Anti-peripheral lymph node addressin antibodies and uses thereof

Abstract

The present disclosure provides, in particular, anti-peripheral lymph node addressin antibodies and antigen-binding fragments thereof. The present disclosure also provides compositions comprising drug-containing polymer particles, which simulate lymphocyte migration in vivo and can specifically deliver immunosuppressive or immunomodulatory drugs to lymphoid tissues and chronic inflammatory sites where T cell activation and T cell-mediated damage occur; such compositions include antibodies or antigen-binding fragments thereof described in the present disclosure. The present disclosure also includes antibody-drug conjugates and compositions comprising antibody-drug conjugates. Methods for preparing and using these antibodies, antigen-binding fragments thereof, and compositions thereof are also provided.

Description

[0001] Priority declaration

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 804,797, filed on February 13, 2019. The entire contents of the foregoing are incorporated herein by reference. Technical Field

[0003] The present invention relates to anti-peripheral lymph node addressin (PNAd) antibodies, antigen-binding fragments thereof, conjugates thereof, and uses thereof. Background Art

[0004] Peripheral lymph node addressin (PNAd) is a tissue-specific endothelial cell antigen constitutively expressed on high endothelial cell venules (HEV) in both mice and humans. PNAd is a group of endothelial sialomucins—CD34, podocalixin, glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), and 200 kDa sialylated glycoprotein (sgp200)—all of which include sulfated sialylated-Lewis X (sLeX)-like motifs (von Andrian, UH & Mackay, CR, N Engl J Med 343: 1020-1034, 2000). PNAd is expressed on the endothelium of venules in peripheral lymph nodes, tonsils, and almost all inflammatory or lymphomatous lesions, but not in normal tissues of ocular adnexa, thyroid, salivary glands, and lungs (Liu Y, J Clin Exp Hematopathol 44 (1): 33-37, 2004). PNAds play an important role in lymphocyte homing to inflamed lesions and in the biological behavior of lymphoma cells in MALT lymphoma tissues of these organs.

[0005] Inflammation is a key pathogenic process in a large number of epidemics from typical immune-mediated diseases such as rheumatoid arthritis to cancer. From transplant rejection of transplanted organs, to multiple sclerosis in which lymphocytes invade the brain, to atherosclerotic plaques in which leukocytes aggravate lipid-induced small and large vessel damage, immune or inflammatory responses are important for the development of these diseases. The past decade has witnessed the major progress in the development of novel immunosuppressive drugs. These drugs play an important role in ensuring successful organ transplantation, and have greatly improved the prognosis of patients with life-threatening immune-mediated diseases. However, the use of immunosuppressants is hindered by a large number of adverse drug reactions that lack selectivity and are often observed. Summary of the invention

[0006] The present disclosure provides anti-peripheral lymph node addressin (PNAd) antibodies, or PNad-binding fragments thereof, and methods of use thereof.

[0007] In one aspect, the disclosure features an anti-Peripheral Lymph Node Addressin (PNAd) antibody, or a PNad-binding fragment thereof, comprising: (a) a heavy chain variable region (VH), wherein the VH comprises the following: a VH complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:3, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:4, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:5; and (b) a light chain variable region (VL), wherein the VL comprises the following: a VL CDR1 comprising the amino acid sequence of SEQ ID NO:8, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:9, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:10.

[0008] In some embodiments, VH comprises the following: a VH framework region 1 (FR1) comprising the amino acid sequence shown in SEQ ID NO:11, a VH FR2 comprising the amino acid sequence shown in SEQ ID NO:12, a VH FR3 comprising the amino acid sequence shown in SEQ ID NO:13, and a VH FR4 comprising the amino acid sequence shown in SEQ ID NO:14.

[0009] In some embodiments, VL comprises the following: VLFR1 comprising the amino acid sequence shown in SEQ ID NO:15, VL FR2 comprising the amino acid sequence shown in SEQ ID NO:16, VL FR3 comprising the amino acid sequence shown in SEQ ID NO:17, and VL FR4 comprising the amino acid sequence shown in SEQ ID NO:18.

[0010] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, VL comprises the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO: 2 and VL comprises the amino acid sequence shown in SEQ ID NO: 7.

[0011] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a murine antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0012] In some embodiments, the antibody, or its PNad binding fragment, comprises human heavy and light chain constant regions. In some embodiments, the antibody, or its PNad binding fragment, comprises a heavy chain constant region of the isotype immunoglobulin mu (IgM).

[0013] In some embodiments, the PNad binding fragment is selected from the group consisting of a single-chain Fv fragment (scFv), a F(ab') fragment, a F(ab) fragment, and a F(ab')2 fragment.

[0014] In some embodiments, the antibody, or its PNad binding fragment, is conjugated to an agent. In some embodiments, the agent is an imaging agent, a cytotoxic agent, or a drug. In some embodiments, the agent is a drug-containing polymer particle. In some embodiments, the drug-containing polymer particle comprises an immunosuppressive or immunomodulatory drug. In some embodiments, the conjugation to the agent is covalent conjugation. In some embodiments, the conjugation to the agent is conjugation through a linker. In some embodiments, the linker is polyethylene glycol.

[0015] Also provided herein are polynucleotides comprising nucleic acid sequences encoding the aforementioned antibodies or PNad-binding fragments thereof.

[0016] Further provided herein are polynucleotides comprising a nucleic acid sequence encoding a heavy chain variable region (VH), wherein VH comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO: 1. In some examples, the polynucleotide is operably linked to a promoter.

[0017] In addition, provided herein are polynucleotides comprising a nucleic acid sequence encoding a heavy chain, wherein the heavy chain comprises a heavy chain variable region (VH), wherein VH comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO: 1. In some cases, the polynucleotide is operably linked to a promoter.

[0018] Also provided herein are polynucleotides comprising a nucleic acid sequence encoding a light chain variable region (VL), wherein VL comprises the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO: 6. In some cases, the polynucleotide is operably linked to a promoter.

[0019] Also provided herein are polynucleotides comprising a nucleic acid sequence encoding a light chain, wherein the light chain comprises a light chain variable region (VL), wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO: 6. In some cases, the polynucleotide is operably linked to a promoter.

[0020] Also provided herein is a vector comprising a promoter operably linked to the aforementioned polynucleotide comprising a nucleic acid sequence encoding: (i) a VH comprising the amino acid sequence shown in SEQ ID NO:2 operably linked to the promoter; or (ii) a VL comprising the amino acid sequence shown in SEQ ID NO:7.

[0021] Also provided herein is a vector comprising a promoter operably linked to the aforementioned polynucleotide comprising a nucleic acid sequence encoding a heavy chain, the heavy chain comprising a VH comprising the amino acid sequence shown in SEQ ID NO:2.

[0022] Also provided herein is a vector comprising a promoter operably linked to the aforementioned polynucleotide comprising a nucleic acid sequence encoding a light chain, the light chain comprising a VL comprising the amino acid sequence shown in SEQ ID NO:7.

[0023] Also provided herein is a vector comprising: (i) a first promoter operably linked to the aforementioned polynucleotide comprising a nucleic acid sequence encoding a heavy chain, wherein the heavy chain includes a VH comprising the amino acid sequence shown in SEQ ID NO:2; and (ii) a second promoter operably linked to the aforementioned polynucleotide comprising a nucleic acid sequence encoding a light chain, wherein the light chain includes a VL comprising the amino acid sequence shown in SEQ ID NO:7.

[0024] Also provided herein are host cells comprising one or more of the foregoing polynucleotides operably linked to a promoter(s).

[0025] Also provided herein are host cells comprising one or more of the aforementioned vectors.

[0026] Also provided herein is a method for producing an anti-Peripheral Lymph Node Addressin (PNAd) antibody or a PNad-binding fragment thereof, comprising: (a) culturing the aforementioned host cell; and (b) isolating the antibody from the culture.

[0027] Also provided herein is a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned antibody or PNad binding fragment thereof and a pharmaceutically acceptable carrier.

[0028] Also provided herein is a method of inhibiting a lymphocyte-mediated immune response in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the aforementioned pharmaceutical composition.

[0029] Also provided herein are methods of treating autoimmune diabetes in a subject in need thereof or delaying the progression of autoimmune diabetes in a subject in need thereof, administering to the subject a therapeutically effective amount of the aforementioned pharmaceutical composition.

[0030] Also provided herein are methods of treating lymphocyte-mediated inflammation in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of the aforementioned pharmaceutical composition.

[0031] Also provided herein is a method for treating a malignant tumor expressing a peripheral lymph node addressin (PNAd) in a subject in need thereof or reducing the metastasis of a malignant tumor expressing a peripheral lymph node addressin (PNAd) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the aforementioned pharmaceutical composition. In some embodiments, the malignant tumor expressing PNad is a lymphoma. In some embodiments, the malignant tumor expressing PNad is a cancer. In some cases, the cancer is breast cancer.

[0032] Also provided herein is a method of delivering a drug to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the aforementioned pharmaceutical composition, wherein the drug delivered to the subject is a drug of drug-containing polymer particles conjugated to an antibody or PNad binding fragment thereof of the pharmaceutical composition. In some embodiments, the drug is delivered to one or more lymphoid tissues or one or more sites of chronic inflammation in the subject.

[0033] The details of one or more embodiments of the invention are shown in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1A -B: MHA112 monoclonal antibody (mAb) recognizes high endothelial venules (HEV) in mice and humans. Figure 1A ) Low magnification image of immunofluorescence staining showing HEV staining with MHA112 mAb. Scale bar, 500 μm. ( Figure 1B ) High magnification images of immunofluorescence staining showing HEV staining with MHA112 mAb in mouse lymph nodes (LN) and human tonsils. Scale bar, 100 μm.

[0035] Figure 2 : MHA112 mAb typing. MHA112 mAb isotype was determined by typing enzyme-linked immunosorbent assay (ELISA). The absorbance was recorded at 450 nm.

[0036] Figure 3A -B: Transport of MHA112 mAb-conjugated nanoparticles (NPs) to LNs. Figure 3A) Fluorescence images showing the distribution of MHA112 mAb-conjugated NPs (MHA112-IR800-NPs) in different LNs. NPs alone (IR800-NPs) were used as negative controls and MECA79-NPs (MECA79_IR800-NPs) were used as positive controls. ( Figure 3B ) Immunofluorescence analysis of MECA79-NP and MHA112-NP distribution in axillary LNs. HEV staining is green, and IR800 dye in NPs is red. Scale bar, 50 μm.

[0037] Figure 4 : MHA112 mAb, but not MECA79 mAb, blocks T cell homing to LN. Flow cytometric analysis of labeled T cells homing to LN in control (PBS, MECA79) and MHA112 mAb-treated groups. Data from three mice / group (n=3) show changes in the percentage of labeled T cells and are summarized in the bar graph. *p<0.05, **p<0.01, ***p<0.001, t-test, ns: not significant.

[0038] Figure 5 : MHA112 sequence. The sequences of the MHA112 heavy and light chain variable regions (VH and VL, respectively) are shown, depicting the framework regions (FR1-FR4) and the IMGT complementarity determining regions (CDR1-CDR3). VH = SEQ ID NO: 2; VH FR1-FR4 = SEQ ID NO: 11-14, respectively; VH CDR1-3 = SEQ ID NO: 3-5, respectively; VL = SEQ ID NO: 7; VL FR1-FR4 = SEQ ID NO: 15-18, respectively; VL CDR1-3 = SEQ ID NO: 3-5, respectively.

[0039] Figure 6 : Determination of binding affinity of MECA79 and MHA112 mAbs by ELISA. Different concentrations of MECA79 and MHA112 were tested against CHO-PNAd cells. Kd was calculated. MHA112 was found to have better binding affinity than MECA79.

[0040] Figure 7: Breast tumor growth curves show that the growth of 4T1 mouse breast tumors in BALB / c-WT mice treated with free paclitaxel and MHA112-paclitaxel (equivalent to 160ng free paclitaxel injected intravenously every two days after transplantation for 33 days) (n=12) was significantly slower than that of the control group treated with PBS (n=12). Data are expressed as mean + / - SD. **P<0.01, ***P<0.001. The triangles on the x-axis represent the injection points. The MHA112-paclitaxel group had significantly smaller tumors compared to the free paclitaxel and untreated groups.

[0041] Figure 8 : Fluorescence micrographs of breast tumor draining lymph nodes (DLN) show that HEV and LYVE1+ lymphatic vessels (green) are less expanded in the MHA112-paclitaxel group compared to the control or free paclitaxel group. Pan-cytokeratin (red) indicates that 4T1 metastasizes in the TDLN. Scale bar: 100 μm. The MHA112-paclitaxel group showed significantly less tumor metastasis and lymphatic expansion compared to the free paclitaxel and untreated groups.

[0042] Fig. 9 : Fluorescence micrographs of breast tumor DLNs demonstrate less severe fibrosis (as represented by collagen I, collagen IV, and laminin) in the MHA112-paclitaxel group compared to the control or free paclitaxel groups. Scale bar: 100 μm. The MHA112-paclitaxel group showed significantly less lymph node fibrosis compared to the free paclitaxel and untreated groups.

[0043] Fig.10 : Survival curves show significantly longer survival in the 4T1 breast tumor hepatoportal metastasis model receiving MHA112-paclitaxel (n=5) compared to the control and free paclitaxel groups. ***P<0.001. Triangles indicate injection days.

[0044] Fig.11 : Survival curves of panc02 pancreatic tumors in the pancreas showed significantly longer survival for MHA112-paclitaxel (n=5) compared to the control and free paclitaxel groups (n=5). DETAILED DESCRIPTION

[0045] I. Definitions

[0046] It should be noted that the term indefinite article "a" or "an" entity refers to one or more of that entity; for example, "an antibody" is understood to mean one or more antibodies. As such, the terms indefinite articles "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

[0047] As used herein, the term "polypeptide" is intended to include the singular "polypeptide" as well as the plural "polypeptides", and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, rather than a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other terms used to refer to one or more chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms.

[0048] The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. The polypeptide is derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. The polypeptide can be produced in any manner, including by chemical synthesis.

[0049] The size of the polypeptides described herein can be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides that do not have a defined three-dimensional structure but adopt a large number of different conformations are referred to as unfolded. As used herein, the term glycoprotein refers to a protein coupled to at least one carbohydrate moiety, which is attached to the protein through oxygen-containing or nitrogen-containing side chains of amino acid residues such as serine residues or asparagine residues.

[0050] An "isolated" polypeptide or fragment, variant, or derivative thereof is a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be removed from the polypeptide's native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purposes described herein, as are native or recombinant proteins that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0051] As polypeptides, fragments, derivatives, analogs or variants of the aforementioned polypeptides, and any combination thereof are also included. When referring to antibodies or antibody polypeptides described herein, the terms "fragment", "variant", "derivative" and "analog" include any polypeptide that retains at least some of the antigen-binding properties of the corresponding natural binding molecules, antibodies, or polypeptides. In addition to the specific antibody fragments discussed in other parts of this article, the fragments of the polypeptides described herein include proteolytic fragments and deletion fragments. The variants of the antibodies and antibody polypeptides described herein include fragments as described above, and also include polypeptides with amino acid sequences that have been changed due to amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions), deletions, or insertions. Variants may be natural or non-natural. Non-naturally occurring variants may be produced using mutagenesis techniques known in the art. Variant polypeptides may include conservative or non-conservative amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions), deletions or additions. Derivatives of PNAd specific binding molecules, such as antibodies and antibody polypeptides described herein, are polypeptides that have been changed to show additional features not found in natural polypeptides. Examples include fusion proteins. Variant polypeptides may also refer to "polypeptide analogs" in this article. As used herein, a "derivative" of a binding molecule or fragment thereof, an antibody, or an antibody polypeptide refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those peptides that contain one or more naturally occurring amino acid derivatives of the 20 standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.

[0052] The term "polynucleotide" is intended to include singular nucleic acids as well as plural nucleic acids, and refers to isolated nucleic acid molecules or constructs, for example, messenger RNA (mRNA) or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid fragments present in a polynucleotide, for example, DNA or RNA fragments. "Isolated" nucleic acid or polynucleotide refers to a nucleic acid molecule, DNA or RNA that has been removed from its native environment. For example, a recombinant polynucleotide of an antibody contained in an encoding vector is considered to be isolated for the purposes described herein. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or solution-purified (partially or substantially) polynucleotides. Isolated RNA molecules include in vivo and in vitro RNA transcripts of the polynucleotides described herein. The isolated polynucleotides or nucleic acids described herein also include such molecules produced synthetically. In addition, polynucleotides or nucleic acids may be regulatory elements or may include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0053] As used herein, "coding region" is a nucleic acid region composed of codons translated into amino acids. Although "stop codon" (TAG, TGA or TAA) is not translated into amino acids, it can be considered as a part of the coding region, but any flanking sequences such as promoters, ribosome binding sites, transcription terminators and introns are not part of the coding region. Two or more coding regions described herein may be present in a single polynucleotide construct, such as on a single vector, or in respective polynucleotide constructs, such as on respective (different) vectors. In addition, any vector may contain a single coding region or may include two or more coding regions, such as a single vector may encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, respectively. In addition, the vectors, polynucleotides or nucleic acids described herein may encode heterologous coding regions, fused or not fused to nucleic acids encoding binding molecules, antibodies, or fragments, variants or derivatives thereof. Heterologous coding regions include, but are not limited to, specific elements or motifs, such as secretory signal peptides or heterologous functional domains.

[0054] In some embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, the polynucleotide comprising a nucleic acid encoding a polypeptide may generally include a promoter and / or other transcription or translation control elements operably associated with one or more coding regions. Operable association is when a gene product such as a coding region of a polypeptide is associated with one or more regulatory sequences, so that the expression of the gene product is under the influence or control of the regulatory sequence (one or more). If the induction of promoter function leads to the transcription of the mRNA encoding the desired gene product, and if the nature of the connection between the two DNA fragments does not interfere with the ability of the expression regulatory sequence to guide the expression of the gene product or the ability of the DNA template to be transcribed, the two DNA fragments (e.g., a polypeptide coding region and a promoter associated therewith) are "operably associated" or "operably connected". Therefore, if the promoter can affect the transcription of the nucleic acid, the promoter region can be operably associated with the nucleic acid encoding the polypeptide. The promoter can be a cell-specific promoter that only directs the substantial transcription of DNA in a predetermined cell. In addition to the promoter, other transcription control elements such as enhancers, operators, repressors, and transcription termination signals can be operably associated with the polynucleotide to guide cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.

[0055] Various transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as, but not limited to, promoters and enhancer fragments from cytomegalovirus (immediate early promoter, linked to intron-A), simian virus 40 (early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes, such as actin, heat shock protein, bovine growth hormone, and rabbit β-globulin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers, and lymphokine-inducible promoters (e.g., promoters induced by interferons or interleukins).

[0056] Similarly, various translation control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly, internal ribosome entry sites, or IRES, also known as CITE sequences).

[0057] In some embodiments, the polynucleotides described herein are RNA, for example, in the form of messenger RNA (mRNA).

[0058] The polynucleotides and nucleic acid coding regions described herein may be associated with additional coding regions encoding secretory or signal peptides, which direct the secretion of the polypeptides encoded by the polynucleotides described herein. According to the signal hypothesis, proteins secreted by mammalian cells have signal peptides or secretory leader sequences, which are cut from mature proteins once the growing protein chain passes through the rough endoplasmic reticulum. Those skilled in the art will recognize that polypeptides secreted by vertebrate cells usually have a signal peptide fused to the N-terminus of the polypeptide, which is cut from a complete or "full-length" polypeptide to produce a secretory or "mature" form of the polypeptide. In some embodiments, natural signal peptides such as immunoglobulin heavy chain or light chain signal peptides are used, or functional derivatives of sequences that retain the ability to direct the secretion of polypeptides operably associated with functional derivatives of the sequence. Alternatively, heterologous mammalian signal peptides, or functional derivatives thereof, may be used. For example, a wild-type leader sequence can replace the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0059] Unless otherwise indicated, the terms "disorder" and "disease" are used interchangeably herein.

[0060] As used herein, "binding molecules" primarily refer to antibodies, and fragments thereof, but may also refer to other non-antibody molecules that bind to PNAds, including but not limited to hormones, receptors, ligands, major histocompatibility complex (MHC) molecules, chaperones such as heat shock proteins (HSPs), and cell-cell adhesion molecules such as cadherins, integrins, C-type lectins, and members of the immunoglobulin (Ig) superfamily. Therefore, for clarity only and without limiting the scope of the present disclosure, most of the following embodiments are discussed with respect to antibodies and antibody-like molecules that represent specific embodiments of binding molecules for development of therapeutic and diagnostic agents.

[0061] The terms "antibody" and "immunoglobulin" are used interchangeably herein. An antibody or immunoglobulin is a PNad binding molecule that comprises at least the variable domain of a heavy chain, and typically at least the variable domains of a heavy chain and a light chain. The basic immunoglobulin structure in vertebrate systems is relatively well understood; see, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988).

[0062] As will be discussed in more detail below, the term "immunoglobulin" encompasses various major classes of polypeptides that can be biochemically identified. It will be appreciated by those skilled in the art that heavy chains are classified as gamma, muon, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, which is IgG, IgM, IgA, IgG, or IgE, respectively. For example, immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1 are well characterized and known to confer functional specialization. In view of the present disclosure, the respective modified versions of these classes and isotypes are easily distinguishable to those skilled in the art, and therefore, are within the scope of the present disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, and the following discussion will generally be directed to the IgG class of immunoglobulin molecules. Regarding IgG, it is a standard immunoglobulin molecule comprising two identical light chain polypeptides of molecular weight about 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically connected by disulfide bonds in a "Y" configuration, wherein the light chain begins to bracket the heavy chain at the mouth of the "Y" and continues through the variable region.

