Compositions and methods for treating netosis-associated disease

Abstract

The disclosure relates generally to the treatment of NETosis associated diseases or conditions. The disclosure also relates to anti-PAL antibodies, and to methods of using these anti-PAL antibodies to treat NETosis associated diseases or conditions.

Description

Attorney File Reference: MBI.0009COMPOSITIONS AND METHODS FOR TREATING NETOSIS-ASSOCIATED DISEASEREFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM[1] The official copy of the Sequence Listing is submitted concurrently with the specification as an xml file, made with WIPO Sequence Version 2.1.0, via EFS-Web, with a file name of “MCI005.xml”, a creation date of August 6, 2025, and a size of 84 kilobytes. The Sequence Listing filed via EFS-Web is part of the specification and is incorporated in its entirety by reference herein.BACKGROUND OF THE DISCLOSURE[2] As the most abundant white blood cell group in humans, neutrophils play an essential role in the body’s innate immunity caused by infection. An excessive activation of neutrophils can lead to the development of multiple organ dysfunction syndromes. Activated neutrophils will also release nuclear DNA and form a network structure containing nucleus, cytoplasm, and granular protein, also known as the neutrophil extracellular traps (NETs). Excessive NETs formation can damage the microcirculation, promote immunothrombosis, and contribute to many pathologies. Numerous investigations during the last decade have revealed the molecular mechanism of NETosis, including the NADPH oxidase 2 (NOX)-dependent and -independent NETosis.[3] It is an object of the disclosure to provide treatments that ameliorate and / or cure diseases caused by overactive NETosis. It is also an object of the disclosure to provide tests to detect overactive NETosis, and / or identify those patients amendable to treatment with the compositions disclosed herein.SUMMARY[4] The disclosure relates to methods of reducing NETosis using anti-poly-N-acetyl lactosamine (anti-PAL) antibodies. The disclosure also relates to methods of treating NETosis associated-diseases using anti-poly-N-acetyl lactosamine (anti-PAL) antibodies. The nucleic acid and amino acid sequences of the variable region heavy chains and light chains for some of these anti-PAL antibodies are disclosed herein. The antibodies can be monoclonal, and can be fully human antibodies, chimeric antibodies, or CDR-grafted antibodies. The antibodies can be full length or antibody fragments. Antibody fragments include any of the well-known formats or types, including for example, antigen-binding fragments (Fab), single chain variable fragments (scFv) and “third generation” (3G) fragments.[5] The anti-PAL antibodies can have a heavy chain variable sequence of SEQ ID NO: 1, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68 or 71, and a light chain variable sequence of SEQ ID NO: 2 or 74. The anti-PAL antibodies can be full length antibodies having a heavy chain sequence of SEQ ID NOs: 3, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48,Attorney File Reference: MBI.000951, 54, 57, 60, 63, 66, 69 or 72, and a light chain sequence of SEQ ID NO: 4 or 75. The anti-PAL antibodies disclosed herein also include CDR grafted antibodies made using the CDRs of any of the foregoing antibodies. The anti-PAL antibodies disclosed herein can also include HA1 A and / or mAb 216.[6] The anti-PAL antibodies can be full length antibodies such as, for example, an IgM, IgG (e.g., IgGl, IgG2, IgG3, or IgG4), an IgM, an IgA, an IgD, or an IgE. The anti-PAL antibody can be an antibody fragment such as, for example, a Fab, F(ab’)2, single chain antibody (scFv), Fv, or other antibody fragments made from recombinant nucleic acids encoding fragments of the antibody chains. The antibody fragments can also be made by digestion of an anti-CDIM antibody to generate a smaller fragment. The anti-PAL antibody can be obtained from a B-cell, a plasma cell, a B memory cell, a pre-B-cell or a progenitor B-cell.[7] Compositions and formulations described here can comprise one or more of the anti-PAL antibodies for administration to a subject. The compositions with the anti-PAL antibodies can also include other drugs or agents for treatment of the subject. For example, the anti-PAL antibody compositions can include analgesics, other antiviral or antibacterial drugs, other antiviral or antibacterial antibodies, anti-inflammatory drugs, and / or agents that reduce symptoms caused by sepsis.[8] Methods described herein use the anti-PAL antibodies to reduce NETOsis. The methods and anti-PAL antibodies can be used to treat subjects with NETosis-associated diseases and thereby reduce symptoms in a subject. Such NETosis-associated diseases include, for example, systemic lupus erythematosus (SLE), rheumatoid arthritis, acute lung injury, thrombosis formation, gout, small-vessel vasculitis, atherosclerosis, COVID19, Acute Respiratory Distress Syndrome, and inflammatory bowel disease. The methods and anti-PAL antibodies can also be used prophylactically to reduce, inhibit, or prevent NETosis-associated diseases in a subject (e.g., a subject susceptible to a NETosis-associated disease).BRIEF DESCRIPTION OF THE FIGURES[9] FIG. 1 shows anti-PAL antibody decrease the release of nucleic acids from neutrophils in a NETosis model.

[0010] FIG. 2 shows anti-PAL antibody binds to permeabilized neutrophils and not intact neutrophils.

[0011] FIG. 3 shows anti-PAL antibody binds to granules in neutrophils.

[0012] FIG. 4 shows a dose response for anti-PAL antibody inhibition of elastase release from neutrophils in a NETosis model.Attorney File Reference: MBI.0009

[0013] FIG. 5 shows the effect from time of addition of anti-PAL antibody on inhibition of elastase release from neutrophils in a NETosis model.

[0014] FIG. 6 shows anti-PAL antibody reduces reactive oxygen species (ROS) generated by neutrophils.

[0015] FIG. 7 shows the overlap of the PAL antigen with neutrophil vesicles.

[0016] FIG. 8 shows the overlap of PAL antigen with different neutrophil vesicle types.DETAILED DESCRIPTION

[0017] Before the various embodiments are described, it is to be understood that the teachings of this disclosure are not limited to the particular embodiments described, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present teachings will be limited only by the appended claims.

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present teachings, some exemplary methods and materials are now described.

[0019] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation. Numerical limitations given with respect to concentrations or levels of a substance are intended to be approximate, unless the context clearly dictates otherwise. Thus, where a concentration is indicated to be (for example) 10 micrograms (“pg”), it is intended that the concentration be understood to be at least approximately or about 10 pg.

[0020] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present teachings. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.DefinitionsAttorney File Reference: MBI.0009

[0021] As used herein, an “antibody” refers to a protein functionally defined as a binding protein and structurally defined as comprising an amino acid sequence that is recognized as being derived from the framework region of an immunoglobulin encoding gene of an animal producing antibodies. An antibody can consist of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0022] A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.

[0023] IgM antibodies assemble tetramers into five or six units (i.e. mostly as pentamers but also hexamers occur) which are each comprised of two heavy-chains (mu-chains) and two light chains, bound together by disulfide bonds and a so-called J-chain. The structure of a pentamer IgM is shown below:

[0024] Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into an Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciateAttorney File Reference: MBI.0009 that fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized using recombinant DNA methodologies. Preferred antibodies include VH-VL dimers, including single chain antibodies (antibodies that exist as a single polypeptide chain), such as single chain Fv antibodies (sFv or scFv) in which a variable heavy and a variable light region are joined together (directly or through a peptide linker) to form a continuous polypeptide. The single chain Fv antibody is a covalently linked VH-VL heterodimer which may be expressed from a nucleic acid including VH- and VL- encoding sequences either joined directly or joined by a peptide-encoding linker (e.g., Huston, et al. Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). While the VH and VL are connected to each as a single polypeptide chain, the VH and VL domains associate non-covalently. Alternatively, the antibody can be another fragment. Other fragments can also be generated, including using recombinant techniques. For example, Fab molecules can be displayed on phage if one of the chains (heavy or light) is fused to g3 capsid protein and the complementary chain exported to the periplasm as a soluble molecule. The two chains can be encoded on the same or on different replicons; the two antibody chains in each Fab molecule assemble post-translationally and the dimer is incorporated into the phage particle via linkage of one of the chains to g3p (see, e.g., U.S. Pat. No: 5,733,743). The scFv antibodies and a number of other structures converting the naturally aggregated, but chemically separated light and heavy polypeptide chains from an antibody V region into a molecule that folds into a three-dimensional structure substantially similar to the structure of an antigen-binding site are known to those of skill in the art (see e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778). In some embodiments, the scFv is a diabody as described in Holliger et al., Proc. Nat’ 1 Acad. Sci. vol. 90, pp. 6444-6448 (1993), which is incorporated by reference in its entirety for all purposes. In some embodiments, antibodies include all those that have been displayed on phage or generated by recombinant technology using vectors where the chains are secreted as soluble proteins, e.g., scFv, Fv, Fab, pr (Fab')2 or generated by recombinant technology using vectors where the chains are secreted as soluble proteins. Antibodies can also include diantibodies and miniantibodies.

[0025] Antibodies of described herein also include heavy chain dimers, such as antibodies from camelids. Since the VH region of a heavy chain dimer IgG in a camelid does not have to make hydrophobic interactions with a light chain, the region in the heavy chain that normally contacts a light chain is changed to hydrophilic amino acid residues in a camelid. VH domains of heavy-chain dimer IgGs are called VHH domains.Attorney File Reference: MBI.0009

[0026] In camelids, the diversity of antibody repertoire is determined by the complementary determining regions (CDR) 1, 2, and 3 in the VH or VHH regions. The CDR3 in the camel VHH region is characterized by its relatively long length averaging 16 amino acids (Muyldermans et al., 1994, Protein Engineering 7(9): 1129). This is in contrast to CDR3 regions of antibodies of many other species. For example, the CDR3 of mouse VH has an average of 9 amino acids.

[0027] Libraries of camelid-derived antibody variable regions, which maintain the in vivo diversity of the variable regions of a camelid, can be made by, for example, the methods disclosed in U.S. Patent Application Ser. No. 20050037421, published Feb. 17, 2005.

[0028] As used herein, the term “naturally occurring” means that the components are encoded by a single gene that was not altered by recombinant means and that pre-exists in an organism, e.g., in an antibody library that was created from naive cells or cells that were exposed to an antigen.

[0029] As used herein, the term “antigen” refers to substances that are capable, under appropriate conditions, of inducing a specific immune response and of reacting with the products of that response, such as, with specific antibodies or specifically sensitized T-lymphocytes, or both. Antigens may be soluble substances, such as toxins and foreign proteins, or particulates, such as bacteria and tissue cells; however, only the portion of the protein or polysaccharide molecule known as the antigenic determinant (epitopes) combines with the antibody or a specific receptor on a lymphocyte. More broadly, the term “antigen” may be used to refer to any substance to which an antibody binds, or for which antibodies are desired, regardless of whether the substance is immunogenic. For such antigens, antibodies may be identified by recombinant methods, independently of any immune response.

[0030] As used herein, the term “epitope” refers to the site on an antigen or hapten to which specific B cells and / or T cells respond. The term is also used interchangeably with “antigenic determinant” or “antigenic determinant site.” Epitopes include that portion of an antigen or other macromolecule capable of forming a binding interaction that interacts with the variable region binding pocket of an antibody.

[0031] As used herein, the term “binding specificity” of an antibody refers to the identity of the antigen to which the antibody binds, preferably to the identity of the epitope to which the antibody binds.

[0032] As used herein, the term “chimeric polynucleotide” means that the polynucleotide comprises regions which are wild-type and regions which are mutated. It may also mean that the polynucleotide comprises wild-type regions from one polynucleotide and wild-type regions from another related polynucleotide.Attorney File Reference: MBI.0009

[0033] As used herein, the term “complementarity-determining region” or “CDR” refer to the art- recognized term as exemplified by the Kabat and Chothia. CDRs are also generally known as hypervariable regions or hypervariable loops (Chothia and Lesk (1987) J Mol. Biol. 196: 901; Chothia et al. (1989) Nature 342: 877; E. A. Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.) (1987); and Tramontane et al. (1990) J Mol. Biol. 215: 175). “Framework region” or “FR” refers to the region of the V domain that flank the CDRs. The positions of the CDRs and framework regions can be determined using various well known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, e.g., Johnson et al., supra; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J. Mol. Biol 1997, 273(4)). Definitions of antigen combining sites are also described in the following: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan l;29(l):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin, et al, Methods Enzymol., 203, 121-153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M. J. E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996).

[0034] The term “conservative substitution” refers to substitution of an amino acid in a polypeptide with a functionally, structurally or chemically similar natural or unnatural amino acid. In certain embodiments, the following groups each contain natural amino acids that are conservative substitutions for one another:1) Glycine (Gly / G), Alanine (Ala / A);2) Isoleucine (Ile / I), Leucine (Leu / L), Methionine (Met / M), Valine (Val / V);3) Phenylalanine (Phe / F), Tyrosine (Tyr / Y), Tryptophan (Trp / W);4) Serine (Ser / S), Threonine (Thr / T), Cysteine (Cys / C);5) Asparagine (Asn / N), Glutamine (Gln / Q);6) Aspartic acid (Asp / D), Glutamic acid (Glu / E); and7) Arginine (Arg / R), Lysine (Lys / K), Histidine (His / H).Attorney File Reference: MBI.0009

[0035] As used herein, the term “hapten” is a small molecule that, when attached to a larger carrier such as a protein, can elicit an immune response in an organism, e.g., such as the production of antibodies that bind specifically to it (in either the free or combined state). A “hapten” is able to bind to a preformed antibody, but may fail to stimulate antibody generation on its own. In the context of this disclosure, the term “hapten” includes modified amino acids, either naturally occurring or non-naturally occurring. Thus, for example, the term “hapten” includes naturally occurring modified amino acids such as phosphotyrosine, phosphothreonine, phosphoserine, or sulphated residues such as sulphated tyrosine (sulphotyrosine), sulphated serine (sulphoserine), or sulphated threonine (sulphothreonine); and also include non-naturally occurring modified amino acids such as p-nitro-phenylalanine.

[0036] As used herein, the term “heterologous” when used with reference to portions of a polynucleotide indicates that the nucleic acid comprises two or more subsequences that are not normally found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences, e.g., from unrelated genes arranged to make a new functional nucleic acid. Similarly, a “heterologous” polypeptide or protein refers to two or more subsequences that are not found in the same relationship to each other in nature.

[0037] As used herein, the term “immunological response” to a composition or vaccine is the development in the host of a cellular and / or antibody-mediated immune response to a composition or vaccine of interest. Usually, an “immunological response” includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and / or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and / or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and / or a lowered viral titer in the infected host.

[0038] As used herein, the term “isolated” refers to a nucleic acid or polypeptide separated not only from other nucleic acids or polypeptides that are present in the natural source of the nucleic acid or polypeptide, but also from polypeptides, and preferably refers to a nucleic acid or polypeptide found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same. The terms “isolated” and “purified” do not encompass nucleic acids or polypeptides present in their natural source.Attorney File Reference: MBI.0009

[0039] As used herein, the term “mammal” refers to warm-blooded vertebrate animals all of which possess hair and suckle their young.

[0040] As used herein, “percentage of sequence identity” and “percentage homology” are used interchangeably herein to refer to comparisons among polynucleotides or polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (z.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv Appl Math. 2:482, 1981; by the homology alignment algorithm of Needleman and Wunsch, J Mol Biol. 48:443, 1970; by the search for similarity method of Pearson and Lipman, Proc Natl Acad Sci. USA 85:2444, 1988; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., J. Mol. Biol. 215:403-410, 1990; and Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1977; respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. BLAST for nucleotide sequences can use the BLASTN program with default parameters, e.g., a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. BLAST for amino acid sequences can use the BLASTP program with default parameters, e.g., a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc Natl Acad Sci. USA 89: 10915, 1989). Exemplary determination of sequence alignment andAttorney File Reference: MBI.0009% sequence identity can also employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

[0041] The term “pharmaceutically acceptable” refers to a substance (e.g., an active ingredient or an excipient) that is suitable for use in contact with the tissues and organs of a subject without excessive irritation, allergic response, immunogenicity and toxicity, is commensurate with a reasonable benefit / risk ratio, and is effective for its intended use. A “pharmaceutically acceptable” excipient or carrier of a pharmaceutical composition is also compatible with the other ingredients of the composition.

[0042] As used herein, the terms “protein”, “peptide”, “polypeptide” and “polypeptide fragment” are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.

[0043] As used herein, the term “protracting moiety” means a molecule that can be attached to a polypeptide (e.g., an antibody light and / or heavy chain) to increase its molecular weight so that the polypeptide’s residence time in the blood (and / or serum) is increased. For example, the protracting moiety can increase the serum / blood half-life of the polypeptide. The increased half-life or residence time can be the result of, for example, reduced glomerular filtration by the kidney, and / or reduced uptake by the liver, and / or reduced binding and removal by immune binding proteins, etc. A protracting moiety can be, for example, another polypeptide, a polymer (e.g., synthetic polymer, natural polymer, etc.), etc.

[0044] As used herein, the term “purified” means that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like. In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99.8% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).

[0045] As used herein, the term “recombinant nucleic acid” refers to a nucleic acid in a form not normally found in nature. That is, a recombinant nucleic acid is flanked by a nucleotide sequence not naturally flanking the nucleic acid or has a sequence not normally found in nature. Recombinant nucleic acids can be originally formed in vitro by the manipulation of nucleic acid by restriction endonucleases, or alternatively using such techniques as polymerase chain reaction. It is understoodAttorney File Reference: MBI.0009 that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.

[0046] As used herein, the term “recombinant polypeptide” refers to a polypeptide expressed from a recombinant nucleic acid, or a polypeptide that is chemically synthesized in vitro.

[0047] As used herein, the term “recombinant variant” refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using recombinant DNA techniques. Guidance in determining which amino acid residues may be replaced, added, or deleted without abolishing activities of interest, such as enzymatic or binding activities, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology.

[0048] Preferably, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, i.e., conservative amino acid replacements. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0049] The term “stringent hybridization conditions” refers to hybridizing in 50% formamide at 5XSSC at a temperature of 42 °C and washing the filters in 0.2XSSC at 60 °C. (1XSSC is 0.15M NaCl, 0.015M sodium citrate.) Stringent hybridization conditions also encompasses low ionic strength and high temperature for washing, for example 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50 °C; hybridization with a denaturing agent, such as formamide, for example, 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 °C; or 50% formamide, 5XSSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5XDenhardt' s solution, sonicated salmon sperm DNA (50 pg / ml), 0.1% SDS, and 10% dextran sulfate at 42 °C, with washes at 42 °CAttorney File Reference: MBI.0009 in 0.2XSSC (sodium chloride / sodium citrate) and 50% formamide at 55 °C, followed by a high- stringency wash consisting of 0.1XSSC containing EDTA at 55 °C.

