METHODS AND COMPOSITIONS FOR IMMUNOASSAYS

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Patent Application Serial No. 61/031,545, filed February 26, 2008.

[0002] This application is also a continuation-in-part of co-pending U.S. Patent Application Serial No. 11/912,992, filed October 29, 2007, which is the U.S. national phase of Patent Cooperation Treaty No. PCT/US2006/016844, filed April 29, 2006, which is a continuation-in-part of U.S. Patent Application Serial No. 09/865,281, filed May 29, 2001, now abandoned, which is a continuation-in-part of U.S. Patent Application Serial No. 09/070,907 filed May 4, 1998, now U.S. Patent No. 6,238,667.

[0003] This application is also a continuation-in-part of co-pending U.S. Patent Application Serial No. 11/119,404, filed April 29, 2005.

[0004] This application is also a continuation-in-part of co-pending U.S. Patent Application Serial No. 10/652,864, filed August 29, 2003, which claims priority from U.S. Provisional Patent Application Serial No. 60/407,421, filed August 30, 2002. [0005] This application is also a continuation-in-part of co-pending U.S. Patent Application Serial No. 12/144,361, filed June 23, 2008.

[0006] The disclosures of the aforementioned applications are incorporated herein by reference in their entireties.

TECHNICAL FIELD OF THE INVENTION

[0007] The present invention relates to antibodies, methods of making the same, and methods of using the antibodies in the detection, prevention, and/or treatment of a variety of disease conditions. In specific embodiments, the invention relates to immunoassays using antibodies conjugated to autophilic peptides.

BACKGROUND OF THE INVENTION

[0008] Antibodies have emerged as a major therapeutic tool for the treatment of chronic diseases, such as cancer and autoimmune disorders. Notable success stories include Herceptin® in the treatment of breast cancer and Rituxan® in the treatment of non-Hodgkin's lymphoma. A key advantage of antibodies in the treatment of disease lies in their ability to target disease-causing cells or molecules, while sparing healthy tissues and normal products

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of the body. However, antibodies that exhibit desired specificities in laboratory studies often fail in pre-clinical and clinical evaluations because of inefficient targeting, low therapeutic efficacy, and/or unacceptable side effects.

[0009] It is known that a major mechanism by which therapeutic antibodies are effective against their target cells is by inducing cell death, i.e., antibody-induced apoptosis. Such induced apoptosis is typically triggered by crosslinking receptors that are part of the cell's apoptosis signal pathway. For example, crosslinking the B-cell antigen receptor by means of antibodies induces apoptosis in B-cell tumors (Ghetie M., et al., 1997). Crosslinking of cellular receptors also increases the binding avidity of an antibody to its target antigen, and thus is likely to increase all cell surface-dependent therapeutic mechanisms, such as complement-mediated killing and complement-dependent opsonization and phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), as well as enhanced inhibition of cell growth or alterations in metabolic pathways within cells through increased binding to and blockade of cellular receptors when using antibodies targeted to cellular receptors. [0010] A rare class of self-binding antibodies, variously known as "autophilic antibodies" or "autobodies", has been identified in Nature. They are capable of forming dimers and/or polymers through noncovalent interactions with self. One example of an autophilic antibody is TEPC- 15, which targets a normally cryptic determinant of phosphorylcholine on apoptotic cells and atherosclerotic lesions (Binder, J., et al., 2003; Kang, C-Y, et al., 1988). Dimerization or multimerization may be induced only after the modified antibody attaches to its cell surface target, i.e., "differential oligomerization". In solution, an autophilic antibody can be in equilibrium between its monomeric and dimeric forms (Kaveri S., et al., 1990). [0011] Autophilic antibodies belong to a larger class of antibodies, referred to herein as "Super Antibodiesâ„¢." Super- antibodies, as used herein, exhibit one or more beneficial properties in addition to the antigen binding properties usually associated with antibodies (Kohler H., et al., 1998; Kohler H., 2000). Specifically, the referenced class of super- antibodies comprises antibodies having catalytic, adjuvant, membrane-penetrating, and/or autophilic properties, and includes molecules that afford superior targeting and therapeutic properties. Such super-antibodies are considered chimeric and typically comprise an antibody or antibody fragment covalently linked to at least one non-antibody moiety, such as a peptide, which has catalytic, adjuvant, membrane-penetrating, and/or autophilic properties. The conjugation of certain peptides to antibodies has been shown to increase the potency of antibodies, e.g., in inducing apoptosis (Zhao, et al. 2001 ; Zhao, et al 2002a; Zhao, et al.

2002b). The conjugation chemistry used in previous studies has utilized the nucleotide binding site (Pavlinkova, et al. 1997) or the carbohydrate moiety of antibodies as the site of specific attachment (Award, et al. 1994).

[0012] In efforts to enhance antigen detection and/or therapeutic efficacy of known antibodies, many hybrid molecules comprising two distinct covalently linked domains have been proposed. For instance, U.S. Patent No. 5,219,996 (issued to Bodmer et al.) proposes changing an amino acid residue of an antibody molecule to a cysteine residue and then coupling an effector or reporter molecule to the antibody through the cysteine thiol group. U.S. Patent No. 5,191,066 (issued to Bieniarz et al.) proposes periodate oxidation of a carbohydrate molecule in the Fc region of an immunoglobulin and attaching a disulfide compound thereto. U.S. Patent No. 6,218,160 (issued to Duan) proposes site-specific conjugation of an enzyme to an antibody by formation of a dihydrazone bridge therebetween. U.S. Patent No. 5,596,081 (issued to Haley et al.) discloses a method for site-specific attachment of a purine or purine analog photoaffuiity compound to an antibody molecule. U.S. Patent No. 6,238,667 (issued to Kohler) proposes photochemically cross-linking an azido-peptide molecule to an antibody at a purine or tryptophan affinity site on the antibody. U.S. Patent Pub. No. 2005/0033033 (Kohler et al.) proposes a super- antibody for inhibiting cell apoptosis, wherein the super-antibody comprises an anti-caspase antibody conjugated to a membrane transporter peptide. U.S. Patent Pub. No. 2003/0103984 (Kohler) discloses a fusion protein comprising antibody and peptide domains in which the peptide domain can have autophilic activity. U.S. Patent No. 6,482,586 (issued to Arab et al.) proposes covalent hybrid compositions for use in intracellular targeting. U.S. Patent No. 6,406,693 (issued to Thorpe et al.) proposes antibodies and conjugates for cancer treatment by binding to aminophospholipid on the luminal surface of tumor blood vessels. U.S. Patent No. 6,780,605 (issued to Frostegard) proposes a method of diagnosing cardiovascular disease that employs antibodies specific for platelet activating factor. U.S. Patent No. 6,716,410 (issued to Witztum et al.) proposes a treatment for atherosclerosis that employs a monoclonal antibody having specific binding affinity for oxidized low density lipoprotein (oxLDL), which is covalently linked to a therapeutic agent, e.g., a thrombolytic agent. U.S. Patent Pub. No. 2003/0143226 (Kobayashi et al.) proposes a monoclonal antibody having specific binding affinity for an oxidized LDL receptor, which inhibits binding of oxLDL to the receptor. [0013] The above approaches are proposed to enhance the antigen detection ability and/or therapeutic efficacy of antibodies, which are not sufficiently effective in locating or killing

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their targets in either their native or "humanized" states. Still, there continues to be a need for enhancing the detection, prevention and/or treatment of many diseases using suitably modified antibodies. An object of the present invention is to address the foregoing needs with suitably prepared super- antibodies.

[0014] Immunoassays using specific antibodies to detect an analyte are used in many applications including medical and scientific research as well as in clinical medicine. Immunoassays are rapid, relatively inexpensive and selective, affording dependable results. However, sensitivity of an immunoassay is highly dependent on particular antibody characteristics. Where an antibody is insufficiently sensitive, an immunoassay using the antibody is limited in application.

[0015] Thus, there is a continuing need for highly sensitive antibodies and methods for their use.

SUMMARY OF THE INVENTION

[0016] The present invention affords novel super-antibodies having autophilic, membrane-penetrating, adjuvant, and/or catalytic properties. A super- antibody contemplated by the present invention comprises immunoglobulin (Ig) and non-immunoglobulin (non-Ig) domains, wherein at least one non-Ig domain is covalently attached to the Ig domain, preferably as a chemically formed hybrid molecule, i.e., an immunoconjugate. The immunoglobulin domain can comprise a polyclonal antibody, monoclonal antibody, Fab fragment, or F(ab') ₂ fragment, which imparts specific binding affinity for an antigenic determinant. The non-Ig domain is an organic chemical moiety that imparts, or augments, autophilic, membrane-penetrating, adjuvant, and/or catalytic properties to the immunoconjugate, but which does not contain an azido, purine or pyrimidine group. Preferably, the non-Ig domain comprises a peptide having autophilic, membrane -penetrating, adjuvant, and/or catalytic properties.

[0017] Autophilic antibodies described herein behave as monomeric antibodies when not bound to an antigen. Binding of an autophilic antibody to an antigen induces dimerization and/or multimerization of autophilic antibodies, a process termed Dynamic Cross Linking (DXL).

[0018] Another aspect of the present invention is directed to a method of making novel super- antibodies .

[0019] Methods of the present invention include molecular biological techniques to generate a recombinant chimeric autophilic antibody. In particular embodiments, a recombinant chimeric autophilic antibody of the present invention includes at least one autophilic peptide.

[0020] Autophilic antibodies are provided according to embodiments of the present invention which include an immunoglobulin component and an autophilic peptide fused thereto. Autophilic antibodies are provided according to embodiments of the present invention which include an immunoglobulin component having a binding affinity for a CD20 antigen an autophilic peptide fused thereto. The immunoglobulin component can be an antibody heavy chain and/or an antibody light chain. In particular embodiments, the immunoglobulin component is chimeric, including immunoglobulin portions derived from two or more sources or species.

[0021] Autophilic antibodies are provided according to embodiments of the present invention wherein immunoglobulin component and autophilic peptide are expressed as a fusion protein. The autophilic peptide is preferably expressed at the C-terminus of the immunoglobulin component in particular embodiments of the present invention.

[0022] Optionally, the autophilic peptide includes a peptide selected from SEQ ID No. 1,

SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 10 and SEQ ID No. 11, SEQ ID No. 14 and may also be an autophilic peptide having a substantially identical amino acid sequence to any of these.

[0023] In a particular embodiment, the immunoglobulin component includes chimeric

1F5. In a particular embodiment, the immunoglobulin component includes rituximab.

[0024] Expression vectors are provided according to embodiments of the present invention which encode a chimeric heavy chain and/or a chimeric light chain, and an autophilic peptide. At least one protein expressed from the expression vector is a fusion protein including a chimeric heavy chain and/or a chimeric light chain, fused to an autophilic peptide.

[0025] In particular embodiments of the present invention, the chimeric heavy chain includes a variable heavy chain of an anti-CD20 antibody such as mouse monoclonal 1F5 anti-CD20 antibody and rituximab anti-CD20 antibody.