[0063] Light chains are classified as kappa or lambda (κ, λ). Each heavy chain can be combined with either κ or λ light chain. Usually, when immunoglobulins are produced by hybridomas, B cells or genetically engineered host cells, light chains and heavy chains are covalently bound to each other, and the "tails" of the two heavy chains are bound to each other by covalent disulfide bonds or non-covalent connections. In the heavy chain, the amino acid sequence extends (runs) from the N-terminal of the fork end of the Y configuration to the C-terminal at the bottom of each chain.

[0064] Both the light chain and the heavy chain are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties, such as secretion, transplacental migration (transplacental mobility), Fc receptor binding, complementary binding, etc. By convention, the numbering of the constant region domains increases as the constant region domains are away from the antigen binding site or amino terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CL domains actually include the carboxyl termini of the heavy chain and the light chain, respectively.

[0065] As shown above, variable regions allow antibodies to selectively identify and specifically bind to epitopes on antigens. That is, a subset of the VL domain and VH domain or complementary determining region (CDR) of a combined antibody is used to form a variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms an antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three CDRs on each VH and VL chain. Any antibody or immunoglobulin fragment containing enough structure to specifically bind to PNad is interchangeably designated as a "binding fragment" or "immunospecific fragment" herein.

[0066] In naturally occurring antibodies, the antibody contains six hypervariable regions present on each antigen binding domain, sometimes referred to as "complementarity determining regions" or "CDRs", which are short, non-contiguous amino acid sequences that are specifically positioned to form an antigen binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The "CDRs" are flanked by four relatively conservative "framework" regions or "FRs" that display low intermolecular variability. The framework regions primarily adopt a β-sheet conformation and the CDRs form loops that connect the β-sheet structure and in some cases form a portion of the β-sheet structure. Thus, the framework regions serve to form a scaffold that is provided for positioning the CDRs in the correct orientation by interchain, non-covalent interactions. The antigen binding domain formed by positioning the CDRs defines a surface that is complementary to an epitope on an immunoreactive antigen. This complementary surface promotes non-covalent binding of the antibody to its cognate epitope. One skilled in the art can readily identify the amino acids comprising the CDRs and framework regions, respectively, for any given heavy or light chain variable region because they have been precisely defined; see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196 (1987), 901-917, which are incorporated herein by reference in their entirety.

[0067] In the case of two or more definitions of terms used and / or accepted in the art, unless explicitly stated to the contrary, the definition of the term used herein is intended to include all of these meanings. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-continuous antigen binding sites found in the variable regions of both heavy and light chain polypeptides. This specific region has been described by Kabat et al., US Pat. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia and Lesk, J. Mol. Biol., 196 (1987), 901-917, which are incorporated herein by reference, wherein when compared to each other, the definition includes an overlap or subset of amino acid residues. However, the application of any definition to refer to the CDR of an antibody or its variant is intended to fall within the scope defined and used herein. Suitable amino acid residues including CDRs defined by the respective references cited above are shown in Table 1 below as a comparison. The exact number of residues comprising a specific CDR varies depending on the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, one skilled in the art routinely determines which residues comprise a particular hypervariable region or CDR of an antibody of the human IgG subclass.

[0068] Table 1: CDR Definition 1

[0069] Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-65 52-58 VH CDR3 95-102 95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VL CDR3 89-97 91-96

[0070] 1 The numbering of all CDR definitions in Table 1 is according to the numbering convention set forth by Kabat et al. (see below).

[0071] Kabat et al. also define a numbering system applicable to the variable domain sequence of any antibody. Those skilled in the art can clearly assign the "Kabat numbering" system to any variable domain sequence, without relying on any experimental data exceeding the sequence itself. As used herein, "Kabat numbering" refers to the numbering system shown in "Sequence of Proteins of Immunological Interest" (1983) by Kabat et al., US Pat. of Health and Human Services. Unless otherwise noted, the numbering of specific amino acid residue positions in antibodies described herein or their antigen-binding fragments, variants or derivatives is according to the Kabat numbering system, however, it is theoretical and cannot be equally applied to each antibody described herein. For example, depending on the position of the first CDR, subsequent CDRs may shift in either direction.

[0072] Antibodies or antigen-binding fragments thereof, immunospecific fragments, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, murinized or chimeric antibodies, single chain antibodies, epitope binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies disclosed herein). ScFv molecules are known in the art and are described, e.g., in U.S. Pat. No. 5,892,019. The immunoglobulin or antibody molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0073] In some embodiments, the antibody is not an IgM or a derivative thereof having a pentavalent structure. In particular, IgM is less useful than IgG and other bivalent antibodies or corresponding binding molecules in specific applications, especially therapeutic uses, because IgMs, due to their pentavalent structure and lack of affinity maturation, generally show non-specific cross-reactivity and very low affinity.

[0074] In some embodiments, the antibody is an IgM or a derivative thereof having a pentavalent structure.

[0075] In some embodiments, the antibody is not a polyclonal antibody, ie, it consists essentially of one specific antibody species rather than being a mixture obtained from a plasma immunoglobulin sample.

[0076] Antibody fragments, including single-chain antibodies, may comprise only the variable region(s) or a combination of the variable region(s) with all or part of the following: hinge region, CH1, CH2, and CH3 domains. PNad binding fragments that also comprise any combination of the variable region(s) with hinge region, CH1, CH2, and CH3 domains are also provided herein. The antibodies described herein or their immunospecific fragments may be from any animal source including birds and mammals. In some embodiments, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be of condricthoid origin (e.g., from sharks).

[0077] In some embodiments, the antibody is a monoclonal antibody isolated from a human. Optionally, the framework region of the human antibody is aligned and adopted according to the pertinent human germ line variable region sequence in the database; see, for example, Vbase (vbase.mrc-cpe.cam.ac.uk) hosted by the MRC Centre for Protein Engineering (Cambridge, UK). For example, it is believed that the amino acid potentially deviates from the true germ line sequence because the PCR primer sequence is mixed in during the cloning process. Compared with artificially produced human-like antibodies such as single-chain antibody fragments (scFv) from phage display antibody libraries or xenogeneic mice, the human monoclonal antibodies described herein are characterized in that (i) the human immune response is obtained, rather than the immune response of animal substitutes, that is, the antibody is produced in response to the natural PNAd of its relevant conformation in the human body, (ii) the individual has been protected or at least important for the presence of PNAd, and (iii) since the antibody is of human origin, the risk of cross-reaction to self-antigens is minimized. Thus, as used herein, the terms "human monoclonal antibody", "human monoclonal autoantibody", and "human antibody" are used to refer to PNad binding molecules of human origin, i.e., they are isolated from, for example, B cells or their hybridomas, or their cDNAs are cloned directly from mRNA of human cells, such as human memory B cells. Human antibodies are still "human" even if amino acid substitutions are made in the antibody, for example, to improve binding properties.

[0078] As described below, antibodies derived from human immunoglobulin libraries or from transgenic animals for one or more human immunoglobulins and transgenic animals that do not express endogenous immunoglobulins are denoted as human-like antibodies, e.g., in U.S. Pat. No. 5,939,598 to Kucherlapati et al., to distinguish them from truly human antibodies described herein.

[0079] For example, the pairing of heavy and light chains of human-like antibodies, such as synthetic and semisynthetic antibodies typically isolated from phage display, does not necessarily reflect the original pairing as it occurred in the original human B cells. Therefore, the Fab and scFv fragments commonly used in the prior art, obtained from recombinant expression libraries, are considered artificial, with all possible associated effects on immunogenicity and stability.

[0080] As used herein, the term "heavy chain portion" includes an amino acid sequence derived from an immunoglobulin heavy chain. The polypeptide comprising the heavy chain portion contains at least one of the following: a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, the binding polypeptide used in the methods described herein may include a polypeptide chain containing a CH1 domain; a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain containing a CH1 domain and a CH3 domain; a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide described herein includes a polypeptide chain containing a CH3 domain. In addition, the binding polypeptide used in the methods described herein may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As indicated above, those skilled in the art will appreciate that these domains (eg, the heavy chain portions) can be modified such that they are altered in amino acid sequence from a naturally occurring immunoglobulin molecule.

[0081] In certain antibodies, or antigen-binding fragments, variants, or derivatives thereof disclosed herein, the heavy chain portion of one polypeptide chain of the multimer is identical to the heavy chain portion of the second polypeptide chain of the multimer. Alternatively, the monomers containing the heavy chain portions described herein are not identical. For example, each monomer may contain a different target binding site, forming, for example, a bispecific antibody or a diabody.

[0082] In some embodiments, the antibodies, or antigen-binding fragments, variants, or derivatives thereof disclosed herein are composed of a single polypeptide chain, such as scFv, and are expressed intracellularly (intrabodies) for potential in vivo therapeutic and diagnostic applications.

[0083] The heavy chain portion of the binding polypeptide used in the diagnosis and treatment methods disclosed herein can be derived from different immunoglobulin molecules. For example, the heavy chain portion of the polypeptide can include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain portion can include a hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In another example, the heavy chain portion can include a chimeric hinge that is partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0084] As used herein, the term "light chain portion" includes an amino acid sequence derived from an immunoglobulin light chain. In some embodiments, the light chain portion comprises at least one of a VL or CL domain.

[0085] The minimum size of the peptide or polypeptide epitope of an antibody is considered to be about 4 to 5 amino acids. The peptide or polypeptide epitope may comprise at least 7 amino acids, at least 9 amino acids, or between at least about 15 to about 30 amino acids. Since CDR can identify antigenic peptides or polypeptides in its tertiary form, the amino acids comprising the epitope do not have to be continuous, and in some cases, may not even be on the same peptide chain. In some embodiments, the peptide or polypeptide epitope recognized by the antibody described herein may comprise at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or about 5 to about 30, about 10 to about 30, or about 15 to about 30 continuous or non-continuous amino acids of a PNAd.

[0086] "Specific binding" or "specific identification", which are used interchangeably herein, generally means that a binding molecule such as an antibody binds to an epitope through its antigen binding domain, and means that some complementarity between the antigen binding domain and the epitope is required for binding. According to this definition, when an antibody binds to an epitope through its antigen binding domain, the antibody is said to "specifically bind" to the epitope more easily than it binds to a random, unrelated epitope. It is understood by those skilled in the art that an antibody can specifically bind to, or specifically identify, an isolated polypeptide consisting of or comprising amino acid residues corresponding to a linear portion of a non-continuous epitope. The term "specificity" is used herein to define the relative affinity of a particular antigen to bind to certain epitopes. For example, antibody "A" can be considered to have a higher specificity for a given epitope than antibody "B", or antibody "A" can be said to bind to epitope "C" with a higher specificity than it does for a related epitope "D".

[0087] When present, the term "immunological binding properties" or other binding properties of an antibody to an antigen, in all its grammatical forms, refers to the specificity, affinity, cross-reactivity, and other binding properties of the antibody.

[0088] By "preferentially binds", it is meant that a binding molecule, such as an antibody, specifically binds to an epitope more readily than it binds to a related, similar, homologous, or analogous epitope. Thus, an antibody that "preferentially binds" to a given epitope will be more likely to bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.

[0089] By way of non-limiting example, if a binding molecule, such as an antibody, binds to a second epitope with a dissociation constant (K D ) Small K D If a binding molecule such as an antibody binds to a first epitope, then the binding molecule, such as an antibody, can be considered to preferentially bind to the first epitope. In another non-limiting example, if the antibody binds to the first epitope with a K of 100% compared to the antibody for the second epitope, then the binding molecule, such as an antibody, can be considered to preferentially bind to the first epitope. DAn antibody may be considered to bind preferentially to a first antigen if the antibody binds to the first epitope with an affinity that is at least one order of magnitude less than that of the antibody for the second epitope. D If an antibody binds the first epitope with an affinity that is at least two orders of magnitude less than the first epitope, then the antibody can be considered to preferentially bind the first epitope.

[0090] In another non-limiting example, a binding molecule, such as an antibody, is considered to preferentially bind to a first epitope if the binding molecule, such as an antibody, binds to the first epitope with a k(off) that is less than the dissociation rate (k(off)) of the antibody for the second epitope. In another non-limiting example, a binding molecule, such as an antibody, is considered to preferentially bind to the first epitope if the binding molecule, such as an antibody, binds to the first epitope with an affinity that is at least one order of magnitude less than the k(off) of the antibody for the second epitope. In another non-limiting example, a binding molecule, such as an antibody, is considered to preferentially bind to the first epitope if the binding molecule, such as an antibody, binds to the first epitope with an affinity that is at least two orders of magnitude less than the k(off) of the antibody for the second epitope.

[0091] A binding molecule, such as an antibody, is said to competitively inhibit binding of a reference antibody to a given epitope if the binding molecule, such as an antibody, preferentially binds to a given epitope to the extent that it blocks binding of a reference antibody to the epitope to the extent that it blocks binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art, such as a competition ELISA assay. An antibody can be said to competitively inhibit binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%. One skilled in the art will appreciate that binding of an antibody to its epitope can also be competitively inhibited by binding molecules that are not antibodies.

[0092] As used herein, the term "affinity" refers to a measurement of the strength of binding of a single epitope to a CDR of a binding molecule such as an immunoglobulin molecule; see, e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd edition (1988), pp. 27-28. As used herein, the term "avidity" refers to the overall stability of the complex between a population of immunoglobulins and an antigen, i.e., the functional binding strength of an immunoglobulin mixture to an antigen; see, e.g., Harlow pp. 29-34. Avidity relates to the affinity of a single immunoglobulin molecule with a specific epitope in a population, as well as the valence of the immunoglobulin and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen having a highly repetitive epitope structure such as a polymer will be one of high avidity. The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method; see, for example, Berzofsky et al., "Antibody-Antigen Interactions" In Fundamental Immunology, Paul, WE, Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, WH Freeman and Company New York, NY (1992), and the methods described herein. Conventional methods for determining the affinity of an antibody for an antigen include ELISA, RIA, and surface plasmon resonance. The affinity of a particular antibody-antigen interaction determined may vary if measured under different conditions, such as salt concentration, pH, etc. Thus, affinity and, for example, K D The determination of other antigen binding parameters such as IC50 is preferably performed using standardized solutions of antibody and antigen, and standardized buffer.

[0093] Binding molecules such as antibodies or antigen-binding fragments, variants or derivatives thereof described herein may also be described or specified in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of an antibody specific for one antigen to react with a second antigen; a determination of the association between two different antigenic substances. Thus, an antibody is cross-reactive if it binds to an epitope other than the epitope that induced its formation. Cross-reactive epitopes typically contain many of the same complementary structural features as the inducing epitope, and in some cases, may actually fit better than the original.

[0094] For example, certain antibodies have some degree of cross-reactivity because they bind to related, but not identical, epitopes, e.g., epitopes that are at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identical to a reference epitope (as calculated using methods known in the art and described herein). An antibody is referred to as having little or no cross-reactivity if it does not bind to an epitope that is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identical to a reference epitope (as calculated using methods known in the art and described herein). An antibody is considered to be "highly specific" for a particular epitope if it does not bind to any other analog, ortholog, or homolog of the epitope.

[0095] Binding molecules such as the antibodies or antigen-binding fragments, variants or derivatives thereof described herein may also be described or specified in terms of their binding affinity to PNAd.

[0096] As previously mentioned, the subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino acid terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is located at the amino terminal end of the hinge region of an immunoglobulin heavy chain molecule.

[0097] As used herein, the term "CH2 domain" includes the portion of the heavy chain molecule extending from about residue 244 to residue 360 ​​of an antibody, for example, using conventional coding schemes (residues 244 to 360, Kabat coding system; and residues 231-340, EU coding system; see Kabat EA et al., cited above). The CH2 domain is unique in that it is not closely paired with another domain. Instead, two N-linked branched sugar chains are inserted between the two CH2 domains of the intact native IgG molecule. It is well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 residues.

[0098] As used herein, the term "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain and the CH2 domain. The hinge region contains about 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. The hinge region can be subdivided into three different domains: the upper, middle, and lower hinge domains; see Roux et al., J. Immunol. 161 (1998), 4083.

[0099] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by disulfide bonds and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 232 using the Kabat numbering system (position 226 or 229, EU numbering system).

[0100] As used herein, the terms "connected", "fused" or "fusion" are used interchangeably. These terms refer to the joining of two or more elements or components by any method including chemical bonding or recombinant methods. "In-frame fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous long ORF in a manner that maintains the correct translation reading frame of the original ORF. Therefore, a recombinant fusion protein is a single protein containing two or more fragments of polypeptides encoded by the original ORFs (whose fragments are usually not so connected in nature). Although the reading frame is thus made continuous throughout the fused fragment, the fragments can be physically or spatially separated by, for example, in-frame linker sequences. For example, a polynucleotide encoding the CDRs of an immunoglobulin variable region may be fused in frame, but separated by a polynucleotide encoding at least one immunoglobulin framework region or an additional CDR region, as long as the "fused" CDRs are co-translated as part of a continuous polypeptide.

[0101] The term "expression" as used herein refers to the process of producing biochemicals such as RNA or polypeptides by genes. The process includes any manifestation of the functional presence of genes in cells, including but not limited to gene knockdown and transient expression and stable expression. It includes but is not limited to the transcription of genes to messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA products, and the translation of such mRNA to polypeptides (one or more). If the final desired product is a biochemical, expression includes the production of biochemicals and any precursors. The expression of a gene produces a "gene product". As used herein, a gene product can be a nucleic acid, such as a messenger RNA produced by transcription of a gene, or a polypeptide, which is translated from a transcript. The gene products described herein also include nucleic acids with post-transcriptional modifications such as polyadenylation, or polypeptides with post-translational modifications such as methylation, glycosylation, addition of lipids, association with other protein subunits, and proteolytic cleavage.

[0102] As used herein, the term "sample" refers to any biological material obtained from a subject or patient. On the one hand, the sample may include blood, plasma, or urine. In other aspects, the sample may include whole blood, plasma, B cells enriched from blood samples, and cultured cells (e.g., B cells from a subject). The sample may also include living tissue or a tissue sample including neural tissue. On the other hand, the sample may include a lysate of intact cells and / or cells. The blood sample may be collected by methods known in the art. On the one hand, the precipitation may be resuspended by vortexing at 4°C in 200 μl buffer (20 mM Tris, pH.7.5, 0.5% Nonidet, 1 mM EDTA, 1 mM PMSF, 0.1 M NaCl, IX Sigma protease inhibitors, and IX Sigma phosphatase inhibitors 1 and 2). The suspension may be kept on ice for 20 minutes, vortexed intermittently. After centrifugation at 15,000 x g for 5 minutes at about 4°C, the supernatant of aliquots is stored at about -70°C.

[0103] As used herein, the term "treatment" or "treatment" refers to therapeutic treatment and preventive or preventive measures, wherein the purpose is to prevent or slow down (reduce) undesirable physiological changes or conditions described herein. Favorable or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in disease extent, stabilization (i.e., no worsening) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (partially or completely), whether detectable or undetectable. "Treatment" can also mean prolonged survival compared to the expected survival if not receiving treatment. Those in need of treatment include those already suffering from a condition or disorder, and those prone to suffering from a condition or disorder, or those in which the manifestation of a condition or disorder will be prevented.

[0104] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, e.g., a human patient, for whom diagnosis, prognosis, prevention, or treatment is desired.

[0105] A composition or method described herein that comprises components or steps may also consist essentially of, or consist of, those components or steps.

[0106] II. Antibodies

[0107] Anti-peripheral lymph node addressin (PNAd) antibodies and antigen-binding fragments thereof are provided. Derivatives or variants of anti-PNAd antibodies or antigen-binding fragments thereof are also provided herein. Conjugates comprising such antibodies or their PNad binding fragments or derivatives or variants thereof are also provided herein. In some embodiments, the antibodies or their PNad binding fragments described herein show binding characteristics and / or biological properties as outlined for antibody MHA112 as described in the Examples section below. As described in the Examples section below, the anti-PNAd antibody MHA112 shows improved binding affinity (Example 6) and improved ability to block T cells from homing to lymph nodes (Example 4) for cells expressing PNad. Administration of drugs conjugated to MHA112 results in reduced tumor growth, reduced tumor metastasis, and reduced tumor-draining lymph node fibrosis (Example 7) in addition to increased survival time (Example 8). In addition, administration of drugs conjugated to MHA112 also increases survival time in a pancreatic tumor model (Example 9).

[0108] Antibodies and antigen-binding fragments thereof are characterized by comprising in their variable regions, e.g., the binding domain Figure 5 At least one complementarity determining region (CDR) of the VH and / or VL variable region of any of the amino acid sequences described in . The corresponding nucleotide sequences encoding the variable regions identified above are shown in Table 4 below. Exemplary descriptions of the CDRs of the above amino acid sequences of the VH and / or VL regions are given in Figure 5 and Table 2. The corresponding framework regions of the CDRs in Table 2 are shown in Table 3. However, as discussed below, those skilled in the art are familiar with the fact that CDRs may be used additionally or alternatively, and in the case of CDR2 and CDR3, their amino acid sequences are similar to Figure 5 Those shown in may differ by 1, 2, 3 or even more amino acids.

[0109] Table 2: IMGT CDR sequences

[0110]

[0111] Table 3: Framework region sequences

[0112]

[0113] Table 4: Variable region sequences

[0114]

[0115] In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise at least one CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-5 and 8-10, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-5 and 8-10. In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise 1, 2, 3, 4, 5 or 6 CDRs comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-5 and 8-10, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-5 and 8-10 (see, e.g., Table 2).

[0116] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 3, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or pNad-binding fragment thereof comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 3, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 5, and further comprises a light chain variable region (VL) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 10.

[0117] In some embodiments, the antibody or PNad-binding fragment thereof comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 8, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody or PNad-binding fragment thereof comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 8, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 10, and further comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 3, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 5.