[0050] The term “substantially homologous” or “substantially identical” in the context of two polypeptides or polynucleotides refers to two or more sequences or subsequences that have at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid or nucleic acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. The terms “substantially homologous” or “substantially identical” can mean at least about 70% amino acid or nucleic acid residue identity. The term “substantially homologous” or “substantially identical” can mean at least about 85% amino acid or nucleic acid residue identity. The substantial homology or identity can exist over a region of the sequences that is at least about 20, 30, 40, 50, 100, 150 or 200 residues in length. The sequences can be substantially homologous or identical over the entire length of either or both comparison biopolymers.

[0051] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. AppL Math., 2:482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988); by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wisconsin); or by visual inspection.

[0052] One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle, J. Mol. Evol., 35:351-360 (1987). The method used is similar to the method described by Higgins and Sharp, CABIOS, 5: 151-153 (1989). The program can align up to about 300 sequences, each having a maximum length of about 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can beAttorney File Reference: MBI.0009 compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. Another algorithm that is useful for generating multiple alignments of sequences is Clustal W (see, e.g., Thompson et al., Nucleic Acids Research, 22:4673-4680

[1994] ).

[0053] Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol., 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction is halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults, e.g., a wordlength (W) of 11, an expectation (E) of 10, M = 5, N = -4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults, e.g., a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89: 10915

[1989] ).

[0054] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5787

[1993] ). One measure of similarity provided by the BLAST algorithm is the smallest sum probability [P(N)], which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In certain embodiments, a polynucleotide is considered similar to a reference sequence if the smallest sum probability in a comparison of the test polynucleotide to the reference polynucleotide is less than about 0.1, 0.01 or 0.001.Attorney File Reference: MBI.0009

[0055] A polypeptide can be substantially homologous or identical to a second polypeptide if the two polypeptides differ only by conservative amino acid substitutions. Two nucleic acid sequences can be substantially homologous or identical if the two polynucleotides hybridize to each other under stringent conditions, or under highly stringent conditions, as described herein.

[0056] The term “therapeutically effective amount” refers to an amount of a compound that, when administered to a subject, is sufficient to prevent, reduce the risk of developing, delay the onset of, slow the progression or cause regression of the medical condition being treated, or to alleviate to some extent the medical condition or one or more symptoms or complications of that condition. The term “therapeutically effective amount” also refers to an amount of a compound that is sufficient to elicit the biological or medical response of a cell, tissue, organ, system, animal or human which is sought by a researcher, veterinarian, medical doctor or clinician.

[0057] The singular terms “a”, “an”, and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Numerical limitations given with respect to concentrations or levels of a substance, such as an antigen, are intended to be approximate. Thus, where a concentration is indicated to be at least (for example) 200 pg, it is intended that the concentration be understood to be at least approximately “about” or “about” 200 pg.Anti-PAL Antibodies

[0058] Antibodies are immune binding proteins that are structurally defined as comprising an amino acid sequence recognized as being derived from the framework region of an immunoglobulin. An antibody can consist of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The immunoglobulin genes can include, for example, the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Antibody light chains can be classified as either kappa or lambda. Antibody heavy chains can be classified as gamma, mu, alpha, delta, or epsilon, which in turn can define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0059] Antibodies can exist as intact immunoglobulins or as a number of well-known fragments. Pepsin digests an antibody below the disulfide linkages in the hinge region and can produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into Fab' monomers. The Fab' monomer can be an Fab with part of the hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993), which is incorporated by reference in its entirety for all purposes). Antibody fragments can also beAttorney File Reference: MBI.0009 synthesized de novo either chemically or by utilizing recombinant DNA methodology. Antibodies can include VH-VL dimers, including single chain antibodies (antibodies that exist as a single polypeptide chain), diabodies, or single chain Fv antibodies (sFv or scFv) in which a variable heavy and a variable light region are joined together (directly or through a peptide linker) to form a continuous polypeptide, (e.g., Huston, et al. Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988, which is incorporated by reference in its entirety for all purposes). Antibodies can also include other fragments, including, for example, Fab molecules displayed on phage if one of the chains (heavy or light) is fused to g3 capsid protein and the complementary chain exported to the periplasm as a soluble molecule, (e.g., U.S. Pat. No: 5,733,743, which is incorporated by reference in its entirety for all purposes). The antibody can be an scFv antibody or a number of other structures converting the naturally aggregated, but chemically separated light and heavy polypeptide chains from an antibody V region into a molecule that folds into a three dimensional structure substantially similar to the structure of an antigen-binding site are known to those of skill in the art (e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778, which are all incorporated by reference in their entirety for all purposes). The scFv can be a diabody as described in Holliger et al., Proc. Nat’l Acad. Sci. vol. 90, pp. 6444-6448 (1993), which is incorporated by reference in its entirety for all purposes. Antibodies include all those that have been displayed on phage or generated by recombinant technology using vectors where the chains are secreted as soluble proteins, e.g., scFv, Fv, Fab, pr (Fab')2. Antibodies can also include miniantibodies. The antibody can be obtained from a B-cell, a plasma cell, a B memory cell, a pre-B-cell or a progenitor B-cell.

[0060] The antibodies can be monoclonal, and can be fully human antibodies, chimeric antibodies, or CDR-grafted antibodies. The antibodies can be full length or and antibody fragment. Antibody fragments include any of the well-known formats or types, including for example, antigen-binding fragments (Fab), single chain variable fragments (scFv) and “third generation” (3G). Nelson, MAbs 2010, 2:77-83, doi: 10.4161 / mabs.2.1.10786, which is incorporated by reference in its entirety for all purposes. F(ab')2, Fab, Fab' and Fv are examples of antigen-binding fragments that can be generated from the variable region of IgG and IgM.

[0061] VH4-34 are antibodies with a variable heavy chain 4-34 gene segment encoded in humans that intrinsically self-reacts with I / i carbohydrates expressed by erythrocytes using a specific motif in their framework region 1 (FWR1). VH4-34 antibodies are intrinsically autoreactive and bind conserved I / i carbohydrate self-epitope expressed at the surface of red blood cells and other cell types. The Ala-Val-Tyr (AVY) motif in VH4-34 FWR1 is responsible for I / i binding that is independent of IgH CDR3 or associated light chain; therefore, mutations in the AVY motif abrogate self-reactivity. Another unusual characteristic of VH4-34-encoded antibodies is the presence of anAttorney File Reference: MBI.0009Asn-X-Ser N-glycosylation site (NHS) in the CDR2 region that allows modulation of antibody avidity to the cognate antigen.

[0062] Poly-reactivity / cross-reactivity allows antibodies to bind and neutralize bacteria or viruses of related strains. For instance, antibody responses generated during flu season may potentially be protective for a related virus strain in the next season. Multispecificity for anti-gut commensal IgA that is enriched in CD27-IgA+ B cells may allow antibodies to recognize related molecular variants of common bacteria strains. Similarly, we also found that polyreactive antibodies expressed by CD27+IgG+ and CD27+IgA+ B cells were enriched in antibacterial clones. In addition, polyreactivity can increase binding to pathogens expressing antigens at very low density. Polyreactivity can enhance binding of anti-gpl20 antibodies that recognize one of the 10-15 gpl20 molecules expressed on the surface of HIV virions.

[0063] Immunoglobulin M ( IgM ) is constructed of five or six units (i.e. mostly as pentamers but also hexamers occur) which are each comprised of two heavy-chains (mu-chains) and two light chains, bound together by disulfide bonds and a so-called J-chain.

[0064] Anti-poly-N-acetyl lactosamine antibodies bind to the antigen poly-N-acetyl lactosamine (PAL) type 2 determinant, with or without a terminal sialic acid has a three-dimensional structural conformation and is sensitive to the enzyme endo-beta-galactosidase. The PAL epitope can have no branching or substitutions, and it can be attached to a glycolipid or a glycoprotein. On glycoproteins, the epitope could branch off a mannose framework (e.g., enzyme MGAT4), or could be a long chain branching off a “large 1” structure, but is normally at least about four hexose moieties in a straight chain (i.e., type 2) after the branch Gal [31-4 GlcNac [31-3 Gal [31-4 Glc 1; at least about six hexoses for good affinity; and least about twelve hexoses in the longest form. PAL can have the structure:The chain is made by enzymes (e.g., B3GNT1, B4GALT1), which add alternate sugars to the epitope. PAL is found on nearly all peripheral B lymphocytes and splenic B lymphocytes and on certain cultured B cell lymphoma lines. The epitope is also found on primary B cell lymphomas of various histopathologic classifications, and on the cells of some solid tumors. In some aspects, the PAL antigen can be capped by sialic acid.Attorney File Reference: MBI.0009

[0065] Anti -PAL antibody MC001 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRRAWGASVNFDYWGQGTLV TVSS (SEQ ID NO: 1)Anti -PAL antibody MC001 can have a variable region comprised of a light chain with the amino acid sequence of:DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITFGQGTRLEIKR (SEQ ID NO: 2)A full length, heavy chain of the IgM format for antibody MC001 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRRAWGASVNFDYWGQGTLV TVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPS VLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVF VPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTY KVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLT KSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNS GERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPA DVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHE ALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 3)A full length, light chain of the IgM format for antibody MC001 can have the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITFGQGTRLEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4)

[0066] The heavy chain CDRs for the anti -PAL antibody MC001 are:

[0067] CDRH1 : FSGYYWS (SEQ ID NO: 5)

[0068] CDRH2: EINHSGSTNYNPSLKS (SEQ ID NO: 6)

[0069] CDRH3 : GRMAWGASVN (SEQ ID NO: 7)

[0070] This light chain CDRs for the anti -PAL antibody MC001 are:

[0071] CDRL1 : RASQSISSYLN (SEQ ID NO: 8)

[0072] CDRL2: AASSLQS (SEQ ID NO: 9)

[0073] CDRL3: QQSYSTP (SEQ ID NO: 10)Attorney File Reference: MBI.0009

[0074] Anti-PAL antibody MC002 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRMARGASVNFDYWGQGTLV TVSS (SEQ ID NO: 11)Anti-PAL antibody MC002 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC002 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRMARGASVNFDYWGQGTLV TVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPS VLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVF VPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTY KVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLT KSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNS GERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPA DVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHE ALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 12)A full length, light chain of the IgM format for antibody MC002 can have the amino acid sequence of SEQ ID NO: 4.

[0075] The heavy chain CDR1 and 2 for the anti-PAL antibody MC002 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: GRMARGASVN (SEQ ID NO: 13)The light chain CDRs for the anti-PAL antibody MC002 are the same as the light chain of MC001.

[0076] Anti-PAL antibody MC003 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRRARGASVNFDYWGQGTLVT VSS (SEQ ID NO: 14)Anti-PAL antibody MC003 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC003 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRRARGASVNFDYWGQGTLVTAttorney File Reference: MBI.0009VSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSV LRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVP PRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYK VTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKS TKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGE RFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPAD VFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEA LPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 15)A full length, light chain of the IgM format for antibody MC003 can have the amino acid sequence of SEQ ID NO: 4.

[0077] The heavy chain CDR1 and 2 for the anti-PAL antibody MC003 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: GRRARGASVN (SEQ ID NO: 16)The light chain CDRs for the anti-PAL antibody MC003 are the same as the light chain of MC001.

[0078] Anti-PAL antibody MC004 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRGMAWGASVNFDYWGQGTLV TVSS (SEQ ID NO: 17)Anti-PAL antibody MC004 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC004 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRGMAWGASVNFDYWGQGTLV TVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPS VLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVF VPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTY KVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLT KSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNS GERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPA DVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHE ALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 18)A full length, light chain of the IgM format for antibody MC004 can have the amino acid sequence of SEQ ID NO: 4.Attorney File Reference: MBI.0009

[0079] The heavy chain CDR1 and 2 for the anti-PAL antibody MC004 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDR3H: RGMAWGASVN (SEQ ID NO: 19)The light chain CDRs for the anti-PAL antibody MC004 are the same as the light chain of MC001.

[0080] Anti-PAL antibody MQ005 or MC005 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRMAWGASVNFDYWGQGTLV TVSS (SEQ ID NO: 20)Anti-PAL antibody MQ005 or MC005 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MQ005 or MC005 can have the amino acid sequence:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRMAWGASVNFDYWGQGTLV TVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPS VLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVF VPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTY KVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLT KSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNS GERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPA DVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHE ALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 21)A full length, light chain of the IgM format for antibody MQ005 or MC005 can have the amino acid sequence OF SEQ ID NO: 4

[0081] The heavy chain CDR1 and 2 for the anti-PAL antibody MQ005 or MC005 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: RGMAWGASVN (SEQ ID NO: 22)The light chain CDRs for the anti-PAL antibody MQ005 or MC005 are the same as the light chain ofMCOOl.

[0082] Anti-PAL antibody MC006 can have a variable region comprised of a heavy chain with the amino acid sequence of:Attorney File Reference: MBI.0009QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRRMAWGASVNFDYWGQGTLV TVSS (SEQ ID NO: 23)Anti-PAL antibody MC006 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC006 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRRMAWGASVNFDYWGQGTLV TVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPS VLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVF VPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTY KVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLT KSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNS GERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPA DVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHE ALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 24)A full length, light chain of the IgM format for antibody MC006 can have the amino acid sequence of SEQ ID NO: 4

[0083] The heavy chain CDR1 and 2 for the anti-PAL antibody MC006 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: RRMAWGASVN (SEQ ID NO: 25)The light chain CDRs for the anti-PAL antibody MC006 are the same as the light chain of MC001.

[0084] Anti-PAL antibody MC007 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRGMARGASVNFDYWGQGTLV TVSS (SEQ ID NO: 26)Anti-PAL antibody MC007 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC007 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRGMARGASVNFDYWGQGTLV TVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPS VLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFAttorney File Reference: MBI.0009VPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTY KVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLT KSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNS GERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPA DVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHE ALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 27)A full length, light chain of the IgM format for antibody MC007 can have the amino acid sequence of SEQ ID NO: 4.

[0085] The heavy chain CDR1 and 2 for the anti-PAL antibody MC007 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: RGMARGASVN (SEQ ID NO: 28)The light chain CDRs for the anti-PAL antibody MC007 are the same as the light chain of MC001.

[0086] Anti-PAL antibody MC008 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRGRARGASVNFDYWGQGTLVT VSS (SEQ ID NO: 29)Anti-PAL antibody MC008 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC008 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRGRARGASVNFDYWGQGTLVT VSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSV LRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVP PRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYK VTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKS TKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGE RFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPAD VFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEA LPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 30)A full length, light chain of the IgM format for antibody MC008 can have the amino acid sequence of SEQ ID NO: 4.

[0087] The heavy chain CDR1 and 2 for the anti-PAL antibody MC008 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:Attorney File Reference: MBI.0009CDRH3 : RGRARGASVN (SEQ ID NO: 31)The light chain CDRs for the anti -PAL antibody MC008 are the same as the light chain of MC001.

[0088] Anti-PAL antibody MC009 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRRGARGASVNFDYWGQGTLVT VSS (SEQ ID NO: 32)Anti-PAL antibody MC009 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC009 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRRGARGASVNFDYWGQGTLVT VSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSV LRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVP PRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYK VTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKS TKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGE RFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPAD VFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEA LPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 33)A full length, light chain of the IgM format for antibody MC009 can have the amino acid sequence of SEQ ID NO: 4.

[0089] The heavy chain CDR1 and 2 for the anti-PAL antibody MC009 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: RRGARGASVN (SEQ ID NO: 34)The light chain CDRs for the anti-PAL antibody MC009 are the same as the light chain of MC001.

[0090] Anti-PAL antibody MC010 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAGRAWGASVNFDYWGQGTLV TVSS (SEQ ID NO: 35)Anti-PAL antibody MC010 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC010 can have the amino acid sequence:Attorney File Reference: MBI.0009QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAGRAWGASVNFDYWGQGTLV TVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPS VLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVF VPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTY KVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLT KSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNS GERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPA DVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHE ALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 36)A full length, light chain of the IgM format for antibody MC010 can have the amino acid sequence of SEQ ID NO: 4.

[0091] The heavy chain CDR1 and 2 for the anti -PAL antibody MC010 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: AGRAWGASVN (SEQ ID NO: 37)The light chain CDRs for the anti -PAL antibody MC010 are the same as the light chain of MC001.

[0092] Anti -PAL antibody MC011 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRGRAWGASVNFDYWGQGTLV TVSS (SEQ ID NO: 38)Anti -PAL antibody MC011 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC011 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRGRAWGASVNFDYWGQGTLV TVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPS VLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVF VPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTY KVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLT KSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNS GERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPA DVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHE ALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 39)Attorney File Reference: MBI.0009A full length, light chain of the IgM format for antibody MC011 can have the amino acid sequence of SEQ ID NO: 4.

[0093] The heavy chain CDR1 and 2 for the anti -PAL antibody MC011 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: RGRAWGASVN (SEQ ID NO: 40)The light chain CDRs for the anti -PAL antibody MC011 are the same as the light chain of MC001.

[0094] Anti -PAL antibody MC012 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGETNHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARTAWGSSIFDYWGQGTLVTV SS (SEQ ID NO: 41)Anti-PAL antibody MC012 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC012 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGETNHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARTAWGSSIFDYWGQGTLVTV SSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVL RGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPP RDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKV TSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKST KLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGER FTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVF VQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALP NRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 42)A full length, light chain of the IgM format for antibody MC012 can have the amino acid sequence of SEQ ID NO: 4.

[0095] The heavy chain CDR1 and 2 for the anti -PAL antibody MC012 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: ARTAWGSSI (SEQ ID NO: 43)The light chain CDRs for the anti -PAL antibody MC012 are the same as the light chain of MC001.

[0096] Anti-PAL antibody MC013 can have a variable region comprised of a heavy chain with the amino acid sequence of:Attorney File Reference: MBI.0009QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARRAWGSSIFDYWGQGTLVTV SS (SEQ ID NO: 44)Anti -PAL antibody MC013 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC013 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARRAWGSSIFDYWGQGTLVTV SSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVL RGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPP RDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKV TSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKST KLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGER FTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVF VQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALP NRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 45)A full length, light chain of the IgM format for antibody MC013 can have the amino acid sequence of SEQ ID NO: 4.

[0097] The heavy chain CDR1 and 2 for the anti -PAL antibody MC013 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: ARRAWGSSI (SEQ ID NO: 46)The light chain CDRs for the anti -PAL antibody MC013 are the same as the light chain of MC001.

[0098] Anti -PAL antibody MC014 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARTARGSSIFDYWGQGTLVTVS S (SEQ ID NO: 47)Anti-PAL antibody MC014 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC014 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARTARGSSIFDYWGQGTLVTVS SGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLR GGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRAttorney File Reference: MBI.0009DGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTS TLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTK LTCL VTDLTTYD S VTISWTRQNGEAVKTHTNISESHPNATF S AVGEASICEDDWNSGERF TCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVF VQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALP NRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 48)A full length, light chain of the IgM format for antibody MC014 can have the amino acid sequence of SEQ ID NO: 4.