[0026] In particular embodiments of the present invention, the chimeric heavy chain includes a human gamma constant heavy chain.

[0027] Expression vectors are provided according to embodiments of the present invention which include a nucleic acid sequence encoding a chimeric immunoglobulin heavy chain linked to an autophilic peptide and a nucleic acid sequence encoding a chimeric light chain of an immunoglobulin. The nucleic acid sequences are operably linked to a transcription promoter. The nucleic acid sequence encoding the chimeric immunoglobulin heavy chain linked to an autophilic peptide is separated from the nucleic acid sequence encoding the chimeric light chain of an immunoglobulin by an internal ribosome entry site

(IRES) such that expression of the nucleic acid sequences produces the chimeric light chain of an immunoglobulin and a fusion protein of the chimeric heavy chain and the autophilic peptide which combine to form an autophilic antibody.

[0028] Optionally, the chimeric heavy chain encoded by a nucleic acid in an expression vector of the present invention includes SEQ ID No. 26, SEQ ID No. 28, or a substantially identical chimeric heavy chain.

[0029] Optionally, the chimeric heavy chain encoded by a nucleic acid in an expression vector of the present invention includes SEQ ID No. 27, SEQ ID No. 45 or a substantially identical chimeric heavy chain- autophilic peptide fusion protein.

[0030] Both the chimeric light chain and the chimeric heavy chain can be expressed as fusion proteins including an autophilic peptide.

[0031] A method of generating a fusion protein which includes an antigen binding region and an autophilic peptide is provided according to embodiments of the present invention expressing the fusion protein from an expression construct encoding the fusion protein. In particular embodiments, the fusion protein forms a heavy chain of an autophilic antibody.

[0032] Isolated host cells transformed with an inventive expression vector described herein are provided according to embodiments of the present invention.

[0033] In a method of the invention, a photoactivatable organic molecule is covalently linked to an immunoglobulin at a site on the immunoglobulin having binding affinity for the organic molecule. The mutual attraction of Ig and photoactivatable organic molecule favors contact and coupling of the two entities upon exposure to activating radiation. Preferably, the organic molecule contains a chromophore, such as an aromatic hydrocarbon moiety, other than a purine or pyrimidine group, susceptible to photoactivation. Also, an azido group need not be present in the molecule.

[0034] Preferably, an aromatic hydrocarbon moiety (AHM) of the invention, which is photoactivatable, is a single ring or polynuclear aiyl or heterocycle. Inclusive of such

moieties are substituted benzene, naphthalene, anthracene, phenanthrene, pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, thiazole, pyridine, indole, benzofuran, thionaphthene, quinoline, or isoquinoline groups. Conveniently, an AHM is present in the photoactivatable organic molecule as part of a side chain of an amino acid residue. Exemplary of such amino acid residues are tryptophan, tyrosine, histidine, and phenylalanine, which have indole, phenol, imidazole, and phenyl side chains, respectively. A tryptophan residue is most preferred.

[0035] A super- antibody of the invention can also be conjugated with one or more non- autophilic peptides to add functionality. For instance, a super- antibody can bear a membrane- penetrating peptide sequence, which facilitates translocation of the antibody across the cell membrane where it can bind to an intracellular target. In a specific embodiment, the membrane-penetrating peptide comprises at least one MTS peptide or MTS-optimized peptide. Additionally, an autophilic super- antibody can be conjugated with a membrane- penetrating peptide sequence, thereby imparting both functionalities to the antibody. [0036] In another aspect of the present invention, a super- antibody having specific binding affinity for atherosclerotic plaques, which permits detection, prevention and/or treatment of atherosclerosis, is contemplated. For example, an autophilic super-antibody is capable of binding an antigenic determinant of atherosclerotic plaques, e.g., ox-LDL, and can dimerize or oligomerize once specifically bound to its antigenic determinant. In this way, uptake of ox-LDL by macrophages can be effectively blocked or reduced, thereby inhibiting chronic inflammation associated with atherosclerosis.

[0037] In specific embodiments, an autophilic peptide of the immunoconjugate comprises a T15, T15E, T15-scr2, R24, R24-charged, or other optimized amino acid sequence. Preferably, the immunoglobulin and/or peptide domains of the super-antibody are humanized to improve tolerance in a patient.

[0038] A pharmaceutical composition is also contemplated, which contains one or more super- antibodies and a pharmaceutically acceptable carrier. Due to its superior avidity, a super- antibody of the invention can be administered to a patient in a dosage similar to, or less than, that practicable for the corresponding non-autophilic antibody.

[0039] In another aspect of the invention, an assay of cells undergoing apoptosis can be performed by contacting the cells with a super- antibody of the invention. The super- antibody specifically binds to an antigenic determinant of a cell undergoing apoptosis and can be visualized by a reporter molecule or secondary antibody. Exemplary of antigenic

determinants associated with apoptosis are membrane-bound phosphorylcholine and phosphatidylserine.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 compares the internalization of MTS conjugated antibodies and non-

MTS conjugated antibodies using anti-caspase 3 antibodies;

[0041] Figure 2 depicts the effect of chemotherapeutic drug (actinomycin D) on cell death in the presence and absence of MTS-conjugated (Sab) antibody;

[0042] Figure 3 depicts enhanced binding of anti-CD20 antibodies conjugated with Tl 5 peptide;

[0043] Figure 4 depicts improved binding of anti-CD20 antibodies conjugated with Tl 5 peptide at low concentrations of antibody;

[0044] Figure 5 depicts improved binding of anti-CD20 antibodies conjugated with Tl 5 peptide to DHL-4 cells at high concentrations of antibody;

[0045] Figure 6 depicts enhanced induction of apoptosis of tumor cells with mouse anti-

CD20 conjugated with Tl 5 peptide;

[0046] Figure 7 compares the binding of anti-GM2 antibody and Tl 5 conjugated anti-

GM2 antibody to ganglioside GM2;

[0047] Figure 8 illustrates the self-binding activity of anti-GM2 antibody and Tl 5 conjugated anti-GM2 antibody;

[0048] Figure 9 demonstrates binding specificity of T15 conjugated anti-GM2 antibody to different gangliosides;

[0049] Figure 10 depicts differences in cell surface binding of anti-GM2 antibody and

T15 conjugated anti-GM2 antibody to Jurkat cells;

[0050] Figure 11 depicts the effect of anti-GM2 antibody and Tl 5 conjugated anti-GM2 antibody on Jurkat cell growth;

[0051] Figure 12 compares the efficacy of autophilic peptide conjugation to an affinity site on an antibody (nucleotide) vs. a non-affinity site (CHO - carbohydrate) using anti-GM2;

[0052] Figure 13 depicts enhanced apoptosis of tumor cells using anti-GM2 antibody conjugated with Tl 5 peptide;

[0053] Figure 14 compares the binding of Herceptin® (upper panel) and the autophilic peptide conjugated form of Herceptin (lower panel) to small cell lung cancer cell;

[0054] Figure 15 depicts photo-conjugation of biotin-amino acids to monoclonal OKT3 antibody. A panel of biotin-amino acids were mixed with the monoclonal antibody OKT3 at concentration from 20-50 μMol and exposed to UV for 2 minutes. The reacted mixture was dot-blotted with avidin-HRP and scanned. Color intensity is indicated at the y-axis;

[0055] Figure 16. Panel A: Titration of biotin-tryptophan photo-conjugation to chimeric anti-GM2 antibody. Chimeric anti-GM2 was photo-biotinylated with Trp peptide at different molarities. ELISA wells were incubated with chimeric biotinylated anti-GM2 blocked and developed with avidin-HRP. Panel B: Photobiotinylation of humanized anti-Her2/neu

(Herceptin) with Trp-biotin peptide under different pH, ELISA as in Panel A;

[0056] Figure 17. Denaturation of photo-biotinylated anti-GM2 antibody. Detection of biotin on denatured/renatured antibody in ELISA as in Fig. 16 A;

[0057] Figure 18. Panel A: Comparison of single versus multiple biotin anti-GM3 antibody. ELISA wells were coated with ganglioside, single and multiple biotin anti-GM3 was added and developed with avidin-HRP. Panel B: Comparison of single versus multiple biotin chimeric anti-Gm2 antibody to Gm2. Comparison of single versus multiple biotin antibody, ELISA as in Fig. 19;

[0058] Figure 19 compares chemically biotinylated with photo-biotinylated antibodies.

Commercial NHS-biotin rabbit anti-mouse (Sigma) and NHS-biotin anti-GM2 are compared with photobiotinylated antibodies, ELISA as in Fig. 16;

[0059] Figure 20 compares detection sensitivity of photo- and chemically biotinylated chimeric anti-glycolyl GM3 binding to glycolyl GM3 monoganglioside, ELISA as in Fig. 19;

[0060] Figure 21 demonstrates antigen specific binding of photobiotinylated anti-glycolyl

GM3 to monogangliosides GMl, GM2, GM3 and glycolyl GM3, ELISA as in Fig. 20;

[0061] Figure 22 illustrates a proposed mechanism by which an autophilic antibody of the present invention, which is immunospecific for ox-LDL, can inhibit chronic inflammation leading to atherosclerosis;

[0062] Figure 23 is a schematic representation of structures of the chimeric 1F5 (chlF5) and DXL 1F5 (chlF5 -DXL) antibodies;

[0063] Figure 24 shows a comparison of binding of chlF5 to DXL-chlF5 to JOK-I cells using FACS on fixed cells;

[0064] Figures 25A-25F show a comparison of induction of apoptosis by chlF5 and

DXL-chlF5 on Raji (A-C) and Ramos (D-F) cells. Panels A and D cells only, B and E chlF5,

C and F DXL-chlF5;

[0065] Figures 26A-26C show a comparison of CDC using chlF5 and DXL-chlF5. Panel

A, Raji, B, Ramos, C, JOK-I;

[0066] Figures 27A-27B show a comparison of ADCC using chlF5 and DXL-chlF5.

Panel A, Raji, B, Ramos;

[0067] Figures 28A-28B show a comparison of inhibition of proliferation, Panel A, Raji,

B, Ramos, with chlF5 and DXL-chlF5;

[0068] Figure 29 is a graph showing results of an ELISA comparing a non-modified anti- Her2 antibody (trastuzumab) to the same anti-Her2 antibody conjugated to an autophilic peptide;

[0069] Figure 30 is a series of graphs showing results of surface plasmon resonance detection in a Her2/neu receptor binding assay comparing binding of a non-modified anti- Her2 antibody (trastuzumab) and the same anti-Her2 antibody conjugated to an autophilic peptide;

[0070] Figure 31 is a series of graphs showing results of FACS detection in a Her2/neu receptor binding assay comparing binding of a control antibody, a non-modified anti-Her2 antibody (trastuzumab), to the same anti-Her2 antibody conjugated to an autophilic peptide, in MDA MB 231 cells which express only low levels of an Her2/neu receptor; [0071] Figure 32 is a series of graphs showing cells classified as "high" Her2 expressers or "low" Her2 expressers;

[0072] Figure 33 is a series of graphs showing increased sensitivity of detection of low expressing Her2 cells;

[0073] Figure 34 is a graph showing increased binding of the anti-CD20 antibody rituximab conjugated to an autophilic peptide (DXL625) compared to rituximab (RITUXAN) without the autophilic peptide;

[0074] Figure 35 is a graph showing results of an ELISA comparing a non-modified antibacterial antigen antibody to the same antibody conjugated to an autophilic peptide; [0075] Figure 36 is a graph showing results of an ELISA comparing a non-modified anti- glycolic GM3 antibody (1X7) to the same antibody conjugated to an autophilic peptide (DXLIX7);

[0076] Figure 37 is a graph showing results of an electrochemical assay comparing a non- modified anti-prostate specific antigen antibody to the same antibody conjugated to an autophilic peptide;

[0077] Figure 38 is a graph showing results of an ELISA for analyte CD32B binding assay using A, chimeric 2B6 antibody and autophilic peptide conjugate; B, humanized 2B6 antibody and autophilic peptide conjugate; and

[0078] Figure 39 is a graph showing more sensitive detection of EGFR analyte iusing the anti-EGFR antibody-autophilic peptide conjugate.