[0118] In some embodiments, an antibody or antibody-binding fragment thereof described herein may comprise a VH comprising: (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 3; (ii) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 4; and (iii) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, an antibody or antibody-binding fragment thereof described herein may comprise a light chain variable region (VL) comprising: (i) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 8; (ii) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 9; and (iii) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody or its PNad-binding fragment may further comprise a heavy chain variable region comprising: VH CDR1 of SEQ ID NO: 3; VH CDR2 of SEQ ID NO: 4; and VHCDR3 of SEQ ID NO: 5, and may further comprise a light chain variable region comprising: VL CDR1 of SEQ ID NO: 8; VLCDR2 of SEQ ID NO: 9; and VL CDR3 of SEQ ID NO: 10.

[0119] In some embodiments, an antibody or antigen-binding fragment thereof described herein may comprise a heavy chain variable region comprising: a VH CDR1 comprising or consisting of SEQ ID NO:3; a VH CDR2 comprising or consisting of SEQ ID NO:4; and a VH CDR3 comprising or consisting of SEQ ID NO:5.

[0120] In some embodiments, an antibody or antigen-binding fragment thereof described herein may comprise a light chain variable region comprising: a VL CDR1 comprising or consisting of SEQ ID NO:8; a VL CDR2 comprising or consisting of SEQ ID NO:9; and a VL CDR3 comprising or consisting of SEQ ID NO:10.

[0121] In some embodiments, the antibodies or antigen-binding fragments thereof described herein may comprise a heavy chain variable region comprising: a VH CDR1 comprising or consisting of SEQ ID NO: 3; a VH CDR2 comprising or consisting of SEQ ID NO: 4; and a VH CDR3 comprising or consisting of SEQ ID NO: 5, and may further comprise a light chain variable region comprising: a VL CDR1 comprising or consisting of SEQ ID NO: 8; a VL CDR2 comprising or consisting of SEQ ID NO: 9; and a VL CDR3 comprising or consisting of SEQ ID NO: 10.

[0122] In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise a VH comprising a heavy chain framework region 1 (FR1) comprising the amino acid sequence shown in SEQ ID NO:11, a heavy chain FR2 comprising the amino acid sequence shown in SEQ ID NO:12, a heavy chain FR3 comprising the amino acid sequence shown in SEQ ID NO:13, and a heavy chain FR4 comprising the amino acid sequence shown in SEQ ID NO:14.

[0123] In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise a VL comprising a light chain FR1 comprising the amino acid sequence shown in SEQ ID NO:15, a light chain FR2 comprising the amino acid sequence shown in SEQ ID NO:16, a light chain FR3 comprising the amino acid sequence shown in SEQ ID NO:17, and a light chain FR4 comprising the amino acid sequence shown in SEQ ID NO:18.

[0124] In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise a VH comprising: a VH CDR1 comprising SEQ ID NO: 3 or consisting thereof; a VH CDR2 comprising SEQ ID NO: 4 or consisting thereof; and a VH CDR3 comprising SEQ ID NO: 5 or consisting thereof, wherein the VH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 2. In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise a VH comprising: a VH CDR1 comprising SEQ ID NO: 3 or consisting thereof; a VH CDR2 comprising SEQ ID NO: 4 or consisting thereof; and a VH CDR3 comprising SEQ ID NO: 5 or consisting thereof, wherein the VH has 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 2. In some cases, the amino acid substitutions are conservative amino acid substitutions.

[0125] In some embodiments, an antibody or antigen-binding fragment thereof described herein comprises a VL comprising: a VL CDR1 comprising SEQ ID NO: 8 or consisting thereof; a VL CDR2 comprising SEQ ID NO: 9 or consisting thereof; and a VL CDR3 comprising SEQ ID NO: 10 or consisting thereof, wherein the VL has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 7. In some embodiments, an antibody or antigen-binding fragment thereof described herein comprises a VL comprising: a VL CDR1 comprising SEQ ID NO: 8 or consisting thereof; a VL CDR2 comprising SEQ ID NO: 9 or consisting thereof; and a VL CDR3 comprising SEQ ID NO: 10 or consisting thereof, wherein the VL has 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 7. In some cases, the amino acid substitutions are conservative amino acid substitutions.

[0126] In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise: (a) a VH comprising: a VH CDR1 comprising or consisting of SEQ ID NO: 3; a VH CDR2 comprising or consisting of SEQ ID NO: 4; and a VH CDR3 comprising or consisting of SEQ ID NO: 5, wherein the VH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2, and (b) a VL comprising: a VL CDR1 comprising or consisting of SEQ ID NO: 8; a VL CDR2 comprising or consisting of SEQ ID NO: 9; and a VL CDR3 comprising or consisting of SEQ ID NO: 10, wherein the VL has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise: (a) a VH comprising: a VH CDR1 comprising or consisting of SEQ ID NO: 3; a VH CDR2 comprising or consisting of SEQ ID NO: 4; and a VH CDR3 comprising or consisting of SEQ ID NO: 5, wherein VH has 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 2; and (b) a VL comprising: a VLCDR1 comprising or consisting of SEQ ID NO: 8; a VL CDR2 comprising or consisting of SEQ ID NO: 9; and a VLCDR3 comprising or consisting of SEQ ID NO: 10, wherein VL has 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the substitutions are conservative substitutions. "Conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced by an amino acid residue with a similarly charged side chain. Families of amino acid residues with similarly charged side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0127] In some embodiments, variants of the antibodies described herein (e.g., antibodies or antigen-binding fragments thereof having amino acid substitutions, additions, or deletions of VH and / or VL relative to the amino acid sequences shown in SEQ ID NOs: 2 and 7, respectively) retain the ability to bind to PNad and / or retain one or more properties of the antibody MHA112 described in the examples below.

[0128] In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise a heavy chain variable region (VH) comprising, or consisting of, an amino acid sequence of SEQ ID NO: 2. In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise a light chain variable region (VL) comprising, or consisting of, an amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise a heavy chain variable region (VH) comprising, or consisting of, an amino acid sequence of SEQ ID NO: 2, and further comprise a light chain variable region (VL) comprising, or consisting of, an amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibodies or antigen-binding fragments thereof comprise a VH of SEQ ID NO: 2 and a VL of SEQ ID NO: 7.

[0129] In some embodiments, the antibodies described herein comprise a heavy chain comprising a VH described herein. In some cases, the heavy chain comprises an IgM constant region. In some cases, the heavy chain comprises a human IgM constant region.

[0130] In some embodiments, the antibodies described herein comprise a light chain comprising a VL described herein. In some cases, the light chain comprises a kappa constant region. In some cases, the light chain comprises a human kappa constant region. In some cases, the light chain comprises a lambda constant region. In some cases, the light chain comprises a human lambda constant region.

[0131] In some embodiments, the antibodies described herein comprise a heavy chain comprising a VH described herein and a light chain comprising a VL described herein, wherein the heavy chain comprises an IgM (e.g., human IgM) constant region and the light chain comprises a κ (e.g., human κ) constant region. In some embodiments, the antibodies described herein comprise a heavy chain comprising a VH described herein and a light chain comprising a VL described herein, wherein the heavy chain comprises an IgM (e.g., human IgM) constant region and the light chain comprises a κ (e.g., human κ) constant region.

[0132] Alternatively, the antibodies described herein are antibodies or antigen-binding fragments, derivatives or variants thereof that compete for binding to PNAd with antibodies having VH CDRs 1-3 as shown in SEQ ID NOs: 3-5, respectively. Alternatively, the antibodies described herein are antibodies or antigen-binding fragments, derivatives or variants thereof that compete for binding to PNAd with antibodies having VL CDRs 1-3 as shown in SEQ ID NOs: 8-10, respectively. Alternatively, the antibodies described herein are antibodies or antigen-binding fragments, derivatives or variants thereof that compete for binding to PNAd with antibodies having VH CDRs 1-3 as shown in SEQ ID NOs: 3-5, respectively, and VL CDRs 1-3 as shown in SEQ ID NOs: 8-10, respectively. Alternatively, the antibodies described herein are antibodies or antigen-binding fragments, derivatives or variants thereof that compete for binding to PNAd with antibodies having VH and / or VL as shown in SEQ ID NOs: 2 and 7, respectively. These antibodies may be human, rodent (e.g., mouse), chimeric, or humanized, particularly for therapeutic applications.

[0133] Optionally, the antibody described herein is an antibody or antigen-binding fragment, derivative or variant thereof that binds to the same epitope as an anti-PNAd antibody containing the VH of SEQ ID NO:2 and the VL of SEQ ID NO:7.

[0134] Competition between antibodies is determined by an assay in which the test immunoglobulin inhibits specific binding of a reference antibody to a conventional antigen, such as PNAd. Various types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay; see Stahli et al., Methods in Enzymology 9 (1983), 242-253; solid phase direct biotin-avidin EIA; see Kirkland et al., J. Immunol. 137 (1986), 3614-3619 and Cheung et al., Virology 176 (1990), 546-552; solid phase direct labeling assay, solid phase direct labeling sandwich assay; see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor, 1990. Press (1988); direct solid-phase labeling RIA using I125 tags; see Morel et al., Molec.Immunol.25 (1988), 7-15 and Moldenhauer et al., Scand.J.Immunol.32 (1990), 77-82. Typically, such assays involve purified PNAd or aggregates thereof using cells bound to a solid surface or carrying any of these, unlabeled test immunoglobulins and labeled reference immunoglobulins, i.e., human monoclonal antibodies described herein. Competitive inhibition is determined by measuring the amount of labels bound to a solid surface or cells in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. In some embodiments, competitive binding assays are performed under conditions as described for ELISA assays in the accompanying examples. Antibodies identified by competition assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent antibodies sufficiently close to the epitope bound by the reference antibody to cause steric hindrance. Typically, when the competing antibody is present in excess, it will inhibit specific binding of the reference antibody to the conventional antigen by at least 50% or 75%. Thus, the present invention also relates to antibodies, or antigen-binding fragments, variants or derivatives thereof, wherein the antibody competitively inhibits binding of a reference antibody comprising VH of SEQ ID NO: 2 and VL of SEQ ID NO: 7 to PNad.

[0135] In some embodiments, provided herein are isolated polypeptides comprising an immunoglobulin heavy chain variable region (VH), wherein at least one of the VH-CDRs of the heavy chain variable region or at least two of the VH-CDRs of the heavy chain variable region are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference heavy chain VH-CDR1, VH-CDR2, or VH-CDR3 amino acid sequence from an antibody disclosed herein (see, e.g., Table 2 for VH CDR sequences). Alternatively, the VH-CDR1, VH-CDR2, and VH-CDR3 regions of the VH are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference heavy chain VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequence from an antibody disclosed herein (see, e.g., Table 2 for VH CDR sequences). Thus, the heavy chain variable regions disclosed herein may have VH-CDR1, VH-CDR2, and VH-CDR3 that are related to the sequences of Table 2 (i.e., SEQ ID NOs: 3-5). Although Table 2 shows VH-CDRs defined by the IMGT system, other CDR definitions, such as VH-CDRs defined by the Kabat or Chothia systems, may also be included in the present invention and can be easily identified by those skilled in the art using the sequences shown in Tables 2-4. In some embodiments, the amino acid sequence of the reference VH CDR1 is SEQ ID NO: 3; the amino acid sequence of the reference VH CDR2 is SEQ ID NO: 4; and the amino acid sequence of the reference VH CDR3 is SEQ ID NO: 5.

[0136] The isolated polypeptides described herein may comprise an immunoglobulin heavy chain variable region (VH), wherein the VH-CDR1, VH-CDR2, and VH-CDR3 regions have polypeptide sequences identical to the VH-CDR1, VH-CDR2, and VH-CDR3 sequences shown in Table 2 (i.e., SEQ ID NOs: 3-5). In some embodiments, the amino acid sequence of VH CDR1 is SEQ ID NO: 3; the amino acid sequence of VH CDR2 is SEQ ID NO: 4; and the amino acid sequence of VH CDR3 is SEQ ID NO: 5.

[0137] In addition, the isolated polypeptide may comprise an immunoglobulin heavy chain variable region (VH), wherein the VH-CDR1, VH-CDR2, and VH-CDR3 regions have polypeptide sequences identical to the VH-CDR1, VH-CDR2, and VH-CDR3 sequences shown in Table 2 (i.e., SEQ ID NOs: 3-5), except for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in any VH-CDR. In some embodiments, the amino acid substitutions are conservative. In some embodiments, the amino acid sequence of VH CDR1 is SEQ ID NO: 3; the amino acid sequence of VH CDR2 is SEQ ID NO: 4; and the amino acid sequence of VH CDR3 is SEQ ID NO: 5.

[0138] The isolated polypeptide may comprise an immunoglobulin light chain variable region (VL), wherein at least one of the VL-CDRs of the light chain variable region or at least two of the VL-CDRs of the light chain variable region are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference light chain VL-CDR1, VL-CDR2, or VL-CDR3 amino acid sequence from an antibody disclosed herein (see, e.g., Table 2 for VL CDR sequences). Alternatively, the VL-CDR1, VL-CDR2, and VL-CDR3 regions of the VL are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference light chain VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequence from an antibody disclosed herein (see, e.g., Table 2 for VL CDR sequences). Thus, the light chain variable region may have VL-CDR1, VL-CDR2, and VL-CDR3 polypeptide sequences related to the sequences of Table 2 (i.e., SEQ ID NOs: 8-10). Although Table 2 shows VH-CDRs defined by the IMGT system, other CDR definitions, such as VH-CDRs defined by the Kabat or Chothia systems, may also be included in the present invention. In some embodiments, the amino acid sequence of the reference VL CDR1 is SEQ ID NO: 8; the amino acid sequence of the reference VL CDR2 is SEQ ID NO: 9; and the amino acid sequence of the reference VL CDR3 is SEQ ID NO: 10.

[0139] Also provided herein are isolated polypeptides comprising an immunoglobulin light chain variable region (VL), wherein the VL-CDR1, VL-CDR2, and VL-CDR3 regions have polypeptide sequences identical to the VL-CDR1, VL-CDR2, and VL-CDR3 sequences of Table 2. In some embodiments, the amino acid sequence of VL CDR1 is SEQ ID NO: 8; the amino acid sequence of VL CDR2 is SEQ ID NO: 9; and the amino acid sequence of VL CDR3 is SEQ ID NO: 10.

[0140] The isolated polypeptide may also comprise an immunoglobulin light chain variable region (VL), wherein the VL-CDR1, VL-CDR2, and VL-CDR3 regions have the same polypeptide sequences as the VL-CDR1, VL-CDR2, and VL-CDR3 of Table 2, except for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in any VL-CDR. In some embodiments, the amino acid substitutions are conservative. In some embodiments, the amino acid sequence of VL CDR1 is SEQ ID NO: 8; the amino acid sequence of VL CDR2 is SEQ ID NO: 9; and the amino acid sequence of VL CDR3 is SEQ ID NO: 10.

[0141] Immunoglobulin or its encoding cDNA can be modified. Therefore, the method described herein may include any one of the steps (one or more) of producing chimeric antibodies, humanized antibodies, single-chain antibodies, Fab fragments, bispecific antibodies, fusion antibodies, labeled antibodies or analogs of any of these. The corresponding method is known to those skilled in the art and is described in, for example, Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor (1988). When the derivatives of the antibody are obtained by phage display technology, the surface plasmon resonance used in the BIAcore system can be used to increase the efficacy of phage antibodies bound to the same epitope as any of the antibodies described herein (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). The generation of chimeric antibodies is described in, for example, International Application WO89 / 09622. Methods for producing humanized antibodies are described in, for example, European application EP-A1 0 239 400 and international application WO90 / 07861. Further sources of antibodies used according to the present invention are referred to as xenoantibodies. The general principles for producing xenoantibodies such as human-like antibodies in mice are described in, for example, international applications WO91 / 10741, WO94 / 02602, WO96 / 34096 and WO96 / 33735. As described above, the antibodies described herein can exist in various forms other than complete antibodies; including, for example, Fv, Fab, and F(ab)2, and in the form of single chains; see, for example, international application WO88 / 09344. Antibodies produced by such methods are also provided herein.

[0142] The antibodies described herein or their corresponding immunoglobulin chains (one or more) may be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and / or recombination(s) and / or any other modification(s) known in the art, alone or in combination. Methods for introducing such modifications into the underlying DNA sequence of the amino acid sequence of the immunoglobulin chain are well known to those skilled in the art; see, for example, Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) NY and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1994). Modifications of the antibodies described herein include chemical and / or enzymatic derivatization at one or more of the constituent amino acids, including side chain modifications, backbone modifications, and N- and C-terminal modifications including acetylation, hydroxylation, methylation, amidation, and attachment of carbohydrate or lipid moieties, and cofactors. Similarly, the present invention includes the production of chimeric proteins comprising the described antibodies or some fragments thereof fused at the amino terminus to a heterologous molecule, such as an immunostimulatory ligand, at the carboxyl terminus; for details of the corresponding technology, see, for example, International Application WO00 / 30680.

[0143] In addition, provided herein are peptides including those containing binding molecules as described above, such as the CDR3 region containing the variable region of any of the aforementioned antibodies, particularly the CDR3 of the heavy chain, because it is often observed that the heavy chain CDR3 (HCDR3) is a region with a greater degree of variability and is primarily involved in antigen-antibody interactions. Such peptides can be easily synthesized or produced by recombinant means to produce binding agents as described herein. Such methods are known to those skilled in the art. Peptides can be synthesized, for example, using a commercially available automatic peptide synthesizer. Peptides can also be produced by introducing DNA expressing the peptide into an expression vector and transforming cells with the expression vector to produce a recombinant technique for producing the peptide.

[0144] Thus, described herein are binding molecules, e.g., that bind to the human anti-PNAd antibodies described herein and exhibit the properties mentioned, i.e., antibodies or binding fragments thereof that specifically identify (bind to) PNad. Such antibodies and binding molecules can be tested for binding specificity and affinity by ELISA and immunoblotting and immunohistochemistry as described herein, see, e.g., the Examples.

[0145] As an alternative to obtaining immunoglobulins directly from cultures of immortalized B cells or B memory cells, the immortalized cells described herein can be used as a source of rearranged heavy and light chain loci for subsequent expression and / or genetic manipulation. Rearranged antibody genes can be reverse transcribed from suitable mRNA to produce cDNA. As required, the heavy chain constant region can be exchanged or eliminated as a whole with the heavy chain constant region of different isotypes. Variable regions can be connected to encode single-chain Fv regions. Multiple Fv regions can be connected to confer binding ability to more than one target or chimeric heavy and light chain combinations can be used. Once genetic material is available, the design of the above-mentioned analogs that maintain their ability to bind to the desired target is clear. Methods for cloning antibody variable regions and producing recombinant antibodies are known to those skilled in the art and are described in, for example, Gilliland et al., Tissue Antigens 47 (1996), 1-20; Doenecke et al., Leukemia 11 (1997), 1787-1792.

[0146] Once the appropriate genetic material is obtained and modified as necessary to encode the analogs, the coding sequences, including those encoding the minimum variable regions of the heavy and light chains, can be inserted into an expression system contained on a vector transfected into a standard recombinant host cell. A variety of such host cells can be used; however, for efficient processing, mammalian cells are contemplated. Typical mammalian cell lines useful for this purpose include, but are not limited to, CHO cells, HEK 293 cells, or NSO cells.

[0147] Then, the production of antibodies or analogs begins with culturing the modified recombinant host under conditions suitable for the growth of host cells and the expression of the coding sequence. The antibody is then recovered by isolating the antibody from the culture. The expression system is designed to include a signal peptide so that the resulting antibody is secreted into the culture medium; however, intracellular production is also possible.

[0148] According to the above, the present invention also relates to polynucleotides encoding antibodies or equivalent binding molecules described herein. In some embodiments, the polynucleotide encodes at least the variable region of the immunoglobulin chain of the above-mentioned antibody. Typically, the variable region encoded by the polynucleotide comprises at least one complementary determining region (CDR) of the VH and / or VL of the variable region of the antibody.

[0149] Those skilled in the art will readily appreciate that the variable domains of antibodies with the above-mentioned variable domains can be used to construct other polypeptides or antibodies of desired specificity and biological functions. Therefore, polypeptides and antibodies comprising at least one or more CDRs, such as all CDRs, of the above-mentioned variable domains are provided herein, and advantageously have the same or similar binding properties as the antibodies described in the attached examples. It is known to those skilled in the art that binding affinity can be enhanced by amino acid substitutions in CDRs or in hypervariable loops overlapping with CDR portions as defined by Kabat (Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917); see, e.g., Riechmann, et al., Nature 332 (1988), 323-327. Therefore, antibodies wherein one or more of the CDRs mentioned herein comprise one or more, but not more than two amino acid substitutions are also provided herein. In some embodiments, the antibodies described herein comprise two or all three CDRs of the variable region as shown in Table 2 in one or two of their immunoglobulin chains.