[0099] The heavy chain CDR1 and 2 for the anti -PAL antibody MC014 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: ARTARGSSI (SEQ ID NO: 49)The light chain CDRs for the anti -PAL antibody MC014 are the same as the light chain of MC001.

[0100] Anti -PAL antibody MC015 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARRARGSSIFDYWGQGTLVTVS S (SEQ ID NO: 50)Anti -PAL antibody MC015 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC015 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARRARGSSIFDYWGQGTLVTVS SGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLR GGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPR DGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTS TLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTK LTCL VTDLTTYD S VTISWTRQNGEAVKTHTNISESHPNATF S AVGEASICEDDWNSGERF TCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVF VQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALP NRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 51)A full length, light chain of the IgM format for antibody MC015 can have the amino acid sequence of SEQ ID NO: 4.

[0101] The heavy chain CDR1 and 2 for the anti -PAL antibody MC015 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:Attorney File Reference: MBI.0009CDRH3: ARRARGSSI (SEQ ID NO: 52)The light chain CDRs for the anti -PAL antibody MC015 are the same as the light chain of MC001.

[0102] Anti -PAL antibody MC016 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRRTAWGSSIFDYWGQGTLVTVS S (SEQ ID NO: 53)Anti -PAL antibody MC016 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC016 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRRTAWGSSIFDYWGQGTLVTVS SGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLR GGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPR DGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTS TLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTK LTCL VTDLTTYD S VTISWTRQNGEAVKTHTNISESHPNATF S AVGEASICEDDWNSGERF TCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVF VQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALP NRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 54)A full length, light chain of the IgM format for antibody MC016 can have the amino acid sequence of SEQ ID NO: 4.

[0103] The heavy chain CDR1 and 2 for the anti -PAL antibody MC016 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: RATAWGSSI (SEQ ID NO: 55)The light chain CDRs for the anti -PAL antibody MC016 are the same as the light chain of MC001.

[0104] Anti -PAL antibody MC017 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRRTAWGSSIFDYWGQGTLVTVS S (SEQ ID NO: 56)Anti -PAL antibody MC017 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC017 can have the amino acid sequence:Attorney File Reference: MBI.0009QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRRTAWGSSIFDYWGQGTLVTVS SGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLR GGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPR DGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTS TLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTK LTCL VTDLTTYD S VTISWTRQNGEAVKTHTNISESHPNATF S AVGEASICEDDWNSGERF TCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVF VQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALP NRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 57)A full length, light chain of the IgM format for antibody MC017 can have the amino acid sequence of SEQ ID NO: 4.

[0105] The heavy chain CDR1 and 2 for the anti -PAL antibody MC017 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: RRTAWGSSI (SEQ ID NO: 58)The light chain CDRs for the anti -PAL antibody MC017 are the same as the light chain of MC001.

[0106] Anti -PAL antibody MC018 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRATARGSSIFDYWGQGTLVTVS S (SEQ ID NO: 59)Anti -PAL antibody MC018 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC018 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRATARGSSIFDYWGQGTLVTVS SGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLR GGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPR DGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTS TLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTK LTCL VTDLTTYD S VTISWTRQNGEAVKTHTNISESHPNATF S AVGEASICEDDWNSGERF TCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVF VQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALP NRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 60)Attorney File Reference: MBI.0009A full length, light chain of the IgM format for antibody MC018 can have the amino acid sequence of SEQ ID NO: 4.

[0107] The heavy chain CDR1 and 2 for the anti -PAL antibody MC018 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: RAT ARGS SI (SEQ ID NO: 61)The light chain CDRs for the anti -PAL antibody MC018 are the same as the light chain of MC001.

[0108] Anti -PAL antibody MC019 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRARARGSSIFDYWGQGTLVTVS S (SEQ ID NO: 62)Anti -PAL antibody MC019 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC019 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRARARGSSIFDYWGQGTLVTVS SGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLR GGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPR DGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTS TLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTK LTCL VTDLTTYD S VTISWTRQNGEAVKTHTNISESHPNATF S AVGEASICEDDWNSGERF TCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVF VQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALP NRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 63)A full length, light chain of the IgM format for antibody MC019 can have the amino acid sequence of SEQ ID NO: 4.

[0109] The heavy chain CDR1 and 2 for the anti -PAL antibody MC019 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: RARARGSSI (SEQ ID NO: 64)The light chain CDRs for the anti -PAL antibody MC019 are the same as the light chain of MC001. [HO] Anti-PAL antibody MC020 can have a variable region comprised of a heavy chain with the amino acid sequence of:Attorney File Reference: MBI.0009QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRRTARGSSIFDYWGQGTLVTVS S (SEQ ID NO: 65)Anti-PAL antibody MC020 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC020 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRRTARGSSIFDYWGQGTLVTVS SGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLR GGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPR DGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTS TLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTK LTCL VTDLTTYD S VTISWTRQNGEAVKTHTNISESHPNATF S AVGEASICEDDWNSGERF TCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVF VQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALP NRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 66)A full length, light chain of the IgM format for antibody MC020 can have the amino acid sequence of SEQ ID NO: 4.[Hl] The heavy chain CDR1 and 2 for the anti-PAL antibody MC020 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: RRTARGSSI (SEQ ID NO: 67)The light chain CDRs for the anti-PAL antibody MC020 are the same as the light chain of MC001.

[0112] Anti-PAL antibody MC021 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGARAWGSSIFDYWGQGTLVTV SS (SEQ ID NO: 68)Anti-PAL antibody MC021 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC021 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGARAWGSSIFDYWGQGTLVTV SSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVL RGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPAttorney File Reference: MBI.0009RDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKV TSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKST KLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGER FTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVF VQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALP NRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 69)A full length, light chain of the IgM format for antibody MC021 can have the amino acid sequence of SEQ ID NO: 4.

[0113] The heavy chain CDR1 and 2 for the anti -PAL antibody MC021 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:CDRH3: GARAWGSSI (SEQ ID NO: 70)The light chain CDRs for the anti -PAL antibody MC021 are the same as the light chain of MC001.

[0114] Anti-PAL antibody MC022 can have a variable region comprised of a heavy chain with the amino acid sequence of:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRARAWGSSIFDYWGQGTLVTV SS (SEQ ID NO: 71)Anti-PAL antibody MC022 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 2.A full length, heavy chain of the IgM format for antibody MC022 can have the amino acid sequence: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRARAWGSSIFDYWGQGTLVTV SSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVL RGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPP RDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKV TSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKST KLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGER FTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVF VQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALP NRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 72)A full length, light chain of the IgM format for antibody MC022 can have the amino acid sequence of SEQ ID NO: 4.

[0115] The heavy chain CDR1 and 2 for the anti-PAL antibody MC022 are the same as the heavy chain of MC001. The heavy chain CDR3 has the sequence:Attorney File Reference: MBI.0009CDR3: RARAWGSSI (SEQ ID NO: 73)The light chain CDRs for the anti -PAL antibody MC022 are the same as the light chain of MC001.

[0116] Anti-PAL antibody MC023 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 1.Anti-PAL antibody MC023 can have a variable region comprised of a light chain with the amino acid sequence:DIQMTQSPSTLSASVGDRVTITCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYGVSNRFS GSKSGNTASLTISGLAAEDEADYYCSSYTSSSTLVVFGGGTKLTVLG (SEQ ID NO: 74) A full length, heavy chain of the IgM format for antibody MC023 can have the amino acid sequence OF SEQ ID NO: 3.A full length, light chain of the IgM format for antibody MC023 can have the amino acid sequence: DIQMTQSPSTLSASVGDRVTITCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYGVSNRFS GSKSGNTASLTISGLAAEDEADYYCSSYTSSSTLVVFGGGTKLTVLGQPKAAPSVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSL TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 75)

[0117] The light chain CDRs for the anti-PAL antibody MC023 are:CDRL1 : TGTSSDVGGYNYVS (SEQ ID NO: 76)CDRL2: GVSNRFS (SEQ ID NO: 77)CDRL3: SSYTSSSTL (SEQ ID NO: 78)

[0118] Anti-PAL antibody MC024 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 11.Anti-PAL antibody MC024 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC024 can have the amino acid sequence OF SEQ ID NO: 12.A full length, light chain of the IgM format for antibody MC024 can have the amino acid sequence of SEQ ID NO: 75.

[0119] Anti-PAL antibody MC025 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 14.Anti-PAL antibody MC025 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC025 can have the amino acid sequence OF SEQ ID NO: 15.Attorney File Reference: MBI.0009A full length, light chain of the IgM format for antibody MC025 can have the amino acid sequence of SEQ ID NO: 75.

[0120] Anti-PAL antibody MC026 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 17.Anti-PAL antibody MC026 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC026 can have the amino acid sequence OF SEQ ID NO: 18.A full length, light chain of the IgM format for antibody MC026 can have the amino acid sequence of SEQ ID NO: 75.

[0121] Anti-PAL antibody MC027 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 20.Anti-PAL antibody MC027 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC027 can have the amino acid sequence OF SEQ ID NO: 21.A full length, light chain of the IgM format for antibody MC027 can have the amino acid sequence of SEQ ID NO: 75.

[0122] Anti-PAL antibody MC028 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 23.Anti-PAL antibody MC028 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC028 can have the amino acid sequence OF SEQ ID NO: 24.A full length, light chain of the IgM format for antibody MC028 can have the amino acid sequence of SEQ ID NO: 75.

[0123] Anti-PAL antibody MC029 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 26.Anti-PAL antibody MC029 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC029 can have the amino acid sequence OF SEQ ID NO: 27.A full length, light chain of the IgM format for antibody MC029 can have the amino acid sequence of SEQ ID NO: 75.Attorney File Reference: MBI.0009

[0124] Anti -PAL antibody MC030 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 29.Anti-PAL antibody MC030 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC030 can have the amino acid sequence OF SEQ ID NO: 30.A full length, light chain of the IgM format for antibody MC030 can have the amino acid sequence of SEQ ID NO: 75.

[0125] Anti-PAL antibody MC031 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 32.Anti-PAL antibody MC031 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC031 can have the amino acid sequence OF SEQ ID NO: 33.A full length, light chain of the IgM format for antibody MC031 can have the amino acid sequence of SEQ ID NO: 75.

[0126] Anti-PAL antibody MC032 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 35.Anti-PAL antibody MC032 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC032 can have the amino acid sequence OF SEQ ID NO: 36.A full length, light chain of the IgM format for antibody MC032 can have the amino acid sequence of SEQ ID NO: 75.

[0127] Anti-PAL antibody MC033 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 38.Anti-PAL antibody MC033 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC033 can have the amino acid sequence OF SEQ ID NO: 39.A full length, light chain of the IgM format for antibody MC033 can have the amino acid sequence of SEQ ID NO: 75.

[0128] Anti-PAL antibody MC034 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 41.Attorney File Reference: MBI.0009Anti-PAL antibody MC034 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC034 can have the amino acid sequence OF SEQ ID NO: 42.A full length, light chain of the IgM format for antibody MC034 can have the amino acid sequence of SEQ ID NO: 75.

[0129] Anti-PAL antibody MC035 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 44.Anti-PAL antibody MC035 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC035 can have the amino acid sequence OF SEQ ID NO: 45.A full length, light chain of the IgM format for antibody MC035 can have the amino acid sequence of SEQ ID NO: 75.

[0130] Anti-PAL antibody MC036 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 47.Anti-PAL antibody MC036 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC036 can have the amino acid sequence OF SEQ ID NO: 48.A full length, light chain of the IgM format for antibody MC036 can have the amino acid sequence of SEQ ID NO: 75.

[0131] Anti-PAL antibody MC037 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 50.Anti-PAL antibody MC037 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC037 can have the amino acid sequence OF SEQ ID NO: 51.A full length, light chain of the IgM format for antibody MC037 can have the amino acid sequence of SEQ ID NO: 75.

[0132] Anti-PAL antibody MC038 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 53.Anti-PAL antibody MC038 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74Attorney File Reference: MBI.0009A full length, heavy chain of the IgM format for antibody MC038 can have the amino acid sequence OF SEQ ID NO: 54.A full length, light chain of the IgM format for antibody MC038 can have the amino acid sequence of SEQ ID NO: 75.

[0133] Anti -PAL antibody MC039 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 56.Anti-PAL antibody MC039 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC039 can have the amino acid sequence OF SEQ ID NO: 57.A full length, light chain of the IgM format for antibody MC039 can have the amino acid sequence of SEQ ID NO: 75.

[0134] Anti-PAL antibody MC040 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 59.Anti-PAL antibody MC040 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC040 can have the amino acid sequence OF SEQ ID NO: 60.A full length, light chain of the IgM format for antibody MC040 can have the amino acid sequence of SEQ ID NO: 75.

[0135] Anti-PAL antibody MC041 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 62.Anti-PAL antibody MC041 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC041 can have the amino acid sequence OF SEQ ID NO: 63.A full length, light chain of the IgM format for antibody MC041 can have the amino acid sequence of SEQ ID NO: 75.

[0136] Anti-PAL antibody MC042 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 65.Anti-PAL antibody MC042 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC042 can have the amino acid sequence OF SEQ ID NO: 66.Attorney File Reference: MBI.0009A full length, light chain of the IgM format for antibody MC042 can have the amino acid sequence of SEQ ID NO: 75.

[0137] Anti-PAL antibody MC043 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 68.Anti-PAL antibody MC043 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC043 can have the amino acid sequence OF SEQ ID NO: 69.A full length, light chain of the IgM format for antibody MC043 can have the amino acid sequence of SEQ ID NO: 75.

[0138] Anti-PAL antibody MC044 can have a variable region comprised of a heavy chain with the amino acid sequence of SEQ ID NO: 71.Anti-PAL antibody MC044 can have a variable region comprised of a light chain with the amino acid sequence of SEQ ID NO: 74A full length, heavy chain of the IgM format for antibody MC044 can have the amino acid sequence OF SEQ ID NO: 72.A full length, light chain of the IgM format for antibody MC044 can have the amino acid sequence of SEQ ID NO: 75.

[0139] CDRs from anti-PAL antibodies MC001 (SEQ ID NO: 5, 6, 7 and 8, 9, 10), MC002 (SEQID NO: 5, 6, 13 and 8, 9, 10), MC003 (SEQ ID NO: 5, 6, 16 and 8, 9, 10), MC004 (SEQ ID NO: 5, 6, 19 and 8, 9, 10), MQ005 or MC005 (SEQ ID NO: 5, 6, 22 and 8, 9, 10), MC006 (SEQ ID NO: 5, 6, 25 and 8, 9, 10), MC007 (SEQ ID NO: 5, 6, 28 and 8, 9, 10), MC008 (SEQ ID NO: 5, 6, 31 and 8, 9, 10), MC009 (SEQ ID NO: 5, 6, 34 and 8, 9, 10), MC010 (SEQ ID NO: 5, 6, 37 and 8, 9, 10), MC011 (SEQ ID NO: 5, 6, 40 and 8, 9, 10), MC012 (SEQ ID NO: 5, 6, 43 and 8, 9, 10), MC013 (SEQ ID NO: 5, 6, 46 and 8, 9, 10), MC014 (SEQ ID NO: 5, 6, 49 and 8, 9, 10), MC015 (SEQ ID NO: 5, 6, 52 and 8, 9, 10), MC016 (SEQ ID NO: 5, 6, 55 and 8, 9, 10), MC017 (SEQ ID NO: 5, 6, 58 and 8, 9, 10), MC018 (SEQ ID NO: 5, 6, 61 and 8, 9, 10), MC019 (SEQ ID NO: 5, 6, 64 and 8, 9, 10), MC020 (SEQ ID NO: 5, 6, 67 and 8, 9, 10), MC021 (SEQ ID NO: 5, 6, 70 and 8, 9, 10), MC022 (SEQ ID NO: 5, 6, 73 and 8, 9, 10), MC023 (SEQ ID NO: 5, 6, 7 and 76, 77, 78), MC024 (SEQ ID NO: 5, 6, 13 and 76, 77, 78), MC025 (SEQ ID NO: 5, 6, 16 and 76, 77, 78),MC026 (SEQ ID NO: 5, 6, 19 and 76, 77, 78), MC027 (SEQ ID NO: 5, 6, 22 and 76, 77, 78),MC028 (SEQ ID NO: 5, 6, 25 and 76, 77, 78), MC029 (SEQ ID NO: 5, 6, 28 and 76, 77, 78),MC030 (SEQ ID NO: 5, 6, 31 and 76, 77, 78), MC031 (SEQ ID NO: 5, 6, 34 and 76, 77, 78),MC032 (SEQ ID NO: 5, 6, 37 and 76, 77, 78), MC033 (SEQ ID NO: 5, 6, 40 and 76, 77, 78),Attorney File Reference: MBI.0009MC034 (SEQ ID NO: 5, 6, 43 and 76, 77, 78), MC035 (SEQ ID NO: 5, 6, 46 and 76, 77, 78),MC036 (SEQ ID NO: 5, 6, 49 and 76, 77, 78), MC037 (SEQ ID NO: 5, 6, 52 and 76, 77, 78),MC038 (SEQ ID NO: 5, 6, 55 and 76, 77, 78), MC039 (SEQ ID NO: 5, 6, 58 and 76, 77, 78),MC040 (SEQ ID NO: 5, 6, 61 and 76, 77, 78), MC041 (SEQ ID NO: 5, 6, 64 and 76, 77, 78),MC042 (SEQ ID NO: 5, 6, 67 and 76, 77, 78), MC043 (SEQ ID NO: 5, 6, 70 and 76, 77, 78), and MC044 (SEQ ID NO: 5, 6, 73 and 76, 77, 78) can be used to make CDR grafted antibodies known in the art.

[0140] An anti-PAL antibody can also include a variable region made from a nucleic acid encoding a heavy chain that has 99%, 95%, 90%, 80% or 70% sequence identity with a nucleic acid encoding a heavy chain having the amino acid sequence of SEQ ID NO: 1, 3, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, and / or 72, and a nucleic acid encoding a light chain that has 99%, 95%, 90%, 80% or 70% sequence identity with a nucleic acid encoding a light chain having the amino acid sequence of SEQ ID NO: 2, 4, 74, and / or 75. An anti-PAL antibody can also include a variable region made from a nucleic acid encoding a heavy chain that has 99%, 95%, 90%, 80% or 70% sequence identity with the amino acid sequence of SEQ ID NO: 1, 3, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, and / or 72, and a nucleic acid encoding a light chain that has 99%, 95%, 90%, 80% or 70% sequence identity with the amino acid sequence of SEQ ID NO: 2, 4, 74, and / or 75. An anti-PAL antibody can also include a variable region made from a nucleic acid encoding a heavy chain that hybridizes under stringent hybridization conditions with one of the nucleic acids encoding one of the heavy chains (SEQ ID NO: 1, 3, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, and / or 72), and a nucleic acid encoding a light chain that hybridizes under stringent hybridization conditions with one of the nucleic acids encoding one of the light chains (SEQ ID NO: 2, 4, 74, and / or 75).