DETAILED DESCRIPTION OF THE INVENTION [0079] SuperAntibodv Synthesis and Formulations

[0080] It has now been discovered that many immunoglobulins have an affinity for certain photoactivatable aromatic hydrocarbon moieties. Such affinity permits close approach and prolonged contact time between the immunoglobulin (Ig) and the aromatic hydrocarbon moiety (AHM), which in turn facilitates photolytic conjugation of the Ig to an organic molecule bearing the AHM. Without wishing to be bound to any particular theory, it is believed that the attraction between the AHM and an affinity site on the Ig is probably due to van der Waals forces and/or dipole-dipole interactions, which promote the close approach and stacking of parallel aromatic rings.

[0081] In the present invention, a photoactivatable organic compound is covalently linked to an Ig to form an immunoconjugate (super-antibody). Such immunoconjugate is formed by admixing the photoactivatable organic compound and Ig, and subjecting the admixture to photoactivation conditions effective to covalently link the photoactivatable organic compound to the Ig. A photoactivatable organic compound of the present invention contains at least one AHM, which has a binding affinity for the Ig. However, the photoactivatable organic compound does not contain an azido, purine or pyrimidine group, inasmuch as such groups may interact with a different affinity site on the Ig, or may unnecessarily complicate synthesis of the photoactivatable organic compound.

[0082] In a preferred aspect of the invention, in addition to an AHM, a photoactivable organic compound comprises a peptide having self-binding, membrane -penetrating, adjuvant, and/or enzymatic properties. Such peptide can thereby impart its properties to a subsequently formed immunoconjugate. Preferably, a photoactivable organic compound comprising a peptide contains from about 5 to about 30 amino acid residues.

[0083] In a further preferred aspect of the invention, a peptide contains an autophilic amino acid sequence selected from the following group:

NH-ASRNKAND YTTD YSASVKGRFTVSR-COOH (SEQ ID NO: 1),

NH-SKAVSRFNAKGIRYSETNVDTYAS-COOH (SEQ ID NO. 4), NH-GAAVAYISSGGSSINYA-COOH (SEQ ID NO. 5), NH-GKAVAYISSGGSSINYAE-COOH (SEQ ID NO. 6), and NH-ASRNKANDYTTEYSASVKGRFIVSR-COOH (SEQ ID NO. 14) [0084] Alternatively, a peptide contains a membrane-penetrating amino acid sequence selected from the following group:

NH-KGEGAAVLLPVLLAAPG-COOH (SEQ ID NO. 2), and NH-WKGESAAVILPVLIASPG-COOH (SEQ ID NO. 7).

[0085] An AHM covalently linked to a peptide in a photoactivatable organic compound is preferably located at a C- or N-terminus of the peptide so as not to interfere with the desired properties of the peptide. Conveniently, the AHM can be present in an aromatic side chain of an amino acid, such as tryptophan, tyrosine, histidine, and phenylalanine. [0086] As referred to herein, an "immunoglobulin" can be a polyclonal antibody, monoclonal antibody, Fab fragment, or F(ab') ₂ fragment. It is generally preferred that mutual attraction and covalent linkage between the Ig and AHM occurs at an affinity site located in a variable domain of the immunoglobulin. For autophilic peptides, this can ensure close approach and noncovalent interaction between two adjacent Ig molecules on a cell surface. Such coupling of Ig molecules can, in turn, facilitate crosslinking of cellular receptors and promote intracellular signaling. Similarly, for membrane-penetrating peptides, location of the peptide adjacent a cellular receptor for the peptide can facilitate transport of an immunoconjugate into the cell. Binding affinity between the Ig and AHM can be demonstrated, as shown hereinafter, by competitive binding with an aromatic reporter molecule also having affinity for the Ig binding site. In practice, due to a multiplicity of affinity sites on the immunoglobulin, a plurality of photoactivatable organic compounds can be covalently linked to the Ig. Functionally, any type of immunoglobulin can be employed with the present invention, such as those having specific binding affinity for a cancer-related antigen, a caspase enzyme, ox-LDL, or cellular receptor.

[0087] An aromatic hydrocarbon moiety (AHM) of the present invention comprises at least one aryl, polynuclear aryl, heterocycle, or polynuclear heterocycle group. Representative of these different chemical classes are the following functional groups: aryl - benzene; polynuclear aryl - naphthalene, anthracene, and phenanthrene; heterocycle - pyrrole, furan, thiophene, pyrazole, oxazole, thiazole, pyridine, and imidazole,; polynuclear

heterocycle - benzofuran, acridine, thionaphthene, indole, quinoline, and isoquinoline, and geometric isomers thereof. Thus, for embodiments in which a photoactivatable organic compound comprises a peptide covalently bonded to an AHM, the AHM can be present in an amino acid residue of the peptide, e.g., tryptophan (indole), tyrosine (substituted benzene), histidine (imidazole), and phenylalanine (benzene). Representative AHMs are illustrated in Table 1.

[0088] Also contemplated within the invention is a pharmaceutical composition that comprises a pharmacologically effective amount of an instant super- antibody and a pharmaceutically acceptable carrier. Representative of such carriers are saline solution, e.g., 0.15% saline solution.

[0089] In a preferred embodiment, a photoreactive biotinylated tryptophan is inserted into several antibodies to yield biotinylated antibodies. This biotinylation reaction is not inhibited by the presence of ATP, which is a ligand for the conserved nucleotide binding site on antibodies (Rajagopalan, et al., 1996), and suggests that a different affinity site is involved. Moreover, it has been reported that UV energy can induce reactive radicals in heterocyclic compounds, such as tryptophan (Miles, et al. 1985). Thus, in a preferred embodiment of the present invention, UV light is used to covalently attach tryptophan-containing molecules to antibodies at a tryptophan affinity site on the antibodies.

Table 1. Aromatic Hydrocarbon Moieties.

[0090] With the discovery of an affinity of antibodies for AHMs, such as tryptophan, a simple, gentle and rapid method is available to conjugate organic molecules to antibodies. A practical application is the use of multiple biotinylated AMHs to affinity biotinylate antibodies. Additionally, AHM-containing peptides having biological or chemical properties can be conveniently affinity cross-linked to antibodies to create super- antibodies. [0091] Alternative methods of synthesizing antibody conjugates employ chemical or genetic engineering techniques to couple a peptide to an antibody. For instance, a peptide can

be attached by chemical means to an immunoglobulin (whole polyclonal or monoclonal antibody, or fragment thereof) at a carbohydrate site of the Fc portion or to an amino or sulfhydryl group of an antibody. Additionally, a peptide can be coupled to an antibody's variable domain structures by photo-crosslinking an azido-tryptophan or azido-purine to the antibody. In the latter approach, the peptide is believed to attach preferentially to the antibody by photoactivation of the azido group at a tryptophan or purine affinity site. [0092] In a further approach, a chimeric antibody can be expressed, using genetic manipulation techniques, as a fusion protein of an autophilic peptide and a whole immunoglobulin, or fragment thereof. See, e.g., U.S. Patent No. 6,238,667, PCT Publ. WO 9914244, U.S. Patent RE 38,008, U.S. Patent No. 5,635,180, and U.S. Patent No. 5,106,951, the disclosures of which are incorporated herein by reference.

[0093] Autophilic antibodies of the present invention typically comprise antibodies conjugated with one or more peptides having an autophilic sequence. It is believed that an autophilic antibody of the invention can comprise virtually any immunoglobulin. In some embodiments, the antibodies bind to targets implicated in a disease or disorder, where binding of the target has a therapeutic effect on the disease or disorder. The target antigens can include cell-surface antigens, including trans-membrane receptors. In specific embodiments, the Ig component of the antibodies can comprise the monoclonal antibody 5D10 which binds human B-cell receptors, the monoclonal antibody S1C5 which binds murine B-cell receptors, anti-CD20 antibodies such as rituximab (Rituxan®) which binds CD20 on normal and malignant pre-B and mature B lymphocytes, mouse monoclonal antibody 1F5 which is specific for CD-20 on human B-cell lymphomas, tositumab (Bexxar®) which also binds CD20 on B lymphocytes, anti-GM2 which binds human ganglioside GM2 lymphocytes, trastuzumab (Herceptin®) which binds the protein HER2 that is produced by breast cells, anti-caspase antibodies which recognize the caspase proteins involved in apoptosis, humanized TEPC- 15 antibodies which are capable of binding oxidized low density lipoproteins (ox-LDL) and can prevent uptake of oxidized LDL by macrophages, humanized T15-idiotype positive antibodies which bind phosphocholine, and humanized R24 antibodies which recognize the human GD3 ganglioside on melanoma cell surfaces. In further embodiments, the Ig component included in an antibody- autophilic peptide conjugate includes anti-epidermal growth factor receptor (EGFR) antibodies, anti-CD32B antibodies, anti-HLADRl antibodies, anti-CD 19 antibodies, anti-epithelial cell adhesion molecule

(EpCAM) antibodies, antibodies directed to a bacterial antigen such as a staphylococcal antigen, and anti-prostate specific antigen (PSA) antibodies.

[0094] An autophilic antibody of the present invention can comprise any autophilic peptide sequence. The autophilic peptide can also comprise optimized peptide sequences, which may include sequences having enhanced functionality, such as those that act as linkers to enhance display and cross-linking activity of antibodies, or residues that enhance solubility of autophilic sequences.

