGGTKLEIK

Example 3: Production and Analysis of anti-rat Tmem273 Antibody

Preclinical development of therapeutic agents is dependent on having relevant animal models. To address this issue for Tmem273 the murine mC 10, rat IgG2b Fc fusion protein was shipped to Green Mountain Antibodies, Inc., to immunize Norwegian brown rats and generate rat monoclonal antibodies against murine Tmem273 to mirror human Tmem273, the hC10-6Gl monoclonal antibody and its humanized recombinant form h6Gl. There are only 2 amino acid differences between rat (SEQ ID NO: 11) and mouse (SEQ ID NO: 2) Tmem273 in the extracellular domain (FIG. 12).

The rat hybridoma fusion yielded a single monoclonal antibody designated mClO- 22G1. mC10-22Gl is an IgM monoclonal antibody specific for mClO. It did not recognize the rat IgG Fc domain of the fusion protein. For experimental purposes as a control this application included a second rat IgM hybridoma from an unrelated project with Green Mountain Antibodies, Inc., 25H05, autoreactive with the rat IgG Fc domain (FIG. 13).

To investigate whether munne Tmem273 plays a role in IL-13 production an alternative approach was taken using hybridomas to deliver Tmem273 activation signals via the monomeric transmembrane form of IgM naturally present on their cell surfaces, with exogenous anti-CD3 antibody immobilized by hybridoma Fc receptors: a cytokine-focused modification of a redirected lysis assay. 22G1 and 24H5 hybridomas share the same mouse myeloma fusion partner. This approach avoided potential steric hindrance issues associated with the secreted pentameric form of IgM and issues with purification of IgM monoclonal antibodies necessary to do conventional costimulation assays with immobilized antibodies on tissue culture plastic. In the "‘redirected cytokine assay" the readout murine T cell clone was 8sAg-l, a CD8 T cell clone that coproduced IFN-y and IL- 13 when activated (Johnson RM, et al., Immunology 2014: 142:248-57). For the assay, 22G1 and 24H5 hybridoma cells were washed immediately prior to seeding experimental wells at 50k per well in a 96 well tissue culture plate. 8sAg-l T cell clone cells were added at 15k per well and the RPMI1640 culture medium (10% FBS, 5xl0-5M 2-me. 1% Glutamax I, 12.5ug/ml gentamicin) was supplemented with 1 r]g/ml recombinant murine IL-7 (R&D Systems cat# 407ML005), with anti-CD3 at two different concentrations (0.5 & 0.25 pg/ml) (Biolegend cat# 100314). Cell culture supernatants were collected on experimental day 4 and levels of IFN-y (Invitrogen cat# ENMM700 & ENMM700B) and IL-13 (Ebioscience # 14-7139-81 & 13-7138-81) determined by ELISA with recombinant mouse IFN-y (R&D systems cat# 485-MI-100) and IL-13 standards (Ebioscience cat# 14-8131-62). Detection was with streptavidin-HRP and TMB substrate (450 nm).

Similar to the human data, antibody binding of murine Tmem273 in setting of an anti- CD3 TCR activating signal specifically enhanced the production of IL-13 relative to IFN-y, thereby increasing the IL-13/IFN-y ratio and confirming that mouse and human Tmem273 have similar immunobiology' (FIG. 14).

The complementarit -determining regions (CDR) of rat IgM monoclonal mC10-22GI were determined by sequencing and are presented below. Heavy Chain

DNA Sequence (SEQ ID NO: 32) caggtgcagctgaaggagtctgatggaggcttagtgcagcctggaaggtccctaaaactctcctgtgcagcctcaggattcactttcag tgactattacatggcctgggtccgccaggctccaacgaaggggctggagtgggtcgcaaccattagttatgatggtagtagcacttact atcgagactccgtgaagggccgattcactatctccagagataatgcaaaaagcaccctatacctgcaaatggacagtctgaggtctga ggacacggccacttattactgtgcaagggaatactacggcgttatggatgcctggggtcaaggagcttcagtcactgtctcctcag

Predicted Protein Sequence (SEQ ID NO: 31)

Complementarity) determining regions (CDRs) are underlined.

OVOLKESDGGLVQPGRSLKLSCAASGFTFSDYYMAWVROAPTKGLEWVATISYDGS

STYYRDSVKGRFTISRDNAKSTLYLOMDSLRSEDTATYYCAREYYGVMDAWGOGA

SVTVSS

Light Chain

DNA Sequence (SEQ ID NO: 37) gatgttgtgatgacacaaactccagtctccctgtctgtaagccttggaaatcaagcctctatctcttgtaggtctagtcagagccttttccat agtgatggaaacacatatttgtcatggtacctccagaagcccggccagtctccacagctcctcatctacttggcttccaaccgattttctg gggtctcaaacaggttcagtggaagtgggtcagggacagatttcacactcaagatcagcagagtagagcctgaggatttgggagttta ttactgcttccaacatacacatttaccattcacgttcggctcagggacgaagttggaaataaaac

Predicted Protein Sequence (SEQ ID NO: 36)

Complementarity determining regions (CDRs) are underlined.

DVVMTOTPVSLSVSLGNOASISCRSSOSLFHSDGNTYLSWYLQKPGOSPOLLIYLASN

RFSGVSNRFSGSGSGTDFTLKISRVEPEDLGVYYCFQHTHLPFTFGSGTKLEIK

The mC10-22Gl monoclonal antibody heavy and light chain sequences were sent to

Absolute Antibody, LTD to engineer a mouse-ized recombinant mC10-22Gl monoclonal antibody of the IgG2b subclass (depleting) using their proprietary Prometheus™ software platform. The sequences are presented below. mC10-m22Gl- Heavy Chain

DNA sequence (SEQ ID NO: 40)

ATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCGCTAGCTCAAGTGC

AGCTGAAGGAGTCAGACGGCGGCCTGGTGCAGCCGGGCAGAAGCCTGAAGCTG

AGCTGCGCCGCAAGCGGCTTCACCTTCAGCGACTACTACATGGCCTGGGTGAGA

CAAGCCCCCACCAAGGGCCTGGAGTGGGTGGCCACCATCAGCTACGACGGCAGC

AGCACCTACTACAGAGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGACAAC GCCAAGAGCACCCTGTACCTGCAGATGGACAGCCTGAGAAGCGAGGACACCGCC ACCTACTACTGCGCTAGAGAGTACTACGGCGTGATGGACGCCTGGGGCCAAGGC GCAAGCGTGACCGTCTCGAGCGCCAAAACCACCCCTCCATCCGTCTACCCTCTCG CCCCCGGCTGCGGCGACACCACTGGATCATCCGTGACTTCCGGATGCCTGGTCAA GGGATACTTCCCGGAGCCGGTCACTGTGACCTGGAACTCCGGTTCACTGTCATCA TCCGTCCACACCTTTCCGGCCCTGCTGCAGTCGGGCTTGTACACCATGAGCAGCA GCGTGACCGTGCCATCCTCGACCTGGCCTAGCCAAACCGTGACTTGCTCCGTGGC ACACCCTGCGTCGTCCACTACTGTGGACAAGAAGCTGGAGCCGTCCGGACCTATC TCCACCATTAACCCCTGCCCGCCCTGCAAGGAATGTCACAAGTGTCCCGCTCCCA

ATCTTGAGGGAGGGCCCAGCGTGTTCATTTTCCCTCCTAACATTAAGGATGTGCT GATGATCTCCCTGACTCCCAAAGTGACATGCGTGGTGGTGGACGTGTCAGAAGA TGACCCGGACGTCCAGATCAGCTGGTTCGTGAACAACGTGGAAGTGCATACGGC GCAGACCCAGACTCACCGCGAGGACTATAACAGCACCATCAGGGTCGTGTCCAC CCTGCCGATTCAGCACCAGGACTGGATGTCCGGGAAGGAGTTCAAGTGCAAGGT CAACAACAAGGACCTCCCATCCCCGATCGAACGGACCATCTCGAAGATCAAGGG CCTCGTGCGGGCCCCTCAAGTGTACACGCTGCCGCCACCGGCCGAGCAGCTGTCG CGGAAGGACGTGTCCCTTACCTGTCTCGTCGTGGGTTTTAACCCCGGAGATATTT

CGGTGGAGTGGACCAGCAACGGCCACACCGAAGAGAACTACAAGGATACCGCC CCGGTGCTGGACTCCGACGGGTCCTACTTCATCTACTCCAAGCTGAATATGAAAA CCTCTAAGTGGGAAAAGACTGATAGCTTCTCGTGCAACGTCAGACATGAAGGCT TGAAGAACTACTACCTGAAAAAGACTATCTCCCGCTCGCCCGGAAAG

Protein sequence (signal peptide) (SEQ ID NO: 39)

MPLLLLLPLLWAGALAQVQLKESDGGLVQPGRSLKLSCAASGFTFSDYYMAWVR QAPTKGLEWVATISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYY CAREYYGVMDAWGQGASVTVSSAKTTPPSVYPLAPGCGDTTGSSVTSGCLVKGYFP EPVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTT VDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVV VDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEF KCKVNNKDLPSPIERTISKIKGLVRAPQVYTLPPPAEQLSRKDVSLTCLVVGFNPGDIS VEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKN YYLKKTISRSPGK mC10-m22Gl - kappa Light Chain

DNA sequence (SEQ ID NO: 43) ATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCGCTAGCTGACGTGG TGATGACACAGACCCCCGTGAGCCTGAGCGTGAGCCTGGGCAACCAAGCAAGCA TCAGCTGCAGAAGCTCTCAGAGCCTGTTCCACAGCGACGGCAACACCTACCTGA GCTGGTACCTGCAGAAGCCTGGGCAGAGCCCTCAGCTGCTGATCTACCTGGCCTC CAATAGGTTCAGCGGCGTCAGCAATCGCTTCAGCGGTAGTGGCAGCGGCACCGA CTTCACCCTGAAGATCAGCAGAGTGGAGCCTGAGGACCTGGGCGTGTACTACTG CTTTCAGCACACCCACCTGCCCTTCACCTTCGGCAGCGGAACCAAACTCGAGATC AAGCGCGCAGATGCTGCTCCTACCGTGAGCATCTTCCCGCCGTCCAGCGAACAAC TCACTAGCGGAGGCGCGTCAGTGGTCTGCTTCCTTAACAATTTCTACCCTAAGGA CATCAACGTCAAGTGGAAGATTGACGGATCGGAACGCCAGAACGGAGTGCTGAA CTCATGGACTGATCAGGATTCCAAAGACTCGACTTACTCCATGTCCAGCACCCTG ACCCTGACCAAAGACGAGTACGAAAGGCACAACTCGTACACGTGCGAAGCCACC CACAAGACTTCCACCTCGCCCATCGTGAAGTCCTTCAATCGCAATGAGTGC

Protein sequence (SEQ ID NO: 42)

MPLLLLLPLLWAGALADVVMTQTPVSLSVSLGNQASISCRSSQSLFHSDGNTYLSW YLQKPGQSPQLLIYLASNRFSGVSNRFSGSGSGTDFTLKISRVEPEDLGVYYCFQHTH LPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDG SERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF NRNEC

That antibody engineering work was successful based on production of the recombinant mC10-m22Gl mouse IgG2b antibody (FIG. 15) and retention of antigen specificity.