[0150] As known to those skilled in the art, binding molecules such as antibodies described herein, or antigen-binding fragments, variants, or derivatives thereof may include a constant region that mediates one or more effector functions. For example, the binding of the C1 component of complement to the constant region of an antibody may activate the complement system. The activation of complement is important in opsonization and the lysis of cell pathogens. The activation of complement also stimulates immune responses and may involve autoimmune hypersensitivity reactions. In addition, antibodies bind to receptors on various cells through the Fc region, wherein the Fc receptor binding site on the antibody Fc region binds to the Fc receptor (FcR) on the cell. There are many Fc receptors that are specific for different types of antibodies, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors), and IgM (mu receptors). The binding of antibodies to Fc receptors on the cell surface triggers many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, killer cell lysis of antibody-coated target cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

[0151] Thus, some embodiments described herein include antibodies, or antigen-binding fragments, variants, or derivatives thereof, in which at least a portion of one or more constant region domains has been deleted or otherwise altered to provide desired biochemical properties, e.g., reduced effector function, ability to non-covalently dimerize, increased ability to localize at the site of a PNAd, reduced serum half-life, or increased serum half-life compared to an intact, unaltered antibody of approximately the same immunogenicity. For example, some antibodies used in the diagnostic and therapeutic methods described herein are domain-deleted antibodies comprising a polypeptide chain similar to an immunoglobulin heavy chain, but lacking at least a portion of one or more heavy chain domains. For example, in certain antibodies, a complete domain of the constant region of the modified antibody will be deleted, e.g., all or part of the CH2 domain is deleted. In other embodiments, certain antibodies used in the diagnostic and therapeutic methods described herein have a constant region, e.g., an IgM heavy chain constant region, that is altered to eliminate glycosylation, referred to elsewhere herein as aglycosylated or "agly" antibodies. Such "aglycosylated" antibodies can be prepared enzymatically as well as by engineering the common glycosylation site(s) in the constant region. Without being limited by theory, it is believed that "aglycosylated" antibodies may have improved in vivo safety and stability characteristics. Methods for producing aglycosylated antibodies with desired effector functions can be found, for example, in International Application WO2005 / 018572, which is incorporated herein in its entirety.

[0152] In certain antibodies described herein, or antigen-binding fragments, variants, or derivatives thereof, the Fc portion may be mutated using techniques known in the art to reduce effector function. For example, deletion or inactivation of the constant region domain (by point mutation or other methods) may reduce Fc receptor binding of circulating modified antibodies, thereby increasing PNAd localization. In other cases, constant region modifications consistent with the present invention mitigate complementary binding, thereby reducing serum half-life and nonspecific association of conjugated cytotoxins. However, other modifications of the constant region may be used to modify disulfide bonds or oligosaccharide moieties, allowing for enhanced localization due to increased antigen specificity or antibody flexibility. The resulting physiological properties, bioavailability, and other biochemical effects of the modifications, such as PNAd localization, biodistribution, and serum half-life, may be easily determined and quantified using known immunological techniques without excessive experimentation.

[0153] In some antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein, the Fc portion may be mutated or exchanged for alternative protein sequences to increase cellular uptake of the antibody, for example by enhancing receptor-mediated endocytosis of the antibody by Fcγ receptors, LRP, or Thy1 receptors, or by "SuperAntibody Technology" that is believed to enable antibodies to shuttle into living cells without damaging them (Expert Opin. Biol. Ther. (2005), 237-241). For example, the production of fusion proteins of the antibody binding region and the cognate protein ligand of a cell surface receptor or bispecific or multispecific antibodies having specific sequences that bind to PNad and a cell surface receptor can be engineered using techniques known in the art.

[0154] In some of the antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein, the Fc portion may be mutated or exchanged for an alternative protein sequence, or the antibody may be chemically modified to increase its blood-brain barrier penetration.

[0155] Modified forms of the antibodies described herein, or their antigen-binding fragments, variants, or derivatives can be made from complete precursors or parent antibodies using techniques known in the art. Exemplary techniques are discussed in more detail herein. The antibodies described herein, or their antibody-binding fragments, variants, or derivatives can be prepared or produced using techniques known in the art. In some embodiments, the antibody molecules or fragments thereof are "recombinantly produced," i.e., produced using recombinant DNA technology. Exemplary techniques for preparing antibody molecules or fragments thereof are described in more detail elsewhere herein.

[0156] The antibodies, or antibody binding fragments, variants, or derivatives described herein also include derivatives modified, for example, by covalent binding of any type of molecule to the antibody, such that the covalent binding does not prevent the antibody from specifically binding to its cognate epitope. For example, without being limited by theory, antibody derivatives include, for example, those modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protection / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, derivatives may include one or more atypical amino acids.

[0157] In some embodiments, the antibodies, or antigen binding fragments, variants, or derivatives described herein will not cause a harmful immune response in the treated animal, e.g., a human. In some embodiments, the binding molecules, e.g., antibodies, or antigen binding fragments thereof, described herein, are derived from a patient, e.g., a human patient, and subsequently used in the same species from which they were derived, e.g., a human, to reduce or minimize the occurrence of a harmful immune response.

[0158] Deimmunization can also be used to reduce the immunogenicity of antibodies. As used herein, the term "deimmunization" includes changing antibodies to modify T cell epitopes; see, for example, International Applications WO98 / 52976 and WO00 / 34317. For example, the VH and VL sequences from the starting antibody are analyzed and "mapped" from human T cell epitopes from each V region, showing the location of epitopes associated with complementary determining regions (CDRs) and other key residues within the sequence. Individual T cell epitopes from T cell epitope mapping are analyzed to identify alternative amino acid substitutions with low risk of changing the activity of the final antibody. A series of optional VH and VL sequences containing a combination of amino acid substitutions are designed, and these sequences are subsequently integrated into a series of binding polypeptides, such as PNad-specific antibodies or immunospecific fragments thereof, for use in the diagnosis and treatment methods described herein, and then tested for their function. Typically, variant antibodies between 12 and 24 are produced and tested. The complete heavy and light chain genes containing the modified V region and the human C region are then cloned into an expression vector and the plasmid is subsequently introduced into a cell line for the production of full antibodies. The antibodies are then compared in appropriate biochemical and biological assays to identify the optimal variant.

[0159] Monoclonal antibodies are prepared using a variety of techniques known in the art, including the use of hybridomas, recombination, and phage display techniques, or combinations thereof. For example, monoclonal antibodies are produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Second Edition (1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas Elsevier, NY, 563-681 (1981), which references are incorporated herein by reference in their entirety. The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, rather than a method for producing it. Therefore, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology.

[0160] In the well-known hybridoma method (Kohler et al., Nature 256 (1975), 495), relatively short-lived, or lethal lymphocytes from mammals, such as B cells derived from a murine subject as described herein, are fused with an immortal tumor cell line (e.g., a myeloma cell line), thereby producing hybrid cells or "hybridomas" that are immortal and capable of producing the genetically encoded antibodies of the B cells. The resulting hybrids are separated into single genetic strains by selection, dilution, and regrowth with individual strains containing specific genes for forming individual antibodies. They produce antibodies that are homologous to the desired antigen and are referred to as "monoclonal" in reference to their pure genetic lineage.

[0161] The hybridoma cells thus prepared are inoculated and grown in a suitable culture medium containing one or more substances that inhibit the growth or survival of unfused, parental myeloma cells. It will be appreciated by those skilled in the art that agents, cell lines and culture media for hybridoma formation, screening and growth are commercially available from many sources, and standardized methods are well established. Typically, the culture medium in which the hybridoma cells are grown is tested for the production of monoclonal antibodies against the desired antigen. The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) as described herein. After identifying hybridoma cells that produce antibodies of the desired specificity, affinity and / or activity, clones can be subcloned by limiting dilution and growth by standard methods; see, for example, Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp 59-103 (1986). It will be further appreciated that the monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites fluid, serum by conventional purification methods such as, for example, protein A, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0162] In some embodiments, lymphocytes can be selected and variable genes can be separated by micromanipulation. For example, peripheral blood mononuclear cells can be separated from immune or naturally immune mammals such as humans and cultured in vitro for about 7 days. The culture is screened for specific immunoglobulins that meet the screening conditions. Cells from positive wells can be separated. Single B cells that produce Ig can be separated by FACS or by identifying a single B cell that produces Ig in a complement-mediated hemolytic plaque assay. B cells that produce Ig can be micromanipulated to a tube and VH and VL genes can be amplified using, for example, RT-PCR. VH and VL genes can be cloned into antibody expression vectors and transfected into cells (e.g., eukaryotic or prokaryotic cells) for expression.

[0163] Alternatively, cell lines producing antibodies can be selected and cultured using techniques known to those skilled in the art. Such techniques are described in various laboratory manuals and original publications. In this regard, techniques suitable for the methods and compositions described hereinafter are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), which is incorporated herein by reference in its entirety, including supplementary files.

[0164] Antibody fragments that identify specific epitopes can be produced by known techniques. For example, Fab and F(ab')2 fragments can be produced recombinantly using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments) or by proteolytic cleavage of immunoglobulin molecules. F(ab')2 fragments contain the variable region, the constant region of the light chain, and the CH1 domain of the heavy chain. Such fragments are sufficient for use, for example, in immunodiagnostic procedures involving conjugating the immunospecific portion of an immunoglobulin to a detection agent such as a radioisotope.

[0165] In some embodiments, the antibodies described herein include at least one heavy chain or light chain CDR of an antibody molecule. In another embodiment, the antibodies described herein include at least two CDRs from one or more antibody molecules. In another embodiment, the antibodies described herein include at least three CDRs from one or more antibody molecules. In another embodiment, the antibodies described herein include at least four CDRs from one or more antibody molecules. In another embodiment, the antibodies described herein include at least five CDRs from one or more antibody molecules. In another embodiment, the antibodies described herein include at least six CDRs from one or more antibody molecules. Exemplary antibody molecules include at least one CDR that can be included in the subject antibody described herein.

[0166] The antibodies described herein can be produced by any method known in the art for synthesizing antibodies, in particular, by chemical synthesis or recombinant expression techniques as described herein.

[0167] In some embodiments, the antibodies described herein, or antigen-binding fragments, variants, or derivatives thereof, comprise synthetic constant regions in which one or more domains are partially or completely deleted ("domain-deleted antibodies"). In some embodiments, compatible modified antibodies will comprise domain-deleted constructs or variants in which the entire CH2 domain has been removed. For other embodiments, the deleted domains may be replaced by short connecting peptides to provide flexibility and freedom of movement of the variable region. Those skilled in the art will appreciate that such constructs are particularly preferred due to the regulatory properties of the CH2 domain on the catabolism rate of the antibody. Domain-deleted constructs can be obtained using vectors encoding IgG1 human constant domains, see, e.g., International Applications WO02 / 060955 and WO02 / 096948A2. The vector is engineered to delete the CH2 domain and provide a synthetic vector for expressing a domain-deleted IgG1 constant region.

[0168] In some embodiments, the antibodies described herein, or antigen-binding fragments, variants, or derivatives thereof, are minibodies. Minibodies can be prepared using methods described in the art, see, for example, U.S. Pat. No. 5,837,821 or International Application No. WO 94 / 09817.

[0169] In some embodiments, the antibodies described herein, or their antigen-binding fragments, variants, or derivatives, comprise immunoglobulin heavy chains with deletions or substitutions of some or even single amino acids, as long as it allows association between monomer subunits and maintains binding to PNAd. For example, mutations of single amino acids in selected regions of the CH2 domain may be sufficient to substantially reduce Fc binding, thereby increasing PNAd localization. Similarly, it may be desirable to simply delete a portion of one or more constant region domains that control effector functions to be regulated (e.g., complement binding). Such partial deletions of the constant region may improve the selected properties of the antibody while leaving other desired functions associated with the target constant region domain intact. In addition, as described above, the constant region of the disclosed antibody may be synthesized by mutation or substitution of one or more amino acids that enhance the properties of the resulting construct. In this regard, the activity provided by the conserved binding site (e.g., Fc binding) may be destroyed while substantially maintaining the configuration and immunogenicity characteristics of the modified antibody. Still another embodiment includes adding one or more amino acids to the constant region to enhance desired properties such as effector functions or to provide more cytotoxins or carbohydrate attachment. In such embodiments, it may be desirable to insert or replicate specific sequences derived from selected constant region domains.

[0170] The present invention also provides antibodies comprising variants (including derivatives) of antibody molecules described herein (e.g., VH regions and / or VL regions), consisting essentially of them, or consisting of them, wherein the antibodies or fragments thereof immunospecifically bind to PNads. Standard techniques known to those skilled in the art can be used to introduce mutations in the nucleotide sequence encoding the antibody, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. In some embodiments, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to a reference VH region, VH-CDR1, VH-CDR2, VH-CDR3, VL region, VL-CDR1, VL-CDR2, or VL-CDR3. Alternatively, mutations can be introduced randomly into all or part of the coding sequence, e.g., by saturation mutagenesis, and the resultant mutants screened for biological activity to identify mutations that maintain activity (e.g., the ability to bind PNad).

[0171] For example, mutations can be introduced only in the framework region of the antibody molecule or only in the CDR region. The introduced mutations can be silent mutations or natural missense mutations, for example, no effect on the ability of the antibody to bind to antigen, or have less effect, in fact, some such mutations will not change any amino acid sequence. These types of mutations can be useful for optimizing codon usage or improving antibody production in hybridomas. Codon-optimized coding regions encoding antibodies described herein are disclosed elsewhere herein. Alternatively, non-natural missense mutations can change the ability of antibodies to bind to antigens. The positioning of most silent mutations or natural missense mutations may be in the framework region, while the positioning of most non-natural missense mutations may be in the CDR, although this is not an absolute requirement. Those skilled in the art can design and test mutant molecules with desired properties such as no change in antigen binding activity or no change in binding activity (e.g., improvement in antigen binding activity or change in antibody specificity). After mutagenesis, the encoded protein can be conventionally expressed, and the function and / or biological activity of the encoded protein (e.g., the ability to immunospecifically bind to at least one epitope of PNad) can be determined using the techniques described herein or by conventional modification techniques known in the art.

[0172] Agents that bind to PNad, such as, but not limited to, antibodies that bind to PNad described herein, can be characterized using any in vivo or in vitro model. It is readily understood by those skilled in the art that agents that bind to PNad described herein (e.g., antibodies) can be characterized in the mouse model described herein.

[0173] III. Polynucleotides encoding antibodies

[0174] Also provided herein are polynucleotides encoding antibodies described herein, or their Fabs, variants, or derivatives. Polynucleotides encoding antibodies, or their Fabs, variants, or derivatives may be composed of any polyribonucleotides or polydeoxyribonucleotides, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides encoding antibodies, or their Fabs, variants, or derivatives may be composed of single-stranded or double-stranded DNA, DNA as a mixture of single-stranded or double-stranded regions, single-stranded and double-stranded RNA, and RNA as a mixture of single-stranded or double-stranded regions, hybrid molecules comprising DNA and RNA, which hybrid molecules may be single-stranded or, more typically, double-stranded or a mixture of single-stranded and double-stranded regions. In addition, polynucleotides encoding antibodies, or their Fabs, variants, or derivatives may be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. Polynucleotides encoding antibodies, or their Fabs, variants, or derivatives may also include one or more modified bases or DNA or RNA backbones modified for stability or for other purposes. "Modified" bases include, for example, triacylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA; thus, "polynucleotide" includes chemically, enzymatically, or metabolically modified forms.

[0175] The isolated polynucleotide encoding a non-natural variant of a polypeptide derived from an immunoglobulin (e.g., an immunoglobulin heavy chain portion or a light chain portion) can be produced by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin so that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques such as site-directed mutagenesis or PCR-mediated mutagenesis. In some embodiments, conservative amino acid substitutions are made at one or more non-essential amino acid residues.

[0176] As is well known, RNA can be extracted and precipitated by guanidine isothiocyanate, followed by centrifugation or chromatography, from original B cells, hybridoma cells, or from other transformed cells by standard techniques. Desirably, RNA can be separated from total RNA by standard techniques, for example, chromatography to oligo dT cellulose. Suitable technology is familiar in the art. In some embodiments, the cDNA of the light chain and heavy chain of the encoding antibody can be prepared according to well-known methods using reverse transcriptase or DNA polymerase, simultaneously or separately. PCR can be initiated by a common constant region primer based on the amino acid sequence of disclosed heavy chain and light chain DNA or by a more specific primer. As discussed above, PCR can also be used for separating DNA clones encoding antibody light chain and heavy chain. In this case, the library can be screened by a common primer or, for example, a larger homologous probe such as a human constant region primer.

[0177] DNA, typically plasmid DNA, can be isolated from cells using techniques known in the art, restriction mapped and sequenced according to standard well-known techniques such as those described in detail in the aforementioned references relating to recombinant DNA technology. Of course, DNA can be synthesized according to the present invention at any point during the isolation process or subsequent analysis.

[0178] In some embodiments, provided herein is an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH), wherein at least one CDR of the heavy chain variable region or at least two VH-CDRs of the heavy chain variable region are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference heavy chain VH-CDR1, VH-CDR2, or VH-CDR3 amino acid sequence from an antibody disclosed herein. Alternatively, the VH-CDR1, VH-CDR2, or VH-CDR3 region of the VH is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference heavy chain VH-CDR1, VH-CDR2, or VH-CDR3 amino acid sequence from an antibody disclosed herein. Thus, according to this embodiment, the heavy chain variable region of the antibodies or antigen-binding fragments thereof described herein has Figure 5 In some embodiments, the amino acid sequence of the reference VH CDR1 is SEQ ID NO:3; the amino acid sequence of the reference VH CDR2 is SEQ ID NO:4; and the amino acid sequence of the reference VH CDR3 is SEQ ID NO:5.

[0179] In some embodiments, provided herein are isolated polynucleotides comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH), wherein the VH-CDR1, VH-CDR2, and VH-CDR3 regions have the same or similar amino acid sequence as those of the immunoglobulin heavy chain variable region (VH), except for one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in any VH-CDR. Figure 5 In some embodiments, the amino acid sequence of VH CDR1 is SEQ ID NO:3; the amino acid sequence of VH CDR2 is SEQ ID NO:4; and the amino acid sequence of VH CDR3 is SEQ ID NO:5.

[0180] In another embodiment, provided herein is an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL), wherein at least one of the VL-CDRs of the light chain variable region or at least two of the VL-CDRs of the VL-CDRs of the light chain variable region are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference light chain VL-CDR1, VL-CDR2, or VL-CDR3 amino acid sequence from an antibody disclosed herein. Alternatively, the VL-CDR1, VL-CDR2, or VL-CDR3 region of the VL is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference light chain VL-CDR1, VL-CDR2, or VL-CDR3 amino acid sequence from an antibody disclosed herein. Thus, according to this embodiment, the light chain variable region of the antibodies or antigen-binding fragments thereof described herein has Figure 5 In some embodiments, the amino acid sequence of the reference VL CDR1 is SEQ ID NO: 8; the amino acid sequence of the reference VL CDR2 is SEQ ID NO: 9; and the amino acid sequence of the reference VL CDR3 is SEQ ID NO: 10.

[0181] In another embodiment, provided herein is an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL), wherein the VL-CDR1, VL-CDR2, and VL-CDR3 regions have the same amino acid sequence as that of the VH-CDR except for one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in any VH-CDR. Figure 5In some embodiments, the amino acid sequence of VL CDR1 is SEQ ID NO: 8; the amino acid sequence of VL CDR2 is SEQ ID NO: 9; and the amino acid sequence of VL CDR3 is SEQ ID NO: 10.

[0182] In another embodiment, provided herein is an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH), wherein the VH-CDR1, VH-CDR2, and VH-CDR3 regions have the same Figure 5 In some embodiments, the amino acid sequence of VH CDR1 comprises SEQ ID NO: 3; the amino acid sequence of VH CDR2 comprises SEQ ID NO: 4; and the amino acid sequence of VH CDR3 comprises SEQ ID NO: 5. In some embodiments, the amino acid sequence of VH CDR1 consists of SEQ ID NO: 3; the amino acid sequence of VH CDR2 consists of SEQ ID NO: 4; and the amino acid sequence of VH CDR3 consists of SEQ ID NO: 5.

[0183] In another embodiment, provided herein is an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL), wherein the VL-CDR1, VL-CDR2, and VL-CDR3 regions have the same Figure 5 In some embodiments, the amino acid sequence of VL CDR1 is SEQ ID NO: 8; the amino acid sequence of VL CDR2 is SEQ ID NO: 9; and the amino acid sequence of VL CDR3 is SEQ ID NO: 10. In some embodiments, the amino acid sequence of VL CDR1 consists of SEQ ID NO: 8; the amino acid sequence of VL CDR2 consists of SEQ ID NO: 9; and the amino acid sequence of VL CDR3 consists of SEQ ID NO: 10.

[0184] As known in the art, " sequence identity " between two polypeptides or two polynucleotides is determined by comparing the amino acid or nucleotide sequence of a polypeptide or polynucleotide with the sequence of a second polypeptide or polynucleotide. When discussed in this article, methods known in the art and computer programs / software can be used to determine whether any particular polypeptide has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity with another polypeptide, for example, but not limited to, BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489 local homology algorithm to find the best homology fragment between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence described herein (e.g., an antibody or antigen-binding fragment thereof described herein), the parameters are, of course, set so that the entire length of the reference polypeptide sequence is covered to calculate the percent identity and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.

[0185] In some embodiments, the polynucleotide comprises, consists essentially of, or consists of a nucleic acid having a polynucleotide sequence of a VH or VL region of an anti-PNAd antibody shown in Table 4. In this regard, it is readily understood by those skilled in the art that a polynucleotide encoding at least a light chain and / or a heavy chain variable domain may encode the variable domains of two immunoglobulin chains or only one immunoglobulin chain. In addition, provided herein are polynucleotides comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID: 2 (e.g., a nucleotide sequence of SEQ ID NO: 1) and / or a nucleotide sequence encoding an amino acid sequence of SEQ ID: 7 (e.g., a nucleotide sequence of SEQ ID NO: 6), or consisting thereof. In a specific embodiment, provided herein are polynucleotides comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID: 2 (e.g., a nucleotide sequence of SEQ ID NO: 1), or consisting thereof. In another specific embodiment, provided herein are polynucleotides comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID: 7 (e.g., a nucleotide sequence of SEQ ID NO: 6), or consisting thereof.

[0186] In some embodiments, provided herein are isolated polynucleotides comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 95% identical to a reference heavy chain VH. In some embodiments, the amino acid sequence of the reference heavy chain variable region is SEQ ID NO: 2.