[0095] The present invention contemplates a method of producing an autophilic conjugate of the invention in which a template peptide has been modified to enhance the crosslinking potential of the autophilic antibodies as described above. In one embodiment of the invention, such functionally enhanced peptides are determined by producing a series of synthetic peptides with substitutions at each amino acid position within the template sequence and then testing this library of peptides for autophilic binding or for binding to the original peptide sequence. Those peptides with superior binding to the original sequence are then conjugated to immunoglobulins and the resultant conjugates are tested for potency, specificity, and the unwanted ability to induce aggregation. In one specific embodiment, the Tl 5 peptide sequence is altered and modified sequences are selected for enhanced function. [0096] In another embodiment of the invention, the self -binding potential of a peptide can be enhanced by increasing complementarity of the sequence, such as described in U.S. Patent No. 4,863,857 (issued to Blalock et al.), which is incorporated herein by reference. The self- binding potential and/or toleration of a peptide can also be enhanced by humanizing a self- binding peptide sequence derived from non-human animals. Humanizing a peptide sequence involves optimizing the sequence for expression or functionality in humans. Examples and methods of humanizing peptides and proteins have been described elsewhere (Roque-Navarro et al., 2003; Caldas et al., 2003; Leger et al., 1997; Isaacs and Waldmann, 1994; Miles et al. 1989; Veeraraghavan et al., 2004; Dean et al., 2004; Hakenberg et al., 2003; Gonzales et al., 2004; and H. Schellekens, 2002).

[0097] In a preferred embodiment, an autophilic peptide comprises the Tl 5 peptide, which originally comprised regions of CDR2 and FR3 of the murine germline-encoded S107/TEPC15 antibody. The Tl 5 peptide comprises the amino acid sequence: ASRNKANDYTTDYSASVKGRFIVSR (SEQ ID NO.: 1) (Kang C-Y, et al., 1988). Its autophilic property has been shown to be antigen-independent, thereby suggesting attachment of the peptide to monomeric antibodies can impart autophilic and increased avidity properties

to the antibodies (Kaveri S., et al., 1991). The T15 peptide can be photo-crosslinked to an aromatic hydrocarbon moiety or nucleotide affinity site of the immunoglobulin to produce the autophilic antibody. Alternatively, the Tl 5 peptide can be crosslinked to a carbohydrate site of the Fc portion or to an amino or sulfhydryl group of the immunoglobulin. Also, the autophilic antibody can be conveniently expressed as a fusion protein of the T15 peptide and whole immunoglobulin, or fragment thereof. In other specific embodiments, an autophilic peptide can comprise the scrambled T15 sequence (T15-scr2), which comprises the amino acid sequence NH-SKA VSRFNAKGIRYSETNVDTYAS-COOH (SEQ ID NO. 4), the peptide R24 comprising the sequence NH-GAA V AYISSGGSSINYA-COOH (SEQ ID NO. 5), the peptide R24-charged comprising the sequence NH-GKA V AYISSGGSSINYAE- COOH (SEQ ID NO. 6), and any modifications to such peptides which optimize or enhance the binding and therapeutic effectiveness of antibodies.

[0098] In further preferred embodiments, an autophilic peptide comprises the T15E peptide, NH- ASRNKAND YTTEYSAS VKGRFIVSR-COOH (SEQ ID NO. 14). The T15E peptide can be photo-crosslinked to an aromatic hydrocarbon moiety or nucleotide affinity site of the immunoglobulin to produce the autophilic antibody. Alternatively, the T15E peptide can be crosslinked to a carbohydrate site of the Fc portion or to an amino or sulfhydryl group of the immunoglobulin. Also, the autophilic antibody can be conveniently expressed as a fusion protein of the T15E peptide and whole immunoglobulin, or fragment thereof.

[0099] The attachment of autophilic peptide to a monomeric antibody can impart autophilic and increased avidity properties to the antibody (Y. Zhao, and H. Kohler, 2002). In specific embodiments, the antibody can be a humanized version of an orthologous antibody, which acquires increased or optimized binding and effectiveness when conjugated to an autophilic peptide, such as one containing the Tl 5 sequence. Methods of humanizing antibodies have been previously described. See, e.g., U.S. Patent No. 5,639,641 (issued to Pedersen et al.), U.S. Patent No. 5,498,531 (issued to Jarrell), U.S. Patent Nos. 6,180,370 and 5,693,762 (issued to Queen et al.), which are incorporated herein by reference. [00100] Autophilic antibody conjugates of the present invention can also comprise one or more other bioactive or functional peptides, which confer additional functionality on the antibody conjugates. For example, the antibody conjugate can comprise an antibody that bears a Tl 5 autophilic peptide and an MTS membrane translocation peptide (Y. Zhao et al., 2003; Y. Lin et al., 1995). In a specific embodiment, the MTS translocation peptide can have

the amino acid sequence KGEGAA VLLPVLLAAPG (SEQ ID NO. 2). In another embodiment, the translocation peptide can be an optimized MTS peptide, comprising the amino acid sequence WKGESAA VILPVLIASPG (SEQ ID NO. 7). The Tl 5 peptide provides autophilicity to the conjugate, and the MTS sequence facilitates entry of the antibody into cells. Such a conjugate can target, for example, cancer cells for radio- immunotherapy, when its antibody region targets a primarily intracellular, tumor-associated antigen, such as carcino-embryonic antigen (CEA). See, e.g., U.S. Patent No. 6,238,667, which is incorporated herein by reference. The autophilic conjugate, upon administration, targets CEA-bearing, colon carcinoma cells, is internalized by translocation of the antibody mediated by the MTS peptide, and is enabled to bind to the more prevalent intracellular form of CEA. Crosslinking of CEA antibody with, for instance, a therapeutic isotope such as ¹³¹ I can be retained in a cell longer than unmodified, labeled antibody and can deliver a higher radioactive dose to the tumor. In addition, such therapeutic isotopes as ⁿ I, which release beta particles of short path length and are not normally considered useful for therapy, can, when delivered intracellularly in closer proximity to the nucleus, be efficacious against certain targets, especially those of lymphoid origin and accessible in the blood and lymph tissues. Other categories of secondary, bioactive or functional peptides include peptides capable of binding to receptors, and peptide mimetics, capable of binding to a distinctive antigen or epitope of the same antigen, targeted by the primary antigen combining site. [00101] Autophilic antibodies conjugated with one or more other functional peptides may also be useful for targeting intracellular antigens. Such antigens could include tumor associated antigens and viral proteins. For example, an autophilic antibody specific for viral proteins which is conjugated with a self-binding peptide and a MTS peptide can also be used to bind to intracellular viral proteins and prevent production of viruses. The antibody can be internalized through the MTS peptide, and can be optimized to bind intracellular viral proteins (Zhao, Y., et al. 2003). Many other functional peptides may also be conjugated to the autophilic antibodies to increase functionality.

[00102] The invention also relates to compositions comprising a super- antibody of the invention and a pharmaceutically acceptable carrier. Conjugate autophilic antibodies can bind non-covalently with other autophilic antibodies when bound to their target antigen(s). However, premature formation of dimers or multimers of the antibodies may lead to difficulties in manufacturing, such as during purification and concentration, as well as drawbacks in administration, which may lead to side effects. As such, compositions

containing autophilic antibody-peptide conjugates of the invention are formulated to reduce this dimerizing potential and maximize monomeric properties while in solution and before administration. For example, it has been found that solution dimerization can be reduced or mitigated by using a hypertonic composition. In some embodiments, salt concentrations of 0.5M or more, low levels of SDS or other various detergents such as those of an anionic nature (see U.S. Patent No. 5,151,266, which is incorporated herein by reference), or modifications of the antibody to decrease its isoelectric point, for example through the use of succinyl anhydride (see U.S. Patent No. 5,322,678, which is incorporated herein by reference), can be used to formulate compositions. Disease Detection, Prevention and Treatment

[00103] A method of enhancing apoptosis, complement fixation, effector cell-mediated killing of targets, or preventing the development of, or enhancement of, a disease state, is also contemplated, which employs a super-antibody of the invention or a composition comprising the super-antibody. In one embodiment, an autophilic conjugate of the invention, or a composition containing an autophilic conjugate of the invention, is administered to a subject. Once administered, the antibodies bind to target cells and enhance apoptosis, complement fixation, effector cell-mediated killing of targets, or prevent target antigens or cells from stimulating the development of, or further enhancing, a disease state. In a further embodiment, allowing time for the autophilic conjugate to bind to target cells and enhance apoptosis, complement fixation, effector cell-mediated killing of targets, or prevent target antigens or cells from further enhancing a disease state, and for the autophilic conjugate to be cleared from normal tissues, a second anti-autophilic peptide antibody can be administered. For example, if an autophilic conjugate contains a non-native autophilic peptide, such as the murine Tl 5 sequence, an anti-T15 peptide antibody can be administered, which recognizes and binds to antibodies conjugated with the Tl 5 sequence. This allows binding to and enhancement of apoptosis of pre-localized super-antibodies. Additionally, a template autophilic peptide can be modified to enhance the crosslinking potential of the autophilic antibodies as described above.

[00104] In another aspect of the invention, a method of potentiating apoptosis of targeted cells of a patient comprises administering a first autophilic antibody-peptide conjugate, or a composition containing an autophilic antibody-peptide conjugate, and a second antibody, or composition containing the second antibody, which recognizes the autophilic peptide domain of the conjugate. In this embodiment, the antibody-peptide conjugate recognizes an antigen

on a target cell. Owing to its homodimerization property, the antibody- peptide conjugate can bind more avidly to the target than the corresponding antibody lacking the autophilic peptide domain. This is likely due to the ability to crosslink antigen at the surface of target cells. Moreover, whenever the autophilic antibodies bind to two or more antigens, with those antigens being brought in close proximity and crosslinked, due to the autophilic property of the antibodies, an apoptosis signal within the cell can be triggered. In those instances when the peptide domain of the conjugate presents an exposed epitope, a second antibody, specific for the autophilic peptide, can be administered, bind to the modified antibody, and enhance the process of crosslinking and even cause temporary clearance of the target antigen. As an example, if the target antigen is a receptor, clearance from the cell surface, endocytosis, and degradation will subsequently require synthesis of new receptor protein, meaning that the biological function of the receptor will be more effectively inhibited for a longer period than using either a simple blocking antibody or small molecule inhibitor. Alternatively, the second antibody can bear a radiolabel or other potentially therapeutic substance, so that when administered, it can attack the targeted cells. Since the autophilic peptide is present on only a small number of immunoglobulins and may be derived from another organism, the secondary antibody should have specificity for antibodies bearing the autophilic peptide. Thus, antibody specific to the autophilic peptide will have the requisite selectivity to be used in vivo. [00105] In another aspect of the invention, a patient who suffers from a disease or condition responsive to antibody therapy is administered at least one autophilic antibody of the invention in an amount effective to alleviate symptoms of the disease or condition. A disease or condition contemplated for treatment by an antibody of the invention can be a malignancy, neoplasm, cancer, atherosclerosis, auto-immune disorder, Alzheimer's disease or other neurodegenerative condition, graft or transplantation rejection, or any other disease or condition responsive to antibody therapy.

[00106] Atherosclerosis is a major cause of fatal and chronic vascular diseases that include stroke, heart failure and disruption of circulation in other organs and sites. There is increasing evidence that atherosclerosis is a chronic inflammatory disease. Recent findings indicate that oxidized lipids, especially phospholipids but also oxysterols, generated during LDL oxidation or within oxidatively stressed cells, are triggers for many of the events seen in developing lesions (Libby, P., et al., 2003). Oxidized phospholipids in ox-LDL are ligands for scavenger receptors on macrophages (Horkko, S., et al., 2000). Thus, ox-LDL and its products, including but not limited to the oxidized phospholipids and oxysterols, are initiating factors to

which the artery wall and its component cells respond. The classical lipid hypothesis and the new inflammation hypothesis should be jointly considered part of the pathogenetic pathway in atherosclerosis.