To confirm that mouse-ized mC10-22Gl (m22Gl) recombinant monoclonal antibody retained antigen specificity, it was compared to the parent rat IgM antibody 22G1 for binding to mClO and hClO fusion proteins. Nunc maxisorp plates with coated with mClO and hClO (2 pg/ml in PBS, 50 pl/well = 100 qg/well, overnight at 4°C). Wells were blocked with PBS containing 1.5% BSA and 0.05% Tween 20, then incubated with serial dilutions of parent and mouse-ized monoclonal antibodies from 0.5 - 0.02 r|g mcAb /well. Detection of the original rat IgM monoclonal antibody 22G1 was with goat anti-rat IgM- HRP (minimal cross reactivity, Jackson ImmunoResearch cat# 112-035-075). The mouse-ized m22Gl recombinant antibody was detected with goat anti-mouse light chain-HRP (minimal cross reactivity, Jackson ImmunoResearch cat# 115-035-174). The anti-rat IgM-HRP detection antibody was diluted 1:6000 in blocking buffer; the anti-mouse light chain-HRP 1:2000. Recombinant mouse-ized m22Gl retained binding specificity for mClO.

Neither the parent rat IgM monoclonal 22G1 nor mouse-ized recombinant m22Gl monoclonal antibody cross react with human Tmem273 (hClO) (FIG. 16A-FIG. 16B). Without wishing to be bound by theory, is likely that 22G1 and m22Gl have moderate rather than high affinity for Tmem273 and furthermore that the absence of mClO-specific IgG monoclonal antibodies in the large rat fusion suggested maturation to high affinity antibodies likely generated autoimmune B cells with antibody recognizing rat Tmem273 that were eliminated.

Without wishing to be bound by theory', mC10-m22Gl could serve as the foundational antibody for ex vivo phage display antibody affinity maturation to generate antimouse Tmem273 extracellular domain specific recombinant monoclonal antibodies with affinities comparable to hC10-h6Gl.

As a preliminary safety study, recombinant mC10-m22Gl antibody was produced in quantity (50 mg) for in vivo testing, and an isotype control recombinant mouse IgG2b purchased from Syd Labs (cat# PA007130-20) as a control. Twenty C57BL/6 female mice were pretreated 1 week prior to Chlamydia muridarum vaginal infection with 2.5 mg of medroxyprogesterone subcutaneously. On day 0 mice were infected vaginally with 50k IFU (infection forming units) of C. muridarum. On day 7 infected mice were divided into two experimental groups of 10 mice each. One group of mice received 0.5 mg of mouse IgG2b isotype control recombinant antibody IP (intraperitoneal), the other mC10-m22Gl (mouse- ized anti-murine Tmem273) IP. Each group received a second 0.5 mg IP dose of the same recombinant antibody 48h later for a total of 1 mg recombinant antibody per mouse. Mice were serially weighed, and reproductive tract immunopathology (hydrosalpinx) scored 32 days post infection.

The incidence of hydrosalpinx per total exposed oviducts was not statistically different between the two experimental groups (50% control IgG2b; 35% m22Gl). In the setting of an active chlamydia infection mouse weights did not differ between the two experimental groups (FIG. 17). There were no observed differences in gross pathology (lungs, liver, heart, kidneys) during end of experiment autopsies on day 32.

Example 4: Analysis of Tmem273

In the present example, the Human Protein Atlas was used to analyze single cell types that express Tmem273 mRNA as well as to analyze tissue expression of Tmem273 protein.

FIG. 18 presents Tmem273 mRNA signals analysis of different cell types from human tissues with signal >_20 nTPM in brain, adipose tissue, endothelial cells, and hematopoietic cells.

Example 5: Flow Cytometry Analysis of Tmem273

The present example describes flow cytometry staining of Tmem273 on peripheral blood T cells from Healthy Control (HC), Early stage SSc, and Late stage SSc subjects. Briefly, fresh human blood samples were collected in BD K2EDTA lOcc tubes, diluted 1: 1 with PBS, added to 10 ml Lymphoprep™ tubes, and spun at 800g x 20 min at 25°C. Resulting buffy coat w as added to 3 ml RPMI160 medium supplemented with Glutamax I, 5X10'⁵M 2-mercaptoethanol, 25ug/ml gentamicin, and 10% FBS (RPMI-CM) and was pelleted at 1500 rpm for 5 min in a clinical centrifuge. Media was aspirated, and the pellet was suspended in 4 ml RPMI-CM, underlay ed with 4 ml Histopaque 1077™, and spun 1500g x 9 minutes. Twice purified buffy coat was collected and then added to 5 ml RPMI-CM, which was then subsequently brought up to 12 ml total volume, counted, then pelleted (1500rpm x 5 min). After pelleting, the pellet was w ashed lx with 3 ml RPMI-CM, then finally suspended at 6xl0⁶cells/ml. 83 ul of cell suspension and 0.5xl0⁶ cells were added to v-bottom wells containing anti-CD3 (brilliant violet 421, antibody UCHT1 Biolegend cat#300434), anti-CD4 (APC/Fire 750, antibody A161A1, Biolegend cat# 357426), anti- CD8a (Alexa 488, antibody HIT8a, Biolegend cat#30091 ), and anti-Tmem273 coupled to Alexa 546 ( ThermoFisher Alexa Fluor 546 Antibody Labeling Kit, cat# A20183) in 75 ul total volume RPMI-CM and incubated for 40 minutes at 4°C. CD3, CD4, CD8 antibodies were used according to manufacturer protocols (2.5ul each per 0.5xl0⁶ cells); anti-Tmem273 hclO-6Gl-alexa546 used at 0.5 ug per 0.5xl0⁶ cells. After 40 minutes PBMC were pelleted at 400g x 5 minutes in tabletop centrifuge at 4°C, then suspended in 100 ul PBS containing live/dead blue stain (LIVE/DEAD Fixable Blue Dead Cell Stain Kit, cat# L34961, according to manufacturer protocol) and incubated for 30 minutes on ice. Finally, 100 ul RPMI-CM wsas added to each well, PBMCs were pelleted in tabletop centrifuge, aspirated, and resuspended in 150 ul 1% paraformaldehyde. After resuspension, the resuspended pellets were stored at 4°C and analyzed within 5 days using a CytoFLEX LX Flow Cytometer. Analysis was performed with FlowJo vl0.2 software. The gating strategy used was Lymphocytes -^single cells- live cells- CD3 (30,000 events) CD4 vs CD8.