[0141] In one aspect, the isolated antigen binding protein binds to PAL, and comprises a heavy chain CDR3 sequence X1X2X3AX4GX5SX6X7, wherein:XI is an G, A, or an R;X2 is an R, a G, or an A. X3 is an M, an T. or a R; X4 is an R, a W, or a Y: X5 is an A, an S or a G; X6 is an I, a V, or a Y; andAttorney File Reference: MBI.0009X7 is an N, or no amino acid; and wherein there is one, and not more than one, Arginine within positions 1 through 3 (relative to heavy chain variable region, positions 98 through 100, position 97 being the invariable Arginine preceding the CDR3 region.

[0142] In another aspect, the isolated antigen binding protein binds to PAL, and comprises a heavy chain CDR3 sequence X1X2X3AX4GX5SX6X7, wherein:XI is an G, A, or an R;X2 is an R, a G, or an A.X3 is an M, an T. or a R;X4 is an R, or a W:X5 is an A, oran S;X6 is an I, or a V; andX7 is an N, or no amino acid; and wherein there is one, and not more than one, Arginine within positions 1 through 3 (relative to heavy chain variable region, positions 98 through 100, position 97 being the invariable Arginine preceding the CDR3 region.

[0143] The heavy and light chain CDRs described above, and optionally FR amino acids from the corresponding light or heavy chain can be used to make CDR grafted antibodies as described below.

[0144] An anti-PAL antibody can bind to PAL with an affinity (Kd) of less than one micromolar. An anti-PAL antibody can bind with an affinity of at least 1 pM, or at least 100 nM, or at least 10 nM.

[0145] The anti-PAL antibodies can include human MAb 216 was prepared by diffusion of uninvolved spleen lymphocytes from a patient with nodular lymphoma. The cells were incubated in vitro with LPS and fused to the heteromyeloma line SHMD33. This antibody was found to be mu, lambda using peroxidase-labeled chain-specific antibodies (Cal Tag, South San Francisco, Calif.). Nucleotide analysis of the heavy chain showed it was encoded by the VH 4.21 gene. A deposit of the hybridoma cell line secreting mAb 216 was made with the ATCC in Rockville, Md. USA, as Deposit No. HB 11659, on Jun. 14, 1994.

[0146] The anti-PAL antibodies can include a HA1 A made employing human lymphocytes which have been sensitized with lipid A from a Gram-negative bacterium. Particularly, the J5 mutant of E. coli 0111 :B4 provides a lipopolysaccharide consisting solely of the core determinants. A human host may be sensitized with J5 vaccine prepared from E. coli J5 bacterial cells as described in Ziegler. Desirably, patients are chosen who are intended for a splenectomy, where the spleen will be removed shortly after the immunization program. The vaccine may be used by itself or inAttorney File Reference: MBI.0009 conjunction with adjuvants to enhance the immune response. Conveniently, the host may be immunized subcutaneously at one or more sites with injections of about 0.5 to 2 ml, usually about 1 ml, where the vaccine will induce a hemagglutinating antibody titer in the range of about 1 :32 to 1 :256. One or more booster vaccinations may be given, generally not more than about two booster vaccinations, where the vaccinations may be separated by from about 48 hr to about three weeks.

[0147] The first fusion with the spleen cells of patient D. M. employed unstimulated B-cells and SHM-D33 in a 1 :4 ratio. The cell fusion employed was the method described by Oi and Herzenberg (1979) In: Selected Methods in Cellular Immunology (B. B. Mishell and S. M. Shiigi, eds.) San Francisco: W. J. Freeman Publishers, pp. 351-372, which is incorporated by reference in its entirety for all purposes. In a second set of fusions, EBV-transformed lymphoblastoid cells were fused with the following heteromyeloma cell lines: SHM-D29, -D-33 (G3), -D36 (G7), -D39, -D49, -D42, - D70, -A6 (H4) and with the parental mouse myeloma line, X63-Ag8,653 ("653"). subcloning of the producer clones had been carried out and testing of these revealed that subclones 2G5 and 1E8 derived from the fusion with SHM-D33 (G3 ); 1F10, 1C2 and 2G9 derived from the fusion with SHM-A6 (H4); and 1B7, 1E3, 1G1, 2F9, 2E6 and 2D7 derived from the fusion with 653 cells were specific anti-J5 endotoxin antibody producers. The hybridoma C5(1F1O) was deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, on Jan. 5, 1984 and given Accession No. HB8669.

[0148] The anti-PAL antibodies can also include the antibodies disclosed in U.S. Patent No. 5,426,046 and U.S. Patent 5,417,972, each of which is incorporated by reference in its entirety for all purposes.

[0149] The anti-PAL antibodies disclosed herein can also be used to make chimeric antigen receptors for arming T-cells for treatment of bacterial infections and / or sepsis. For example, the antibodies can be formatted into a single chain structure recombinantly combined with appropriate transmembrane and signaling components to make a chimeric antigen receptor.Nucleic Acids

[0150] The disclosure also relates to nucleic acids that encode, at least in part, the individual peptides, polypeptides, and proteins described herein. The nucleic acids may be natural, synthetic or a combination thereof. The nucleic acids may be RNA, mRNA, DNA or cDNA.

[0151] Nucleic acids also include expression vectors, such as plasmids, or viral vectors, or linear vectors, or vectors that integrate into chromosomal DNA. Expression vectors can contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of cells. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria. In eukaryotic host cells, e.g., mammalianAttorney File Reference: MBI.0009 cells, the expression vector can be integrated into the host cell chromosome and then replicate with the host chromosome. Similarly, vectors can be integrated into the chromosome of prokaryotic cells.

[0152] Expression vectors also generally contain a selection gene, also termed a selectable marker. Selectable markers are well-known in the art for prokaryotic and eukaryotic cells, including host cells of the disclosure. Generally, the selection gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. In some aspects, an exemplary selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Other selectable markers for use in bacterial or eukaryotic (including mammalian) systems are well-known in the art.

[0153] An example of a promoter that is capable of expressing a transgene encoding an immune binding protein in a mammalian host cell is the EFla promoter. The native EFla promoter drives expression of the alpha subunit of the elongation factor- 1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. The EFla promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving expression from transgenes cloned into a lentiviral vector. See, e.g., Milone et al., Mol. Ther. 17(8): 1453-1464 (2009), which is incorporated by reference in its entirety for all purposes. Another example of a promoter is 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 the simian virus 40 (SV40) early promoter, mouse mammary tumor virus promoter (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, phosphoglycerate kinase (PGK) promoter, MND promoter (a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer, see, e.g., Li et al., J. Neurosci. Methods vol. 189, pp. 56-64 (2010) which is incorporated by reference in its entirety for all purposes), an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters suchAttorney File Reference: MBI.0009 as, but not limited to, the actin promoter, the myosin promoter, the elongation factor- la promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention is not limited to the use of constitutive promoters.

[0154] Inducible promoters are also contemplated herein. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, a tetracycline promoter, a c-fos promoter, the T-REx system of ThermoFisher which places expression from the human cytomegalovirus immediate-early promoter under the control of tetracycline operator(s), and RheoSwitch promoters of Intrexon. Karzenowski, D. et al., BioTechiques 39: 191-196 (2005); Dai, X. et al., Protein Expr. Purif 42:236-245 (2005); Palli, S. R. et al., Eur. J. Biochem. 270: 1308-1515 (2003); Dhadialla, T. S. et al., Annual Rev. Entomol. 43:545-569 (1998); Kumar, M. B, et al., J. Biol. Chem. 279:27211- 27218 (2004); Verhaegent, M. et al., Annal. Chem. 74:4378-4385 (2002); Katalam, A. K., et al., Molecular Therapy 13 : S 103 (2006); and Karzenowski, D. et al., Molecular Therapy 13 : S194 (2006), U.S. Patent Nos. 8,895,306, 8,822,754, 8,748,125, 8,536,354, all of which are incorporated by reference in their entirety for all purposes.

[0155] Expression vectors typically have promoter elements, e.g., enhancers, to regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0156] Control regions suitable for a bacterial host cell can be used in the expression vector. Suitable control regions for directing transcription of the nucleic acid constructs include, for example, the control regions obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene, the tac promoter, or the T7 promoter.

[0157] In some embodiments, control regions for filamentous fungal host cells, include control regions obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor mieheiAttorney File Reference: MBI.0009 aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alphaamylase, Aspergillus niger o Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid control regions thereof. Exemplary yeast cell control regions can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GALI), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3- phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3 -phosphoglycerate kinase.

[0158] Exemplary control regions for insect cells include, among others, those based on polyhedron, PCNA, OplE2, OplEl, Drosophila metallothionein, and Drosophila actin 5C. In some embodiments, insect cell promoters can be used with Baculoviral vectors.

[0159] Exemplary control regions for plant cells include, among others, those based on cauliflower mosaic virus (CaMV) 35S, polyubiquitin gene (PvUbil and PvUbi2), rice (Oryza saliva) actin 1 (OsActl) and actin 2 (OsAct2) control regions, the maize ubiquitin 1 (ZmUbil) control region, and multiple rice ubiquitin (RUBQ1, RUBQ2, rubi3) control regions.

[0160] The expression vector can contain one or more selectable markers, which permit selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis. or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol (Example 1) or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Embodiments for use in an Aspergillus cell include the amdS and pyrG genes of Aspergillus nidulans ox Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.

[0161] It may be desirable to modify the polypeptides described herein. One of skill will recognize many ways of generating alterations in a given nucleic acid construct to generate variant polypeptides Such well-known methods include site-directed mutagenesis, PCRAttorney File Reference: MBI.0009 amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide (e.g., in conjunction with ligation and / or cloning to generate large nucleic acids) and other well-known techniques (see, e.g., Gillam and Smith, Gene 8:81-97, 1979; Roberts et al., Nature 328:731-734, 1987, which is incorporated by reference in its entirety for all purposes). The recombinant nucleic acids encoding the polypeptides herein can be modified to provide preferred codons which enhance translation of the nucleic acid in a selected organism.

[0162] The polynucleotides described herein also include polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides described herein. Polynucleotides according to the invention can have at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence identity to a polynucleotide of the disclosure. The disclosure also provides the complement of the polynucleotides including a nucleotide sequence that has at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence identity to a polynucleotide encoding a polypeptide recited above. The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions which can routinely isolate polynucleotides of the desired sequence identities.

[0163] Nucleic acids which encode protein analogs or variants in accordance with those described herein (z.e., wherein one or more amino acids are designed to differ from the wild type polypeptide) may be produced using site directed mutagenesis or PCR amplification in which the primer(s) have the desired point mutations. For a detailed description of suitable mutagenesis techniques, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and / or Current Protocols in Molecular Biology, Ausubel et al., eds, Green Publishers Inc. and Wiley and Sons, N.Y (1994), each of which is incorporated by reference in its entirety for all purposes. Chemical synthesis using methods well known in the art, such as that described by Engels et al., Angew Chem Inti Ed. 28:716-34, 1989 (which is incorporated by reference in its entirety for all purposes), may also be used to prepare such nucleic acids.

[0164] Amino acid “substitutions” for creating variants can be preferably the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, z.e., conservative amino acid replacements. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids includeAttorney File Reference: MBI.0009 alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0165] The nucleic acids described herein can be linked to another nucleic acid so as to be expressed under control of a suitable promoter. The nucleic acid can be also linked to, in order to attain efficient transcription of the nucleic acid, other regulatory elements that cooperate with a promoter or a transcription initiation site, for example, a nucleic acid comprising an enhancer sequence, a polyA site, or a terminator sequence. In addition, a gene that can be a marker for confirming expression of the nucleic acid (e.g. a drug resistance gene, a gene encoding a reporter enzyme, or a gene encoding a fluorescent protein) may be incorporated.

[0166] When the nucleic acid described herein is introduced into a cell ex vivo, the nucleic acid of may be combined with a substance that promotes transference of a nucleic acid into a cell, for example, a reagent for introducing a nucleic acid such as a liposome or a cationic lipid, in addition to the aforementioned excipients. Alternatively, a vector carrying the nucleic acid can also be useful. Particularly, a composition in a form suitable for administration to a living body which contains the nucleic acid of the present invention carried by a suitable vector is suitable for in vivo gene therapy.Host Cells

[0167] Nucleic acids encoding an immune binding protein described herein (e.g., an antibody) can be cloned into an appropriate expression vector for expression of immune binding protein in a host cell. Host cells include, for example, bacterial, fungi, or mammalian host cells. The host cell can be a bacterium including, for example, Bacillus, such as B. lichenformis or B. sublilis: Pantoea, such as P. cilrea: Pseudomonas, such as P. alcaligenes,' Streptomyces, such as S. lividans or S. rubiginosus,' Escherichia, such as E. coli Enter obacter, Streptococcus,' Archaea, such as Methanosarcina mazer, or Corynebacterium, such as C. glutamicum.

[0168] The host cells can be fungi cells, including, but not limited to, fungi of the genera Saccharomyces, Klyuveromyces, Candida, Pichia, Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces, or Schizosaccharomyces. In some embodiments, the host cell is a fungi, including, among others, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, Yarrowia lipolytica, and the like. The eukaryotic cells can be algal, including but not limited to algae of the genera Chlorella, Chlamydomonas, Scenedesmus,Attorney File Reference: MBI.0009Isochrysis, Dunaliella, Tetraselmis, Nannochloropsis, or Prototheca. The algae can be a green algae, red algae, glaucophytes, chlorarachniophytes, euglenids, chromista, or dinoflagellates.

[0169] The eukaryotic cells can be mammalian cells, such as mouse, rat, rabbit, hamster, porcine, bovine, feline, or canine. The mammalian cells can be cells of primates, including but not limited to, monkeys, chimpanzees, gorillas, and humans. The mammalians cells can be mouse cells, as mice routinely function as a model for other mammals, most particularly for humans (see, e.g., Hanna, J. et al., Science 318: 1920-23, 2007; Holtzman, D.M. et al., J Clin Invest. 103(6):R15-R21, 1999; Warren, R.S. et al., J Clin Invest. 95: 1789-1797, 1995; each publication is incorporated by reference in its entirety for all purposes). Animal cells include, for example, fibroblasts, epithelial cells (e.g., renal, mammary, prostate, lung), keratinocytes, hepatocytes, adipocytes, endothelial cells, and hematopoietic cells. In some embodiments, the animal cells are adult cells (e.g., terminally differentiated, dividing or non-dividing) or embryonic cells (e.g., blastocyst cells, etc.) or stem cells. The animal cell can be a cell line derived from an animal or other source, such as a Chinese hamster ovary cell line (CHO cell), or murine myeloma cell lines (NS0, Sp2 / 0), or human cell lines including, for example, HEK293, HT-1080, or PER.C6.

[0170] The mammalian cell can be a cell found in the circulatory system of a mammal, including humans. Exemplary circulatory system cells include, among others, red blood cells, platelets, plasma cells, T-cells, natural killer cells, B-cells, macrophages, neutrophils, or the like, and precursor cells of the same. As a group, these cells are defined to be circulating eukaryotic cells of the disclosure. The mammalian cells can be derived from any of these circulating eukaryotic cells. The immune binding proteins described herein may be used with any of these circulating cells or cells derived from the circulating cells. The mammalian cell can be a T-cell or T-cell precursor or progenitor cell. The mammalian cell can be a helper T-cell, a cytotoxic T-cell, a memory T-cell, a regulatory T-cell, a natural killer T-cell, a mucosal associated invariant T-cell, a gamma delta T cell, or a precursor or progenitor cell to the aforementioned. The mammalian cell can be a natural killer cell, or a precursor or progenitor cell to the natural killer cell. The mammalian cell can be a B-cell, or a plasma cell, or a B-cell precursor or progenitor cell. The mammalian cell can be a neutrophil or a neutrophil precursor or progenitor cell. The mammalian cell can be a megakaryocyte or a precursor or progenitor cell to the megakaryocyte. The mammalian cell can be a macrophage or a precursor or progenitor cell to a macrophage.

[0171] A source of cells can be obtained from a subject. The subject may be any living organism. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleuralAttorney File Reference: MBI.0009 effusion, spleen tissue, and tumors. Any number of T cell lines available in the art, may be used. T cells can be obtained from a unit of blood collected from a subj ect using any number of techniques known to the skilled artisan, such as Ficoll separation. Cells from the circulating blood of an individual can be obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. The cells can be washed with phosphate buffered saline (PBS). In an alternative aspect, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation.

[0172] Plant cells can be cells of monocotyledonous or dicotyledonous plants, including, but not limited to, alfalfa, almonds, asparagus, avocado, banana, barley, bean, blackberry, brassicas, broccoli, cabbage, canola, carrot, cauliflower, celery, cherry, chicory, citrus, coffee, cotton, cucumber, eucalyptus, hemp, lettuce, lentil, maize, mango, melon, oat, papaya, pea, peanut, pineapple, plum, potato (including sweet potatoes), pumpkin, radish, rapeseed, raspberry, rice, rye, sorghum, soybean, spinach, strawberry, sugar beet, sugarcane, sunflower, tobacco, tomato, turnip, wheat, zucchini, and other fruiting vegetables (e.g. tomatoes, pepper, chili, eggplant, cucumber, squash etc.), other bulb vegetables (e.g., garlic, onion, leek etc.), other pome fruit (e.g. apples, pears etc.), other stone fruit (e.g., peach, nectarine, apricot, pears, plums etc.), Arabidopsis, woody plants such as coniferous and deciduous trees, an ornamental plant, a perennial grass, a forage crop, flowers, other vegetables, other fruits, other agricultural crops, herbs, grass, or perennial plant parts (e.g., bulbs; tubers; roots; crowns; stems; stolons; tillers; shoots; cuttings, including un-rooted cuttings, rooted cuttings, and callus cuttings or callus-generated plantlets; apical meristems etc.). The term “plants” refers to all physical parts of a plant, including seeds, seedlings, saplings, roots, tubers, stems, stalks, foliage and fruits.CDR Grafted Antibodies

[0173] CDR grafted forms of antibodies are chimeric immunoglobins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain CDR sequences derived from one immunoglobulin grafted into the framework sequences of a second immunoglobulin. CDR grafted antibodies include immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient antibody are replaced by residues from a CDR of another antibody (donor antibody) having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the recipient antibody are replaced by corresponding residues from the donor antibody.Attorney File Reference: MBI.0009CDR grafted antibodies may also comprise residues which are found neither in the recipient antibody nor in the donor CDR or framework sequences. In general, the CDR grafted antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a donor antibody and all or substantially all of the FR regions are those of a recipient antibody consensus sequence. The CDR grafted antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a recipient antibody [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992), all of which are incorporated by reference in their entirety for all purposes],

[0174] Humanization is one type of CDR grafting to make a chimeric antibody. Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non- human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988), all of which are incorporated by reference in their entirety for all purposes], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567 which is incorporated by reference in its entirety for all purposes), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequences from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.Multispecific Antibodies

[0175] It may be desirable to generate multispecific (e.g. bispecific) antibodies having binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes one of which can be PAL. Bispecific antibodies may also be used to localize antibacterial agents. These antibodies possess an PAL-binding arm and an arm which binds the cytotoxic agent (e.g., saporin, anti-interferon-alpha, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0176] According to another approach for making bispecific antibodies, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part ofAttorney File Reference: MBI.0009 the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. See WO96 / 27011 published Sep. 6, 1996, which is incorporated by reference in its entirety for all purposes.