[00107] One aspect of the present invention aims to block the inflammatory pathway, thereby halting further plaque formation in patients with high cholesterol and lipid levels. In a preferred embodiment, a mouse Tl 5 antibody is "humanized" into a therapeutic antibody to treat vascular diseases in humans. Humanization of non-human antibodies may require extensive re-shaping of the antibody molecule, which can result in loss or reduction of antibody specificity and affinity. By conjugating an autophilic peptide to a humanized Tl 5 antibody, its superb targeting for ox-LDL can be restored, thereby blocking uptake of ox- LDL by macrophages and inhibiting chronic inflammation associated with atherosclerosis. A humanized Tl 5 specific for ox-LDL thereby mimics the human body's autoantibody response to the same antigen, which may be diminished in immune-compromised individuals. [00108] Accordingly, a general method of preventing or treating atherosclerosis in a patient comprises administering to the patient a super-antibody having specific binding affinity for oxidized low density lipoprotein (ox-LDL) and autophilic properties. The super- antibody is administered at a dose effective to block or reduce uptake of ox-LDL by macrophages, thereby inhibiting chronic inflammation associated with atherosclerosis. Preferably, the immunoconjugate specifically binds phosphorylcholine and expresses the Tl 5 idiotype. The immunoconjugate can be humanized, and preferably contains an autophilic peptide sequence, such as SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 14.

[00109] According to the principles of the present invention, a super- antibody, or a composition containing a super-antibody, is preferably administered in one or more dosage amounts substantially identical to, or lower than, those practicable for unmodified antibodies. Thus, in the treatment of a lymphoma or a breast cancer, an autophilic antibody of the invention can be administered in one or more dose amounts substantially identical to, or less than, the doses used for rituximab or trastuzumab. For example, treatment with trastuzumab (a humanized monoclonal anti-HER2/neu antibody) in a patient with HER2 ⁺ breast cancer employs an antibody concentration of about 10 mg/ml. Intravenous infusion over 90 minutes provides a total initial dose of 250 mg on day 0. Beginning at day 7, 100 mg is administered weekly for a total of 10 doses. The dosing regimen is reduced gradually from 250 mg to 100 mg to a maintenance dose of 50 mg per week. Similar or lower dosage regimens to that for

trastuzumab can be employed with autophilic antibodies, with any adjustments being well within the capabilities of a skilled practitioner.

[00110] In a preferred embodiment, a super- antibody of the present invention has a specific binding affinity for oxLDL. Exemplary of an antibody domain of the super- antibody is the monoclonal antibody 1K17, as described by U.S. Patent No. 6,716,410 (issued to Witztum et al.), the pertinent disclosure of which is incorporated herein by reference. When modified with an autophilic peptide according to the principles of the present invention, the resulting superior avidity of the autophilic antibody can enhance the binding property of the antibody absent the peptide. An autophilic antibody can localize to oxLDL of atherosclerotic plaques, whereupon it can be employed to detect the situs of the plaque when used with a label, reporter molecule, or secondary antibody, and the like. Alternatively, an autophilic antibody can be employed to coat the site of oxLDL deposition, thereby preventing further accumulation of plaque. In yet another aspect, an autophilic antibody can be employed to direct an anti-plaque agent, e.g., a thrombolytic or antioxidant agent.

[00111] Witztum et al. have reported that a human antibody fragment (Fab), referred to as IKl 7, binds to an epitope of ox-LDL and a breakdown product, MDA-LDL, but not native LDL. Moreover, they propose the Fab can inhibit uptake of ox-LDL by macrophages, presumably by binding to an epitope on ox-LDL that is recognized by macrophage scavenger receptors. The Fab is therefore proposed to inhibit atherogenesis by blocking the inflammatory response. These authors also report that anti-ox-LDL human antibodies express the so-called T15 idiotype (Shaw, P., et al, 2000). The T15 idiotype was originally described as being specific for phosphorylcholine (Lieberman, et al., 1974). Previously, it was discovered that the T15 idiotype is autophilic, i.e., they self-associate as noncovalent dimers (Kaveri, S., et al., 2000). By coupling the autophilic T15 peptide to a humanized T15/S107 antibody, the self- binding properties of the Tl 5 antibody and its avidity can be restored. [00112] Upon showing that the Tl 5 antibody is biologically equivalent to the human anti- phosphorylcholine antibodies known to bind to ox-LDL and inhibit inflammation initiated by macrophages, the efficacy of the Tl 5 antibody in preventing and/or treating atherosclerosis is demonstrated. A proposed mode of action of the Tl 5 antibody is schematically indicated in Fig. 22 (modified from Steinberg, Nature Medicine, 2002, 8: 12311).

[00113] The present invention is also for a method of detecting a disease state, such as the presence of atherosclerotic plaques in a patient's vascular system. Such method comprises administering to a patient an immunoconjugate of the present invention, which has a specific

binding affinity for oxidized low density lipoprotein (ox-LDL). The immunoconjugate also has autophilic properties. Sites of immunoconjugate concentration in the patient's vascular system are then detected, thereby localizing and visualizing the atherosclerotic plaques. Preferably, the immunoconjugate binds phosphorylcholine and/or expresses the T15 idiotype. More preferably, the immunoconjugate bears an autophilic peptide having an aforementioned amino acid sequence.

[00114] A method of detecting cells undergoing apoptosis, which may be indicative of a disease state, is also contemplated. For example, when an antigenic determinant of a cell surface is represented by membrane -bound phosphorylcholine or phosphatidylserine, the cell can be contacted with an autophilic immunoconjugate of the invention, which has specific binding affinity for the antigenic determinant. The presence or absence of immunoconjugate bound to the cell is then detected. Previously described autophilic peptides can be used. Such methods as flow cytometry, fluorescent microscopy, histological staining, or in vivo imaging are particularly preferred for conducting detection. To facilitate these, the immunoconjugate may be labeled with fluorescein.

[00115] Additionally, an in vitro assay of apoptosis can be used to screen multiple antigen- positive target cell lines, and if possible, fresh isolates of antigen-positive cells. A non- modified antibody is incubated with a secondary (antiimmunoglobulin) antibody to enhance the potential for cross-linking. Cells may be enumerated by pre-labeling, such as with ¹ Cr or ¹³¹ I-UDR, or by FACS analysis using indicators of apoptosis. Positive results in this assay predict a positive outcome using an autophilic immunoconjugate. However, negative results in the assay do not necessarily mean that subsequent conjugation with an autophilic peptide will not improve one or more antibody effector properties.

[00116] Autophilic antibodies of the present invention have a higher potential for forming dimers in vitro under laboratory conditions, such as in solution with PEG. This laboratory characteristic correlates with crosslinking ability upon binding to a cell-surface target and higher therapeutic potency through such mechanisms as triggering apoptosis. This characteristic can be used to identify natural SuperAntibodies and to screen for proper conjugation of self -binding peptides to a non- autophilic antibody. Suitable animal models for testing efficacy of the aforementioned autophilic antibodies include severely compromised immunodeficient (SCID) mice or nude mice bearing human tumor xenografts. [00117] Scientific and technical terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art unless otherwise defined herein.

Such terms are found defined and used in context in various standard references illustratively including J. Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; F.M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002; D.L. Nelson and M.M. Cox, Lehninger Principles of Biochemistry, 4th Ed., W.H. Freeman & Company, 2004; Herdewijn, P. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004; J. P. Sundberg and T.Ichiki, Eds., Genetically Engineered Mice Handbook, CRC; 2005; M. H. Hofker and J. van Deursen, Eds., Transgenic Mouse Methods and Protocols, Humana Press, 2002; and A. L. Joyner, Gene Targeting: A Practical Approach, Oxford University Press, 2000.

[00118] Antibodies, antigen binding fragments and methods for their generation are known in the art and such antibodies, antigen binding fragments and methods are described in further detail, for instance, in Antibody Engineering, Kontermann, R. and Dubel, S. (Eds.), Springer, 2001; Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; Ausubel, F. et al., (Eds.), Short Protocols in Molecular Biology, Wiley, 2002, particularly chapter 11 ; J. D. Pound (Ed.) Immunochemical Protocols, Methods in Molecular Biology, Humana Press; 2nd ed., 1998; B.K.C. Lo (Ed.), Antibody Engineering: Methods and Protocols, Methods in Molecular Biology, Humana Press, 2003; and Kohler, G. and Milstein, C, Nature, 256:495-497 (1975).

[00119] In embodiments of the present invention, a recombinant chimeric autophilic antibody is provided which includes a fusion protein including an autophilic peptide fused to at least a portion of an immunoglobulin. Figure 23 shows a schematic representation of the structures of an unmodified antibody and a "DXL" autophilic antibody including an autophilic peptide at the C-terminus of the immunoglobulin heavy chain. [00120] An autophilic peptide included in a recombinant chimeric autophilic antibody is an autophilic peptide which includes the amino acid sequence SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 14, or a substantially identical amino acid sequence. An amino acid sequence which is substantially identical to the 25-mers of SEQ ID Nos.l and 14 has at least 20 contiguous amino acids, more preferably at least 22 contiguous amino acids, having an amino acid sequence at least 70%, 80%, 85%, 90% and more preferably 95%, 96%, 97%, 98%, 99% or 100% identical to 20 or more contiguous amino acids of the identified autophilic amino acid sequence. An amino acid sequence which is substantially identical to

the 17-mers of SEQ ID Nos.5 and 6 has at least 13 contiguous amino acids, more preferably at least 15 contiguous amino acids, having an amino acid sequence at least 70%, 80%, 85%, 90% and more preferably 95%, 96%, 97%, 98%, 99% or 100% identical to 13 or more contiguous amino acids of the identified autophilic amino acid sequence. A peptide which is substantially identical to an autophilic peptide retains a substantially similar or better autophilic function compared to the reference autophilic peptide with which it is substantially identical.

[00121] Percent identity is determined by comparison of amino acid or nucleic acid sequences, including a reference sequence and a putative homologue sequence. Algorithms used for determination of percent identity illustratively include the algorithms of S. Karlin and S. Altshul, PNAS, 90:5873-5877, 1993; T. Smith and M. Waterman, Adv. Appl. Math. 2:482-489, 1981, S. Needleman and C. Wunsch, J. MoI. Biol., 48:443-453, 1970, W. Pearson and D. Lipman, PNAS, 85:2444-2448, 1988 and others incorporated into computerized implementations such as, but not limited to, GAP, BESTFIT, FASTA, TFASTA; and BLAST, for example incorporated in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.) and publicly available from the National Center for Biotechnology Information.

[00122] Multimers of autophilic peptides can be used in particular embodiments of the present invention. Exemplary multimers having spacer amino acids disposed between the autophilic peptides are shown as SEQ ID No. 10, SEQ ID No. 11.