Referring now⁷ to FIG. 19A-FIG, 19C, FIG. 19A-FIG. 19C present data and results related to the flow cytometry staining of Tmem273. FIG. 19A presents the gating strategy for the flow cytometry data. FIG. 19B presents representative flow data showing Tmem273 positive frequency in CD4 and CD8 T cell subsets. FIG. 19B presents the absolute number of Tmem273 positive cells per 30,000 T cells in CD4 and CD8 subsets for the first 7 subjects enrolled (3 HC, 4 SSc) in an ongoing clinical investigation comparing Tmem273 T cell frequency in healthy controls and rheumatoid arthritis subjects to SSc subjects (20 subjects each arm). The preliminary’ data show elevated numbers of Tmem273 positive CD4 T cells in the peripheral blood of SSc subjects compared to healthy controls. Based on this trend a cohort of 9HC and 12SSc subjects with a similar Tmem273 frequency distribution would be statistically significant (p = 0.03; Mann- Whitney test), supporting the current 20:20:20 investigation design as sufficiently powered to reach a meaningful conclusion. Additionally, existing data as is demonstrated that the humanized hc!0-6Gl antibody bound a unique subset of CD4 and CD8 T cells in human peripheral blood. The large number of Tmem273 pos CD4 T cells in an SSc subject with early disease is particularly appealing for a biotherapeutic based on depletion of Tmem273 cells. The preliminary data limited to peripheral blood suggests a more prominent role for the Tmem273 pos CD4 subset in SSc. rather than Tmem273 pos CD8 subset. Without wishing to be bound by theory, it is postulated that the Tmem273 positive T cell subset(s) in SSc skin lesions may not be proportionally represented in peripheral blood.

Example 6: Mouse Model Study

In the present example, a mouse model study was performed in which the effects of administration of an anti-murine antibody to Tmem273 were analyzed. FIG. 20A presents a schematic representation of the study. Mice in experimental groups of 3 were sensitized with 20 ug ovalbumin IP in alum adjuvant on days 0 & 7. On day 21 mice in experimental groups of 3 received either recombinant isotype control (Con) IgG2b monoclonal antibody or recombinant anti-Tmem273 antibody m22Gl (22G1). All mice were challenged with inhaled ovalbumin on days 26, 27, 28. On day 30 lungs were harvested for histology and T lymphocyte isolation.

The relative number of Tmem273 positive T cells in the CD4 & CD8 T cells subsets in lung tissue was determined by flow cytometry using the same gating strategy as for human flow studies in FIG. 19A. Additionally, the same staining and flow protocols as used for the human cells of Example 5 were used in the present example, except that 10,000 CD3 evens were captured in the present example. The antibodies used were: CD3 brilliant violet 421, antibody 17A2 (biolegend cat# 100227); CD4 APC/Fire 750, antibody GK.1.5 (biolegend, cat#100459); CD8a alexa 488, antibody 53-6.7 (biolegend, cat#100723); and Tmem273 alexa 546, antibody m22Gl coupled with same kit as hC10-6Gl. Data are presented in FIG. 20B as absolute number per 10,000 T cells. Mean and SD for 3 mice in each group are presented for each T cell subset. * = p value <0.05; *** = p value <0.0005 (Students two-tailed t test).

Referring now to FIG. 20B, FIG. 20B presents a graphical representation of results demonstrating that IP administration of anti-murine antibody Tmem273 resulted in expansion of Tmem273 positive CD4 and CD8 T cells. The relative proportion of Tmem273 pos CD4 and CD8 T cells in lung tissue supports the proposition that the tissue resident Tmem273 CD4: CD8 ratio may differ from peripheral blood.

A gross autopsy was performed at the end of the study. No gross pathology was seen in kidneys, spleen, liver, or bowel in any of the mice, either control or those treated with m22Gl antibody. Referring to FIG. 21, tri chrome staining of lungs from mice that received either recombinant isotype control antibody (Con) or anti-Tmem273 antibody (m22Gl) on day 21, are ordered from lowest (inset A) to highest (inset F) absolute number Tmem273 positive T cells recovered from lung tissue. # Tmem273 positive CD4 and CD8 per 10,000 T cells; indicated for each inset (CD4. CD8). Inset A) Con 1 (280, 290), Inset B) Con 2 (321, 267), Inset C) Con 3 (304, 201), Inset D) m22Gl-2 (467, 338), Inset E) m22Gl-3 (555, 435), Inset F) m22Gl-l (613, 513). The expansion of Tmem273 positive T cells shown in FIG. 20B correlated with worsening lung pathology. Combined the flow and histology data demonstrated that the Tmem273 phenotype is associated with immunopathology. If m22Gl driven Tmem273 T cell expansion enhanced immunopathology, then altering the Fc domain to be reliably depleting (e.g. coupling to cytotoxic drug or bispecific with anti-CD3) would likely prevent immunopathology.

Enumerated Embodiments

In some aspects, the present invention is directed to the following non-limiting embodiments:

Embodiment 1 : An isolated and/or recombinant antibody or antigen binding fragment thereof which specifically binds to transmembrane protein 273 (Tmem273), wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the VH comprises: (a) a complementarity' determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12; a CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 14; (b) a CDR1 comprising the amino acid sequence of SEQ ID NO: 58; a CDR2 comprising the amino acid sequence of SEQ ID NO: 59; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 60; (c) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 71; (d) a CDR1 comprising the amino acid sequence of SEQ ID NO: 80; a CDR2 comprising the amino acid sequence of SEQ ID NO: 81; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 82; (e) a CDR1 comprising the amino acid sequence of SEQ ID NO: 91; a CDR2 comprising the amino acid sequence of SEQ ID NO: 92; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 93; or (f) a CDR1 comprising the amino acid sequence of SEQ ID NO: 102; a CDR2 comprising the amino acid sequence of SEQ ID NO: 103; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 104; the VL comprises: (g) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17; a CDR2 comprising the amino acid sequence of KVS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19; (h) a CDR1 comprising the amino acid sequence of SEQ ID NO: 64; a CDR2 comprising the amino acid sequence of YTS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 65; (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 75; a CDR2 comprising the amino acid sequence of YTS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 76; (j) a CDR1 comprising the amino acid sequence of SEQ ID NO: 86; a CDR2 comprising the amino acid sequence of KVS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 87; (k) a CDR1 comprising the amino acid sequence of SEQ ID NO: 97; a CDR2 comprising the amino acid sequence of FAS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 98; or (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 108; a CDR2 comprising the amino acid sequence of LVS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 109.