[0177] Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, which is incorporated by reference in its entirety for all purposes, along with a number of cross-linking techniques.

[0178] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) (which is incorporated by reference in its entirety for all purposes) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. In yet a further embodiment, Fab'-SH fragments directly recovered from E. coli can be chemically coupled in vitro to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217 225 (1992) (which is incorporated by reference in its entirety for all purposes).

[0179] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5): 1547 1553 (1992), which is incorporated by reference in its entirety for all purposes. The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized toAttorney File Reference: MBI.0009 form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444 6448 (1993) which is incorporated by reference in its entirety for all purpose, has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152:5368(1994) which is incorporated by reference in its entirety for all purposes. Alternatively, the bispecific antibody may be a “linear antibody” produced as described in Zapata et al. Protein Eng. 8(10): 1057 1062 (1995) which is incorporated by reference in its entirety for all purposes.

[0180] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991) which is incorporated by reference in its entirety for all purposes.Modified Antibodies

[0181] An anti -PAL antibody can include a moiety that extends a half-life (T1 / 2) or / and the duration of action of the antibody. The moiety can extend the circulation T1 / 2, blood T1 / 2, plasma T1 / 2, serum T1 / 2, terminal T1 / 2, biological T1 / 2, elimination T1 / 2 or functional T1 / 2, or any combination thereof, of the antibody.

[0182] An anti-PAL antibody may be modified by a single moiety. Alternatively, an anti-PAL antibody may be modified by two or more substantially similar or identical moieties or two or more moieties of the same type. An anti-PAL antibody may include two or more moieties of different types, or two or more different types of moieties. Two or more anti-PAL antibodies can also be attached to one moiety. The attachment between the anti-PAL antibody and the moiety can be covalent or noncovalent.

[0183] A polypeptide moiety can be recombinantly fused to the N-terminus or the C-terminus of the heavy chain or the light chain of an anti-PAL antibody, optionally via a linker. The linker may contain about 4-30 amino acid residues. The linker may contain from about 6 or 8 amino acid residues to about 20 amino acid residues, or from about 6 or 8 amino acid residues to about 15 amino acid residues.

[0184] A protracting moiety can be human serum albumin (HSA) or a portion thereof (e.g., domain III) that binds to the neonatal Fc receptor (FcRn). The HSA or FcRn-binding portion thereof canAttorney File Reference: MBI.0009 optionally have one or more mutations that confer a beneficial property or effect. In some embodiments, the HSA or FcRn-binding portion thereof has one or more mutations that enhance pH-dependent HSA binding to FcRn or / and increase HSA half-life, such as K573P or / and E505G / V547A. A protracting moiety can be an unstructured polypeptide.

[0185] A protracting moiety can be a carboxy-terminal peptide (CTP) derived from the P-subunit of human chorionic gonadotropin (hCG). In the human body, the fourth, fifth, seventh and eight serine residues of the 34-aa CTP of hCG-P typically are attached to O-glycans terminating with a sialic acid residue.

[0186] A protracting moiety can be 1, 2, 3, 4, 5 or more moi eties of a synthetic polymer. The synthetic polymer can be biodegradable or non-biodegradable. Biodegradable polymers useful as protracting moieties include, but are not limited to, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA). Non-biodegradable polymers useful as protracting moieties include without limitation polyethylene glycol) (PEG), polyglycerol, poly(A-(2-hydroxypropyl)methacrylamide) (PHPMA), polyoxazolines and poly(A-vinylpyrrolidone) (PVP). A synthetic polymer can be polyethylene glycol (PEG). PEGylation can be done by chemical or enzymatic, site-specific coupling or by random coupling.

[0187] The protracting moieties can also include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, proly propylene oxide / ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

[0188] The individual mass (e.g., average molecular weight), or the total mass, of the one or more synthetic polymer moieties can be about 10-50, 10-20, 20-30, 30-40 or 40-50 kDa, or about 10, 20,Attorney File Reference: MBI.000930, 40 or 50 kDa. The individual mass (e.g., average MW), or the total mass, of the one or more synthetic polymer moieties also can be greater than about 50 kDa, such as about 50-100, 50-60, 60- 70, 70-80, 80-90 or 90-100 kDa, or about 60, 70, 80, 90 or 100 kDa. Moreover, the mass (e.g., average MW) of an individual synthetic polymer moiety can be less than about 10 kDa, such as about 1-5 or 5-10 kDa, or about 5 kDa. The individual mass (e.g., average MW), or the total mass, of the one or more synthetic polymer (e.g., PEG) moieties can be about 20 or 40 kDa.Pharmaceutical Compositions

[0189] Antibodies specifically binding PAL identified herein, as well as other immune binding proteins identified by the screening assays disclosed hereinbefore, can be administered for the treatment of various disorders in the form of pharmaceutical compositions.

[0190] Pharmaceutical compositions generally are prepared according to current good manufacturing practice (GMP), as recommended or required by, e.g., the Federal Food, Drug, and Cosmetic Act §501(a)(2)(B) and the International Conference on Harmonisation Q7 Guideline.

[0191] Pharmaceutical compositions / formulations can be prepared in sterile form. For example, pharmaceutical compositions / formulations for parenteral administration by injection or infusion generally are sterile. Sterile pharmaceutical compositions / formulations are compounded or manufactured according to pharmaceutical-grade sterilization standards known to those of skill in the art, such as those disclosed in or required by the United States Pharmacopeia Chapters 797, 1072 and 1211, and 21 Code of Federal Regulations 211.

[0192] Pharmaceutically acceptable excipients and carriers include pharmaceutically acceptable substances, materials and vehicles. Non-limiting examples of types of excipients include liquid and solid fillers, diluents, binders, lubricants, glidants, surfactants, dispersing agents, disintegration agents, emulsifying agents, wetting agents, suspending agents, thickeners, solvents, isotonic agents, buffers, pH adjusters, absorption-delaying agents, stabilizers, antioxidants, preservatives, antimicrobial agents, antibacterial agents, antifungal agents, chelating agents, adjuvants, sweetening agents, flavoring agents, coloring agents, encapsulating materials and coating materials. The use of such excipients in pharmaceutical formulations is known in the art. For example, conventional vehicles and carriers include without limitation oils (e.g., vegetable oils such as olive oil and sesame oil), aqueous solvents {e.g., saline, buffered saline (e.g., phosphate-buffered saline [PBS]) and isotonic solutions (e.g., Ringer’s solution)}, and organic solvents (e.g., dimethyl sulfoxide [DMSO] and alcohols [e.g., ethanol, glycerol and propylene glycol]). Except insofar as any conventional excipient or carrier is incompatible with an anti-PAL antibody or a fragment thereof, the disclosure encompasses the use of conventional excipients and carriers in formulations containing an anti-PAL antibody or a fragment thereof. See, e.g., Remington: The Science andAttorney File Reference: MBI.0009Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (Philadelphia, Pennsylvania) (2005); Handbook of Pharmaceutical Excipients, 5th Ed., Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association (2005); Handbook of Pharmaceutical Additives, 3rd Ed., Ash and Ash, Eds., Gower Publishing Co. (2007); and Pharmaceutical Preformulation and Formulation, Gibson, Ed., CRC Press (Boca Raton, Florida) (2004).

[0193] Appropriate formulation can depend on various factors, such as the route of administration chosen. Potential routes of administration of a pharmaceutical composition comprising an antiPAL antibody or a fragment thereof include without limitation oral, parenteral (including intradermal, subcutaneous, intramuscular, intravascular, intravenous, intraarterial, intraperitoneal, intramedullary, intrathecal and topical), intracavitary, and topical (including dermal / epicutaneous, transdermal, mucosal, transmucosal, intranasal [e.g., by nasal spray or drop], intraocular [e.g., by eye drop], pulmonary [e.g., by oral or nasal inhalation], buccal, sublingual, rectal [e.g., by suppository], and vaginal [e.g., by suppository]). Topical formulations can be designed to produce a local or systemic therapeutic effect. In certain embodiments, an anti-PAL antibody or a fragment thereof is administered parenterally (e.g., intravenously, subcutaneously, intramuscularly or intraperitoneally) by injection (e.g., as a bolus) or by infusion over a period of time.

[0194] Excipients and carriers that can be used to prepare parenteral formulations include without limitation solvents (e.g., aqueous solvents such as water, saline, physiological saline, buffered saline [e.g., phosphate-buffered saline], balanced salt solutions [e.g., Ringer’s BSS] and aqueous dextrose solutions), isotonic / iso-osmotic agents (e.g., salts [e.g., NaCl, KC1 and CaC12] and sugars [e.g., sucrose]), buffering agents and pH adjusters (e.g., sodium dihydrogen phosphate [monobasic sodium phosphate] / di sodium hydrogen phosphate [dibasic sodium phosphate], citric acid / sodium citrate and L-histidine / L-histidine HC1), and emulsifiers (e.g., non-ionic surfactants such as polysorbates [e.g., polysorbate 20 and 80] and pol oxamers [e.g., pol oxamer 188]). Protein formulations and delivery systems are discussed in, e.g., A. J. Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, 3rd Ed., CRC Press (Boca Raton, Florida) (2015).

[0195] The excipients can optionally include one or more substances that increase protein stability, increase protein solubility, inhibit protein aggregation or reduce solution viscosity, or any combination or all thereof. Examples of such substances include without limitation hydrophilic amino acids (e.g., arginine and histidine), polyols (e.g., myo-inositol, mannitol and sorbitol), saccharides {e.g., glucose (including D-glucose [dextrose]), lactose, sucrose and trehalose}, osmolytes (e.g., trehalose, taurine, amino acids [e.g., glycine, sarcosine, alanine, proline, serine, L- alanine and y-aminobutyric acid], and betaines [e.g., trimethylglycine and trimethylamine N-Attorney File Reference: MBI.0009 oxide]), and non-ionic surfactants {e.g., alkyl polyglycosides, ProTek® alkylsaccarides (e.g., a monosaccharide [e.g., glucose] or a disaccharide [e.g., maltose or sucrose] coupled to a long-chain fatty acid or a corresponding long-chain alcohol), and polypropylene glycol / polyethylene glycol block co-polymers (e.g., poloxamers [e.g., PluronicTM F-68], and Genapol® PF-10 and variants thereof)}. Because such substances increase protein solubility, they can be used to increase protein concentration in a formulation. Higher protein concentration in a formulation is particularly advantageous for subcutaneous administration, which has a limited volume of bolus administration (e.g., < about 1.5 mL). In addition, such substances can be used to stabilize proteins during the preparation, storage and reconstitution of lyophilized proteins. Formulations and excipients for inhalation delivery are known in the art including, for example, those described in US Patent No. 5,898,028 which is hereby incorporated by reference in its entirety for all purposes.

[0196] For parenteral (e.g., intravenous, subcutaneous or intramuscular) administration, a sterile solution or suspension of an anti-PAL antibody in an aqueous solvent containing one or more excipients can be prepared beforehand and can be provided in, e.g., a pre-filled syringe. Alternatively, an anti-PAL antibody can be dissolved or suspended in an aqueous solvent that can optionally contain one or more excipients prior to lyophilization (freeze-drying). Shortly prior to parenteral administration, the lyophilized anti-PAL antibody stored in a suitable container (e.g., a vial) can be reconstituted with, e.g., sterile water that can optionally contain one or more excipients. If the anti-PAL antibody is to be administered by infusion (e.g., intravenously), the solution or suspension of the reconstituted anti-PAL antibody can be added to and diluted in an infusion bag containing, e.g., sterile saline (e.g., about 0.9% NaCl).

[0197] Excipients that enhance transmucosal penetration of smaller proteins include without limitation cyclodextrins, alky saccharides (e.g., alkyl glycosides and alkyl maltosides [e.g., tetradecylmaltoside]), and bile acids (e.g., cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, glycodeoxycholic acid, chenodeoxycholic acid and dehydrocholic acid).

[0198] Excipients that enhance transepithelial or transdermal penetration of smaller proteins include without limitation chemical penetration enhancers (CPEs, including fatty acids [e.g., oleic acid]), cell-penetrating peptides {CPPs, including arginine-rich CPPs [e.g., polyarginines such as R6-R11 (e.g., R6 and R9) and TAT -related CPPs such as TAT(49-57)] and amphipathic CPPs [e.g., Pep-1 and penetratin]}, and skin-penetrating peptides (SPPs, such as the skin-penetrating and cellentering [SPACE] peptide). Transdermal penetration of smaller proteins can be further enhanced by use of a physical enhancement technique, such as iontophoresis, cavitational or non-cavitational ultrasound, electroporation, thermal ablation, radio frequency, microdermabrasion, microneedles or jet injection. US 2007 / 0269379 provides an extensive list of CPEs. F. Milletti, Drug Discov.Attorney File Reference: MBI.0009Today, 17:850-860 (2012) is a review of CPPs. R. Ruan et al., Ther. Deliv., 7:89-100 (2016) discuss CPPs and SPPs for transdermal delivery of macromolecules, and M. Prausnitz and R. Langer, Nat. Biotechnol., 26: 1261-1268 (2008) discuss a variety of transdermal drug-delivery methods.

[0199] An anti-PAL antibody can be delivered from a sustained-release composition. As used herein, the term “sustained-release composition” encompasses sustained-release, prolonged- release, extended-release, slow-release and controlled-release compositions, systems and devices. Protein delivery systems are discussed in, e.g., Banga (supra). A sustained-release composition can deliver a therapeutically effective amount of an anti-PAL antibody over a prolonged time period. In some embodiments, a sustained-release composition delivers an anti-PAL antibody over a period of at least about 3 days, 1 week, 2 weeks, 3 weeks, 1 month (4 weeks), 6 weeks, 2 months, 3 months or longer. A sustained-release composition can be administered, e.g., parenterally (e.g., intravenously, subcutaneously or intramuscularly).

[0200] A sustained-release composition of a protein can be in the form of, e.g., a particulate system, a lipid or oily composition, or an implant. Particulate systems include without limitation nanoparticles, nanospheres, nanocapsules, microparticles, microspheres and microcapsules. Nanoparticulate systems generally have a diameter or an equivalent dimension smaller than about 1 pm. In certain embodiments, a nanoparticle, nanosphere or nanocapsule has a diameter or an equivalent dimension of no more than about 500, 400 or 300 nm, or no more than about 200, 150 or 100 nm. In some embodiments, a microparticle, microsphere or microcapsule has a diameter or an equivalent dimension of about 1-200, 100-200 or 50-150 pm, or about 1-100, 1-50 or 50-100 pm. A nano- or microcapsule typically contains the therapeutic agent in the central core, while the therapeutic agent typically is dispersed throughout a nano- or microparticle or sphere. In certain embodiments, a nanoparticulate system is administered intravenously, while a microparticulate system is administered subcutaneously or intramuscularly.

[0201] In some embodiments, a sustained-release particulate system or implant is made of a biodegradable polymer or / and a hydrogel. In certain embodiments, the biodegradable polymer comprises lactic acid or / and glycolic acid [e.g., an L-lactic acid-based copolymer, such as poly(L- lactide-co-glycolide) or poly(L-lactic acid-co-D,L-2-hydroxyoctanoic acid)]. Non-limiting examples of polymers of which a hydrogel can be composed include polyvinyl alcohol, acrylate polymers (e.g., sodium polyacrylate), and other homopolymers and copolymers having a relatively large number of hydrophilic groups (e.g., hydroxyl or / and carboxylate groups). The biodegradable polymer of the particulate system or implant can be selected so that the polymer substantially completely degrades around the time the period of treatment is expected to end, and so that the byproducts of the polymer’s degradation, like the polymer, are biocompatible.Attorney File Reference: MBI.0009

[0202] Alternatively, a sustained-release composition of a protein can be composed of a non- biodegradable polymer. Examples of non-biodegradable polymers include without limitation poloxamers (e.g., poloxamer 407). Sustained-release compositions of a protein can be composed of other natural or synthetic substances or materials, such as hydroxyapatite.

[0203] Sustained-release lipid or oily compositions of a protein can be in the form of, e.g., liposomes, micelles (e.g., those composed of biodegradable natural or / and synthetic polymers, such as lactosomes), and emulsions in an oil.

[0204] A sustained-release composition can be formulated or designed as a depot, which can be injected or implanted, e.g., subcutaneously or intramuscularly. A depot can be in the form of, e.g., a polymeric particulate system, a polymeric implant, or a lipid or oily composition. A depot formulation can comprise a mixture of a protein and, e.g., a biodegradable polymer [e.g., poly(lactide-co-glycolide)] or a semi-biodegradable polymer (e.g., a block copolymer of lactic acid and PEG) in a biocompatible solvent system, whether or not such a mixture forms a particulate system or implant.

[0205] The anti-PAL antibodies can be administered at a dose of from about 2.5 to about 3000 mg / m2or, from about 25 to 1000 mg / m2, or in particular, about 75, 150, 300 or 600 mg / m2. In additional aspects, the antibody is administered at a dose of from about 0.25 mg / kg to about 100 mg / kg, and more preferably, at about 1.25, 2.5, 5, 10, or 20 mg / kg. The anti-PAL antibody is typically administered on a weekly basis, and in Some embodiments, more frequently than once per week, as often as once per day. Additional agents can be administered in an amount of 10-375 mg / m per week for four weeks, or 0.4-20 mg / kg per week for 2 to 10 weeks in form of a combination therapy. In an aspect, anti-PAL antibodies are currently administered to a patient daily as monotherapy in an amount from about 0.25 mg / kg to about 100 mg / kg. In another aspect, anti-PAL antibodies are administered to a patient daily in combination therapy with a second agent selected from the agents disclosed below in an amount from about from about 0.15 mg / kg to about 50 mg / kg. In an aspect, a dose of 10 mg, 25 mg, 50 mg, 100 mg, 250 mg, or 500 mg of anti-PAL antibody is administered to a patient.

[0206] A pharmaceutical composition can be presented in unit dosage form as a single dose wherein all active and inactive ingredients are combined in a suitable system, and components do not need to be mixed to form the composition to be administered. The unit dosage form generally contains an effective dose of the therapeutic agent. A representative example of a unit dosage form is a single-use pen comprising a pre-filled syringe, a needle and a needle cover for parenteral (e.g., intravenous, subcutaneous or intramuscular) injection of the therapeutic agent.Attorney File Reference: MBI.0009

[0207] Alternatively, a pharmaceutical composition can be presented as a kit in which the therapeutic agent, excipients and carriers (e.g., solvents) are provided in two or more separate containers (e.g., ampules, vials, tubes, bottles or syringes) and need to be combined to form the composition to be administered. The kit can contain instructions for storing, preparing and administering the composition (e.g., a solution to be injected intravenously or subcutaneously).Methods for Making Immune Binding Proteins

[0208] Suitable host cells for making immune binding proteins recombinantly (e.g., anti-PAL antibodies) include higher eukaryote cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV 1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture. Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather. Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138. ATCC CCL 75): human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562. ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells: and a human hepatoma line (Hep G2).