[00123] In embodiments of the present invention, a nucleic acid expression construct is provided which encodes a DNA sequence encoding an autophilic peptide inserted in-frame with a DNA sequence encoding at least a portion of an immunoglobulin for use in producing a recombinant chimeric autophilic antibody.

[00124] In specific embodiments, a DNA sequence encoding SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 14, or a substantially identical autophilic peptide is inserted in- frame with a DNA sequence encoding an immunoglobulin heavy chain and/or immunoglobulin light chain. The fusion protein expressed from the DNA sequence contains an immunoglobulin heavy chain and/or immunoglobulin light chain having SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 14, or a substantially identical autophilic peptide at the C-terminus or N-terminus. In preferred embodiments, SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 14, or a substantially identical autophilic peptide is disposed at the C-terminus of the immunoglobulin heavy chain and/or immunoglobulin light chain.

[00125] Recombinant chimeric autophilic antibodies provided according to embodiments of the present invention include a chimeric immunoglobulin heavy chain and/or a chimeric immunoglobulin light chain, and fused to an autophilic peptide.

[00126] A chimeric autophilic antibody of the invention can comprise virtually any chimeric immunoglobulin. In some embodiments, the antibodies bind to targets implicated in a disease or disorder, where binding of the target has a therapeutic effect on the disease or disorder. The target antigens can include cell-surface antigens, including trans -membrane receptors.

[00127] In particular embodiments, a chimeric autophilic antibody of the invention includes a chimeric immunoglobulin heavy chain and/or a chimeric immunoglobulin light chain. A chimeric autophilic antibody of the invention preferably includes a human constant heavy chain and/or a human constant light chain. A chimeric autophilic antibody of the invention preferably includes a human gamma constant heavy chain region and/or a human kappa constant light chain region.

[00128] Nucleic acids encoding immunoglobulin heavy chains or immunoglobulin light chains are well-known and any of various nucleic acids encoding immunoglobulin heavy chains or immunoglobulin light chains can be used to produce a recombinant chimeric autophilic antibody of the present invention. Specific nucleic acids are described herein which encode human constant heavy chain and/or a human constant light chains, particularly human gamma constant heavy chains and human kappa constant light chains.

[00129] Nucleic acids encoding human gamma constant heavy chains and/or human kappa constant light chains can be obtained from commercial sources, such as vector pAc-k-

CH3, available from Progen Biotechnik GmbH. Nucleic acids encoding protein and/or peptides described herein, including human gamma constant heavy chains and/or human kappa constant light chains, can be produced using recombinant techniques such as by cloning or synthesis.

[00130] Particular immunoglobulin constant heavy chains and/or immunoglobulin kappa constant light chains, are described, for instance, in U.S. Patent Nos. 5,736,137; 6,194,551;

6,528,624; 6,538,124; 6,737,056; 7,122,637; 7,151,164; 7,183,387; 7,297,775; 7,332,581;

7,335,742; 7,355,008; 7,364,731 and 7,371,826.

[00131] In specific embodiments, a chimeric autophilic antibody of the invention includes a variable heavy chain and/or a variable light chain derived from: the monoclonal antibody

5D10 which binds human B-cell receptors, the monoclonal antibody S1C5 which binds

murine B-cell receptors, anti-CD20 antibodies such as rituximab (Rituxan®) which binds CD20 on normal and malignant pre-B and mature B lymphocytes, mouse monoclonal antibody 1F5 which is specific for CD-20 on human B-cell lymphomas, tositumab (Bexxar®) which also binds CD20 on B lymphocytes, anti-GM2 which binds human ganglioside GM2 lymphocytes, trastuzumab (Herceptin®) which binds the protein HER2 that is produced by breast cells, anti-caspase antibodies which recognize the caspase proteins involved in apoptosis, humanized TEPC- 15 antibodies which are capable of binding oxidized low density lipoproteins (ox-LDL) and can prevent uptake of oxidized LDL by macrophages, humanized T15-idiotype positive antibodies which bind phosphocholine, and humanized R24 antibodies which recognize the human GD3 ganglioside on melanoma cell surfaces. [00132] Rituximab antibodies and their properties are described, for example, in McLaughlin P, et al., J Clin Oncol. 1998 Aug;16(8):2825-33; Edwards JC, et al., N Engl J Med. 2004 Jun 17;350(25):2572-81; Braendstrup P, et al., Am J Hematol. 2005 Apr;78(4):275-80; Binder M, et al., Blood. 2006 Sep 15;108(6):1975-8; and Burton C, et al.., N Engl J Med. 2003 Jun 26;348(26):2690-l.

[00133] Particular autophilic antibodies according to embodiments of the present invention include a chimeric immunoglobulin heavy chain having a variable heavy chain of an anti- CD20 immunoglobulin.

[00134] For example, a chimeric autophilic antibody of the present invention includes chimeric immunoglobulin gamma heavy chain including the variable heavy chain of monoclonal antibody 1F5 and a human gamma constant heavy chain conjugated to an autophilic peptide. SEQ ID No. 28 is an amino acid sequence of a chimeric immunoglobulin heavy chain including the variable heavy chain of monoclonal antibody 1F5 and a human gamma constant heavy chain. Thus, in particular embodiments of the present invention, a chimeric autophilic antibody includes SEQ ID No. 28 or a substantially identical amino acid sequence.

[00135] A substantially identical amino acid sequence of an immunoglobulin component has an amino acid sequence at least 70%, 80%, 85%, 90% and more preferably 95%, 96%, 97%, 98%, 99% or greater % identical to an amino acid sequence disclosed herein in particular embodiments of the present invention, wherein the substantially identical protein retains a substantially similar or better function compared to the reference protein with which it is substantially identical.

[00136] SEQ ID No. 26 is an amino acid sequence of a chimeric immunoglobulin heavy chain including the variable heavy chain of monoclonal antibody 1F5 and a human gamma constant heavy chain conjugated to the T15E autophilic peptide. An immunoglobulin gamma heavy chain portion of an anti-CD20 antibody included in a recombinant chimeric autophilic antibody has amino acid sequence SEQ ID No. 26 or a substantially identical amino acid sequence in particular embodiments of the present invention.

[00137] A chimeric immunoglobulin gamma heavy chain portion of an anti-CD20 antibody included in a recombinant chimeric autophilic antibody has amino acid sequence SEQ ID No. 45 or a substantially identical amino acid sequence in particular embodiments of the present invention.

[00138] SEQ ID NO.45: Chimeric immunoglobulin heavy chain portion of an anti-CD20 autophilic antibody including an N-terminal leader and T15E at the C-terminus

MGWSCI I LFLVATATGVQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGA IYPGNGDT SYNQKFKGKATLTVDKSS STAYMQLS SLTSED SAVYFCARWYYSNSYWYFDVWGTGTTVTVSGP SVFPLAP SSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVP SS SLGTQTYICNVNHKP SN TKVDKKAEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTI SKAKGQPREPQVYTLPP SRDEL TKNQVSLTCLVKGFYP SD IAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGKGAAASRNKANDYTTEYSASVKGRFIVSR

[00139] SEQ ID NO.47: Chimeric immunoglobulin heavy chain portion of an anti-CD20 autophilic antibody without the N-terminal leader and T15E at the C-terminus

QAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGD TSYNQKFKGKATLT VDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSGPSVFPLAPS SKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDK

HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK

[00140] A chimeric immunoglobulin kappa light chain portion of an anti-CD20 antibody included in a recombinant chimeric autophilic antibody has amino acid sequence SEQ ID No.

46 or a substantially identical amino acid sequence in particular embodiments of the present invention.

[00141] SEQ ID NO. 46: Chimeric immunoglobulin light chain kappa portion of an anti-

CD20 autophilic antibody including a leader.

MGWSCI I LFLVATATGVQIVLSQSPAI LSASPGEKVTMTCRAS SSVSYMHWYQQKPGS SPKPWI YAP SNLASGVP ARFSGSGSGTSYSLTI SRVEAEDAATYYCQQWSFNPPTFGAGTKLELKRTVAAP SVFIFPP SDEQLKSGTASWC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPV TKSFNR

[00142] SEQ ID NO. 48: Chimeric immunoglobulin light chain kappa portion of an anti- CD20 autophilic antibody without the leader.

QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGV PARFSGSGSGTSYS LTI SRVEAEDAATYYCQQWSFNPPTFGAGTKLELKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNR

[00143] SEQ ID NO. 49: Variable region of the immunoglobulin light chain kappa portion of an anti-CD20 autophilic antibody.

QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGV PARFSGSGSGTSYS LTISRVEAEDAATYYCQQWSFNPPT

[00144] An anti-CD20 antibody immunoglobulin heavy chain includes a chimeric gamma heavy chain including the variable region of monoclonal antibody 1F5 and human gamma constant heavy chain region including amino acid sequence SEQ ID No. 28 or a substantially identical amino acid sequence in particular embodiments of the present invention.

SEQ ID No. 28

QVQLRQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLE WIGAIYPGNGDTSYNQKFKG KATLTADKSSSTA YMQLSSLTSEDSA VYYCARSHYGSNYVD YFD YWGQGTTLTVSSASTKGPSVFPLA PSSKSTSGGTAALGCL VKD YFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL P APIEK TISKAKGQPREPQVYTLPPSREEVTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQAA

[00145] In a particular embodiment, an anti-CD20 antibody immunoglobulin gamma heavy chain has amino acid sequence SEQ ID No. 27 or a substantially identical amino acid sequence in particular embodiments of the present invention.