Embodiment 2: The antibody or antigen binding fragment thereof of embodiment 1, wherein the antibody or antigen binding fragment thereof comprises: (a) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 12; the CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 17; the CDR2 comprising the amino acid sequence of KVS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 19; (b) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 58; the CDR2 comprising the amino acid sequence of SEQ ID NO: 59; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 60; and the Vi, comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 64; the CDR2 comprising the amino acid sequence of YTS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 65; (c) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 69; the CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 71; and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 75; the CDR2 comprising the amino acid sequence of YTS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 76; (d) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 80; the CDR2 comprising the amino acid sequence of SEQ ID NO: 81; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 82; and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 86; the CDR2 comprising the amino acid sequence of KVS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 87; (e) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 91; the CDR2 comprising the amino acid sequence of SEQ ID NO: 92; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 93; and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 97; the CDR2 comprising the amino acid sequence of FAS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 98; or (f) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 102; the CDR2 comprising the amino acid sequence of SEQ ID NO: 103; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 104; and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 108; the CDR2 comprising the amino acid sequence of LVS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 109.

Embodiment 3 : The antibody or antigen binding fragment thereof of embodiment 1 or embodiment 2. wherein the antibody or antigen binding fragment specifically binds to the extracellular domain of Tmem273.

Embodiment 4: The antibody or antigen binding fragment thereof of any one of embodiments 1-3, wherein the antibody specifically binds to mouse (SEQ ID NO: 2) and/or human (SEQ ID NO: 1) Tmem273.

Embodiment 5 : The antibody or antigen binding fragment thereof of any one of embodiments 1-4, wherein the antibody or antigen binding fragment thereof comprises a VH comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 15; SEQ ID NO: 61; SEQ ID NO: 72; SEQ ID NO: 83; SEQ ID NO: 94; and SEQ ID NO: 105.

Embodiment 6: The antibody or antigen binding fragment thereof of any one of embodiments 1-5 wherein the antibody or antigen binding fragment thereof comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15: SEQ ID NO: 61; SEQ ID NO: 72; SEQ ID NO: 83; SEQ ID NO: 94; and SEQ ID NO: 105.

Embodiment 7 : The antibody or antigen binding fragment thereof of any one of embodiments 1-6, wherein the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20; SEQ ID NO 66: SEQ ID NO: 77; SEQ ID NO: 88; SEQ ID NO: 99; and SEQ ID NO: 110.

Embodiment 8: The antibody or antigen binding fragment thereof of any one of embodiments 1-7, wherein the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20: SEQ ID NO 66: SEQ ID NO: 77; SEQ ID NO: 88; SEQ ID NO: 99; and SEQ ID NO: 110.

Embodiment 9: The antibody or antigen binding fragment thereof of any one of embodiments 1-8, wherein the antibody or antigen binding fragment thereof comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 20; (b) a VH comprising the amino acid sequence of SEQ ID NO: 61 and a VL comprising the amino acid sequence of SEQ ID NO: 66; (c) a VH comprising the amino acid sequence of SEQ ID NO: 72 and a VL comprising the amino acid sequence of SEQ ID NO: 77; (d) a VH comprising the amino acid sequence of SEQ ID NO: 83 and a VL comprising the amino acid sequence of SEQ ID NO: 88; (e) a VH comprising the amino acid sequence of SEQ ID NO: 94 and a VL comprising the amino acid sequence of SEQ ID NO: 99; or (f) a VH comprising the amino acid sequence of SEQ ID NO: 105 and a VL comprising the amino acid sequence of SEQ ID NO: 110.

Embodiment 10: The antibody or antigen binding fragment thereof of any one of embodiments 1-9, wherein the antibody or antigen binding fragment thereof is humanized.

Embodiment 11 : The antibody or antigen binding fragment thereof of any one of embodiments 1-10, wherein the antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 22.

Embodiment 12: The antibody or antigen binding fragment thereof of any one of embodiments 1-11, wherein the antibody or antigen binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 25.

Embodiment 13 : The antibody or antigen binding fragment thereof of any one of embodiments 1-12, wherein the antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 22 and a light chain comprising the amino acid sequence of SEQ ID NO: 25.

Embodiment 14: The antibody or antigen binding fragment thereof of any one of embodiments 1-13, wherein the antibody or antigen binding fragment thereof comprises a heavy chain and/or a light chain comprising a signal peptide.

Embodiment 15: The antibody or antigen binding fragment thereof of embodiment 14, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 23 and/or the light chain comprises the amino acid sequence of SEQ ID NO: 26.

Embodiment 16: The antibody or antigen binding fragment thereof of any one of embodiments 1-15, wherein the antibody or antigen binding fragment thereof comprises an IgGl, IgG2, IgG3 or IgG4 constant region or fragment thereof.

Embodiment 17 : The antibody or antigen binding fragment thereof of any one of embodiments 1-16, wherein the antibody or antigen binding fragment thereof comprises an IgGl constant region or fragment thereof.

Embodiment 18: The antibody or antigen binding fragment thereof of any one of embodiments 1-17, wherein the antibody or antigen binding fragment thereof is directly or indirectly attached to a detectable label or a therapeutic agent.

Embodiment 19: The antibody or antigen binding fragment thereof of any one of embodiments 1-18, wherein the antigen binding fragment is an Fab fragment, an Fab' fragment, or an F(ab’)2 fragment.