[0209] The host cells used to produce the immune binding proteins described herein (e.g., anti-PAL antibodies) may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPML1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM). Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Pat. No. Re. 30,985, each of which is incorporated by reference in its entirety for all purposes, may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would beAttorney File Reference: MBI.0009 known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[0210] Anti-PAL antibodies made recombinantly as described above, will typically have altered glycosylation from naturally occurring human antibodies. For example, if the anti-PAL antibodies are made in CHO cells, the glycosylation on the full-length antibodies will be non-human, and can change biochemical properties of the antibodies and change the half-life of the antibodies.

[0211] The nucleic acids encoding the anti-PAL antibodies and used to recombinantly produce the anti-PAL antibodies can be codon optimized for the host cell used to recombinantly make the antibodies. The codon optimization can be done on at least one or more codons of the nucleic acid to make a non-naturally occurring nucleic acid. Methods for codon optimizing nucleic acids for recombinant production are well known in the art.Combination Therapies

[0212] As noted above, the methods herein, include administering to the subject one or more additional therapeutic agents in combination with an antibody or other immune binding proteins obtained (e.g., anti-PAL antibody). As used herein, the expression “in combination with” means that the additional therapeutic agents is / are administered before, after, or concurrent with the pharmaceutical composition comprising the immune binding proteins described herein (e.g., anti- PAL antibody). The term “in combination with” also includes sequential or concomitant administration of the immune binding protein described herein and a second therapeutic agent (which could be a different immune binding protein described herein).

[0213] Combination therapies may include an anti-PAL antibody and any additional therapeutic agent that may be advantageously combined with an antibody described herein, or with a biologically active fragment of an antibody described herein. For example, a second or third therapeutic agent may be employed to aid in treatment of NETosis-associate diseases or conditions, for example, anti -inflammation drugs, immune-modulators, analgesics, NSAIDs, anti-cancer drugs, anti-memetics, etc.

[0214] Combination therapies can combine the anti-PAL antibody with an anti-inflammatory including, for example, aspirin, bromfenac, celecoxib, corticosteroids (e.g., betamethasone, cortisone, cyclophosphamide, dexamethasone, ethamethasoneb, etodolac, fenoprofen, flurbiprofen, fludrocortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone), diclofenac, diflunisal, etodolac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, magnesium salicylate, meclofenamate, mefenamic acid, meloxicam, misoprostol, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tolmetin, voltaren, etc.Attorney File Reference: MBI.0009

[0215] The anti-PAL antibodies can be used in combination with lupus treatments such as, for example, anifrolumab-fnia, azathioprine, belimumab, corticosteroids (e.g., betamethasone, cortisone, cyclophosphamide, dexamethasone, ethamethasoneb, etodolac, fenoprofen, flurbiprofen, fludrocortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone), hydroxychloroquine, methotrexate, mycophenolate mofetil, NSAIDS (e.g., those recited above), rituximab, voclosporin, etc.

[0216] The anti-PAL antibodies can be used in combination with drugs for treating irritable bowel disease (e.g., ulcerative colitis, Crohn’s disease) such as, for example, adalimumab, 5- aminosalicylic acid, antibiotics, corticosteroids (e.g., betamethasone, cortisone, cyclophosphamide, dexamethasone, ethamethasoneb, etodolac, fenoprofen, flurbiprofen, fludrocortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone), dicyclomine, etrasimod, laxatives, probiotics, plecanatide, rifaximin, vedolizumab, etc.

[0217] The anti-PAL antibodies can be used in combination with drugs for treating gout such as, for example, NSAIDs such as bromfenac, celecoxib, diclofenac, diflunisal, etodolac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, magnesium salicylate, meclofenamate, mefenamic acid, meloxicam, misoprostol, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tolmetin, voltaren, etc.; corticosteroids (e.g., betamethasone, cortisone, cyclophosphamide, dexamethasone, ethamethasoneb, etodolac, fenoprofen, flurbiprofen, fludrocortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone); allopurinol, analgesics, colchicine, febuxostat, probenecid, etc.

[0218] The anti-PAL antibodies can be used in combination with drugs or treatment for treating small vessel vasculitis such as, for example, avacopan, azathioprine, corticosteroids (e.g., betamethasone, cortisone, cyclophosphamide, dexamethasone, ethamethasoneb, etodolac, fenoprofen, flurbiprofen, fludrocortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone), cyclophosphamide, leflunomide, methotrexate, mycophenolate mofetil, rituximab, tocilizumab, TNF inhibitors (e.g., infliximab, adalimumab, certilizomab or golimumab), plasmapheresis, surgery, etc.

[0219] The anti-PAL antibodies can be used in combination with drugs or treatment for acute lung injury such as, for example, acyclovir, beta-agonists, corticosteroids (e.g., betamethasone, cortisone, cyclophosphamide, dexamethasone, ethamethasoneb, etodolac, fenoprofen, flurbiprofen, fludrocortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone), dazoxiben, granulocyte-macrophage colony-stimulating factor (GM-CSF), inhaled pulmonary vasodilators (e.g., nitric oxide, prostacyclins), interferon B-la, ketoconazole, IL- 10, indomethacin, lisofylline, mesenchymal stromal cells, N-acetylcysteine and procysteine, neuromuscular blockadeAttorney File Reference: MBI.0009(e.g., positive end-expiratory pressure, pancuronium, vecuronium, atracurium, cisatracurium), neutrophil elastase inhibitor, pentoxifylline, prostaglandin El, statins, surfactant, etc.

[0220] Combination therapies can combine the anti-PAL antibody with an anticancer drug or treatment. The anticancer drug or treatment can include, for example, a chemotherapeutic, an antibody, an antibody-drug conjugate, a radiotherapy, an alkylating agent, a plant alkaloid, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, and / or an anti -neoplastic.

[0221] Antibodies and antibody-drug conjugates (ADC) can bind to a tumor associated antigen, including, for example, any of the tumor associate antigens described herein as targets for a CAR. The drug component of the ADC can be, for example, a chemotherapeutic, a radionucleotide, an alkylating agent, a plant alkaloid, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, an anti -neoplastic, and / or an immunotherapy. The drug component of the ADC can be attached to the antibody through a linker which can be cleavable or non-cleavable in nature.

[0222] Alkylating agents can include, for example, mustard gas derivatives (e.g., mechlorethamine, cyclophosphamide, chlorambucil, melphalan, or ifosfamide), ethylenimines (e.g., thiotepa or hexamethylmelamine), alkylsulfonates (e.g., busulfan), hydrazines and triazines (e.g., altretamine, procarbazine, dacarbazine, or temozolomide), nitrosoureas (e.g., carmustine, lomustine or streptozocin), and metal salts (e.g., carboplatin, cisplatin, or oxaliplatin). Plant alkaloids can include, for example, Vinca alkaloids (e.g., vincristine, vinblastine, or vinorelbine), taxanes (e.g., paclitaxel or docetaxel), podophyllotoxins (e.g., etoposide or tenisopide), and camptothecan analogs (e.g., irinotecan or topotecan). Antitumor antibiotics can include, for example, anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, mixoantrone, or idarubicin), and chromomycins (e.g., dactinomycin or plicamycin). Antimetabolites can include, for example, folic acid antagonists (e.g., methotrexate), pyrimidine antagonists (e.g., 5-flurouracil, foxuridine, cytarabine, capecitabine, or gemcitabine), purine antagonists (e.g., 6-mercaptopurine or 6- thioguanine), and adenosine deaminase inhibitors (e.g., cladribine, fludarabine, nelarabine, or pentostatin). Topoisomerase inhibitors can include, for example, topoisomerase I inhibitors (e.g., irinotecan or topotecan) and topoisomerase II inhibitors (e.g., amsacrine, etoposide, etoposide phosphate, or teniposide). Anti-neoplastics can include, for example, ribonucleotide reductase inhibitors (e.g., hydroxyurea), adrenocortical steroid inhibitors (e.g., mitotane), enzymes (e.g., asparaginase or pegaspargase), antimicrotubule agents (e.g., estramustine), and retinoids (e.g., bexarotene, isotretinoin, or tretinoin).

[0223] The drug component can also be an anthracycline, a camptothecin, a tubulin inhibitor, a maytansinoid, a calicheamycin, a pyrrolobenzodiazepine dimer (PBD), an auristatin, a nitrogen mustard, an ethylenimine derivative, an alkyl sulfonate, a nitrosourea, a triazene, a folic acidAttorney File Reference: MBI.0009 analog, a taxane, a COX-2 inhibitor, a pyrimidine analog, a purine analog, an antibiotic, an enzyme inhibitor, an epipodophyllotoxin, a platinum coordination complex, a vinca alkaloid, a substituted urea, a methyl hydrazine derivative, an adrenocortical suppressant, a hormone antagonist, an antimetabolite, an alkylating agent, an antimitotic, an anti -angiogenic agent, a tyrosine kinase inhibitor, an mTOR inhibitor, a heat shock protein (HSP90) inhibitor, a proteosome inhibitor, an HD AC inhibitor, a pro-apoptotic agent, and a combination thereof.

[0224] Specific drugs of use may be selected from the group consisting of 5-fluorouracil, afatinib, aplidin, azaribine, anastrozole, anthracy clines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxy camptothecin, carmustine, celecoxib, chlorambucil, cisplatinum, COX-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin, DM1, DM3, DM4, doxorubicin, 2-pyrrolinodoxorubicine (2-PDox), a pro-drug form of 2-PDox (pro-2-PDox), cyano-morpholino doxorubicin, doxorubicin glucuronide, endostatin, epirubicin glucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptor binding agents, etoposide (VP 16), etoposide glucuronide, etoposide phosphate, exemestane, fmgolimod, floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, flavopiridol, fostamatinib, ganetespib, GDC- 0834, GS-1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, monomethylauristatin F (MMAF), monomethylauristatin D (MMAD), monomethylauristatin E (MMAE), navelbine, neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine, SN- 38, sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide, transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine, vincristine, vinca alkaloids and ZD1839. Preferably, the drug is SN-38.

[0225] In an aspect the combination therapy is a protein conjugate. The protein conjugate can carry a payload that can be a therapeutic, diagnostic, or a reporter. A single molecule of the therapeutic, diagnostic or reporter may be present or two or more molecules may be present. The therapeutic can be a chemotherapeutic including, for example, any of those described herein such as a radionucleotide, an alkylating agent, a plant alkaloid, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, and / or an anti -neoplastic. The payload of the conjugate can be any one or more of these therapeutics, diagnostics and / or reporters. The protein can be aAttorney File Reference: MBI.0009 fragment, a monomer, a dimer, or a multimeric protein. The protein can be an antibody, an antibody fragment or derivative, a single chain antibody, an enzyme, cytokine, chemokine, receptor, blood factor, peptide hormone, toxin, and / or transcription factor.

[0226] Many conjugating reagents can be used to conjugate a payload to a protein. Such reagents may contain at least one functional group capable of reacting with a protein or peptide. For example, the conjugating reagent may comprise a functional group capable of reacting with at least one electrophile or, especially, nucleophile, present in the protein, the functional group being attached to the payload via the linker. Any type of known conjugation reaction may be used to form the conjugate. For example, the reaction can be carried out using the known methods of thiol bonding, amine conjugation, or click chemistry. The reagent may contain a maleimide group, an N-hydroxysuccinimide group, a click-chemistry group, for example an azide or alkyne group, an amine group, a carboxyl group, a carbonyl group, or an active ester group. Other possible approaches include the use of proteins that have been recombinantly engineered with an amino acid specifically for conjugation such as engineered cysteines or non-natural amino acids, and enzymatic conjugation through a specific enzymatic reaction such as with transglutaminase. The reaction site on the protein may be either nucleophilic or electrophilic in nature. Common protein conjugation sites are at lysine or cysteine amino acid residues or carbohydrate moieties. Alternatively, conjugation may occur at a polyhistidine tag which has been attached to a binding protein.

[0227] A conjugating reagent can be advantageously capable of reacting with a nucleophile in a protein and hence becoming chemically bonded thereto. In these examples, the conjugating reagent typically includes at least one leaving group which is lost on reaction with a nucleophile. The conjugating reagent may, for example, include two or more leaving groups. The conjugating reagent can be capable of reacting with two nucleophiles. The conjugating reagent can comprise at least two leaving groups. When two or more leaving groups are present, these may be the same or different. Alternatively, a conjugating reagent may contain a single group which is chemically equivalent to two leaving groups and which single group is capable of reacting with two nucleophiles. Nucleophilic groups include, for example, sulfur atoms and amine groups, and nucleophilic groups in proteins are for example provided by cysteine, lysine or histidine residues. Nucleophilic groups can be a sulfur atom present in a cysteine residue of a protein. Such structures may be obtained by reduction of a disulfide bond in the protein. The nucleophilic group may be an imidazole group in a histidine residue of the protein, e.g., as present in a polyhistidine tag.Attorney File Reference: MBI.0009

[0228] The conjugates can contain a linker which connects the therapeutic, diagnostic or labelling agent to the protein or peptide in the conjugate. The backbone of the linker can be a continuous chain of atoms which runs from the therapeutic, diagnostic or labelling agent at one end to the protein or peptide at the other end. The linker may contain a degradable group, i.e. it may contain a group which breaks under physiological conditions, separating the payload from the protein to which it is, or will be, bonded. Alternatively, the linker is not cleavable under physiological conditions. Where a linker breaks under physiological conditions, it is preferably cleavable under intracellular conditions. Where the target is intracellular, preferably the linker is substantially insensitive to extracellular conditions (i.e. so that delivery to the intracellular target of a sufficient dose of the therapeutic agent is not prohibited).

[0229] Where the linker contains a degradable group, this is generally sensitive to hydrolytic conditions, for example it may be a group which degrades at certain pH values (e.g. acidic conditions). Hydrolytic / acidic conditions may for example be found in endosomes or lysosomes. Examples of groups susceptible to hydrolysis under acidic conditions include hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters and ketals. The degradable linker can also be an acid-cleavable linker or a reducible linker. The reducible linker may comprise a disulfide group. The linker may also contain a group which is susceptible to enzymatic degradation, for example it may be susceptible to cleavage by a protease (e.g. a lysosomal or endosomal protease) or peptidase. For example, it may contain a peptidyl group comprising at least one, for example at least two, or at least three amino acid residues (e.g. Phe- Leu, Gly-Phe-Leu-Gly, Vai-Ala, Val-Cit, Phe-Lys, Glu-Glu-Glu). For example, it may include an amino acid chain having from 1 to 5, for example 2 to 4, amino acids. The enzyme cleavable linker can also comprise a chemical group which can be cleaved or degraded by one or more lysosomal enzymes. Suitable groups include, for example, a valine-citrulline dipeptide group, a phenylalanine-lysine dipeptide group, and a P-glucuronide group.

[0230] When the protein in the protein conjugate is an antibody (e.g., full length, fragment, and / or single chain) one end of the first linker can be covalently attached to the antibody. The antibody- reactive end of the linker can be a site that is capable of conjugation to the antibody through a cysteine thiol or lysine amine group on the antibody, and so can be a thiol-reactive group such as a double bond (as in maleimide) or a leaving group such as a chloro, bromo, or iodo, or an R- sulfanyl group, or an amine-reactive group such as a carboxyl group.

[0231] The immunotherapy can be any immune cell engineered to treat disease, for example, T- cells engineered with a chimeric antigen receptor, a T-cell receptor, and / or a payload, natural killer cells engineered with a receptor and / or a payload, or innate immune cells engineered toAttorney File Reference: MBI.0009 increase or decrease activity. The engineered receptors can bind to tumor associated antigens which are well-known in the art. The engineered receptors can bind a variety of ligands including those described in U.S. Pat. Nos. 5,359,046, 5,686,281 and 6,103,521, each of which is incorporated by reference in its entirety for all purposes.Antibody Conjugates

[0232] The anti-PAL antibodies can also be used to make antibody conjugates with other compounds. Other compounds that can be conjugated to the anti-PAL antibodies include, for example, radioisotopes, radionucleotides, toxins, therapeutic groups, chemotherapeutic groups, and / or drugs. Drugs attached to the anti-PAL antibodies can make antibody drug conjugates (ADC). The drug of the ADC can include, for example, an anti-inflammatory drug, a pro- inflammatory drug, a chemotherapeutic, a radionucleotide, an alkylating agent, a plant alkaloid, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, and / or an anti -neoplastic. The drug component of the ADC can be attached to the antibody through a linker which can be cleavable or non-cleavable in nature.

[0233] The anti-PAL antibody conjugate can carry a payload that can be a therapeutic, a diagnostic, or a reporter. A single molecule of the therapeutic, diagnostic or reporter may be present or two or more molecules may be present conjugated to the anti-PAL antibody. The therapeutic can be a chemotherapeutic including, for example, any of those described herein such as a radionucleotide, an alkylating agent, a plant alkaloid, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, and / or an anti -neoplastic. The payload of the conjugate can be any one or more of these therapeutics, diagnostics and / or reporters. The payload can also be an enzyme, cytokine, chemokine, receptor, blood factor, peptide hormone, toxin, and / or transcription factor. Reporters that can be conjugated to an antibody include, for example, a chromophore, a fluorophore, a fluorescent protein, enzymes, fluorescent proteins, bioluminescent proteins, receptors, antigenic epitopes, transporters, a hapten, an isotope, and / or a radioisotope. In general, a reporter is a moiety or makes a moiety capable of being detected indirectly or directly.

[0234] Many conjugating reagents can be used to conjugate a payload to the anti-PAL antibody. Such reagents may contain at least one functional group capable of reacting with a protein or peptide. For example, the conjugating reagent may comprise a functional group capable of reacting with at least one electrophile or, especially, nucleophile, present in the protein, the functional group being attached to the payload via the linker. Any type of known conjugation reaction may be used to form the conjugate. For example, the reaction can be carried out using the known methods of thiol bonding, amine conjugation, or click chemistry. The reagent may contain a maleimide group, an N-hydroxysuccinimide group, a click-chemistry group, for example an azide or alkyne group,Attorney File Reference: MBI.0009 an amine group, a carboxyl group, a carbonyl group, or an active ester group. Other possible approaches include the use of ana anti-PAL antibody that has been recombinantly engineered with an amino acid specifically for conjugation such as engineered cysteines or non-natural amino acids, and enzymatic conjugation through a specific enzymatic reaction such as with transglutaminase. The reaction site on the protein may be either nucleophilic or electrophilic in nature. Common protein conjugation sites are at lysine or cysteine amino acid residues or carbohydrate moieties. Alternatively, conjugation may occur at a polyhistidine tag which has been attached to an antibody.