[0187] In some embodiments, provided herein are isolated polynucleotides comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 95% identity to a reference light chain VL. In some embodiments, the amino acid sequence of the reference light chain variable region is SEQ ID NO:7.

[0188] Also provided herein are fragments of the polynucleotides described herein, as described elsewhere. In addition, polynucleotides encoding fusion polynucleotides, Fab fragments or other variants as described herein are also included.

[0189] Polynucleotides can be produced or prepared by any method known in the art. For example, if the nucleotide sequence of the antibody is known, the polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides, for example, as described in Kutmeier et al., BioTechniques 17 (1994), 242, which in brief involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and connecting these oligonucleotides, and then amplifying the connected oligonucleotides by PCR.

[0190] Alternatively, polynucleotides encoding antibodies, or antigen-binding fragments, variants, or derivatives thereof, can be generated from nucleic acids from a suitable source. If a clone containing nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, the nucleic acid encoding the antibody can be chemically synthesized or amplified by PCR using synthetic primers that can hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for a particular gene sequence to identify, for example, cDNA clones from a cDNA library encoding the antibody to obtain nucleic acids from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing a PNAd-specific antibody, such as a hybridoma cell selected to express the antibody, or nucleic acids isolated from any tissue or cell expressing a PNAd-specific antibody, such as a hybridoma cell selected to express the antibody, preferably polyA+RNA). The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

[0191] Once the nucleotide sequence or corresponding amino acid sequence of an antibody, or antigen-binding fragment, variant, or derivative thereof is determined, the nucleotide sequence can be manipulated using methods well known in the art for manipulating nucleotide sequences, e.g., recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (see, e.g., the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1990) and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998), both of which are incorporated herein by reference in their entireties), to generate antibodies with different amino acid sequences, e.g., to generate amino acid substitutions, deletions, and / or insertions.

[0192] IV. Expression of Antibody Polypeptides

[0193] After operating the isolated genetic material to provide the antibodies described herein, or their antigen-binding fragments, variants, or derivatives, the polynucleotides encoding the antibodies are usually inserted into expression vectors to be introduced into host cells that can be used to produce the desired amount of antibodies. The recombinant expression of antibodies, or their fragments, derivatives, or analogs, such as the heavy or light chains of antibodies that bind to target molecules, is described herein. Once polynucleotides encoding antibody molecules described herein or heavy or light chains of antibodies, or parts thereof (preferably containing heavy or light chain variable domains) have been obtained, vectors for producing antibody molecules are produced by recombinant DNA techniques using techniques well known in the art. Therefore, methods for preparing proteins by expressing polynucleotides containing antibodies encoding nucleotide sequences are described herein. Methods known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and suitable transcription and translation control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. Therefore, nucleotide sequences comprising operably connected to promoters encoding antibody molecules described herein, or their antigen-binding fragments, or their heavy or light chains, or heavy or light chain variable domains are provided herein. Such vectors may include nucleotide sequences encoding the constant region of an antibody molecule (see, e.g., International Applications WO 86 / 05807 and WO 89 / 01036; and U.S. Pat. No. 5,122,464), and the variable domains of an antibody may be cloned into such vectors for expression of complete heavy or light chains.

[0194] The term "vector" or "expression vector" used herein refers to a vector used as a carrier for introducing a host cell and expressing a desired gene in a host cell according to the methods described herein. It is known to those skilled in the art that such vectors can be easily selected from the group consisting of plasmids, phages, viruses and retroviruses. Generally, the vector described herein will include a selection marker, a suitable restriction site to facilitate the cloning of the desired gene, and the ability to enter eukaryotic or prokaryotic cells and / or replicate in eukaryotic and prokaryotic cells. Many expression vector systems can be used. For example, a class of vectors utilizes DNA elements derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV or MOMLV) or SV40 virus. Others relate to the use of a polycistronic system with an internal ribosome binding site. In addition, cells that have been integrated into their chromosomes by DNA can be selected by introducing one or more markers that allow the selection of transfected host cells. Markers can provide prototrophy, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper for immunodeficient hosts. The selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. In order to optimize the synthesis of mRNA, additional elements are required. These elements may include signal sequences, splicing signals, and transcription promoters, enhancers, and terminator signals.

[0195] In a specific embodiment, the cloned variable region gene can be inserted into an expression vector together with the heavy chain and light chain constant region genes (e.g., human heavy chain and light chain constant region genes) as discussed above. Any expression vector that can cause expression in eukaryotic cells can be used. The example of a suitable vector includes, but is not limited to, plasmid pcDNA3, pHCMV / Zeo, pCR3.1, pEF1 / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAX1, and pZeoSV2 (available from Invitrogen, San Diego, CA) and plasmid pCI (available from Promega, Madison, WI). Generally, if immunoglobulin heavy chain and light chain are routine experiments, such as carried out by a robotic system, a large number of transformed cells are screened for expression of appropriately high levels of those. In U.S. Patent Nos. 5,736,137 and 5,658,570, vector systems are also taught, each of which is incorporated herein by reference in its entirety. This system provides high level expression, eg, >30 pg / cell / day.Other exemplary vector systems are disclosed in, eg, US Pat. No. 6,413,777.

[0196] In other embodiments, antibodies described herein, or their Fabs, variants, or derivatives can be expressed using polycistronic constructs such as those disclosed in U.S. Patent Application Publication No. 2003-0157641A1, which are incorporated herein by reference in their entirety. In these expression systems, multiple target gene products such as the heavy and light chains of antibodies can be produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of antibodies. Compatible IRES sequences are disclosed in U.S. Patent No. 6,193,980, which are also incorporated herein. Those skilled in the art will appreciate that such expression systems can be used to effectively produce a full range of antibodies disclosed in this application.

[0197] More specifically, once a vector or DNA sequence encoding a monomeric subunit of an antibody has been prepared, the expression vector can be introduced into a suitable host cell. Introduction of the plasmid into a host cell can be accomplished by a variety of techniques well known to those skilled in the art. These include, but are not limited to, the use of, for example, The invention relates to the invention of the invention to the transfection of liposome transfection of lipofectamine, protoplast fusion, calcium phosphate precipitation, cell and envelope DNA fusion, microinjection and infection by complete virus. Generally, plasmid is introduced into host by standard calcium phosphate coprecipitation method. Host cells with expression construct grow under the conditions suitable for producing light chain and heavy chain, and detect heavy chain and / or light chain protein synthesis. Exemplary detection techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or fluorescence activated cell sorter analysis (FACS) and immunohistochemistry etc.

[0198] The expression vector is transferred to a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce antibodies for use in the methods described herein. Therefore, a host cell comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof, or a heavy chain or light chain thereof, operably linked to a heterologous promoter is also provided herein. In a specific embodiment, in order to express a double-chain antibody, the vector encoding the heavy chain and the light chain can be co-expressed in a host cell to express a complete immunoglobulin molecule, as described in detail below.

[0199] Host cells can be co-transfected with two expression vectors, a first vector encoding a heavy chain derived polypeptide and a second vector encoding a light chain derived polypeptide as described herein. The two vectors may contain the same selective marker, which can equally express heavy and light chain polypeptides. Alternatively, a single vector encoding both heavy and light chain polypeptides can be used. In such a case, the light chain is advantageously placed after the heavy chain to avoid excess non-toxic heavy chain; see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad. Sci. USA 77 (1980), 2197. The coding sequences of the heavy and light chains may comprise cDNA or genomic DNA.

[0200] As used herein, "host cell" refers to a cell having a vector constructed using recombinant DNA technology and encoding at least one heterologous gene. In the description of the process of isolating antibodies from a recombinant host, unless otherwise specifically indicated, the terms "cell" and "cell culture" can be used interchangeably to indicate the source of the antibody. In other words, the recovery of polypeptides from "cells" can refer to recovery from centrifuged whole cells, or from cell cultures containing culture medium and suspended cells.

[0201] Various host-expression vector systems can be used to express the antibody molecules used in the methods described herein. Such host-expression systems represent vectors that can produce and subsequently purify the target coding sequence, but also represent cells that can in situ express the antibody molecules described herein when transformed or transfected with the appropriate nucleotide coding sequence. These include, but are not limited to, bacteria (e.g., E. coli, Bacillus subtilis) transformed by recombinant phage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) and other microorganisms transformed by recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected by recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; virus, CaMV; tobacco mosaic virus, TMV) infected or transformed by a recombinant plasmid expression vector (e.g., Ti plasmid) containing an antibody coding sequence; or a mammalian cell system (e.g., COS, CHO, NSO, BLK, 293, 3T3 cells) with a recombinant expression construct containing a promoter (e.g., metallothionein promoter) derived from the genome of a mammalian cell, or a promoter (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter) derived from a mammalian virus. In some embodiments, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, are used to express recombinant antibody molecules, particularly for the expression of fully recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, combined with, for example, a major immediate early gene promoter element from human cytomegalovirus, are effective expression systems for antibodies, see, for example, Foecking et al., Gene 45 (1986), 101; Cockett et al., Bio / Technology 8 (1990), 2.

[0202] Host cell lines used for protein expression are usually mammalian in origin; those skilled in the art are considered to have the ability to determine that a particular host cell line is most suitable for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, CHO (Chinese hamster ovary), DG44 and DUXB11 (Chinese hamster ovary lines, DHFR defective (minus)), HELA (human cervical cancer), CVI (monkey kidney line), COS (derivative of CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, WI38, R1610 (Chinese hamster fibroblasts) BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney line), SP2 / O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes) and 293 (human kidney). In a specific embodiment, the host cell line is a CHO or 293 cell. Host cell lines are commonly available from commercial suppliers, the American Tissue Culture Collection, or from the published literature.

[0203] In addition, host cell strains that regulate the expression of inserted sequences or modify and process gene products in a desired specific form can be selected. Modification (e.g., glycosylation) and processing (e.g., cutting) of such protein products are important for the function of the protein. Different host cells can have features and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure the correct modification and processing of the exogenous proteins expressed. For this reason, eukaryotic host cells with appropriate processing, glycosylation of gene products, and phosphorylation of cellular mechanisms for primary transcripts can be used.

[0204] For the long-term, high-yield production of recombinant proteins, preferably stable expression.For example, the cell line of the engineered stable expression antibody molecule.Rather than using the expression vector comprising the viral replication origin, the host cell can be transformed by the DNA and the selective marker controlled by suitable expression control elements (for example, promoter, enhancer, sequence, transcription terminator, polyadenylation (polyadenylation) site, etc.).After introducing foreign DNA, the engineered cells can be allowed to grow 1-2 days in the enriched culture medium, then switch to the selected culture medium. The selective marker in the recombinant plasmid gives the tolerance to selection and allows the cells to stably integrate the plasmid into their chromosome and grow to form a focus (foci), then can be cloned and expanded into a cell line.The method can be advantageously used for the cell line of the engineered stable expression antibody molecule.

[0205] Various selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., Cell 11 (1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48 (1992), 202), and adenine phosphoribosyltransferase (Lowy et al., Cell 22 (1980), 817) genes for tk-, hgprt-, or aprt- cells, respectively. In addition, antimetabolite resistance can be used as the basis for selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77 (1980), 357; O'Hare et al., Proc. Natl. Acad. Sci. USA 78 (1981), 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78 (1981), 2072); neo, which confers resistance to the aminoglycoside G-418 (Goldspiel et al., Clinical Pharmacy 12 (1993), 488-505; Wu and Wu, Biotherapy 3 (1991), 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32 (1993), 573-596; Mulligan, Science 260 (1993), 926-932; and Morgan and Anderson, Ann. Rev. Biochem. 62 (1993), 191-217; TIB TECH 11 (1993), 155-215; and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30 (1984), 147.Conventional methods of recombinant DNA technology known in the art that can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated herein by reference in their entirety.

[0206] The expression level of antibody molecules can be increased by vector amplification. For a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987). When the marker in the vector system expressing the antibody is amplifiable, an increase in the level of inhibitors present in the culture of the host cell will increase the copy number of the marker gene. Since the amplified region is related to the antibody gene, the production of the antibody will also increase; see Crouse et al., Mol. Cell. Biol. 3 (1983), 257.

[0207] In vitro production allows expansion to provide a large amount of desired polypeptides. The technology for mammalian cell culture under tissue culture conditions is known in the art and includes, for example, uniform suspension culture in an airlift reactor or in a continuous agitator reactor, or, for example, in a hollow fiber, microcapsule, immobilized or trapped cell culture on agar microbeads or ceramic cartridges. If necessary and / or desired, solutions of purified polypeptides can be obtained by common chromatography, for example, gel filtration, ion exchange chromatography, chromatography on DEAD-cellulose or (immunity) affinity chromatography, for example, after the preferential biosynthesis of a synthetic hinge region polypeptide or before or after the HIC chromatography described herein.

[0208] Genes encoding the antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein may also be expressed in non-mammalian cells such as bacteria or insect or yeast or plant cells. Bacteria that readily take up nucleic acids include members of the Enterobacteriaceae family, for example, strains of Escherichia coli or Salmonella; Bacillaceae, for example, Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will be further understood that when expressed in bacteria, heterologous polypeptides typically become part of inclusion bodies. Heterologous polypeptides must be isolated, purified, and then assembled into functional molecules. When a tetravalent form of the antibody is desired, the subunits will self-assemble into a tetravalent antibody; see, for example, International Application WO02 / 096948.

[0209] In bacterial systems, many expression vectors can be advantageously selected according to the intended use of the molecule of the expressed antibody. For example, when a large amount of such proteins are produced, when used to produce a pharmaceutical composition of an antibody molecule, it is desirable to instruct a vector for high-level expression of a fusion protein product that is easily purified. Such vectors include, but are not limited to, Escherichia coli expression vector pUR278 (Ruther et al., EMBO J.2 (1983), 1791), in which the antibody coding sequence can be individually connected to a vector with a framework of a lacZ coding region to produce a fusion protein; pIN vector (Inouye & Inouye, Nucleic Acids Res. 13 (1985), 3101-3109; Van Heeke & Schuster, J. Biol. Chem. 24 (1989), 5503-5509); etc. pGEX vectors can also be used to express exogenous polypeptides as fusion proteins with glutathione S-transferase (GST). Typically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to a glutathione-agarose bead matrix followed by elution in the presence of free glutathione. The pGEX vector is designed to include a thrombin or factor Xa protease cleavage site so that the cloned target gene product can be released from the GST moiety.

[0210] In addition to prokaryotes, eukaryotic microorganisms can also be used. Among eukaryotic microorganisms, Saccharomyces cerevisiae or common baker's yeast is most commonly used, although a large number of other strains are generally available, for example, Pichia pastoris. For expression in Saccharomyces, plasmid YRp7 (Stinchcomb et al., Nature 282 (1979), 39; Kingsman et al., Gene 7 (1979), 141; Tschemper et al., Gene 10 (1980), 157) is generally used. This plasmid already contains the TRP1 gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan (ATCC No. 44076 or PEP4-1 (Jones, Genetics 85 (1977), 12)). The presence of trp1 damage as a feature of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0211] In insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) is often used as a vector to express foreign genes. The virus is grown in Spodoptera frugiperda cells. Antibody coding sequences can be cloned individually into non-essential regions of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0212] Once the antibodies described herein have been recombinantly expressed, the whole antibodies described herein, their dimers, individual light and heavy chains, or other immunoglobulin forms can be purified according to standard procedures in the art, including, for example, by chromatography (e.g., ion exchange, affinity, particularly affinity for specific antigens after protein A, and sizing column chromatography), centrifugation, differential solubility, such as ammonium sulfate precipitation, or by any other standard technique for purifying proteins; see, e.g., Scopes, "Protein Purification", Springer Verlag, NY (1982). Alternatively, another method for increasing the affinity of antibodies described herein is disclosed in U.S. Patent Publication 2002-0123057 A1.

[0213] V. Fusion Proteins and Conjugates

[0214] In some embodiments, the antibody polypeptide includes an amino acid sequence or one or more parts that are not usually combined with the antibody. Exemplary modifications are described in more detail below. For example, the single-chain Fv antibody fragment described herein may include a flexible linker sequence, or may be modified to add a functional part (e.g., PEG, a drug, a toxin, or a label such as fluorescent, radioactive, enzyme, nuclear magnetic, and heavy metals, etc.).

[0215] The antibody polypeptides described herein may comprise, consist essentially of, or consist of a fusion protein. A fusion protein may be an immunoglobulin PNAd binding domain comprising, for example, at least one target binding site and at least one heterologous portion, i.e., a portion that is not naturally connected to it in nature. Amino acid sequences may typically be present in separate proteins that are aggregated together in a fusion polypeptide, or they may typically be present in the same protein but placed in a new arrangement in a fusion polypeptide. Fusion proteins may be produced, for example, by chemical synthesis, or by generating and translating polynucleotides in which peptide regions are encoded in a desired relationship.

[0216] The term "heterologous" as applied to polynucleotides and polypeptides means that the polynucleotide or polypeptide is derived from an entity that is different from the entity of the rest of the entity being compared. For example, as used herein, a "heterologous polypeptide" fused to an antibody or antigen-binding fragment, variant or analog thereof is derived from a non-immunoglobulin polypeptide of the same species, or an immunoglobulin or non-immunoglobulin polypeptide of a different species.

[0217] As discussed in more detail elsewhere herein, the antibodies described herein, or antigen-binding fragments, variants, or derivatives thereof, can be further recombinantly fused to heterologous polypeptides or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions at the N- or C-terminus. For example, the antibodies can be recombinantly fused or conjugated to molecules used as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins; see, e.g., International Applications WO92 / 08495; WO91 / 14438; WO89 / 12624; U.S. Patent No. 5,314,995; and European Patent Application EP 0 396 387.

[0218] In some cases, the antibody or its Fab is conjugated to the drug. The method of producing such drug conjugates is known in the art and described herein (see, for example, Examples 7-9 below). In some cases, the drug is an immunosuppressive drug (for examples of immunosuppressive drugs, see, for example, the following chapters "immunosuppressive or immunomodulatory drugs"). In some cases, the drug is paclitaxel. In some cases, the drug is directly conjugated to the antibody or its Fab. In some cases, the drug is conjugated to the antibody or its Fab by a linker. Examples of linkers that can be used to conjugate the drug to the antibody or its Fab described herein include sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) or maleimidoacetyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-vc-PAB). In some cases, the drug is covalently conjugated to the antibody or its Fab. The covalent conjugation of the drug to the antibody or its Fab can be performed as described in the Examples section below. For example, the drug can be chemically modified using, for example, glutaraldehyde to generate a carboxyl group and then activated using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / N-hydroxysulfosuccinimide (NHS) chemistry; the resulting drug preferentially reacts with (i.e., conjugates to) primary amines on the antibody or antigen-binding fragment thereof (e.g., those on lysine).

[0219] The antibodies described herein, or their antigen-binding fragments, variants, or derivatives may be composed of amino acids connected to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than 20 gene-encoded amino acids. Antibodies may be modified by natural processes such as post-translational processing, or by chemical modification techniques well known in the art. Such modifications are well described in basic textbooks and more detailed monographs as well as a large number of research documents. Modifications may occur anywhere in the antibody, including peptide backbones, amino acid side chains, and amino or carboxyl termini, or on moieties such as carbohydrates. It should be understood that the same type of modification may exist at multiple sites in a given antibody to the same or different extents. In addition, a given antibody may contain many types of modifications. For example, as a result of ubiquitination, antibodies may be branched, and they may be cyclic with or without branches. Circular, branched, and branched cyclic antibodies may be derived from post-translational natural processing or may be prepared by synthetic methods. Modifications include acetylation, acylation, ADP ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation and ubiquitination; see, e.g., Proteins-Structure And Molecular Properties, TECreighton, WH Freeman and Company, New York 2nd ed., (1993); Posttranslational Covalent Modification Of Proteins, BC Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182 (1990), 626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).

[0220] Also provided herein are fusion proteins comprising antibodies, or antigen-binding fragments, variants, or derivatives thereof, and heterologous polypeptides. In some embodiments, the fusion protein comprises a polypeptide having any one or more of the amino acid sequences of the VH region of an antibody described herein or any one or more of the amino acid sequences of the VL region of an antibody or its fragment or variant described herein, and a heterologous polypeptide sequence, and is essentially composed of it, or is composed of it. In another embodiment, the fusion protein for the diagnosis and treatment methods disclosed herein comprises a polypeptide having any one, two, or three amino acid sequences of the VH-CDR of an antibody, or its fragment, variant, or derivative, or any one, two, or three amino acid sequences of the VL-CDR of an antibody, or its fragment, variant, or derivative, and a heterologous polypeptide sequence, and is essentially composed of it, or is composed of it. In some embodiments, the fusion protein comprises a polypeptide having an amino acid sequence of the VH-CDR3 of an antibody, or its fragment, derivative, or variant described herein, and a heterologous polypeptide sequence, wherein the fusion protein specifically binds to PNad. In another embodiment, the fusion protein comprises an amino acid sequence of at least one VH region of an antibody described herein and an amino acid sequence of at least one VL region of an antibody or its fragment, derivative or variant described herein, and a polypeptide of a heterologous polypeptide sequence. In some embodiments, the VH and VL regions of the fusion protein correspond to a single-source antibody (or scFv or Fab fragment) that specifically binds to PNad. In still another embodiment, the fusion protein for the diagnosis and treatment methods disclosed herein comprises a polypeptide having any one, two, three or more amino acid sequences of the VH CDR of an antibody and any one, two, three or more amino acid sequences of the VL CDR of an antibody, or its fragment or variant, and a heterologous polypeptide sequence. In some embodiments, two, three, four, five or more VH-CDR (one or more) or VL-CDR (one or more) correspond to a single-source antibody (or scFv or Fab fragment) described herein. Nucleic acid molecules encoding these fusion proteins are also provided herein.