Embodiment 20: An isolated or recombinant antibody or antigen binding fragment thereof which specifically binds to transmembrane protein 273 (Tmem273), wherein the antibody or antigen binding fragment thereof comprises a heavy chain comprising a variable region (VH) chain comprising Vn complementarity determining regions (CDR) CDR1, CDR2 and CDR3 respectively of the amino acid sequences of SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30; and a light chain comprising a variable region (VL) comprising VL complementarity determining regions (CDR) CDR1, CDR2 and CDR3 respectively of the amino acid sequences of SEQ ID NO: 33, LAS, and SEQ ID NO: 35.

Embodiment 21 : The antibody or antigen binding fragment thereof of embodiment 20, wherein the antibody or antigen binding fragment specifically binds to the extracellular domain of Tmem273.

Embodiment 22: The antibody or antigen binding fragment thereof of embodiment 20 or 21, wherein the antibody specifically binds to mouse Tmem273 (SEQ ID NO: 2).

Embodiment 23: The antibody or antigen binding fragment thereof of any one of embodiments 20-22, wherein the antibody or antigen binding fragment thereof comprises a VH comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 31.

Embodiment 24: The antibody or antigen binding fragment thereof of any one of embodiments 20-23, wherein the antibody or antigen binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 31.

Embodiment 25 : The antibody or antigen binding fragment thereof of any one of embodiments 20-24. wherein the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 36.

Embodiment 26: The antibody or antigen binding fragment thereof of any one of embodiments 20-25, wherein the antibody or antigen binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 36.

Embodiment 27 : The antibody or antigen binding fragment thereof of any one of embodiments 20-26, wherein the antibody or antigen binding fragment thereof comprises a VH comprises the amino acid sequence of SEQ ID NO: 31 and a VL comprises the amino acid sequence of SEQ ID NO: 36.

Embodiment 28: The antibody or antigen binding fragment thereof of any one of embodiments 20-27, wherein the antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38.

Embodiment 29: The antibody or antigen binding fragment thereof of any one of embodiments 20-28, wherein the antibody or antigen binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 41.

Embodiment 30: The antibody or antigen binding fragment thereof of any one of embodiments 20-29, wherein the antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 41.

Embodiment 31 : The antibody or antigen binding fragment thereof of any one of embodiments 20-30, wherein the antibody or antigen binding fragment thereof comprises a heavy chain and/or a light chain comprising a signal peptide.

Embodiment 32: The antibody or antigen binding fragment thereof of embodiment 31, wherein the heavy chain comprises an amino acid sequence of SEQ ID NO: 23 and/or the light chain comprises an amino acid sequence of SEQ ID NO: 26.

Embodiment 33: The antibody or antigen binding fragment thereof of any one of embodiments 20-32, wherein the antibody or antigen binding fragment thereof is directly or indirectly attached to a detectable label or a therapeutic agent.

Embodiment 34: The antibody or antigen binding fragment thereof of any one of embodiments 20-33, wherein the antigen binding fragment is an Fab fragment, an Fab' fragment, or an F(ab’)2 fragment.

Embodiment 35: The antibody or antigen binding fragment thereof of any one of embodiments 20-34, wherein the antibody or antigen binding fragment thereof comprises a mouse constant region.

Embodiment 36: The antibody or antigen binding fragment thereof of embodiment 35, wherein the constant region is IgGl, IgG2a, IgG2b, or IgG3, or a fragment thereof.

Embodiment 37: The antibody or antigen binding fragment thereof of embodiment 36, wherein the constant region is IgG2b.

Embodiment 38: A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen binding fragment thereof of any one of embodiments 1-37 and a pharmaceutically effective carrier.

Embodiment 39: An isolated polynucleotide comprising a nucleotide sequence encoding the antibody or antigen binding fragment thereof of any one of embodiments 1-37.

Embodiment 40: A vector comprising the polynucleotide of embodiment 39.

Embodiment 41 : An isolated host cell that recombinantly produces the antibody or antigen binding fragment thereof of any one of embodiments 1-37.

Embodiment 42: A method of treating, ameliorating, and/or preventing a disease or condition associated with Tmem273 expression, wherein the method comprises contacting a cell that is capable of expressing Tmem273 with the antibody or antigen binding fragment thereof of any one of embodiments 1-37 or the pharmaceutical composition of claim 38.

Embodiment 43: The method of embodiment 42, wherein the cell is a T cell.

Embodiment 44: The method of embodiment 43, wherein the T cell is a CD8 T cell.

Embodiment 45: The method of embodiment 44, wherein the CD8 T cell is a CD8yl3 T cell.

Embodiment 46: The method of embodiment 42, wherein the T cell is a CD4 T cell.

Embodiment 47: The method of embodiment 46, wherein the CD4 T cell is a CD4yl3 T cell.

Embodiment 48: The method of any one of embodiments 42-47, wherein the cells express at least one of TNFa, IL-13, IFN- y, and combinations thereof.

Embodiment 49: A method of treating, ameliorating, and/or preventing systemic sclerosis (SSc) in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the antibody or antigen binding fragment of any one of embodiments 1-37 or the pharmaceutical composition of claim 38 to the subject in need thereof.

Embodiment 50: A method of treating, ameliorating, and/or preventing chlamydia in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the antibody or antigen binding fragment of any one of embodiments 1- 37 or the pharmaceutical composition of claim 38 to the subject in need thereof.

Embodiment 51 : A method of treating, ameliorating, and/or preventing asthma in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the antibody or antigen binding fragment of any one of embodiments 1- 37 or the pharmaceutical composition of claim 38 to the subject in need thereof.

Embodiment 52: The method of embodiment 51, wherein the asthma is chronic asthma.

Embodiment 53: A method of treating, ameliorating, and/or preventing fibrosis in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the antibody or antigen binding fragment of any one of embodiments 1- 37 or the pharmaceutical composition of claim 38 to the subject in need thereof.