TABLE 7. Comparison of Heavy Chains of ChlF5-DXL (SEQ ID No. 26) and an alternate anti-CD20 antibody immunoglobulin gamma heavy chain (SEQ ID No. 27)

SEQ ID No. 27 QVQLQQPGAELVKPGAξVKMξCKAξGYTFTξYNMHWVKQTPGRGLEWIGAIYPGNG DTξY 60

SEQ ID No. 26 QVQLRQPGAELVKPGAξVKMξCKAξGYTFTξYNMHWVKQTPGQGLEWIGAIYPGNG DTξY 60

SEQ ID No. 27 NQKFKGKATLTADKξξξTAYMQLξξLTξEDξAVYYCARξTYYGGDW—YFN VWGAGTTVT 118

SEQ ID No. 26 NQKFKGKATLTADKξξξTAYMQLξξLTξEDξAVYYCARξ-HYGξNYVDYFD YWGQGTTLT 119

SEQ ID No. 27 VξAAξTKGPξVFPLAPξξKξTξGGTAALGCLVKDYFPEPVTVξWNξGALTÎ ¾GVHTFPAVL 178

SEQ ID No. 26 VξξAξTKGPξVFPLAPξξKξTξGGTAALGCLVKDYFPEPVTVξWNξGALT ξGVHTFPAVL 179

SEQ ID No. 27 QξξGLYξLξξVVTVPξξξLGTQTYICNVNHKPξNTKVDKKAEPKξCDKTH TCPPCPAPEL 238

SEQ ID No. 26 QξξGLYξLξξVVTVPξξξLGTQTYICNVNHKPξNTKVDKRVEPKξCDKTH TCPPCPAPEL 239

SEQ ID No. 27 LGGPξVFLFPPKPKDTLMIξRTPEVTCWVDVξHEDPEVKFNWYVDGVEVHNAKTKPR EE 298

SEQ ID No. 26 LGGPξVFLFPPKPKDTLMIξRTPEVTCWVDVξHEDPEVKFNWYVDGVEVHNAKTKPR EE 299

SEQ ID No. 27 QYNξTYRVVξVLTVLHQDWLNGKEYKCKVξNKALPAPIEKTIξKAKGQPREPQVYT LPPξ 358

SEQ ID No. 26 QYNξTYRVVξVLTVLHQDWLNGKEYKCKVξNKALPAPIEKTIξKAKGQPREPQVYT LPPξ 359

SEQ ID No. 27 RDELTKNQVξLTCLVKGFYPξDIAVEWEξNGQPENNYKTTPPVLDξDGξFFLYξ KLTVDK 418

SEQ ID No. 26 REEVTKNQVξLTCLVKGFYPξDIAVEWEξNGQPENNYKTTPPVLDξDGξFFLYξ KLTVDK 419

SEQ ID No. 27 ξRWQQGNVFξCξVMHEALHNHYTQKξLξLξPGK 451

SEQ ID No. 26 ξRWQQGNVFξCξVMHEALHNHYTQAAAξRNKANDYTTEYξAξVKGRFIVξR 470

[00146] In a particular embodiment, an anti-CD20 antibody immunoglobulin heavy chain includes a gamma heavy chain variable region including amino acid sequence SEQ ID No. 33 with or without leader sequence, SEQ ID NO. 34 or a substantially identical amino acid sequence in particular embodiments of the present invention.

SEQ ID No . 33

MGW SLILLF LVAVATRVL SQVQLQQPGAELVKPGAS VKMSCKASGYTFTS YNMHWVKQTPGRGLEWIG

AIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDW YFNVWGAGT

TVTVSA

SEQ ID No. 34 MGWSLILLFLVAVATRVLS

[00147] In a particular embodiment, an anti-CD20 antibody immunoglobulin light chain includes a kappa light chain variable region including amino acid sequence SEQ ID No. 37 or a substantially identical amino acid sequence in particular embodiments of the present invention.

SEQ ID No. 37

Met Asp Phe GIn VaI GIn He He Ser Phe Leu Leu He Ser Ala Ser VaI He Met Ser Arg GIy GIn He VaI Leu Ser GIn Ser Pro Ala He Leu Ser Ala Ser Pro GIy GIu Lys VaI Thr Met Thr Cys Arg Ala Ser Ser Ser VaI Ser Tyr He His Trp Phe GIn GIn Lys Pro GIy Ser Ser Pro Lys Pro Trp He Tyr Ala Thr Ser Asn Leu Ala Ser GIy VaI Pro VaI Arg Phe Ser GIy Ser GIy Ser GIy Thr Ser Tyr Ser Leu Thr He Ser Arg VaI GIu Ala GIu Asp Ala Ala Thr Tyr Tyr Cys GIn GIn Trp Thr Ser Asn Pro Pro Thr Phe GIy GIy GIy Thr Lys Leu GIu He Lys

[00148] In a particular embodiment, an anti-CD20 antibody immunoglobulin heavy chain includes a gamma heavy chain variable region including amino acid sequence SEQ ID No. 39 or a substantially identical amino acid sequence in particular embodiments of the present invention. SEQ ID No. 39

MGWSCI I LFLVATATGVQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGA IYPGNGDT SYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARWYYSNSYWYFDVWGTGTTVT VS

[00149] In a particular embodiment, an anti-CD20 antibody immunoglobulin heavy chain includes a gamma heavy chain variable region of monoclonal antibody 1F5 including amino acid sequence SEQ ID No. 41. A substantially identical amino acid sequence has an amino acid sequence at least 70%, 80%, 85%, 90% and more preferably 95%, 96%, 97%, 98%, 99% or greater % identical to SEQ ID No. 41. [00150] SEQ ID No. 41

MAQVQLRQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGN GDTSYNQKFKGKA TLTADKSSSTAYMQLSSLTSEDSAVYYCARSHYGSNYVDYFDYWGQGTLVTVSTG

[00151] In a particular embodiment, an anti-CD20 antibody immunoglobulin light chain includes a kappa light chain variable region of monoclonal antibody 1F5 including amino acid sequence SEQ ID No. 43 or a substantially identical amino acid sequence in particular embodiments of the present invention. SEQ ID No. 43

MAQIVLSQSPAILSASPGEKVTMTCRASSSLSFMHWYQQKPGS SPKPWIYATSNLASGVPARFSGSGSGT SYSLTISRVEAEDAATYFCHQWSSNPLTFGAGTKVEIKRK

[00152] Compositions provided according to embodiments of the present invention include an expression vector encoding an immunoglobulin heavy chain and/or an immunoglobulin light chain; and encoding an autophilic peptide.

[00153] In particular embodiments of the present invention, an expression construct is provided that includes a DNA sequence encoding an autophilic peptide. [00154] The term "expression construct" refers to a recombinant nucleic acid sequence including a nucleic acid sequence encoding a peptide or protein to be expressed. The nucleic acid encoding a peptide or protein to be expressed is operably linked to one or more regulatory nucleic acid sequences that facilitate expression of the peptide or protein to be expressed. Nucleic acid sequences are operably linked when they are in functional relationship. A regulatory nucleic acid sequence is illustratively a promoter, an enhancer, a DNA and/or RNA polymerase binding site, a ribosomal binding site, a polyadenylation signal, a transcription start site, a transcription termination site or an internal ribosome entry site (IRES). An expression construct can be incorporated into a vector, such as an expression vector and/or cloning vector. The term "vector" refers to a recombinant nucleic acid vehicle for transfer of a nucleic acid. Exemplary vectors are plasmids, cosmids, viruses and bacteriophages. Particular vectors are known in the art and one of skill in the art will recognize an appropriate vector for a specific purpose.

[00155] In particular embodiments of the present invention, an expression construct encoding

[00156] An internal ribosome entry site (IRES) is a nucleic acid sequence that permits translation initiation at an internal site in an mRNA. IRES are well-known in the art, for example as described in Pelletier, J. et al., Nature, 334:320-325, 1988; Vagner, S. et al., EMBO Rep., 2:893-898, 2001 ; and Hellen, C. U. et al, Genes Dev. 15:1593-1612, 2001 [00157] Expression constructs according to embodiments of the present invention include, in operable linkage: a promoter, a DNA sequence encoding an autophilic peptide and a transcription termination site. In particular embodiments of the present invention, an expression construct including, in operable linkage: a promoter, a DNA sequence encoding an autophilic peptide and a transcription termination site, is included in an expression vector. Particular expression vectors of the present invention are described herein. [00158] In particular embodiments of the present invention, an expression construct including, in operable linkage: a promoter, a DNA sequence encoding an autophilic peptide and a transcription termination site, is included in a plasmid expression vector. [00159] The term "promoter" is known in the art and refers to one or more DNA sequences that bind an RNA polymerase and allow for initiation of transcription. A promoter nucleic acid sequences is typically positioned upstream (5') of a nucleic acid encoding a peptide or protein to be expressed. One of skill in the art is familiar with various well-known promoters and is able to select a promoter suitable for use in expressing a peptide or protein in a particular environment, such as in a specified cell type. Examples of well-known promoters that can be used include mouse metallothionein- 1 promoter, the long terminal repeat region of Rous Sarcoma virus (RSV promoter), the early promoter of human cytomegalovirus (CMV promoter) and the simian virus 40 (SV40) early promoter. [00160] The term "transcription termination site" refers to a DNA sequence operable to terminate transcription by an RNA polymerase. A transcription termination site is generally positioned downstream (3') of a nucleic acid encoding a peptide or protein to be expressed. [00161] A leader sequence can be used in conjunction with expression of one or more immunoglobulin components included in an autophilic antibody described herein. Leader sequences shown can be modified or replaced with alternative leader sequences if desired. [00162] A specific DNA sequence encoding T15E autophilic peptide ASRNKANDYTTEYSASVKGRFIVSR (SEQ ID NO. 14) is:

[00163] 5' gca agt aga aac aaa get aat gat tat aca aca gag tac agt gca tct gtg aag ggt egg ttc ate gtc tec aga 3' (SEQ ID No. 29)

[00164] A specific DNA sequence encoding Tl 5 autophilic peptide

ASRNKANDYTTDYSASVKGRFIVSR (SEQ ID NO. 1) is:

[00165] 5' gca agt aga aac aaa get aat gat tat aca aca gac tac agt gca tct gtg aag ggt egg ttc ate gtc tec aga 3' (SEQ ID No. 30)

[00166] As will be appreciated by one of skill in the art, the degeneracy of the genetic code is such that more than one nucleic acid will encode a particular autophilic peptide and these alternative sequences are considered within the scope of the present invention.

[00167] In addition, one or more amino acid substitutions, additions or deletions may occur in a particular autophilic peptide amino acid sequence as long as the autophilic properties of the peptide remain.

[00168] In a particular embodiment, an anti-CD20 antibody immunoglobulin heavy chain included in an autophilic antibody of the present invention includes a gamma heavy chain region encoded by nucleic acid sequence SEQ ID No. 31 or a homolog thereof.

[00169] In a particular embodiment, an anti-CD20 antibody immunoglobulin light chain included in an autophilic antibody of the present invention includes a kappa light chain encoded by nucleic acid sequence SEQ ID No. 32 or a homolog thereof.

[00170] A homolog of a nucleic acid sequence disclosed herein encodes an amino acid sequence having at least 70%, 80%, 85%, 90% and more preferably 95%, 96%, 97%, 98%,

99% or greater % identity to the amino acid sequence encoded by the specific nucleic acid sequence referred to. A nucleic acid sequence homolog hybridizes under high stringency hybridization conditions to the reference nucleic acid sequence, or a complement thereof, in particular embodiments of the present invention.

[00171] The terms "hybridizing" and "hybridization" refer to pairing and binding of complementary nucleic acids. Hybridization occurs to varying extents between two nucleic acids depending on factors such as the degree of complementarity of the nucleic acids, the melting temperature, Tm, of the nucleic acids and the stringency of hybridization conditions, as is well known in the art. High stringency hybridization conditions are those which only allow hybridization of highly complementary nucleic acids. Determination of stringent hybridization conditions is routine and is well known in the art, for instance, as described in

J. Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring

Harbor Laboratory Press; 3rd Ed., 2001 ; and F.M. Ausubel, Ed., Short Protocols in Molecular

Biology, Current Protocols; 5th Ed., 2002.

[00172] The term "complementary" refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a "percent complementarity" to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10 or

10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3'-TCGA-5' is 100% complementary to the nucleotide sequence 5'-AGCT-3'. Further, the nucleotide sequence 3'-TCGA- is 100% complementary to a region of the nucleotide sequence 5'-TTAGCTGG-3'.