[0221] Exemplary fusion proteins reported in the literature include T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84 (1987), 2936-2940; CD4 (Capon et al., Nature 337 (1989), 525-531; Traunecker et al., Nature 339 (1989), 68-70; Zettmeissl et al., DNA Cell Biol. USA 9 (1990), 347-353; and Byrn et al., Nature 344 (1990), 667-670); L-selectin (homing receptor) (Watson et al., J. Cell. Biol. 110 (1990), 2221-2229; and Watson et al., Nature 349 (1991), 164-167); CD44 (Aruffo et al., Cell Biol. USA 9 (1990), 347-353; and Byrn et al., Nature 344 (1990), 667-670); 61 (1990), 1303-1313); CD28 and B7 (Linsley et al., J. Exp. Med. 173 (1991), 721-730); CTLA-4 (Lisley et al., J. Exp. Med. 174 (1991), 561-569); CD22 (Stamenkovic et al., Cell 66 (1991), 1133-1144); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88 (1991), 10535-10539; Lesslauer et al., Eur. J. Immunol. 27 (1991), 2883-2886; and Peppel et al., J. Exp. Med. 174 (1991), 1483-1489 (1991); and fusion of IgE receptor a (Ridgway and Gorman, J. Cell. Biol. 115 (1991), Abstract No. 1448).

[0222] As discussed elsewhere herein, the antibodies described herein, or antigen-binding fragments, variants, or derivatives thereof, can be fused to heterologous polypeptides to increase the in vivo half-life of the polypeptides or used in immunoassays using methods known in the art. For example, in some embodiments, PEG is conjugated to the antibodies described herein to increase their in vivo half-life; see, e.g., Leong et al., Cytokine 16 (2001), 106-119; Adv. in Drug Deliv. Rev. 54 (2002), 531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

[0223] In addition, the antibodies described herein, or their antigen-binding fragments, variants, or derivatives can be fused to marker sequences such as peptides to facilitate their purification and detection. In a specific embodiment, the marker amino acid sequence is a six-histidine peptide (HIS), such as a tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), many of which are commercially available. As described in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86 (1989), 821-824, for example, six-histidine is provided to facilitate the purification of the fusion protein. Other peptide tags that are conducive to purification include, but are not limited to, an "HA" tag, which corresponds to an epitope derived from an influenza hemagglutinin protein (Wilson et al., Cell 37 (1984), 767) and a "flag" tag.

[0224] Fusion proteins can be prepared using methods well known in the art; see, for example, U.S. Patent Nos. 5,116,964 and 5,225,538. The precise site for fusion can be chosen empirically to optimize the secretion or binding properties of the fusion protein. The DNA encoding the fusion protein is then transfected into a host cell for expression.

[0225] The antibodies described herein can be used in an unconjugated form or can be conjugated to at least one of a variety of molecules, for example, to improve the therapeutic properties of the molecule, to facilitate target detection, or for imaging or treatment of patients. When purified, the antibodies described herein, or their antigen-binding fragments, variants, or derivatives can be labeled or conjugated before and after purification. Specifically, the antibodies described herein, or their antigen-binding fragments, variants, or derivatives can be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.

[0226] Immunotoxin conjugates including conventional antibodies have been widely described in the art. The toxin can be coupled to the antibody by conventional coupling techniques, or the protein toxin portion containing the immunotoxin can be produced as a fusion protein. Such immunotoxins can be obtained using the antibodies described herein in a corresponding manner. Illustrations of such immunotoxins are those described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and by Fanger, Immunol. Today 12 (1991), 51-54.

[0227] Those skilled in the art will appreciate that, depending on the selected reagent to be conjugated, conjugates may also be assembled using a variety of techniques. For example, conjugates with biotin are prepared by reacting PNad binding polypeptides with activated esters of biotin such as biotin N-succinimidyl esters. Similarly, conjugates with fluorescent markers may be prepared in the presence of coupling agents such as those listed herein, or by reacting with isothiocyanates or fluorescein isothiocyanate. Conjugation of antibodies described herein, or their antigen-binding fragments, variants, or derivatives is prepared in a similar manner.

[0228] Also provided herein are antibodies described herein, or antigen-binding fragments, variants, or derivatives thereof, conjugated to diagnostic or therapeutic agents. Antibodies can be used for diagnosis, e.g., to demonstrate the presence of a PNAd-related disease, to indicate the risk of developing a PNAd-related disease, to monitor the development or progression of a PNAd-related disease, i.e., as part of a clinical testing procedure for, e.g., determining the efficacy of a given treatment and / or prevention regimen. Detection is facilitated by coupling the antibody, or an antigen-binding fragment, variant, or derivative thereof to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions; for metal ions that can be conjugated to antibodies for diagnostics, see, e.g., U.S. Pat. No. 4,741,900. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include 125I, 131I, 111In, or 99Tc.

[0229] The antibody, or antigen-binding fragment, variant, or derivative thereof can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the luminescence that occurs during the chemical reaction. Examples of particularly useful chemiluminescent-labeled compounds are luminol, isoluminol, theromatic acridinium esters, imidazoles, acridinium salts, and oxalates.

[0230] One of the ways in which an antibody, or an antigen-binding fragment, variant, or derivative thereof, can be detectably labeled is by linking it to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)" Microbiological Associates Quarterly Publication, Walkersville, Md., Diagnostic Horizons 2 (1978), 1-7); Voller et al., J. Clin. Pathol. 31 (1978), 507-520; Butler, Meth. Enzymol. 73 (1981), 482-523; Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980); Ishikawa, E. et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo (1981). The enzyme bound to the antibody will react with a suitable substrate, preferably a chromogenic substrate, to produce a chemical moiety that can be detected, for example, by spectrophotometry, fluorescence, or by visual means. Enzymes that can be used to detectably label antibodies include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, saccharifying enzyme, and acetylcholinesterase. In addition, detection can be accomplished by colorimetry, which uses a chromogenic substrate for the enzyme. Detection can also be accomplished by visually comparing the degree of enzymatic reaction of the substrate with a similarly prepared standard.

[0231] Detection can also be performed using any of a variety of other immunoassays. For example, by radiolabeling an antibody, or an antigen-binding fragment, variant, or derivative thereof, it is possible to detect the antibody using a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, (March 1986)), which is incorporated herein by reference. Radioisotopes can be detected using methods including, but not limited to, a gamma-ray counter, a scintillation counter, or an autoradiography method.

[0232] Antibodies, or antigen-binding fragments, variants, or derivatives thereof, can also be detectably labeled with fluorescent emitting metals such as 152Eu, or other lanthanides. These metals can be attached to antibodies using chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0233] Techniques for conjugating various moieties to antibodies, or antigen-binding fragments, variants, or derivatives thereof are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Edition), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In CancerTherapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation AndCytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62 (1982), 119-158.

[0234] As mentioned, in some embodiments, moieties that enhance the stability or efficacy of binding molecules, such as binding polypeptides, such as antibodies or immunospecific fragments thereof, may be conjugated. For example, in some embodiments, PEG may be conjugated to the binding molecules described herein to increase their in vivo half-life. Leong et al., Cytokine 16 (2001), 106; Adv. in Drug Deliv. Rev. 54 (2002), 531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

[0235] A. Drug-containing polymer particles

[0236] Also provided herein are nanoparticles or microparticles conjugated to antibodies or their antigen-binding fragments described herein. The nanoparticles or microparticles described herein can be made of the following materials: (i) biocompatible, i.e., when used in a pharmaceutically relevant amount, no significant adverse reactions will be caused in living animals; (ii) a characteristic functional group (feature functional group) of a lymphocyte homing adhesion molecule or an antibody or its antigen-binding fragment described herein can be covalently linked, (iii) low nonspecific binding to other molecules is shown, and (iv) stable in solution, i.e., particles do not precipitate. Particles can be monodisperse (single crystal of material, e.g., metal per particle) or polydisperse (multiple crystals, e.g., 2, 3, or 4 per particle).

[0237] Many biocompatible particles are known in the art, such as organic or inorganic nanoparticles or microparticles. Liposomes, dendrimers, carbon materials, and polymer micelles are examples of organic particles. Quantum dots may also be used. Inorganic particles include metal particles such as Au, Ni, Pt, and TiO2 nanoparticles or microparticles. Magnetic particles, such as FeO2 with dextran or PEG molecules surrounding them, may also be used. 2+ and / or Fe 3+ The spherical nanocrystals of 10-20 nm with a core. Colloidal gold nanoparticles are described in, for example, Qian et al., Nat. Biotechnol. 26 (1): 83-90 (2008); U.S. Patent Nos. 7060121; 7232474; and U.S. Preliminary Publication No. (USP GPub. No.) 2008 / 0166706. Suitable nanoparticles, and methods for constructing and using multifunctional nanoparticles are discussed in, for example, Anvicens and Marco, Trends Biotech., 26 (8): 425-433 (2008).

[0238] In some cases, the particles are polymer particles. Many biocompatible polymers suitable for producing the polymer particles described herein are known in the art, for example, polylactic-co-glycolic acid (PLGA), polylactic acid, polyglycolide. In a specific embodiment, the polymer particles comprise PLGA polymers. In a specific embodiment, the polymer particles comprise polylactic acid polymers. In a specific embodiment, the polymer particles comprise polyglycolic acid polymers.

[0239] In some embodiments, the polymer particles can be poly(ethylene glycol) (PEGylated), for example, as described in U.S. Pat. Nos. 7,291,598; 5,145,684; 6,270,806; 7,348,030 and others, to reduce blood protein binding, and / or liver and spleen uptake. For example, in some embodiments, the polymer particles comprise PEGylated PLGA polymers. In another example, the polymer particles comprise PEGylated polylactic acid polymers. In still another example, the polymer particles comprise PEGylated polyglycolic acid polymers. PEGylation produces a stealth-like structure to avoid immune identification by macrophages, thereby inhibiting opsonization and enhancing retention of particles in circulation. Simple surface modifications such as PEGylation can increase the circulation half-life of particles from a few minutes to a few hours or tens of hours.

[0240] In some embodiments, the polymer particles can be attached (connected, e.g., covalently conjugated) to an antibody or antigen binding fragment thereof described herein. The antibody or antigen binding fragment thereof can be attached to the polymer particles through a functional group of the polymer of the polymer nanoparticle. For example, the antibody or antigen binding fragment thereof can be conjugated to the polymer particle.

[0241] Nanoparticles or microparticles are attached (connected) to antibodies or antigen-binding fragments thereof described herein by functional group (one or more). In some embodiments, nanoparticles or microparticles are combined with polymers including functional group (one or more), and are also used to keep metal oxides dispersed from each other. The polymer can be a synthetic polymer, such as, but not limited to, polyethylene glycol or silane, a natural polymer, or a derivative of a synthetic or natural polymer, or a combination of these. Useful polymers are hydrophilic. In some embodiments, the polymer "coating" is not a continuous film around the magnetic metal oxide, but a "net" or "cloud" of extended polymer chains attached to and surrounding the metal oxide. The polymer can include polysaccharides and derivatives, including dextran, pullulan, carboxydextran, carboxymethyldextran, and / or reduced carboxymethyldextran. The metal oxide can be a collection of one or more crystals in contact with each other, or is trapped or surrounded by a polymer alone.

[0242] In other embodiments, the nanoparticles or microparticles are combined with non-polymeric functional group compositions. Methods for synthesizing stable, functionalized nanoparticles or microparticles that are not combined with polymers are known, and this is also within the scope of the present invention. Such methods are described in, for example, Halbreich et al., Biochimie, 80 (5-6): 379-90, 1998.

[0243] In some embodiments, immunosuppressive or immunomodulatory drugs described herein can be encapsulated in nanoparticles or microparticles, such as mixed in or under the coating of nanoparticles or microparticles. In some embodiments, immunosuppressive or immunomodulatory drugs described herein can be conjugated to the outside of the surface of nanoparticles or microparticles.

[0244] i. Immunosuppressive or immunomodulatory drugs

[0245] Three typical immunosuppressive drugs, cyclosporine A, rapamycin, and tacrolimus, have been successfully incorporated into the drug-containing particles described herein, showing controlled drug release and inhibition of T cell activity in vitro (Azzi, J., et al., FASEB J 24:3927-3938, 2010; Tang, L., et al., J. Transplantation, 2012:896141, 2012; Tong, R. and Cheng, JJ, Macromolecules 45:2225-2232, 2012).

[0246] In some embodiments, tacrolimus (also known as TAC, FK-509, or fujimycin) is incorporated into the drug-containing particles described herein. Tacrolimus is an immunosuppressive drug used primarily after allogeneic organ transplantation to reduce the host's immune response and reduce the risk of organ rejection. At the cellular level, tacrolimus is a macrolide that can reduce interleukin-2 (IL-2) produced by T cells (Liu J, Farmer J, Lane W, Friedman J, Weissman I, Schreiber S, Cell 66 (4): 807–815, 1991). Other suitable immunosuppressive or immunomodulatory drugs that can be incorporated into the present delivery system include, but are not limited to, mycophenolate (mofetil), cyclosporine, sirolimus, fingolimod, myriocin, cyclophosphamide, corticosteroids (e.g., cortisol, corticosterone, cortisone, or aldosterone), rapamycin, mycophenolate (mofetil), checkpoint inhibitors (e.g., anti-CTLA-4 antibodies, anti-PD1 antibodies, anti-PDL1 antibodies), anti-inflammatory agents (e.g., anti-TNF drugs, such as, for example, adalimumab), chemotherapeutic agents, and monoclonal antibodies such as, for example, anti-CD3 antibodies, anti-CD25 antibodies, anti-IL6 antibodies, CTLA-4-Ig, adalimumab, anti-CD52 antibodies (e.g., alemtuzumab). In some embodiments, paclitaxel is incorporated into the drug-containing particles described herein. In some embodiments, the immunosuppressive or immunomodulatory drug has one or more hydroxyl or thiol groups and can act as a polymerization initiator in the presence of certain catalysts.

[0247] In some embodiments, the drug is a chemotherapeutic agent. Chemotherapy can be, for example, the use of a cytotoxin or cytotoxic agent that is harmful to cells. Examples include paclitaxel, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, mitoxantrone, mithramycin, actinomycin D, and puromycin and its analogs or homologues. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., nitrogen mustard, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). See, e.g., Johnson and Win, Oncoimmunology. 2018 Mar 13;7(4):e1408744; Chowdhury et al., J Intern Med. 2018 Feb;283(2):110-120; Tang et al., Nature Reviews Drug Discovery 17:854–855 (2018).

[0248] ii. Synthesis of drug-containing polymer particles

[0249] There are various methods by which drug-containing nanoparticles or microparticles can be prepared, but in all methods, the result must be a particle having functional groups that can be used to link the particle to the antibodies or antigen-binding fragments thereof described herein.

[0250] In some embodiments, the immunosuppressive or immunomodulatory drugs described herein may be encapsulated in nanoparticles or microparticles, for example, mixed in or under the coating of the nanoparticles or microparticles. Such drug-encapsulated particles can be synthesized using nanoprecipitation methods, for example, as described in Tang, L., et al., J. Transplantation, 2012: 896141, 2012, the contents of which are incorporated herein by reference. Briefly, the drug to be delivered and the PEGylated polymer can be dissolved in a suitable organic solvent. The resulting solution can be added dropwise to a non-solvent such as water under vigorous stirring to form PEGylated drug-encapsulated polymer particles. The particle suspension can then be stirred at room temperature to evaporate the organic solvent. An aliquot of the particle suspension can be centrifuged and the supernatant can be analyzed, for example, by reverse phase HPLC to determine the incorporation efficiency and load of the drug in the particles. The particles can then be purified and collected, for example, by ultrafiltration. Optionally, a lyophilization protectant such as bovine serum albumin (BSA) can be added to the particle solution, which is then lyophilized and stored at -20°C. The size and polydispersity of the resulting particles can be determined by dynamic light scattering (DLS) and / or scanning electron microscopy (SEM).

[0251] In some embodiments, the immunosuppressive or immunomodulatory drugs described herein can be conjugated externally, for example, on the surface of nanoparticles or microparticles. Such drug-conjugated particles can be synthesized using the method described in Azzi, J., et al., FASEB J 24: 3927-3938, 2010, or in International Application Publication WO 2008 / 109483, the contents of which are incorporated herein by reference. Briefly, the drug to be delivered can be mixed with one or more cyclic monomers and a catalyst in a suitable solvent. The drug acts as an initiator in a ring-opening polymerization reaction to form a drug-polymer conjugate, wherein the drug is covalently linked to the polymer. Since the drug is used as an initiator for the polymer, the conjugation efficiency of the drug to the polymer is very high, and the drug loading percentage can be controlled by adjusting the monomer / initiator ratio. The drug-polymer conjugate and the pegylated polymer can then be added dropwise to a non-solvent such as water under vigorous stirring to form pegylated drug-conjugated polymer particles. Then, the particle suspension can be stirred at room temperature to evaporate the organic solvent. The particles can be purified and collected, for example, by ultrafiltration. Optionally, a lyoprotectant such as bovine serum albumin (BSA) can be added to the particle solution, which is then lyophilized and stored at -20° C. The size and polydispersity of the resulting particles can be determined by dynamic light scattering (DLS) and / or scanning electron microscopy (SEM).

[0252] Suitable monomers for ring-opening polymerization include various cyclic monomers, for example, cyclic esters, cyclic carbonates, cyclic siloxanes, cyclic phosphates, cyclic peptides or amino acid derivatives, or cyclic phosphazanes. Exemplary cyclic monomers include lactide or glycolide.

[0253] The polymer particles described herein can be used to deliver any small molecule drug containing at least one functional group capable of initiating a ring-opening polymerization reaction, such as a hydroxyl or thiol group. The drug may contain a plurality of such polymerization initiating groups, for example, a plurality of hydroxyl or thiol groups. In some embodiments, the drug contains only one of such polymerization groups. The hydroxyl group may be a primary, secondary, or tertiary hydroxyl group. Similarly, the thiol group may be a primary, secondary, or tertiary thiol group. The hydroxyl group may also be a phenolic hydroxyl group. In some embodiments, the drug contains one or more non-phenolic hydroxyl groups. In some embodiments, the drug contains one or more non-phenolic hydroxyl groups that are primary or secondary hydroxyl groups. In some embodiments, the drug contains a single non-phenolic hydroxyl group. In some embodiments, the drug contains a single primary or secondary hydroxyl group. In some embodiments, an immunosuppressive or immunomodulatory drug with one or more hydroxyl or thiol groups acts as a polymerization initiator in the presence of certain catalysts.

[0254] A variety of catalysts can be used to promote the formation of drug-containing polymer particles. Exemplary catalysts include a variety of metal oxides (M-OR) and alcohol-metal oxides (RO-M) developed for controlled living polymerization of cyclic monomers (O. Dechy-Cabaret, B. Martin-Vaca, & D. Bourissou, Chemical Reviews 104: 6147-6176, 2004). Metal oxides can be prepared in situ by mixing hydroxyl-containing compounds with active metal complexes such as metal-amide compounds (BM Chamberlain, M. Cheng, DR Moore, TM Ovitt, EB Lobkovsky, GW Coates, J. Am. Chem. Soc. 123: 3229, 2001). For example, a very active catalyst (BDI) MgN (TMS) 2 (Chamberlain, 2001) for the polymerization of lactide can be used. Certain Zn catalysts such as (BDI)ZnN(TMS)2 promote rapid initiation and relatively slow chain growth and can be used as polymerization catalysts. Zn-mediated polymerization of lactide can produce polymers with narrow polydispersity. Other useful catalysts include Ca and Fe catalysts. Since Mg, Zn, Ca, and Fe are elements found in the human body, catalysts containing these elements have better safety than other active catalysts containing Al and Sn. Exemplary Zn, Mg, Ca, and Fe catalysts include organic catalysts described in WO 2008 / 109483. In some embodiments, Zn catalysts such as (BDI)ZnN(TMS)2 are used to initiate drug-containing polymer particles.

[0255] The particles described herein have shown high drug loading (about 50%) and loading efficiency (98-100%), well-controlled drug release kinetics without a burst effect, and excellent controlled particle size with a very narrow particle size distribution (Tong, R. & Cheng, J., Angew. Chem., Int. Ed. 47:4830-4834, 2008; Tong, R. & Cheng, J., J. Am. Chem. Soc. 131:4744-4754, 2009).

[0256] Particle size can be controlled by adjusting reaction conditions, for example, by varying temperature as described in U.S. Pat. No. 5,262, 176. Uniform particle size material can also be produced by fractionating the particles using centrifugation, ultrafiltration, or gel filtration, as described, for example, in U.S. Pat. No. 5,492,814.

[0257] The size of the drug-containing polymer particles described herein can be in the range of about 2nm to about 500μm. In some embodiments, the drug-containing polymer particles have a range of about 0.01μm to about 10μm, for example, an overall size of about 0.05μm to about 5μm, about 0.1μm to about 3μm, about 1μm to about 2.5μm. In some embodiments, the drug-containing polymer particles have an overall size of about 500nm to about 5μm. In some embodiments, the drug-containing polymer particles have an overall size of about 1μm to about 2.5μm. In some embodiments, the drug-containing polymer particles have an overall size of about 200nm to about 800nm. In some embodiments, the drug-containing polymer particles have an overall size of about 2-20nm and are particularly advantageous for delivery to cells.

[0258] In some embodiments, the drug-containing polymer particles disclosed herein can be surface modified to provide functional groups that can be used to connect the particles to the antibodies or antigen-binding fragments thereof described herein. For example, the particles can be functionalized according to a version of the method of Albrecht et al., Biochimie, 80(5-6):379-90, 1998. Dimercaptosuccinic acid can bind to the particles and provide carboxyl functional groups. Functionalization refers to the presence of amino or carboxyl or other reactive groups that can be used to attach the desired moiety to the particles.