The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and/or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

In sum. while this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. An isolated and/or recombinant antibody or antigen binding fragment thereof which specifically binds to transmembrane protein 273 (Tmem273). wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the VH comprises:

(a) a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12; a CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 14;

(b) a CDR1 comprising the amino acid sequence of SEQ ID NO: 58; a CDR2 comprising the amino acid sequence of SEQ ID NO: 59; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 60;

(c) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 70: and a CDR3 comprising the amino acid sequence of SEQ ID NO: 71:

(d) a CDR1 comprising the amino acid sequence of SEQ ID NO: 80; a CDR2 comprising the amino acid sequence of SEQ ID NO: 81; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 82:

(e) a CDR1 comprising the amino acid sequence of SEQ ID NO: 91; a CDR2 compnsing the amino acid sequence of SEQ ID NO: 92: and a CDR3 comprising the amino acid sequence of SEQ ID NO: 93; or

(!) a CDR1 comprising the amino acid sequence of SEQ ID NO: 102; a CDR2 comprising the amino acid sequence of SEQ ID NO: 103; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 104; the VL comprises:

(g) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17; a CDR2 comprising the amino acid sequence of KVS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19;

(h) a CDR1 comprising the amino acid sequence of SEQ ID NO: 64; a CDR2 comprising the amino acid sequence of YTS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 65;

(i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 75; a CDR2 comprising the amino acid sequence of YTS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 76;

(j) a CDR1 comprising the amino acid sequence of SEQ ID NO: 86; a CDR2 comprising the amino acid sequence of KVS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 87;

(k) a CDR1 comprising the amino acid sequence of SEQ ID NO: 97; a CDR2 comprising the amino acid sequence of FAS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 98; or

(l) a CDR1 comprising the amino acid sequence of SEQ ID NO: 108; a CDR2 comprising the amino acid sequence of LVS; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 109.

2. The antibody or antigen binding fragment thereof of claim 1 , wherein the antibody or antigen binding fragment thereof comprises:

(a) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 12; the CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 17; the CDR2 comprising the amino acid sequence of KVS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 19;

(b) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 58; the CDR2 comprising the amino acid sequence of SEQ ID NO: 59; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 60; and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 64; the CDR2 comprising the amino acid sequence of YTS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 65;

(c) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 69; the CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 71; and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 75; the CDR2 comprising the amino acid sequence of YTS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 76;

(d) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 80; the CDR2 comprising the amino acid sequence of SEQ ID NO: 81 ; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 82; and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 86; the CDR2 comprising the amino acid sequence of KVS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 87;

(e) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 91; the CDR2 comprising the amino acid sequence of SEQ ID NO: 92; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 93; and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 97; the CDR2 comprising the amino acid sequence of FAS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 98; or

(I) the VH comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 102; the CDR2 comprising the amino acid sequence of SEQ ID NO: 103; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 104; and the VL comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 108; the CDR2 comprising the amino acid sequence of LVS; and the CDR3 comprising the amino acid sequence of SEQ ID NO: 109.

3. The antibody or antigen binding fragment thereof of claim 1 or claim 2. wherein the antibody or antigen binding fragment specifically binds to the extracellular domain of Tmem273.

4. The antibody or antigen binding fragment thereof of any one of claims 1-3, wherein the antibody specifically binds to mouse (SEQ ID NO: 2) and/or human (SEQ ID NO: 1) Tmem273.

5. The antibody or antigen binding fragment thereof of any one of claims 1-4, wherein the antibody or antigen binding fragment thereof comprises a VH comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 15; SEQ ID NO: 61; SEQ ID NO: 72; SEQ ID NO: 83; SEQ ID NO: 94; and SEQ ID NO: 105.

6. The antibody or antigen binding fragment thereof of any one of claims 1-5, wherein the antibody or antigen binding fragment thereof comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15; SEQ ID NO: 61; SEQ ID NO: 72; SEQ ID NO: 83; SEQ ID NO: 94; and SEQ ID NO: 105.

7. The antibody or antigen binding fragment thereof of any one of claims 1-6, wherein the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 20; SEQ ID NO 66: SEQ ID NO: 77; SEQ ID NO: 88; SEQ ID NO: 99; and SEQ ID NO: 110.

8. The antibody or antigen binding fragment thereof of any one of claims 1-7, wherein the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20; SEQ ID NO 66: SEQ ID NO: 77; SEQ ID NO: 88; SEQ ID NO: 99; and SEQ ID NO: 110.

9. The antibody or antigen binding fragment thereof of any one of claims 1-8, wherein the antibody or antigen binding fragment thereof comprises:

(a) a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 20;

(b) a VH comprising the amino acid sequence of SEQ ID NO: 61 and a VL comprising the amino acid sequence of SEQ ID NO: 66;

(c) a VH comprising the amino acid sequence of SEQ ID NO: 72 and a VL comprising the amino acid sequence of SEQ ID NO: 77;

(d) a VH comprising the amino acid sequence of SEQ ID NO: 83 and a VL comprising the amino acid sequence of SEQ ID NO: 88;

(e) a VH comprising the amino acid sequence of SEQ ID NO: 94 and a VL comprising the amino acid sequence of SEQ ID NO: 99; or

(!) a VH comprising the amino acid sequence of SEQ ID NO: 105 and a VL comprising the amino acid sequence of SEQ ID NO: 110.

10. The antibody or antigen binding fragment thereof of any one of claims 1-9, wherein the antibody or antigen binding fragment thereof is humanized.

11. The antibody or antigen binding fragment thereof of any one of claims 1-10, wherein the antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 22.

12. The antibody or antigen binding fragment thereof of any one of claims 1-11, wherein the antibody or antigen binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 25.

13. The antibody or antigen binding fragment thereof of any one of claims 1-12, wherein the antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 22 and a light chain comprising the amino acid sequence of SEQ ID NO: 25.

14. The antibody or antigen binding fragment thereof of any one of claims 1-13, wherein the antibody or antigen binding fragment thereof comprises a heavy chain and/or a light chain comprising a signal peptide.

15. The antibody or antigen binding fragment thereof of claim 14, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 23 and/or the light chain comprises the amino acid sequence of SEQ ID NO: 26.

16. The antibody or antigen binding fragment thereof of any one of claims 1-15, wherein the antibody or antigen binding fragment thereof comprises an IgGl, IgG2, IgG3 or IgG4 constant region or fragment thereof.