[0235] A conjugating reagent can be advantageously capable of reacting with a nucleophile in an antibody and hence becoming chemically bonded thereto. In these examples, the conjugating reagent typically includes at least one leaving group which is lost on reaction with a nucleophile. The conjugating reagent may, for example, include two or more leaving groups. The conjugating reagent can be capable of reacting with two nucleophiles. The conjugating reagent can comprise at least two leaving groups. When two or more leaving groups are present, these may be the same or different. Alternatively, a conjugating reagent may contain a single group which is chemically equivalent to two leaving groups and which single group is capable of reacting with two nucleophiles. Nucleophilic groups include, for example, sulfur atoms and amine groups, and nucleophilic groups in proteins are for example provided by cysteine, lysine or histidine residues. Nucleophilic groups can be a sulfur atom present in a cysteine residue of a protein. Such structures may be obtained by reduction of a disulfide bond in the protein. The nucleophilic group may be an imidazole group in a histidine residue of the protein, e.g., as present in a polyhistidine tag.

[0236] The conjugates can contain a linker which connects the therapeutic, diagnostic or labelling agent to the antibody in the conjugate. The backbone of the linker can be a continuous chain of atoms which runs from the therapeutic, diagnostic or labelling agent at one end to the antibody at the other end. The linker may contain a degradable group, i.e. it may contain a group which breaks under physiological conditions, separating the payload from the antibody to which it is, or will be, bonded. Alternatively, the linker is not cleavable under physiological conditions. Where a linker breaks under physiological conditions, it is preferably cleavable under intracellular conditions. Where the target is intracellular, preferably the linker is substantially insensitive to extracellular conditions (i.e. so that delivery to the intracellular target of a sufficient dose of the therapeutic agent is not prohibited).

[0237] Where the linker contains a degradable group, this is generally sensitive to hydrolytic conditions, for example it may be a group which degrades at certain pH values (e.g. acidic conditions). Hydrolytic / acidic conditions may for example be found in endosomes or lysosomes. Examples of groups susceptible to hydrolysis under acidic conditions include hydrazones,Attorney File Reference: MBI.0009 semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters and ketals. The degradable linker can also be an acid-cleavable linker or a reducible linker. The reducible linker may comprise a disulfide group. The linker may also contain a group which is susceptible to enzymatic degradation, for example it may be susceptible to cleavage by a protease (e.g. a lysosomal or endosomal protease) or peptidase. For example, it may contain a peptidyl group comprising at least one, for example at least two, or at least three amino acid residues (e.g. Phe-Leu, Gly-Phe-Leu-Gly, Vai-Ala, Val-Cit, Phe-Lys, Glu-Glu-Glu). For example, it may include an amino acid chain having from 1 to 5, for example 2 to 4, amino acids. The enzyme cleavable linker can also comprise a chemical group which can be cleaved or degraded by one or more lysosomal enzymes. Suitable groups include, for example, a valine-citrulline dipeptide group, a phenylalanine-lysine dipeptide group, and a P-glucuronide group.

[0238] The antibody -reactive end of the linker can be a site that is capable of conjugation to the antibody through a cysteine thiol or lysine amine group on the antibody, and so can be a thiolreactive group such as a double bond (as in maleimide) or a leaving group such as a chloro, bromo, or iodo, or an R-sulfanyl group, or an amine-reactive group such as a carboxyl group.NETosis and Other Applications

[0239] Uncontrolled NET formation (NETosis) can result in numerous diseases that adversely affect health. In the recent past, much attention has been directed towards neutrophil extracellular traps (NETs) formation for their relevance in mediating tissue injury, cancer progression, and inflammatory and autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis, acute lung injury, thrombosis formation, gout, cancer metastases, atherosclerosis, COVID19, Acute Respiratory Distress Syndrome, Acute renal injury, small-vessel vasculitis, and inflammatory bowel disease. Histone modifications in NETs can act as a double-edged sword, as they are capable of altering multiple types of neutrophil death, and influencing numerous NET- mediated diseases, such as acute lung injury (ALI), thrombosis, sepsis, systemic lupus erythematosus, and cancer progression. Excessive release or dysfunction of NETs can trigger and amplify inflammatory responses, which can cause tissue damage and a variety of diseases. The components of NETs might become autoantigens, causing inflammation and autoimmune diseases. B lymphocytes, for example, may develop autoantibodies against NET-derived cell-free DNA (cfDNA) in individuals, e.g., individuals with systemic lupus erythematosus (SLE).

[0240] SLE is an autoimmune disease which is characterized by chronic inflammation as a result of the immune system attacking the body’s own tissues and organs. In fact, serum in normal conditions can degrade NETs; however, lupus patients possess autoantibodies that target DNA and histones, and this results in protecting NETs from being degraded by serum nucleases, which resultsAttorney File Reference: MBI.0009 in lupus nephritis. The NET-DNA and -histone protection can be achieved by the complement deposition (e.g., Clq) and activation, which results in the suppression of DNase activity. This results in increased exacerbations in SLE, ranging from rashes to seizures and psychosis. The antiPAL antibody compositions described herein can be administered to lupus patients to inhibit NETosis, thereby reducing or eliminating the symptoms in the patient. Patients with SLE have autoantibodies against histones and cfDNA, which protects NETs from degradation. Furthermore, insufficient clearance of dead cells caused by NETosis may result in increased production of autoantibodies. The presence of DNasel inhibitors / antibodies or the arrest of the link between DNase 1 and NETs by complement component Clq or antibodies of NETs may explain the inefficient degradation of NETs.

[0241] The common feature that exists between different autoimmune diseases is the inflammatory response to autoantigens, such as in patients with lupus, where high levels of antinuclear and anti- DNA antibodies are present, or patients with rheumatoid arthritis where anti-neutrophil cytoplasmic antibodies and plasma autoantibodies against guanosine peptides and histones are prevalent. NETosis participates in progression of rheumatoid arthritis (RA). The synovial fluid of RA patients contains neutrophils, which can generate NETs. The debris of NETs, including elastase, citrulline histone H3, and MPO, have been discovered in the synovial fluid and serum of RA patients. Peptidylarginine deaminases (PAD) play a significant role in the production of NETs. Two PAD enzymes, PAD2 and PAD4, are required for the citrullination of several proteins in NET s, including actin, histone H3, a-enolase, and vimentin, in RA. In this way, B cells can produce anti-citrullinated protein antibodies (ACPAs) with the support of T cells. ACPAs are found in more than two-thirds of the sera of RA patients and are more specific than rheumatoid factors. Moreover, anticyclic citrullinated peptide antibody has emerged as a key biomarker for RA. These citrullinated autoantigens are potent inducers of proinflammatory cytokine release and NET formation. Research evidence shows that specific cytokines such as tumor necrosis factor-alpha (TNF-a), IL- 17A, and IL-8 can induce NETosis in RA neutrophils. NETosis may be a source of autoantigens, and the ensuing ACPAs can cause the development of NETs and a subsequent inflammatory response. The anti-PAL antibody compositions described herein can inhibit NETosis, thereby inhibiting or preventing rheumatoid arthritis.

[0242] NETosis discharges endogenous cellular components that can act as autoantigens, induce host immune responses, and discharge host-associated molecular patterns that augment inflammatory responses, which, in turn, can induce NETosis, leading to the perpetuation of pathological mechanisms. For example, Immune complexes, such as DNA-bacterial peptides released by NETs, are present in the serum of lupus patients and can trigger plasmacytoid dendriticAttorney File Reference: MBI.0009 cells to create interferon through toll-like receptors 9, and interferon-a can induce NETosis. Additionally, a subpopulation of neutrophils, low-density neutrophils, has been identified in lupus patients, and low-density neutrophils are more likely to induce NETosis. In patients with gout, NETs can degrade cytokines and chemokines to attenuate the inflammatory response. The antiPAL antibody compositions described herein can inhibit NETosis, thereby reducing the amount of auto-antigens in the patient, and inhibiting or preventing the disorders and / or diseases.

[0243] Neutrophils are key players to ALI (acute lung injury), which is characterized by an increased microvascular permeability due to the disruption of alveolar-capillary morphology. ALI can be associated with increased CitH3 tissue levels in vivo, and DNase treatment significantly reduces CitH3 levels and degrades NETs. As ALI progresses, lung tissues become damaged and scarred in a process termed “pulmonary fibrosis”. Emerging evidence shows that an HDAC inhibitors (e.g., Trichostatin A, TSA) increases AcH4 and partially attenuates lung fibrosis. The anti-PAL antibodies disclosed herein can reduce NETosis in ALI patients and thereby reduce or eliminate the symptoms from ALI.

[0244] NETosis and thrombosis activated platelets induce NETosis, and NETs act as platelet scaffolds and activate FXII, which binds fibrin to red blood cells to form a dense network, while the NE of NETs and histone G degrade coagulation inhibitors. Thrombosis is also a mechanism by which NETosis accelerates the progression of atherosclerosis, cancer, and COVID-19. NETosis induces thrombosis in the microvasculature, and can damage endothelial cells, causing endothelial dysfunction and attracting monocytes, which can differentiate into macrophages and form foam cells after phagocytosis of large amounts of lipoproteins and cholesterol, gradually forming atherosclerotic plaques. Advanced plaques exposed to neutrophil-derived proteases and ROS lead to plaque instability. Coronary thrombus can consist of activated platelets, neutrophils, and NETs near the platelets. Activated platelets deliver high mobility group box 1 protein to neutrophils, allowing them to participate in autophagy and NET formation. The interaction of thrombin- activated platelets and polymorphonuclear leukocytes at the site of plaque rupture, can lead to the formation of local NETs and the delivery of active tissue factors in acute ST-segment elevation myocardial infarction. The anti-PAL antibodies disclosed herein can reduce NETosis and thereby reduce or eliminate thrombosis formation in the patient.

[0245] Cancer cells can release chemokines to attract neutrophils and induce NETosis. NETs can have a role in tumor recurrence and dissemination. Matrix metallopeptidase 9 and NE in NETs break down extracellular matrix laminin, this triggers the integrin 31 / FAK / ERK / MLCK / YAP signaling cascade, which wakes up dormant cancer cells and stimulates the growth and survival of cancer cells. The DNA of NETs interacts with the transmembrane protein coiled-coil domain-Attorney File Reference: MBI.0009 containing protein 25, and coiled-coil domain-containing protein 25 stimulates the ILK - Parvin pathway, attracting cancer cells to distant metastases. NETs are capable of capturing circulating tumor cells to facilitate cancer dissemination; the mechanism could be related to NET-derived IL- 8 activating the nuclear factor-KB pathway, NETs can also accelerate the conversion of cancer cells epithelial morphology to a mesenchymal phenotype, which confers mobility and invasion. Additionally, NETs not only provide a physical barrier that prevents pathogen transmission but also contribute to immune evasion by preventing cytotoxic cells from entering growing tumors. The anti-PAL antibodies disclosed herein can reduce NETosis in cancer patients and thereby reduce or eliminate these effects.

[0246] NETosis can induce cancer progression and metastasis. Circulating levels of NET components, including cfDNA, NE-DNA, MPO-DNA, and CitH3, are useful biomarkers for several cancers. NET formation can be caused by the hypoxic environment generated by solid tumors with high levels of hypoxia-inducible factor-la. Tumor-derived exosomes from cancer patients in prethrombotic stages can cause NETosis. Tumor cells can also directly induce NETosis by secreting granulocyte-colony stimulating factor. Neutrophil elastase (NE) which is released during NETosis, can promote cancer progression. Matrix metallopeptidase 9 (MMP9), a NETosis product, can promote cancer progression by increasing angiogenesis via the production of vascular endothelial growth factor. Moreover, NE and MMP9 can cleave laminin, which is a significant component of the extracellular matrix (ECM), and ECM degradation to the damaged basement membrane is a prerequisite for tumor cell invasion and metastasis. NETs can induce the production of HMGB1, which activates tumor cells through the TLR9 pathway. C5a can trigger polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) to release NETs, hence aiding cancer cell migration and metastasis. The anti-PAL antibodies disclosed herein can reduce NETosis in cancer patients and thereby reduce or eliminate cancer metastases.

[0247] Platelets produced by NETosis can protect circulating tumor cells (CTCs) from immune cells due to the trapping properties of NETs. Experimental evidence shows that NETs produced by surgical stress-activated platelets may enhance CTC trapping and distant metastasis. Moreover, by depleting or blocking platelets, metastasis can be minimized or stopped. Moreover, NETs can increase tumor cell extravasation by entrapping or attaching them to capillaries. Administering anti-PAL antibodies can disrupt the interaction between platelets, tumor cells, and NETs preventing metastasis.

[0248] Endothelial cells have a limited capacity to internalize NETs, and the persistent presence of NETs can alter endothelial cell-cell contacts through the proteolysis of vascular endothelial- cadherin by neutrophil elastase. This results in increased vascular leakage and transendothelialAttorney File Reference: MBI.0009 albumin passage. In addition, NET-associated elastase can induce a nuclear translocation of junctional P-catenin and promote endothelial-to-mesenchymal transition. NETs can trap circulating lung carcinoma cells and induce the formation of hepatic micro-metastases and gross metastatic disease. The anti-PAL antibodies disclosed herein can reduce NETosis in cancer patients and thereby reduce or eliminate these mechanisms.

[0249] Small-vessel vasculitis (SVV) is an autoimmune disease characterized by the appearance of anti-neutrophil cytoplasmic antibodies (ANCAs) against MPO and PR3. Increased levels of NETs can be found in the blood of SVV patients. Immunofluorescence can reveal NETs in the necrotizing lesions of SVV. Furthermore, IgG antibodies in the sera of SVV patients were more likely to stimulate NET production in vitro than those in the control group. The activity of DNAsel can be decreased, resulting in NET accumulation in SVV patients. In addition, NETs can activate the alternative complement pathway participating in the pathogenesis of ANCA-associated vasculitis (AAV). In addition, a-PR3 and a-MPO ANCAs can trigger NETosis, resulting in a vicious circle. The dysregulated release of NETs may induce endothelial cell damage because of their cytotoxic effect. The anti-PAL antibodies disclosed herein can reduce NETosis in cancer patients and thereby reduce or eliminate the symptoms from SVV.

[0250] Inflammatory bowel diseases (IBDs), which include ulcerative colitis (UC) and Crohn's disease (CD), are a category of gastrointestinal diseases characterized by chronic inflammation. Clinical evidence shows that IBD symptoms include severe diarrhea, fluid loss, abdominal pain, and bleeding. Neutrophils can play a significant role in IBD pathogenesis, and neutrophil condensation in the intestinal mucosa is positively correlated with the severity of UC and CD. Antineutrophil cytoplasmic antibodies (ANCAs) are a key biomarker for IBD, and ANCAs can target neutrophil proteins produced during NETosis.

[0251] Proteins from NETs can cause tissue damage in IBD patients. These proteins are linked to IBD pathology and produce inflammatory responses, extracellular matrix (ECM) degradation, and other severe outcomes. NETs may induce macrophages to release proinflammatory cytokines such as TNF-a, IL-6, and monocyte chemotactic protein- 1, which leads to platelet activation and intestinal damage. PAD, a major NETosis product, plays a role in the pathogenesis of IBD by increasing proinflammatory cytokine levels in conjunction with MPO and decreasing the antiinflammatory cytokine IL-10. The anti-PAL antibodies disclosed herein can reduce NETosis and thereby reduce or eliminate the symptoms from IBD, UC and / or CD.

[0252] Gout is a common type of arthritis characterized by the precipitation of monosodium urate (MSU) crystals in the peripheral joints. NETs can be found in the fluid from acutely inflamed joints in gout patients. In addition, the uninflamed tophi were coated with NETs in patients with gout.Attorney File Reference: MBI.0009Moreover, a recent study indicated that a decrease in GPR105, which is highly expressed in neutrophils and sensitive to MSU, can prevent NETosis and induce apoptosis. Therefore, targeting GPR105 might be a possible therapy for acute gouty arthritis. The anti-PAL antibodies disclosed herein can reduce NETosis in gout patients and thereby reduce or eliminate these symptoms from gout.

[0253] NETosis is proinflammatory and can increase inflammation. Excessive Neutrophil Extracellular Traps (NETs) formation can damage the microcirculation, promote immune- thrombosis and lead to diffuse intravascular coagulation as they facilitate the formation of thrombus as a scaffold for thrombosis. The anti-PAL antibody compositions described herein can inhibit NETosis, thereby reducing the damage from NETs.

[0254] Depending on whether the neutrophil plasma membrane is ruptured and whether it continues to perform phagocytosis, NETosis can be classified into suicidal NETosis (neutrophil dies after expelling the nucleic acid filaments) and vital NETosis (NET expulsion does not disrupt the neutrophil membrane and cells can still phagocytose microbes). Suicidal NETosis is a programmed cell death with neutrophils releasing NETs when the cytoplasmic membrane ruptures, while vital NETosis extrudes NETs in a vesicular outgrowth manner, in which the granulocyte surface membrane is intact, still contains granules, and is phagocytic. Vital NETosis takes approximately 30 min, while suicidal NETosis lasts 3-4 h. NETosis can be induced by bacteria, bacterial toxins, fungi, parasites, phorbol myristate acetate (PMA), lipopolysaccharide, immune complexes, activated platelets, cholesterol crystals, antibodies, interleukin- ip, tumor necrosis factor-a and so on. Distinct stimuli can induce different types of NETosis; for example, PMA often induces suicidal NETosis, while microbes and activated platelets often induce vital NETosis.

[0255] The anti-inflammatory effect of the anti-PAL antibodies is also seen in the anti-NETosis activity of these antibodies using two independent markers, elastase and DNA. Immunofluorescence staining shows binding by the anti-PAL antibodies to neutrophil granules.

[0256] Other indications or uses of the antibodies disclosed here in include, for example, Acute Respiratory Distress Syndrome (ARDS), and certain cancers (e.g., cervical cancer, ovarian cancer, and melanoma). ARDS can be caused by or associated with Sepsis, severe pneumonia, viral infections of the lungs (e.g., SARS-CoV-2), and other pathologies. A mechanical cause of ARDS can be from fluid leaked from the smallest blood vessels in the lungs into the tiny air sacs where blood is oxygenated. Normally, a protective membrane can keep this fluid in the vessels. Severe illness or injury, however, can cause damage to the membrane, leading to the fluid leakage of ARDS. Anti-PAL antibodies can be administered to patients to reduce the severity of ARDs. Anti-Attorney File Reference: MBI.0009PAL antibodies increase survival in a CLP model by changing cytokine levels towards an antiinflammatory state.