[0259] Carboxyl functionalized nanoparticles or microparticles can be prepared, for example, according to the method of Gorman (see WO 00 / 61191). Carboxyl functionalized particles can also be prepared from polysaccharide coated particles by reaction with bromoacetic acid or chloroacetic acid in a strong base to attach carboxyl groups. In addition, carboxyl functionalized particles can be prepared from amino functionalized particles by converting the amino groups to carboxyl groups using reagents such as succinic anhydride or maleic anhydride.

[0260] The particles can also be treated with periodate to form aldehyde groups. The particles containing aldehyde groups can then be reacted with a diamine (e.g., ethylenediamine or hexamethylenediamine), which will produce a Schiff base, followed by reduction with sodium borohydride or sodium cyanoborohydride.

[0261] Dextran coated particles can also be made and crosslinked with, for example, epichlorohydrin. Addition of ammonia will react with epoxy groups to produce amine groups, see Hogemann et al., Bioconjug. Chem. 2000. 11(6):941-6, and Josephson et al., Bioconjug. Chem., 1999, 10(2):186-91.

[0262] The carboxyl-functionalized particles can be converted to amino-functionalized magnetic particles by carboxyl-functionalizing the particles using water-soluble carbodiimides and diamines such as ethylenediamine or hexamethylenediamine.

[0263] Avidin and streptavidin can be attached to the particles for biotinylated binding moieties, such as oligonucleotides or polypeptides. See, for example, Shen et al., Bioconjug. Chem., 1996, 7(3): 311-6. Similarly, biotin can be attached to the particles for avidin-labeled binding moieties.

[0264] In some embodiments, the drug-containing polymer particles can be pegylated, for example, as described in U.S. Pat. Nos. 7,291,598; 5,145,684; 6,270,806; 7,348,030 and others, to reduce blood protein binding, and / or liver and spleen uptake. Pegylation creates a stealth structure to avoid immune recognition by macrophages, thereby achieving inhibited opsonization and enhanced retention of particles in circulation. Simple surface modifications such as pegylation can increase the circulation half-life of particles from a few minutes to a few hours or tens of hours.

[0265] iii. Conjugation Method

[0266] The drug-containing polymer particles described herein are conjugated to the antibodies or antigen-binding fragments thereof described herein.

[0267] In some embodiments, the antibody or antigen-binding fragment thereof can be attached to the drug-containing polymer particle by covalent attachment, such as by a chemical bond between a functional group on the molecule and a functional group on the drug-containing polymer particle. The functional group can be an amino or carboxyl group or other reactive group that can be used to attach a desired moiety to a particle such as an antibody or antigen-binding fragment thereof.

[0268] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be attached to the drug-containing polymer particles via a linker or a binding agent. The linker or binding agent can have a terminal amino group, a carboxyl group, a sulfhydryl group, or a phosphate group. Illustrative examples of useful linkers or binding agents include organic polymers such as polyethylene glycol (PEG) and its derivatives, proteins, and small molecules.

[0269] VI. How to use

[0270] As described above, the present disclosure is based, at least in part, on the development of anti-PNAd antibodies with improved binding affinity for cells expressing PNAd (Example 6) and improved ability to block T cell homing to lymph nodes (Example 4). Administration of the drugs conjugated to the anti-PNAd antibodies described herein resulted in reduced tumor growth, reduced tumor metastasis, and reduced fibrosis of tumor-draining lymph nodes (Example 7), in addition to increased survival time (Example 8). In addition, administration of the drugs conjugated to the anti-PNAd antibodies described herein also increased survival time after cardiac transplantation (Example 9). Therefore, the antibody-drug conjugates described herein and in the following examples can be used to treat local and distant metastases in primary tumors and lymph nodes; this use of the antibodies is not unexpected because it is not known whether metastatic lesions express HEV. Therefore, the present disclosure provides compositions and methods for suppressing lymphocyte-mediated immune responses in subjects in need, treating autoimmune diabetes or delaying the progression of autoimmune diabetes in subjects in need, treating lymphocyte-mediated inflammation in subjects in need, and treating malignant tumors expressing peripheral lymph node addressins (PNAd) or reducing the metastasis of malignant tumors expressing peripheral lymph node addressins (PNAd) in subjects in need. These methods may include identifying a subject in need of treatment and administering one or more compositions described herein to the subject. Subjects in need of treatment may be identified, for example, by a physician. In some embodiments, the antibodies or antigen-binding fragments thereof described herein (or compositions or particles comprising antibodies or antigen-binding fragments thereof (e.g., particles containing drugs)) can be used in methods for suppressing lymphocyte-mediated immune responses in subjects in need, comprising administering to the subject a therapeutically effective amount of the antibodies or antigen-binding fragments thereof described herein (or compositions or particles containing antibodies or antigen-binding fragments thereof (e.g., particles containing drugs)). In some embodiments, the antibodies or antigen-binding fragments thereof described herein (or compositions or particles containing the antibodies or antigen-binding fragments thereof (e.g., particles containing drugs)) can be used in a method of treating autoimmune diabetes or delaying the progression of autoimmune diabetes in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of the antibodies or antigen-binding fragments thereof described herein (or compositions or particles containing the antibodies or antigen-binding fragments thereof (e.g., particles containing drugs)). In some embodiments, the antibodies or antigen-binding fragments thereof described herein (or compositions or particles containing the antibodies or antigen-binding fragments thereof (e.g., particles containing drugs)) can be used in a method of treating lymphocyte-mediated inflammation in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of the antibodies or antigen-binding fragments thereof described herein (or compositions or particles containing the antibodies or antigen-binding fragments thereof (e.g., particles containing drugs)).In some embodiments, the antibodies or antigen-binding fragments thereof described herein (or compositions or particles containing antibodies or antigen-binding fragments thereof (e.g., particles containing drugs)) can be used in a method of treating or reducing metastasis of a malignant tumor expressing PNad in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein (or a composition or particle containing antibodies or antigen-binding fragments thereof (e.g., particles containing drugs)). In some embodiments, the malignant tumor expressing PNad is a lymphoma. In some embodiments, the malignant tumor expressing PNad is a cancer. In some cases, the cancer is breast cancer. In some cases, the cancer is a primary tumor. In some cases, the cancer is a local metastasis. In some cases, the cancer is a local metastasis in a lymph node. In some cases, the cancer is a distant metastasis (i.e., a location away from the primary tumor).

[0271] As described above, the antibodies and antigen-binding fragments thereof described herein are particularly useful for the development of drug-containing polymer particles that mimic lymphocyte migration in vivo, and drugs such as immunosuppressive or immunomodulatory drugs can be specifically delivered to lymphoid tissues and sites of chronic inflammation where T cell activation and T cell-mediated damage occur. Therefore, the present disclosure provides compositions or methods for treating or delaying disease progression in the following: in subjects (e.g., humans) in need of immunosuppression, for example, for autoimmune or inflammatory conditions such as type 1 diabetes or rheumatoid arthritis; and in subjects (e.g., humans) in need of post-transplant immunosuppression, for example, in heart or other organ transplants. The present disclosure also provides compositions and methods for treating or delaying disease progression in subjects with cancer (e.g., lymphoma or breast cancer). The present disclosure also provides methods for treating or delaying the progression of malignant tumors such as cancer, such as lymphomas, in which lymphocyte homing processes play a role in the spread of tumors, and / or in which malignant tumors express PNads by delivering chemotherapeutic drugs to lymphoid tissues.

[0272] These methods may include identifying a subject in need of treatment and administering to the subject one or more compositions described herein. A subject in need of treatment may be identified, for example, by a physician.

[0273] A. Treatment of autoimmune diabetes

[0274] Type 1 diabetes (T1D) is characterized by autoimmune destruction of insulin-producing β cells in the pancreatic islets by autoreactive T cells (Hoglund, P., et al., J Exp Med 189:331-339, 1999). The core of the pathogenesis of autoimmune diabetes is the activation of autoreactive T cells by presentation of islet antigens to T cells by antigen-presenting cells in the draining pancreatic lymph nodes (Roncarolo, MG & Battaglia, M., Nat Rev Immunol 7:585-598, 2007). Many studies have shown that pancreatic lymph nodes play a key role in the pathogenesis of autoimmune diabetes (Katz, JD, Wang, B., Haskins, K., Benoist, C. & Mathis, D. Cell 74:1089-1100, 1993; Hoglund, P., et al., J Exp Med 189, 331-339, 1999; Turley, S., Poirot, L., Hattori, M., Benoist, C. & Mathis, D., J Exp Med 198:1527-1537, 2003; Turley, SJ, Lee, JW, Dutton-Swain, N., Mathis, D. & Benoist, C., Proc Natl Acad Sci USA 102:17729-17733, 2005). After initiation, activated autoreactive T cells are recruited to the pancreas, leading to insulitis.

[0275] The PNAd pathway has been shown to regulate lymphocyte trafficking to lymphoid tissues in NOD mice (Hanninen, A., Salmi, M., Simell, O., Andrew, D., and Jalkanen, S., Blood 88:934-944, 1996; Xu, B., et al., J Exp Med 197:1255-1267, 2003; Xu, B., Cook, RE, and Michie, SA, J Autoimmun. 35:124-129, 2010). In addition, PNAd is upregulated on venous endothelial cells in inflammatory states and autoimmune diseases such as T1D (Michie, SA, Streeter, PR, Bolt, PA, Butcher, EC, and Picker, LJ, Am J Pathol 143: 1688-1698, 1993; Mikulowska-Mennis, A., Xu, B., Berberian, JM, and Michie, SA, Am J Pathol 159: 671-681, 2001; Penaranda, C. and Bluestone, JA, Immunity 31: 534-536, 2009). It has been reported that inflammatory signals and endothelial cell activation during insulitis confer pancreatic islet endothelial morphology and characteristics to HEV expressing PNAd. Thus, the onset of insulitis has been reported to be associated with islet expression of PNAd (Hanninen, A., Salmi, M., Simell, O., Andrew, D. & Jalkanen, S., Blood 88, 934-944, 1996; Faveeuw, C., Gagnerault, M.C., Kraal, G. & Lepault, F., Int Immunol 7: 1905-1913, 1995; Yang, X.D., et al., Proc Natl Acad Sci USA 91: 12604-12608, 1994; Yang, X.D., et al., J Autoimmun 7: 859-864, 1994; Faveeuw, C., Gagnerault, M.C. & Lepault, F., J Immunol 152:5969-5978, 1994; Hanninen, A., Salmi, M., Simell, O. & Jalkanen, S., Diabetes 45:1173-1180, 1996; Dong, H., Burke, SD and Croy, BA, Placenta 29:201-209, 2008).The interaction of leukocytes with endothelial cells through adhesion molecules provides a great opportunity to design various strategies, such as the development of inhibitors or antibodies targeting the trafficking of leukocytes (Mackay, CR, Nat Immunol 9:988-998, 2008).

[0276] The antibodies or antigen-binding fragments thereof described herein, or conjugates thereof, can be used to selectively deliver immunosuppressants to pancreatic lymph nodes and pancreas to effectively inhibit autoreactive T cells and treat or delay the progression of autoimmune diabetes such as type 1 diabetes (T1D). Subjects with some pancreatic β cell function are more likely to benefit from the present treatment methods. Such subjects can be identified, for example, by determining that C-peptide levels in the subject are above a threshold.

[0277] B. Immunosuppression after transplantation

[0278] After tissue or organ transplantation, immune cells are activated for the exogenous antigens present on the allograft. Activated immune cells can move to the transplanted tissue or organ and cause damage there. A small amount of immunosuppressive drugs are directly delivered to the site where immune cells are activated to reduce their activation and increase allograft survival. This targeted delivery of immunosuppressive drugs also reduces the toxicity seen when a large amount of immunosuppressive drugs are administered systemically. Compositions described herein are useful carriers that immunosuppressants are directly delivered to the site of organ transplantation and increase allograft survival.

[0279] C. Treatment of lymphoma

[0280] The compositions described herein can also be used to treat lymphomas, e.g., lymphomas expressing PNAd. The lymphocyte homing process is known to play a role in the spread of some tumors. For example, the spread of non-Hodgkin lymphoma (NHL) is mediated by the lymphocyte homing program (Pals ST, de Gorter DJJ, and Spaargaren M, Blood 110 (9): 3102-3111, 2007). Other exemplary lymphomas that can be treated by the compositions disclosed herein include small lymphocytic lymphoma (SLL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), Burkitt's lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), cutaneous lymphomas such as cutaneous T-cell lymphoma (CLCL), intestinal lymphomas such as intestinal T-cell lymphoma (ITL), nodal T-cell lymphoma, extranodal lymphomas occurring in gut-associated lymphoid tissue or skin, adult T-cell leukemia / lymphoma (ATLL), B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), primary follicular lymphoma (FL), B-cell chronic lymphocytic leukemia (B-CLL), and hairy cell leukemia (HCL).

[0281] In some embodiments, the antibodies or antigen-binding fragment conjugates described herein are used to treat or delay progression of lymphomas that express PNads.

[0282] VII. Pharmaceutical Compositions

[0283] A therapeutically effective amount of one or more antibodies or antigen-binding fragments thereof described herein, or conjugates thereof, may be incorporated into a pharmaceutical composition suitable for delivery to a subject such as a human. Such compositions typically include an antibody or antigen-binding fragment thereof, or conjugates thereof, and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are compatible with drug administration. Such media and agents are known for use in pharmaceutically active substances. Unless any conventional media or agents are incompatible with the active compound, such media may be used in the compositions described herein. Supplementary active compounds may also be incorporated into the composition, for example, ligand degradation inhibitors.

[0284] The pharmaceutical composition can be formulated to be compatible with its intended route of administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate or phosphate, and an agent for adjusting tension, such as sodium chloride or glucose. The pH may be adjusted by an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be encapsulated in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0285] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL TM (castor oil polyoxyethylene ether; BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be a liquid that is easy to inject. It must be stable under manufacturing and storage conditions, and must be able to prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersion, and by using surfactants. The effects of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal (thimerosal). In many cases, it is preferred to include isotonic agents in the composition, for example, sugars, polyols such as mannitol, sorbitol, sodium chloride. The extended absorption of the injectable composition can be achieved by including agents such as aluminum monostearate and gelatin that delay absorption in the composition.

[0286] Sterile injectable solutions can be prepared by incorporating the desired amount of a composition (e.g., an agent described herein) into an appropriate solvent having one or a combination of the above-listed compositions, as required, and then filtering and sterilizing. Typically, dispersions are prepared by mixing the active ingredient into a sterile carrier containing a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any other desired ingredients from its previously sterile filtered solution.

[0287] Oral compositions typically include an inert diluent or edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be mixed with an excipient and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash, wherein the compound in the liquid carrier is administered orally and gargled and spit out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients, or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth or gelatin; excipients, such as starch or lactose, disintegrants, such as alginic acid, PRIMOGEL TM (sodium starch glycolate), or corn starch; lubricants such as magnesium stearate or STEROTES TM ; a glidant, such as colloidal silicon dioxide; a sweetener, such as sucrose or saccharin; or a flavoring, such as mint, methyl salicylate, or orange flavor.

[0288] It can also be administered systemically by transmucosal or transdermal methods. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salve, gel, or cream as known in the art.

[0289] In some embodiments, the active compound (e.g., an antibody or its antigen-binding fragment, or its conjugate) is prepared together with a carrier that protects the compound from rapid elimination from the body, for example, a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers can be used, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters (polyorthoesters), and polylactic acid. The preparation method of such preparations is obvious to those skilled in the art. The material can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0290] The pharmaceutical composition can be included in a container, pack, or dispenser together with instructions for administration. In one aspect, the pharmaceutical composition can be included as part of a kit.

[0291] Generally, the dosage for applying the pharmaceutical composition is conducive to the intended purpose of prevention and / or treatment, without undesirable side effects, such as toxicity, irritation or allergic reactions. Although individual needs can vary, the determination of the preferred range of the effective amount of the preparation is within the technical scope of the art. Human dosage can be easily inferred from animal studies (Katocs et al., Chapter 27 In: "Remington's Pharmaceutical Sciences", 18th edition, Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990). Generally, the dosage required for the preparation providing an effective amount that can be adjusted by those skilled in the art will vary depending on multiple factors, including the age, health, physical condition, body weight, type and degree of disease or illness of the subject, treatment frequency, the nature of simultaneous treatment, as needed, and the nature and scope of the desired effect (one or more) (Nies et al., Chapter 3, In: Goodman & Gilman's "The Pharmacological Basis of Therapeutics", 9th edition, Hardman et al., eds., McGraw-Hill, New York, NY, 1996).

[0292] VIII. Administration

[0293] A therapeutically effective amount of one or more of the compositions described herein can be administered by standard methods, e.g., by one or more routes of administration, e.g., by one or more routes of administration currently approved by the U.S. Food and Drug Administration (FDA; see, e.g., World Wide Web address fda.gov / cder / dsm / DRG / drg00301.htm), e.g., oral, topical, mucosal, or parenteral, e.g., intravenous or intramuscular.

[0294] IX. Kit

[0295] The present invention also includes kits for the methods described herein. In some embodiments, the kit includes one or more doses of the compositions described herein. The compositions, shapes, and dosage forms used for induction and maintenance regimens will vary depending on the needs of the subject. For example, the dosage form can be a parenteral dosage form, an oral dosage form, a delayed or controlled release dosage form, a topical, and a mucosal dosage form, including any combination thereof.

[0296] In specific embodiments, the kit may include one or more of the following in a package or container: (1) one or more doses of a composition described herein; (2) one or more pharmaceutically acceptable adjuvants or excipients (e.g., pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, or inclusion compounds); (3) one or more carriers for administration of the doses; (5) instructions for administration. Embodiments in which two or more, including all, of components (1)-(5) are found in the same container may also be used.

[0297] When a kit is provided, the different components of the included compositions can be packaged in separate containers and mixed immediately prior to use. Such separate packaging of the components can allow for long-term storage without loss of function of the active components. When more than one biologically active agent (e.g., an antibody or antigen-binding fragment thereof, or a conjugate thereof) is included in a particular kit, the biologically active agents can be (1) packaged separately and mixed separately with suitable (similar or different, but compatible) adjuvants or excipients immediately prior to use, (2) packaged together and mixed together immediately prior to use, or (3) packaged separately and mixed together immediately prior to use. If the selected compounds (e.g., antibodies or antigen-binding fragments thereof, or conjugates thereof) will remain stable after mixing, the compounds can be mixed at a time prior to use other than immediately prior to use, including, for example, minutes, hours, days, months, years, and time of manufacture.

[0298] The compositions contained in the kits of the present invention in particular can be provided in any type of container so that the life of the different components is optimally preserved and not absorbed or altered by the material of the container. Suitable materials for these containers can include, for example, glass, organic polymers (e.g., polycarbonate and polystyrene), ceramics, metals (e.g., aluminum), alloys, or any other material commonly used to hold similar reagents. Exemplary containers can include, but are not limited to, test tubes, vials, flasks, bottles, and syringes, etc.

[0299] As described above, the kit may be provided with instructional materials. These instructions may be printed and / or may be provided as an electronically readable medium, including, but not limited to, for example, a floppy disk, CD-RO, DVD, Zip disk, videotape, audiotape, and flash memory device. Alternatively, the instructions may be published on an Internet web page or distributed to users as an e-mail.

[0300] Example

[0301] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0302] Example 1: MHA112 monoclonal antibody (mAb) recognizes HEV in mice and humans

[0303] Sialyl Lewis X Two GlcNAc6ST1 / 2 / 4 triple knockout (KO) mice lacking peripheral lymph node vascular addressin (PNAd) were immunized with a Chinese hamster ovary (CHO) cell line expressing CD34 (sLeX) type glycans. MHA112 single hybridoma clones were identified by HEV immunofluorescence staining. Supernatants of MHA112 hybridoma cultures were collected and used to stain mouse lymph nodes (LN) and human tonsil sections. Figure 1A Low magnification image showing high endothelial venule (HEV) structures of mouse LN identified by MHA112 hybridoma supernatant. High magnification image ( Figure 1B ) shows mouse and human HEV structures stained with MHA112.

[0304] Example 2: MHA112 mAb typing

[0305] Then, a typing enzyme-linked immunosorbent assay (ELISA) was performed to identify the immunoglobulin (Ig) form of the MHA112 mAb. Figure 2 As shown in , MHA112 supernatant was positive in the anti-IgM coated wells. These data indicate that the MHA112 isotype is mouse IgM.

[0306] Example 3: Transport of MHA112 mAb-conjugated NPs to LNs

[0307] MECA79 is a monoclonal ligand that recognizes all known L-selectin ligands on endothelial venules—PNAds, including a subset of CD34, GlyCAM-1, and MAdCAM-1 (Hemmerich, S., Butcher, EC & Rosen, SD, J Exp Med 180, 2219-2226, 1994). MECA79 was produced by using collagenase-dispersed elements of mesenteric and peripheral lymph node stroma as immunogens and selection based on selective staining of peripheral lymph node HEV (Streeter et al., J. Cell. Biol. 107: 1853-1862, 1988).

[0308] MHA112-conjugated, MECA79-conjugated and non-conjugated particles (loaded with IR800 dye) were injected intravenously into mice. Analysis 24 hours after injection showed high signals in LNs of MHA112-NP and positive control MECA79-NP groups compared with mice injected with non-conjugated IR800-NP ( Figure 3A). Sections of axillary LNs were then stained to evaluate the intra-LN distribution of NPs 24 hours after injection. Cell nuclei were stained blue by DAPI (4′,6-diamidino-2-phenylindole). PNads were stained green using MECA79 mAb and NPs were visualized in red. At 24 hours, MECA79-NPs and MHA112-NPs were localized around HEVs. Significantly fewer NPs were detected in the LNs of mice injected with non-target NPs ( Figure 3B ). The transport of MHA112-NPs to LNs was comparable to that of MECA79-NPs, highlighting the specificity of the MHA112 mAb in effectively transporting NPs to LNs.