17. The antibody or antigen binding fragment thereof of any one of claims 1-16, wherein the antibody or antigen binding fragment thereof comprises an IgGl constant region or fragment thereof.

18. The antibody or antigen binding fragment thereof of any one of claims 1-17, wherein the antibody or antigen binding fragment thereof is directly or indirectly attached to a detectable label or a therapeutic agent.

19. The antibody or antigen binding fragment thereof of any one of claims 1-18, wherein the antigen binding fragment is an Fab fragment, an Fab' fragment, or an F(ab’)2 fragment.

20. An isolated or recombinant antibody or antigen binding fragment thereof which specifically binds to transmembrane protein 273 (Tmem273), wherein the antibody or antigen binding fragment thereof comprises a heavy chain comprising a variable region (VH) chain comprising VH complementarity determining regions (CDR) CDR1, CDR2 and CDR3 respectively of the amino acid sequences of SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30; and a light chain comprising a variable region (VL) comprising VL complementarity determining regions (CDR) CDR1, CDR2 and CDR3 respectively of the amino acid sequences of SEQ ID NO: 33, LAS, and SEQ ID NO: 35.

21. The antibody or antigen binding fragment thereof of claim 20, wherein the antibody or antigen binding fragment specifically binds to the extracellular domain of Tmem273.

22. The antibody or antigen binding fragment thereof of claim 20 or 21, wherein the antibody specifically binds to mouse Tmem273 (SEQ ID NO: 2).

23. The antibody or antigen binding fragment thereof of any one of claims 20-22, wherein the antibody or antigen binding fragment thereof comprises a VH comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 31.

24. The antibody or antigen binding fragment thereof of any one of claims 20-23, wherein the antibody or antigen binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 31.

25. The antibody or antigen binding fragment thereof of any one of claims 20-24, wherein the antibody or antigen binding fragment thereof comprises a VL comprising an amino acid sequence at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 36.

26. The antibody or antigen binding fragment thereof of any one of claims 20-25, wherein the antibody or antigen binding fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 36.

27. The antibody or antigen binding fragment thereof of any one of claims 20-26, wherein the antibody or antigen binding fragment thereof comprises a VH comprises the amino acid sequence of SEQ ID NO: 31 and a VL comprises the amino acid sequence of SEQ ID NO: 36.

28. The antibody or antigen binding fragment thereof of any one of claims 20-27, wherein the antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38.

29. The antibody or antigen binding fragment thereof of any one of claims 20-28, wherein the antibody or antigen binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 41.

30. The antibody or antigen binding fragment thereof of any one of claims 20-29, wherein the antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 41.

31. The antibody or antigen binding fragment thereof of any one of claims 20-30, wherein the antibody or antigen binding fragment thereof comprises a heavy chain and/or a light chain comprising a signal peptide.

32. The antibody or antigen binding fragment thereof of claim 31 , wherein the heavy chain comprises an amino acid sequence of SEQ ID NO: 23 and/or the light chain comprises an amino acid sequence of SEQ ID NO: 26.

33. The antibody or antigen binding fragment thereof of any one of claims 20-32, wherein the antibody or antigen binding fragment thereof is directly or indirectly attached to a detectable label or a therapeutic agent.

34. The antibody or antigen binding fragment thereof of any one of claims 20-33, wherein the antigen binding fragment is an Fab fragment, an Fab' fragment, or an F(ab’)2 fragment.

35. The antibody or antigen binding fragment thereof of any one of claims 20-34, wherein the antibody or antigen binding fragment thereof comprises a mouse constant region.

36. The antibody or antigen binding fragment thereof of claim 35, wherein the constant region is IgGl. IgG2a, IgG2b, or IgG3, or a fragment thereof.

37. The antibody or antigen binding fragment thereof of claim 36, wherein the constant region is IgG2b.

38. A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen binding fragment thereof of any one of claims 1-37 and a pharmaceutically effective carrier.

39. An isolated polynucleotide comprising a nucleotide sequence encoding the antibody or antigen binding fragment thereof of any one of claims 1-37.

40. A vector comprising the polynucleotide of claim 39.

41. An isolated host cell that recombinantly produces the antibody or antigen binding fragment thereof of any one of claims 1-37.

42. A method of treating, ameliorating, and/or preventing a disease or condition associated with Tmem273 expression, wherein the method comprises contacting a cell that is capable of expressing Tmem273 with the antibody or antigen binding fragment thereof of any one of claims 1-37 or the pharmaceutical composition of claim 38.

43. The method of claim 42, wherein the cell is a T cell.

44. The method of claim 43, wherein the T cell is a CD8 T cell.

45. The method of claim 44, wherein the T cell is a CD8yl3 T cell.

46. The method of claim 42, wherein the T cell is a CD4 T cell.

47. The method of claim 46, wherein the CD4 T cell is a CD4 y 13 T cell.

48. The method of any one of claims 42-47. wherein the cells express at least one of TNFa, IL-13, IFN- y, and combinations thereof.

49. A method of treating, ameliorating, and/or preventing systemic sclerosis (SSc) in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the antibody or antigen binding fragment of any one of claims 1- 37 or the pharmaceutical composition of claim 38 to the subject in need thereof.

50. A method of treating, ameliorating, and/or preventing chlamydia in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the antibody or antigen binding fragment of any one of claims 1-37 or the pharmaceutical composition of claim 38 to the subject in need thereof.

51. A method of treating, ameliorating, and/or preventing asthma in a subj ect in need thereof, wherein the method comprises administering a therapeutically effective amount of the antibody or antigen binding fragment of any one of claims 1-37 or the pharmaceutical composition of claim 38 to the subject in need thereof.

52. The method of claim 51, wherein the asthma is chronic asthma.

3. A method of treating, ameliorating, and/or preventing fibrosis in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the antibody or antigen binding fragment of any one of claims 1-37 or the pharmaceutical composition of claim 38 to the subject in need thereof.