[0257] Certain IgMs, natural and adaptive, have been isolated from the tumors of patients with cancer; this IgM can eliminate tumors through various mechanisms, such as apoptosis and complement. Natural IgM can have a direct cytotoxic effect on tumor cells, it can recognize tumor- modified cell surfaces (e.g., tumor associated antigens) that develop during tumorigenesis, and it activates complement to destroy nascent transformed cells. VH4.34 can be administered to cancer patients to regress the cancer, or aid in the regression of the cancer.

[0258] The anti-PAL antibody described herein can be used in therapies for cancer, allergies, and autoimmune diseases. The therapy can be administer to males and / or females. The immune binding proteins described herein can be used in therapies to treat subjects with NETOsis-associated diseases or disorders. The anti-PAL antibody described herein can be used prophylactically to provide protection to those individuals who are particularly susceptible to a NETosis-associated disease or particularly susceptible to bad outcomes from such a disease. The anti-PAL antibody described herein can also be used in diagnostic applications. The immune binding proteins described herein can provide information on a subject’s immune state and / or response to a therapy. The immune binding proteins described herein can provide information on a subject’s response to an antibody therapy, small molecule drug therapy, biologic therapy, or cellular immunotherapy.

[0259] Biomarkers that can be used to diagnose NETosis include, for example, one or more of the following: cell-free DNA (cfDNA), nucleosomes, citrullinated histone 3 (citH3), calprotectin, myeloperoxidase (MPO), neutrophil elastase, immune modulators GAS6 and AXL, Serum MPO- DNA complex, and nucleosome levels. See for example, Huckriede et al., Evoluation of NETosis markers and DAMPs have prognostic value in critically ill COVID-19 patients, 2021, Scientific Reports vol. 11, article 15701, Tomas-Perez et al, Increased levels of NETosis biomarkers in high grade serous ovarian cancer patients’ biofluids: potential role in disease diagnosis and management, 2023, Front. Immunol. 14: 1111344, Reshetnyak et al, Markers of NETosis in patients with systemic lupus erythematosus and antiphospholipid syndrome, 2023, Int J Mol Sci 24:9210, each of which is incorporated by reference in its entirety for all purposes.

[0260] Higher levels of NETs have been found in the tracheal aspirate and lung tissues of patients with severe COVID-19, and NET components may elicit an inflammatory response and vascular microthrombosis, resulting in ARDS in COVID-19 patients. Clinical studies have shown that individuals with COVID-19 have increased amounts of cfDNA, MPO-DNA complexes, and CitH3, all of which are important components of NETs. In vitro, serum samples from CO VID-19 patients may stimulate NET formation in control neutrophils. Moreover, cfDNA is linked to acute-phaseAttorney File Reference: MBI.0009 reactants and lactate dehydrogenase, as well as neutrophil count, while CitH3 is linked to platelet levels, supporting a function for NETosis in thrombosis. Notably, SARS-CoV-2 can directly cause spontaneous NET release in vitro. Histones, a major component of NETs, can enhance SARS- CoV-2 infection. Mounting evidence shows that NETosis is associated with thrombosis, which is a significant predictor of disease severity in COVID-19 patients. In COVID-19 patients, researchers discovered inflammatory microvascular thrombosis with NET-associated fibrin and platelets in the heart, lung, and kidney; several neutrophil-platelet aggregates in the patients suffered from COVID-19.

[0261] Acute renal injury (AKI) is a group of clinical syndromes that refers to a sudden and continuous sudden decline in renal function. Research showed that NET release and tubular necrosis caused histone and cytokine release, promoting kidney injury. Henry et al. indicated that intravascular NETosis was related to the pathogenesis of COVID-19-associated AKI and microthrombosis. Administration of anti-PAL antibodies to patients can ameliorate the above symptoms and conditions linked to acute renal injury.

[0262] Studies have suggested that NETosis is associated with pancreatitis. Leppkes et al. found that NETosis promotes pancreatitis by ductal occlusion. Some special components in pancreatic juice, including calcium carbonate crystals and bicarbonate ions, can lead to aggregated NET formation.

[0263] The anti-PAL antibody described herein can be useful for treating subjects with autoimmune diseases or whom have cytokine storm response to pathogen infections. The autoimmune disease can be rheumatoid arthritis, lupus, celiac disease, Sjorgren’s syndrome, polymyalgia rheumatica, multiple sclerosis, ankylosing spondylitis, Type 1 diabetes, and the like. The anti-PAL antibody described herein ameliorate the autoimmune / cytokine storm reaction.

[0264] The treatment of NETosis-associated diseases with the anti-PAL antibodies described herein can be combined with detection of NETosis in patients. Using the above, in combinations or any one of the diagnostics methodologies alone, patients are rapidly diagnosed upon presentation as having a NETosis-associated disease and these patients can treated with the anti-PAL antibodies.

[0265] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.EXAMPLESExample 1. Anti-PAL Antibodies Decrease Release of Nucleic Acids in NETosisAttorney File Reference: MBI.0009

[0266] Neutrophils were purified from human peripheral blood using magnetic sorting using MACSxpress® Whole Blood Neutrophil Isolation Kit from Miltenyi Biotech. Purity of neutrophils was tested using a neutrophil specific marker CD66b (Biolegend) by flow cytometry. Neutrophils were >90% pure.

[0267] Neutrophils underwent NETosis in-vitro by stimulating with phorbol 12-myristate 13- acetate (PMA, 25nM) in the presence of Sytox Green. Sytox, a membrane impermeable DNA binding dye, gives a fluorescent signal when DNA NETs are released during the process of NETosis. Thus, the signal is directly correlated to the amount of extruded DNA. NETosis correlates directly with the amount of PMA used for stimulation. Sytox green signal was measured. The data shows that NETosis, as measured by DNA release, is inhibited by an anti-PAL antibody (MC005). See FIG. 1.Example 2, Binding of anti-PAL Antibody to Permeabilized Neutrophils

[0268] Neutrophils were purified from human peripheral blood using magnetic sorting using MACSxpress® Whole Blood Neutrophil Isolation Kit from Miltenyi Biotech. Purity of neutrophils was tested using a neutrophil specific marker CD66b (Biolegend) by flow cytometry. Neutrophils were >90% pure.

[0269] Neutrophils were fixed and permeabilized (F / P) using a BD Cytofix / Cytoperm™ Fixation / Permeabilization Kit. This process allows antibodies to access proteins inside the neutrophil. Unfixed and fixed / permeabilized neutrophils were then stained with an anti-PAL antibody (MC005) followed by an anti-human IgM-AF-488 secondary antibody. Neutrophils were analyzed by flow cytometry (Millipore, Guava EasyCyte). Mean fluorescence of binding shows that MC005 only binds fixed and permed neutrophils, but not membrane intact neutrophils. Isotype control human IgM does not bind neutrophils. This data shows that the ligand for an anti-PAL antibody (MC005) is present internally in human neutrophils. See FIG. 2.Example 3, Anti-PAL Antibody Binds Internal Antigens in Neutrophils

[0270] Neutrophils were purified, fixed and permeabilized as described Example 1. Cells were stained with anti-PAL antibody (MC005) and anti-hu IgM -AF488 secondary and resuspended in media with propidium iodide (PI, a DNA intercalating agent). Cells were visualized by an ImageXpress Pico (Molecular Devices). Anti-PAL antibody (MC005) binds internal granules in fixed / permeabilized Neutrophils. See FIG. 3.Example 4, Anti-PAL Antibody Inhibits Release of Elastase from Neutrophils

[0271] Neutrophils were purified and stimulated with PMA to induce NETosis and the release of elastase. Different concentrations of anti-PAL antibody (MC005) were added to PMA induced neutrophils. The impact of anti-PAL antibody (MC005) on NETosis was measured using theAttorney File Reference: MBI.0009 functional activity of elastase from the neutrophils. The enzyme activity was measured using a NETosis Assay Kit (Abeam) according to manufacturer’s instructions. Elastase activity was inhibited by anti-PAL antibody (MC005) incubated with pre-activated neutrophils and postactivated neutrophils, showing that anti-PAL antibody (MC005) can be added after stimulation to block NETosis. See FIG. 4 and FIG. 5.Example 5, Anti-PAL Antibody Inhibits Sepsis in Human Patients

[0272] MQ005 (described above) is a human monoclonal antibody of the IgM (p, K) class derived from the VH4- 34 gene and binds a glycan target, straight chain poly-N-acetyl-lactosamine (SC- PNAL) or ‘i-antigen’. In humans, SC-PNAL / i-antigen is expressed strongly on human B- Lymphocytes and human fetal / cord RBCs, but NOT on human adult RBCs. SC-PNAL is also expressed in varying degrees on other immune cell types, such as, activated T-cells, Macrophages and Dendritic cells (ref). It is also detected strongly within neutrophil granules, but not on the neutrophil membrane.

[0273] Pre-clinical animal models show that binding of MQ005 to its ligand on activated immune cells reduces and / or shunts the inflammatory manifestations associated with abnormal immune activation. Changes in innate cell function and decrease in pro-inflammatory cytokines led to significantly increased survival in an animal model of sepsis.

[0274] The trial design follows a standard 3 + 3 dose escalation scheme (Hansen et al., Phase 1 trial design: is 3 + 3 the best?, 2014, Cancer Contr. 21 :200-208, which is incorporated by reference in its entirety for all purposes) where three patients are initially enrolled into a given dose cohort. If there is no dose-limiting toxicity (DLT) seen in any of these participants, the trial proceeds to enroll additional participants into the next higher dose cohort. If one patient manifests a DLT at a specific dose, an additional three individuals are accrued into that same dose cohort. Development of DLTs in two or more out of six patients at a specific dose level indicates that the MTD has been exceeded; further dose escalation is not pursued, and the prior dose level is expanded to six patients; if there is no more than one patient who experiences a DLT among those six patients, that dose level is considered the MTD. The MTD is therefore defined as the highest dose level in which six patients were treated and, at most, one patient experienced a DLT during the first cycle of therapy. If more than six patients are treated at any dose level, the MTD is exceeded if more than one-third of the patients experience a DLT.

[0275] Patients are selected for the trial based on either or: a clinical diagnosis of sepsis plus the utilization of SIRS and SOFA scores to quantitate the degree of sepsis at entry into the trial; patients signs, symptoms and laboratory values evaluated by an Mcure Bio designed Artificial Intelligence Algorithm (Al) to predict clinically significant sepsis. Patients are eligible for inclusion if they areAttorney File Reference: MBI.0009 predicted to have clinically important sepsis which will require supportive therapy by any of these three or a combination of these methods.

[0276] The first 3 eligible patients receive MQ005 at a dose of 1.25 mg / kg followed by a dose in 6 hours of 1.25 mg / kg and then every 12 hrs until sepsis resolves. No more than 3 days of treatment will occur. After 14 days, if no dose limiting toxi cities are reached the next dosing group of 3 patients receiving 2.5 mg / kg in the same regimen. If a DLT is observed in the first 3 patients an additional 3 patients receive the 2.5 mg / kg dose regimen as outlined for the standard 3 + 3 dose escalation as outlined above. The same procedure is then followed for 5.0mg / kg, lOmg / kg, and 20 mg / kg doses.Example 6,

[0277] Neutrophils are isolated from human peripheral blood using Miltenyi kit, catalog # 130- 104-434. The kit isolates untouched neutrophils by magnetic depletion of other cell types on peripheral blood. Isolated neutrophils are plated in 1% bovine serum albumin (BSA) in hanks balanced salt solution (HBSS) with 20 pM DHR 123 (Dihydrorhodamine 123) in 37C CO2 incubator. Cells are then stimulated with 100 pg / mL LPS (lipopolysaccharide from bacteria) and 5 pg / mL FMLP (N-formyl-methionyl-leucyl-phenylalanine, a bacterial tripeptide). Different concentrations of anti-PAL antibody (MC005 or MQ005) are added and cells incubated. The reaction was stopped on ice and cells washed in ice-cold PBS. Cells were fixed and analyzed immediately by flow cytometry.

[0278] FIG. 6 shows a bar graph with the results of the experiment using different amounts of anti- PAL antibody. This shows that Anti-PAL antibody (MC005 or MQ005) reduces generation of ROS in activated neutrophils.Example 7,

[0279] Neutrophils from human whole blood were purified using Miltenyi kit #130-104-434 following manufacturer’s protocol. Cells were checked for purity on flow cytometry using neutrophil specific marker CD66b-AF647. Cells were greater than >93% pure. Neutrophil cells were fixed and permeabilized using a Becton Dickinson(BD) kit# 554722 as per protocol. Cells were then blocked with Fc block reagent (BD #564220). Cells were stained in separate tubes with the following antibodies: Alexa Fluor™ 647 Mouse Anti -Human Neutrophil Elastase (BD #569517) and anti-PAL antibody (MC005); Alexa Fluor™ 647 Mouse Anti -Human Myeloperoxidase (BD # 568914) + anti-PAL antibody (MC005); MMP9 Monoclonal Antibody (L51 / 82), APC (Invitrogen, # MA5-45511) + anti-PAL antibody (MC005); and PE Anti-Lactoferrin antibody [LF5-1D2] (Abeam # ab270649) + anti-PAL antibody (MC005).Attorney File Reference: MBI.0009

[0280] Cells were mounted and images captured at Stanford Imaging facility, Stanford University; on an inverted Zeiss LSM 880 Laser Scanning Confocal Microscope. Images were processed using Zen Blue Carl Zeiss software Desk 3.4. Data is generated to calculate colocalization between two markers using different threshold (fixed and Otsu) potentials and graphs plotted suing Graphpad Prism software.

[0281] This data is shown in FIG. 7 and FIG. 8. Showing that anti-PAL antibody can bind gelatinous granules, secretory granules, and azurophilic granules. FIG. 8 also shows some binding to MPO.

[0282] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0283] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

Attorney File Reference: MBI.0009CLAIMSWe claim:

1. A method for inhibiting NETosis, comprising administering to the subject an agent, wherein said agent is an isolated antigen binding protein which binds to PAL, comprising:(a) a heavy chain comprising a CDRH1 having a sequence shown in SEQ ID NO: 5, a CDRH2 having a sequence shown in SEQ ID NO: 6, and a CDRH3 having a sequence shown in any of SEQ ID NOS: 7, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, or 73; and(b) a light chain comprising (i) a CDRL1 having a sequence shown in SEQ ID NO: 8, a CDRL2 having a sequence shown in SEQ ID NO: 9 and a CDRL3 having a sequence shown in SED ID NO: 10, or (ii) a CDRL1 having a sequence shown in SEQ ID NO:8 or 76, a CDRL2 having a sequence shown in SEQ ID NO:9 or SEQ ID NO:77, and a CDRL3 shown in SEQ ID NO: 10 or 78.

2. The method of claim 1, wherein the isolated antigen binding protein does not substantially cross-react with (ssDNA), double stranded DNA (dsDNA), lipopolysaccharide, cardiolipin, chondroitin and heparan.

3. The method of claim 1, wherein the isolated antigen binding protein further comprises a heavy chain comprising a framework 1 (FR1) shown in any of SEQ ID NOS: 1, 3, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 28, 29, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, or 72.

4. The method of claim 1, wherein the isolated antigen binding ability and bioefficacy are independent of lambda and kappa light chain.

5. The method of claim 1, wherein said isolated binding protein which has bioefficacy independent to J-chain.

6. A method of treating NETosis associated disease or condition in a subject, comprising administering to the subject an agent, wherein said agent is an isolated antigen binding protein which binds to PAL, comprising a heavy chain sequence selected from the group consisting of SEQ ID NOS: 1, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68 or 71, and a light chain sequence selected from the group consisting of SEQ ID NOS: 2 or 74.

7. The method of claim 1 or 6, wherein the isolated antigen binding protein further comprises a heavy chain CDR2 sequence: EINHSGSTNYNPSLKS, numbered (50-65), and wherein His53 may be substituted with Tyrosine at position 53.Attorney File Reference: MBI.00098. The method of claim 1 or 6, wherein the isolated antigen binding protein does not substantially cross-react with (ssDNA), double stranded DNA (dsDNA), lipopolysaccharide, cardiolipin, chondroitin and heparin.

9. The method of claim 1 or 6, wherein said antigen binding protein is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multi-specific antibody, or an antibody fragment thereof.

10. The method of claim 1 or 6, wherein said antibody fragment is a Fab fragment, a Fab’ fragment, a F(ab)2 fragment, a Fv fragment, a diabody, or a single chain antibody molecule.

11. The method of claim 1 or 6, wherein said antigen binding protein is a human antibody.

12. The method of claim 9, wherein said antigen binding protein is a monoclonal antibody.

13. The method of claim 1 or 6, wherein said antigen binding protein is selected from the group consisting of IgA, IgD, IgM, IgG, and IgE.

14. The method of claim 13, wherein said antigen binding protein is an IgM.

15. The method of claim 12, wherein the antigen binding protein comprises a mixture of pentamers and hexamers.

16. The method of claim 1 or 6, wherein the antigen binding protein is mixed with a pharmaceutically acceptable carrier, diluent or adjuvant.

17. A method of inhibiting NETosis, comprising exposing a neutrophil that has been activated for NETosis to an agent, wherein said agent is an isolated antigen binding protein which binds to PAL, comprising:(a) a heavy chain comprising a CDRH1 having a sequence shown in SEQ ID NO: 5, a CDRH2 having a sequence shown in SEQ ID NO: 6, and a CDRH3 having a sequence shown in any of SEQ ID NOS: 7, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, or 73; and(b) a light chain comprising (i) a CDRL1 having a sequence shown in SEQ ID NO: 8, a CDRL2 having a sequence shown in SEQ ID NO: 9 and a CDRL3 having a sequence shown in SED ID NO: 10, or (ii) a CDRL1 having a sequence shown in SEQ ID NO:8 or 76, a CDRL2 having a sequence shown in SEQ ID NO:9 or SEQ ID NO:77, and a CDRL3 shown in SEQ ID NO: 10 or 78.

18. The method of claim 17, wherein the isolated antigen binding protein does not substantially cross-react with (ssDNA), double stranded DNA (dsDNA), lipopolysaccharide, cardiolipin, chondroitin and heparan.Attorney File Reference: MBI.000919. The method of claim 17, wherein the isolated antigen binding protein further comprises a heavy chain comprising a framework 1 (FR1) shown in any of SEQ ID NOS: 1, 3, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 28, 29, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, or 72.

20. The method of claim 17, wherein said antigen binding protein is selected from the group consisting of IgA, IgD, IgM, IgG, and IgE.

21. The method of claim 20, wherein said antigen binding protein is an IgM.

22. The isolated antigen binding protein of claim 17, wherein said antigen binding protein is a human antibody.

23. The method of claim 17, wherein said antigen binding protein is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multi-specific antibody, or an antibody fragment thereof.

24. The method of claim 23, wherein said antigen binding protein is a monoclonal antibody.

25. The method of claim 23, wherein said antibody fragment is a Fab fragment, a Fab’ fragment, a F(ab)2 fragment, a Fv fragment, a diabody, or a single chain antibody molecule.

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