[0309] Example 4: MHA112 mAb blocks T cell homing to LNs

[0310] To test whether MHA112 mAb can block T cell homing to LN in vivo, T cells were labeled with fluorescein isothiocyanate (FITC) fluorescent dye and transferred into naive mice ( mice) and their distribution was evaluated 19 hours later. MHA112 (anti-HEV) reduced the number of transferred T cells entering the axillary (Axi), inguinal (Ing), and brachial (Bra) LNs by more than 25% ( Figure 4 In addition, in the presence of MHA112, the number of T cells entering the mesenteric LN was also reduced by about 12% of the PBS control ( Figure 4 In contrast, MECA79 did not reduce the number of transferred T cells into axillary, inguinal, brachial, or mesenteric LNs ( Figure 4 In the three groups, the number of labeled T cells entering the spleen was unchanged ( Figure 4 ). These data suggest that MHA112 mAb shares binding sites with T cells in HEV and blocks T cell homing in vivo. MHA112 mAb is significantly better at blocking T cell homing than MECA79 antibody.

[0311] Example 5: MHA112 mAb sequence

[0312] Figure 5 The sequences of the MHA112 heavy and light chain variable regions (VH and VL, respectively) are shown in and the framework regions (FR1-FR4) and IMGT complementarity determining regions (CDR1-CDR3) are depicted.

[0313] Materials and Methods for Examples 1 to 5

[0314] Immunization and antibody isolation

[0315] Two GlcNAc6ST1 / 2 / 4 triple KO mice lacking PNad were immunized by a CHO cell line expressing CD34 modified with sLeX-type glycans. After the third immunization, mice were sacrificed for hybridomas. About 500 individual clones were screened by HEV staining. The monoclonal antibody MHA112 against mouse and human HEV was identified.

[0316] Immunohistochemistry

[0317] Fresh LNs were embedded in tissue freezing medium. Cryosections (8 μm thick) were cut for imaging by fluorescence confocal microscopy. MHA112 was used as the primary antibody for tissue staining. Alexa Fluor 488-conjugated anti-mouse IgM was used as the secondary antibody. DAPI was used to stain cell nuclei.

[0318] ELISA

[0319] 96-well ELISA plates were coated with goat anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3 (5 μg / mL) and incubated overnight at 4°C. The plates were then blocked with 5% BSA in Dulbecco's phosphate buffered saline (DPBS) and washed three times with DPBS+0.05% Tween-20 (PBST). MHA112 hybridoma supernatant (1:500) was then added to the wells and incubated for 1 hour at 37°C. The wells were washed again with PBST and incubated for 1 hour at 37°C with horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (Ig) (Invitrogen). After washing three times with PBST, the samples were treated with 3,3',5,5'-tetramethylbenzidine and the absorbance at 450nm was recorded using a VersaMax microplate reader (Molecular Devices Corp.).

[0320] Lymph node transport

[0321] MECA79 and MHA112 mAbs were conjugated to the functional surface of nanoparticles (NPs) using thiol-maleimide chemistry. MECA79 and MHA112 mAbs were pretreated with a mild reducing agent (tris (2-carboxyethyl) phosphine (TCEP), 15 minutes, room temperature) to break the thiol group and immediately mixed with the NP suspension. The maleimide group on the NP was covalently conjugated to the broken thiol of the MECA79 and MHA112 antibodies. MECA79-NPs and MHA112-NPs were stored at 4°C before use. 8-10 week-old C57BL / 6J (JAX#000664) mice were used for biodistribution studies. Samples were administered intravenously by retro-orbital injection. The transport of fluorescent MECA79-IR-NPs and MHA112-IR-NPs was studied using a UVP iBOXExplorer imaging microscope (UVP) equipped with a 750-780 nm excitation filter and an 800 nm long pass emission filter. Multiple LNs including axillary, brachial, inguinal, renal, and mesenteric were harvested and imaged using iBOX.

[0322] T cell blockade

[0323] Spleens were harvested from three C57BL / 6 mice. Single cell suspensions were prepared and cells were enriched for T cell positives according to the manufacturer's instructions, using MACS reagents and columns (Miltenyi Biotech, Bergisch Gladbach, Germany). For injection, T cells were labeled by CellTracker Green (Thermo Fisher) and resuspended in sterile saline, and 5 million T cells were injected per mouse with 100 μl. 2 hours before T cell injection, 200 μg blocking antibodies MHA112 and MECA79 were administered intraperitoneally (ip) per mouse with 200 μl. 19 hours after transfer, mice were euthanized and LNs were processed for flow cytometry.

[0324] Example 6: MHA112 binding affinity

[0325] To compare the affinity of MHA112 with MECA79, a cell-based ELISA was performed. CHO cells expressing PNAd (CHO-PNAd) (2×10 4) were coated on 96-well plates and incubated overnight at 37°C. After washing five times with phosphate-buffered saline (PBS), the plates were fixed with 1% paraformaldehyde (PFA) and blocked by 3% bovine serum albumin (BSA). MECA79 and MHA112 were serially diluted from 1 ng / ml to 300 μg / ml. After washing five times in PBST, horseradish peroxidase- (HRP-) conjugated goat anti-mouse and anti-rat secondary antibodies were added at a dilution of 1:10,000 and incubated for 1 hour at room temperature. Binding was detected by adding TMB (3,3′,5,5′-tetramethylbenzidine) substrate, and the reaction was stopped by adding TMB stop solution (N600, Thermo Scientific). The absorbance signal was read at 450 nm. As Figure 6 As shown in , the fitting curves of the binding affinities of MECA79 and MHA112 were constructed, and the Kd values ​​were 1.41 nM and 0.97 nM, respectively. The data showed that the binding affinity of MHA112 was higher than that of MECA79 (30%) ( Figure 6 ).

[0326] Example 7: MHA112-drug conjugation reduces tumor growth, fibrosis, and metastasis

[0327] Mice were anesthetized with isoflurane and injected subcutaneously in the mammary gland with 4T1 breast tumor cells (1x10 5 cells per mouse). Free paclitaxel or MHA112-paclitaxel was administered intraperitoneally every other day starting 9 days after transplantation for 24 days. Tumor growth was monitored twice a week by electronic calipers. Five weeks after transplantation, the tumor size in the MHA112-paclitaxel group was approximately 3-fold or approximately 2-fold smaller than that in the control group (phosphate-buffered saline treatment) and the free paclitaxel group, respectively (p=0.0001) ( Figure 7 ). These results show the advantages of MHA112-paclitaxel in the treatment of breast tumors in mouse models. The kinetics of drug delivery in LNs are largely unknown. Draining lymph nodes are the main sites of metastasis. These metastatic sites in lymph nodes are extremely difficult to treat even with high-dose systemic administration of chemotherapeutic drugs, which often leads to significant toxicity and intolerance on the part of patients.

[0328] MHA112 has a unique premise to target the hidden metastatic niche in LN. Therefore, the effect of LN delivery of MHA112 in tumor populations in LN was investigated. The extent to which MHA112 can restore the overall structure of LN was also investigated. Tumors are known to produce a fibrotic environment. 4T1 tumor draining lymph nodes (TDLN) were cut by frozen sections and stained with: anti-HEV (sc-19602, SCBT), anti-LYVE1 (ab14917, Abcam), anti-pan-cytokeratin (C2931, Sigma), anti-collagen I (ab34710, Abcam), anti-collagen IV (ab6586, Abcam), and anti-laminin (ab11575, Abcam). DAPI (VECTASHIELD, Vector Laboratories) was used to counterstain cell nuclei. The stained tissue sections were visualized using the EVOSTM FL Auto 2 imaging system. Figure 8 The images in the figure show that TDLN in the control and free paclitaxel groups expanded in the HEV and lymphatic (LYVE1) areas. The expansion of HEV and lymphatic in the MHA112-paclitaxel TDLN was less than that in the other groups. In addition, pan-cytokeratin showed that primary tumor metastasis in the TDLN of the MHA112-paclitaxel group was also significantly reduced compared with the other groups. The structural changes of TDLN were also examined. Fig. 9 As shown in , collagen I, IV and laminin expression levels were lower in MHA112-paclitaxel. This data suggests that TDLN fibrosis has been improved in MHA112-paclitaxel.

[0329] Example 8: MHA112-drug conjugate increases survival in an in vivo metastatic cancer model

[0330] To investigate the effect of MHA112-paclitaxel on tumor metastasis, 4T1 tumor cells were slowly injected into the portal vein of mouse liver (1x10 4 Cells per mouse). Starting 4 hours after transplantation, 160 ng of free paclitaxel or MHA112-paclitaxel was injected intraperitoneally every other day for 20 days. Survival was monitored daily. Fig.10 The survival curves in show that the median survival time (MST) of the control group and the free paclitaxel group was 36 days and 37 days, respectively. The MST in the MHA112-paclitaxel group was significantly longer (p<0.0001), MST=58.

[0331] Example 9: MHA112-drug conjugate increases survival in the pan02 pancreatic cancer model

[0332] Mice were anesthetized with isoflurane and Panc02 pancreatic tumor cells were slowly injected into the pancreas of the mice. For the Panc02 tumor model, 2 million cells (2x10 6 Cells). Starting 4 hours after transplantation, 160 ng of free paclitaxel or MHA112-paclitaxel was injected intraperitoneally every other day for 20 days. Survival was monitored every day. Fig.11 The survival curves in the Figure 4 show that the median survival time (MST) in the control group and the free paclitaxel group was 30 days and 32 days, respectively. The MST in MHA112-paclitaxel was significantly longer (p<0.001), MST=45.

[0333] Materials and Methods for Examples 7 to 9

[0334] Antibody-drug conjugates

[0335] Glutaric anhydride (100 mg, Sigma-Aldrich) and paclitaxel (33 mg, LClaboratories) were prepared in a 4 mL vial, dried under high vacuum for 24 hours and dissolved in 1 mL of pyridine. The solution was stirred at room temperature for 2 hours under an Ar atmosphere. The reaction was quenched for 2 hours by removing the solvent under high vacuum. 2'-glutaryl paclitaxel was purified by reverse HPLC (Phenomenex Luna 5μm C18 250×10.0mm, flow rate 2mL / min, UV 250nm detection) with a gradient solvent system (15% to 75% ACN / H2O with 0.1% formic acid, 40 minutes). 2'-Glutarylpaclitaxel (0.2 mg) dissolved in DMSO (ThermoScientific Fisher) was activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.4 mg, Sigma-Aldrich) and Sulfo-NHS (N-hydroxysulfosuccinimide) (1.1 mg, Thermo Scientific Fisher) in MES buffer (pH 6.0, ThermoScientific Fisher) at room temperature for 15 minutes (final solution; ~1 mL in 10% DMSO). EDC was quenched by 2-mercaptoethanol (1.4 μL, Sigma-Aldrich) for 10 minutes. Immediately, the pH of the solution was increased to ~8 by Na2CO3 (0.1 M, Sigma-Aldrich). MHA112 dissolved in PBS (pH 7.4, Corning) was mixed with activated 2'-glutaryl paclitaxel at room temperature for 2 hours (1:20 molar ratio of MHA112 to paclitaxel, final solution; 10% DMSO). The free paclitaxel was removed by dialysis with 10 kD MWCO (Sigma-Aldrich) for 15 min, twice. TM ,7kDMWCO,Thermo Scientific Fisher) to further purify the solution.

[0336] Other Implementations

[0337] It should be understood that although the invention has been described in conjunction with the detailed description of the invention, the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. An anti-peripheral lymph node addressin (PNAd) antibody or a PNad binding fragment thereof, comprising: (a) a heavy chain variable region (VH), wherein the VH comprises a VH complementarity determining region 1 (CDR1), a VH CDR2 and a VH CDR3, wherein the amino acid sequence of the VH CDR1 is shown in SEQ ID NO:3, the amino acid sequence of the VH CDR2 is shown in SEQ ID NO:4, and the amino acid sequence of the VH CDR3 is shown in SEQ ID NO:5; and (b) a light chain variable region (VL), wherein the VL comprises VL CDR1, VL CDR2 and VL CDR3, wherein the amino acid sequence of the VL CDR1 is shown in SEQ ID NO:8, the amino acid sequence of the VL CDR2 is shown in SEQ ID NO:9, and the amino acid sequence of the VL CDR3 is shown in SEQ ID NO:

10.

2. The anti-PNAd antibody or PNad-binding fragment thereof according to claim 1, wherein the VH comprises the following: a VH framework region 1 (FR1) comprising the amino acid sequence shown in SEQ ID NO: 11, a VH FR2 comprising the amino acid sequence shown in SEQ ID NO: 12, a VH FR3 comprising the amino acid sequence shown in SEQ ID NO: 13, and a VH FR4 comprising the amino acid sequence shown in SEQ ID NO:

14.

3. The anti-PNAd antibody or PNad-binding fragment thereof according to claim 1, wherein the VL comprises the following: a VL FR1 comprising the amino acid sequence shown in SEQ ID NO: 15, a VLFR2 comprising the amino acid sequence shown in SEQ ID NO: 16, a VL FR3 comprising the amino acid sequence shown in SEQ ID NO: 17, and a VL FR4 comprising the amino acid sequence shown in SEQ ID NO:

18.

4. The anti-PNAd antibody or PNad-binding fragment thereof according to claim 2, wherein the VL comprises the following: a VL FR1 comprising the amino acid sequence shown in SEQ ID NO: 15, a VLFR2 comprising the amino acid sequence shown in SEQ ID NO: 16, a VL FR3 comprising the amino acid sequence shown in SEQ ID NO: 17, and a VL FR4 comprising the amino acid sequence shown in SEQ ID NO:

18.

5. The anti-PNAd antibody or PNad-binding fragment thereof according to claim 1, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:

2.

6. The anti-PNAd antibody or PNad-binding fragment thereof according to claim 1, wherein the VL comprises the amino acid sequence shown in SEQ ID NO:

7.

7. The anti-PNAd antibody or PNad-binding fragment thereof according to claim 1, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:2 and the VL comprises the amino acid sequence shown in SEQ ID NO:

7.

8. The anti-PNAd antibody or PNad-binding fragment thereof according to claim 1, which is a monoclonal antibody.

9. The anti-PNAd antibody or PNad-binding fragment thereof according to claim 1, which is a murine antibody. 10 . The anti-PNAd antibody or PNad-binding fragment thereof according to claim 1 , which is a chimeric antibody. The anti-PNAd antibody or PNad-binding fragment thereof according to claim 1 , which is a humanized antibody.

12. The anti-PNAd antibody or PNad-binding fragment thereof according to claim 1, which is an antibody.

13. The anti-PNAd antibody or PNad-binding fragment thereof according to claim 12, comprising human heavy chain and light chain constant regions. 14 . The anti-PNAd antibody or PNad-binding fragment thereof according to claim 12 , comprising a heavy chain constant region of IgM. 15 . The anti-PNAd antibody or PNad-binding fragment thereof according to claim 13 , which comprises the heavy chain constant region of IgM. 16 . The anti-PNAd antibody or PNad-binding fragment thereof according to claim 1 , which is a PNad-binding fragment of the anti-PNAd antibody.

17. The anti-PNAd antibody or PNad-binding fragment thereof according to claim 16, wherein the PNad-binding fragment is selected from the group consisting of a single-chain Fv fragment (scFv), a F(ab') fragment, a F(ab) fragment, and a F(ab')2 fragment.

18. The anti-PNAd antibody or PNad-binding fragment thereof according to any one of claims 1 to 17, which is conjugated to an agent.

19. The anti-PNAd antibody or PNad-binding fragment thereof according to claim 18, wherein the agent is an imaging agent, a cytotoxic agent or a drug.

20. The anti-PNAd antibody or PNad-binding fragment thereof of claim 19, wherein the drug is selected from the group consisting of tacrolimus, mycophenolate mofetil, cyclosporine, sirolimus, fingolimod, myriocin, cyclophosphamide, corticosteroids, rapamycin, checkpoint inhibitors, anti-inflammatory agents, chemotherapeutic agents, anti-CD3 antibodies, anti-CD25 antibodies, anti-IL6 antibodies, or anti-CD52 antibodies.

21. The anti-PNAd antibody or PNad-binding fragment thereof of claim 20, wherein the chemotherapeutic agent is selected from the group consisting of paclitaxel, an antimetabolite, an alkylating agent, an anthracycline, an antibiotic, or an antimitotic agent.

22. The anti-PNAd antibody or PNad-binding fragment thereof of claim 20, wherein the checkpoint inhibitor is an anti-CTLA-4 antibody.

23. The anti-PNAd antibody or PNad-binding fragment thereof of claim 20, wherein the anti-inflammatory agent is adalimumab.

24. The anti-PNAd antibody or PNad-binding fragment thereof according to claim 18, wherein the agent is a drug-containing polymer particle.

25. The anti-PNAd antibody or PNad-binding fragment thereof of claim 24, wherein the drug is selected from the group consisting of tacrolimus, mycophenolate mofetil, cyclosporine, sirolimus, fingolimod, myriocin, cyclophosphamide, corticosteroids, rapamycin, checkpoint inhibitors, anti-inflammatory agents, chemotherapeutic agents, anti-CD3 antibodies, anti-CD25 antibodies, anti-IL6 antibodies, or anti-CD52 antibodies.

26. The anti-PNAd antibody or PNad-binding fragment thereof of claim 25, wherein the chemotherapeutic agent is selected from the group consisting of paclitaxel, an antimetabolite, an alkylating agent, an anthracycline, an antibiotic, or an antimitotic agent.

27. The anti-PNAd antibody or PNad-binding fragment thereof of claim 25, wherein the checkpoint inhibitor is an anti-CTLA-4 antibody.

28. The anti-PNAd antibody or PNad-binding fragment thereof of claim 25, wherein the anti-inflammatory agent is adalimumab.

29. The anti-PNAd antibody or PNad-binding fragment thereof of claim 24, wherein the drug-containing polymer particle comprises an immunosuppressive or immunomodulatory drug.

30. The anti-PNAd antibody or PNad-binding fragment thereof of claim 18, wherein the conjugation is covalent conjugation.

31. The anti-PNAd antibody or PNad-binding fragment thereof according to any one of claims 19 to 29, wherein the conjugation is covalent conjugation.

32. The anti-PNAd antibody or PNad-binding fragment thereof of claim 18, wherein the conjugation is via a linker.

33. The anti-PNAd antibody or PNad-binding fragment thereof according to any one of claims 19 to 29, wherein the conjugation is via a linker.

34. The anti-PNAd antibody or PNad-binding fragment thereof of claim 32, wherein the linker is polyethylene glycol.

35. The anti-PNAd antibody or PNad-binding fragment thereof of claim 33, wherein the linker is polyethylene glycol.

36. A polynucleotide comprising a nucleic acid sequence encoding the anti-PNAd antibody or PNad-binding fragment thereof according to any one of claims 1 to 17.

37. A polynucleotide comprising a nucleic acid sequence encoding a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises the amino acid sequence shown in SEQ ID NO:7, and the VH comprises the amino acid sequence shown in SEQ ID NO:

2.

38. A polynucleotide comprising a nucleic acid sequence encoding a light chain and a heavy chain, wherein the light chain comprises a light chain variable region (VL), wherein the VL comprises the amino acid sequence shown in SEQ ID NO:7, and wherein the heavy chain comprises a heavy chain variable region (VH), wherein the VH comprises the amino acid sequence shown in SEQ ID NO:

2.

39. The polynucleotide according to claim 37 or 38, comprising the nucleic acid sequence shown in SEQ ID NO:6 and the nucleic acid sequence shown in SEQ ID NO:

1.

40. A vector comprising the polynucleotide of claim 37 operably linked to a promoter.

41. A vector comprising the polynucleotide of claim 38 operably linked to a promoter.

42. A host cell comprising the polynucleotide of claim 37, wherein the polynucleotide is operably linked to a promoter.

43. A host cell comprising the polynucleotide of claim 38, wherein the polynucleotide is operably linked to a promoter.

44. A host cell comprising the vector according to claim 40.

45. A host cell comprising the vector according to claim 41.

46. ​​A method for producing an anti-Peripheral Lymph Node Addressin (PNAd) antibody or a PNad binding fragment thereof, comprising: (a) culturing the host cell according to claim 43 or 45; and (b) isolating the anti-PNAd antibody or PNad-binding fragment thereof from the culture.

47. A pharmaceutical composition comprising a therapeutically effective amount of the anti-PNAd antibody or PNad-binding fragment thereof according to any one of claims 1 to 23 and a pharmaceutically acceptable carrier.

48. A pharmaceutical composition comprising a therapeutically effective amount of the anti-PNAd antibody or PNad-binding fragment thereof according to any one of claims 24 to 29 and a pharmaceutically acceptable carrier.

49. Use of the pharmaceutical composition according to claim 47 or 48 in the preparation of a medicament for blocking T cell homing to lymph nodes in a subject in need thereof by the following method, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 47 or 48.

50. Use of the pharmaceutical composition according to claim 47 or 48 in the preparation of a medicament for treating a malignant tumor expressing peripheral lymph node addressin (PNAd) in a subject in need thereof or reducing the metastasis of a malignant tumor expressing peripheral lymph node addressin (PNAd) in a subject in need thereof by the following method, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 47 or 48, wherein the malignant tumor expressing peripheral lymph node addressin (PNAd) is a breast tumor or a pancreatic tumor.

51. Use of the pharmaceutical composition of claim 48 in the preparation of a medicament for delivering a drug to a subject in need thereof by the following method, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 48, wherein the drug delivered to the subject is the drug of the drug-containing polymer particles conjugated to the anti-PNAd antibody or its PNad-binding fragment, wherein the drug is delivered to one or more lymphoid tissues or one or more sites of chronic inflammation in the subject.

52. The use according to any one of claims 49 to 51, wherein the subject is a human.

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