[00173] High stringency hybridization conditions are known in the art and one of skill in the art is able to discern high stringency conditions. Exemplary high stringency conditions include 50% formamide, 5X SSC, 5OmM sodium phosphate, pH 6.8, 0.1% sodium pyrophosphate, 5X Denhardt's solution, 50 micrograms/mL salmon sperm DNA, 0.1% SDS and 10% dextran sulfate at 42°C and a high stringency wash such as a wash in 0.1X

SSCVO.1% w/v SDS at 50 ⁰ C.

[00174] In a particular embodiment, an anti-CD20 antibody gamma immunoglobulin heavy chain variable region included in an autophilic antibody of the present invention includes a gamma immunoglobulin heavy chain variable region encoded by nucleic acid sequence SEQ ID No. 35 or a homolog thereof.

[00175] SEQ ID No. 35

ATGGGTTGGAGCCTCATCTTGCTCTTCCTTGTCGCTGTTGCTACGCGTGTCCTGTC

CCAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGT

GAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGG

GTAAAACAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGA

AATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCA

GACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGAC

TCTGCGGTCTATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTTCA

ATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCA

[00176] SEQ ID No. 36 encodes the exemplary leader sequence having SEQ ID NO. 34.

[00177] SEQ ID No. 36

ATGGGTTGGAGCCTCATCTTGCTCTTCCTTGTCGCTGTTGCTACGCGTGTCCTGTC

C

[00178] In a particular embodiment, an anti-CD20 antibody kappa immunoglobulin light chain variable region included in an autophilic antibody of the present invention includes a kappa immunoglobulin light chain variable region encoded by nucleic acid sequence SEQ ID

No. 38 or a homolog thereof.

[00179] SEQ ID No. 38

ATGGATTTTCAGGTGCAGATTATCAGCTTCCTGCTAATCAGTGCTTCAGTCATAAT

GTCCAGAGGGCAAATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCA

GGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTACATCCAC

TGGTTCCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCA

ACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACTTCTTAC

TCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGC

AGTGGACTAGTAACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATCAAA

[00180] In a particular embodiment, an anti-CD20 antibody gamma immunoglobulin heavy chain variable region included in an autophilic antibody of the present invention includes a gamma immunoglobulin heavy chain variable region encoded by nucleic acid sequence SEQ ID No. 40 or a homolog thereof. [00181] SEQ ID No. 40

ATGGGATGGTCTTGTATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCAGGCC TACCTGCAG CAGTCTGGCGCCGAGCTGGTGCGCCCTGGCGCCTCCGTGAAAATGAGCTGCAAAGCCTCT GGCTAT

GGCCATCTACCCAGGCAACGGCGATACCTCTTACAATCAGAAGTTCAAGGGAAAGGC CACACTGA CAGTGGACAAGTCTTCTAGCACCGCCTACATGCAGCTGAGCAGCCTGACCTCCGAGGATT CCGCCG TGTACTTTTGCGCCAGAGTGGTGTATTATTCCAATTCCTACTGGTACTTCGATGTGTGGG GGACCGG CACAACCGTGACCGTGTCC

[00182] In a particular embodiment, an anti-CD20 antibody gamma immunoglobulin heavy chain variable region included in an autophilic antibody of the present invention includes a monoclonal antibody 1F5 gamma immunoglobulin heavy chain variable region encoded by nucleic acid sequence SEQ ID No. 42 or a homolog thereof. [00183] SEQ ID No. 42

TAG

[00184] In a particular embodiment, an anti-CD20 antibody kappa immunoglobulin light chain variable region included in an autophilic antibody of the present invention includes a monoclonal antibody 1F5 kappa immunoglobulin light chain variable region encoded by nucleic acid sequence SEQ ID No. 44 or a homolog thereof. [00185] SEQ ID No. 44

GCTGGGACAAAGGTGGAAATAAAACGTAAGTAG

[00186] In a particular embodiment, an anti-CD20 antibody kappa immunoglobulin light chain variable region included in an autophilic antibody of the present invention includes a kappa immunoglobulin light chain variable region encoded by nucleic acid sequence SEQ ID No. 50 or a homolog thereof. [00187] SEQ ID No. 50

AGAGTGGAAGCCGAAGACGCCGCCACCTACTACTGCCAGCAGTGGTCTTTCAATCCT CCCACC

[00188] An expression construct of the present invention including a DNA sequence encoding an autophilic peptide can be used to produce an autophilic antibody. [00189] Compositions provided according to embodiments of the present invention include an expression construct encoding a chimeric immunoglobulin heavy chain and/or a chimeric immunoglobulin light chain, and encoding an autophilic peptide. [00190] In specific embodiments, an expression construct encoding a chimeric immunoglobulin heavy chain and/or a chimeric immunoglobulin light chain includes at least a variable heavy chain and/or at least a variable light chain derived from: the monoclonal antibody 5D10 which binds human B-cell receptors, the monoclonal antibody S1C5 which binds murine B-cell receptors, anti-CD20 antibodies such as rituximab (Rituxan®) which binds CD20 on normal and malignant pre-B and mature B lymphocytes, mouse monoclonal antibody IF5 which is specific for CD-20 on human B-cell lymphomas, tositumab (Bexxar®) which also binds CD20 on B lymphocytes, anti-GM2 which binds human ganglioside GM2 lymphocytes, trastuzumab (Herceptin®) which binds the protein HER2 that is produced by breast cells, anti-caspase antibodies which recognize the caspase proteins involved in

apoptosis, humanized TEPC- 15 antibodies which are capable of binding oxidized low density lipoproteins (ox-LDL) and can prevent uptake of oxidized LDL by macrophages, humanized T15-idiotype positive antibodies which bind phosphocholine, and humanized R24 antibodies which recognize the human GD3 ganglioside on melanoma cell surfaces. [00191] As will be appreciated by one of skill in the art, the degeneracy of the genetic code is such that more than one nucleic acid will encode a particular immunoglobulin component and these alternative sequences are considered within the scope of the present invention.

[00192] The chimeric light and heavy chains of autophilic antibodies of the present invention can be expressed together or separately to produce autophilic antibodies. For example, as described herein, expression vectors are constructed encoding chimeric light and/or heavy chains of autophilic antibodies of the present invention. Chimeric light and heavy chains can be encoded by nucleic acids included separate expression vectors, such as in separate plasmids. The plasmids can be used together or separately to express the encoded proteins and produce the autophilic antibodies in particular embodiments. For example, when expressed separately, chimeric light and heavy chains of autophilic antibodies can be purified and combined to form the autophilic antibodies. Alternatively, expressed together, the expressed proteins can combine to form the autophilic antibodies.

[00193] Compositions provided according to embodiments of the present invention include an isolated host cell transformed with an expression vector encoding an immunoglobulin heavy chain having an antigen binding domain and an autophilic peptide. In particular embodiments, the isolated host cell is also transformed with an expression vector encoding an immunoglobulin light chain having an antigen binding domain and the antigen binding domain of the immunoglobulin heavy chain and the antigen binding domain of the immunoglobulin light chain together form an antigen binding site of an anti-CD20 antibody. An isolated host cell for producing a recombinant autophilic antibody of the present invention is in vitro in particular embodiments of the present invention. Expression systems for autophilic antibody expression illustratively include: eukaryotic cells such as mammalian cells, plant cells, insect cells, yeast, and amphibian cells; and prokaryotic expression systems such as bacteria. One of skill in the art is able to select a particular expression system for use in producing a recombinant autophilic antibody. [00194] Immunoassays

[00195] Immunoassays are provided according to the present invention which include contacting an analyte in a biological or environmental sample with an antibody conjugated to an autophilic peptide. A complex formed by the analyte and the antibody conjugated to an autophilic peptide is then detected.

[00196] In particular embodiments, assays of the present invention are characterized by detection of antigens expressed at low levels, such as on a cell surface, using antibodies containing autophilic peptides either naturally or by conjugation of an autophilic peptide to the antibody.

[00197] The use of highly- specifc antibodies is common in many diagnostic applications. The binding of said antibodies may be detected directly by a number of means or, alternatively, secondary antibodies are required for signal enhancement and detection. Previously, the signal detection was directly related to the amount of antibody bound, either mono or divalently bound to target. In the present invention, enhanced signal strength results from polyvalent binding interactions of autophilic peptide-conjugated antibodies bound to a target analyte.

[00198] Whether directly or indirectly detected, autophilic peptide-conjugated antibodies greatly enhance signal detection because of lattice formation and more antibody surrounding the target. Thus, naturally occurring autophilic antibodies and non-naturally occurring autophilic peptide-conjugated antibodies can be directly labeled with a detectable label affording an enhanced signal in an immunoassay. Similarly, indirect labeling, such as labeling of a secondary antibody produces increased signal in an immunoassay since the secondary antibody will bind with increased numbers to the primary autophilic peptide- conjugated antibodies.

[00199] In embodiments of assays of the present invention, a naturally-occurring autophilic antibody or an antibody conjugated to an autophilic peptide is used to enhance signal detection of antigen immobilized on a substrate, such as plastic, in assays such as ELISA.

[00200] In embodiments of assays of the present invention, a naturally-occurring autophilic antibody or an antibody conjugated to an autophilic peptide is used to enhance "off -rate" or increase in avidity of antigen coated or bound to a polymer chip and detected by surface plasmon resonance.

[00201] In embodiments of assays of the present invention, a naturally-occurring autophilic antibody or an antibody conjugated to an autophilic peptide is used to enhance

binding and localization detection of the antibody bound to a target in vivo, such as in a xenograft tumor animal model, by fluorescence or other signal detection method. [00202] In embodiments of assays of the present invention, a naturally-occurring autophilic antibody or an antibody conjugated to an autophilic peptide is used to enhance binding and localization detection of naturally-occurring autophilic antibodies or peptide conjugated autophilic antibodies labeled with spin molecules and detected by magnetic resonance imaging, to enhance signal of autophilic antibodies bound to cell surface targets and detected by FACS. In embodiments of assays of the present invention, a naturally- occurring autophilic antibody or an antibody conjugated to an autophilic peptide is used to enhance the signal obtained upon binding of antibody to antigen as detected by any natural effector mechanism, such as complement activation or cell-mediated cytotoxicity. [00203] Assays according to embodiments of the present invention can include virtually any immunoglobulin conjugated with one or more autophilic peptides for enhanced detection of an analyte.

[00204] The term "analyte" refers to any molecule or compound which is specifically recognized by an antibody conjugated to an autophilic peptide, illustratively including a protein, a peptide, a hapten, a carbohydrate, a lipid, a ganglioside and combinations of these. [00205] In particular embodiments, an analyte can be a mammalian analyte, illustratively including a protein, peptide, hapten, carbohydrate, lipid, ganglioside or combination thereof generated, for instance, by a normal or abnormal cell of a mammal.

[00206] In further particular embodiments, an analyte can be a non-mammalian analyte, such as a protein, peptide, hapten, carbohydrate, lipid, ganglioside or combination thereof, generated by a microorganism, such as a bacterium or a virus.

[00207] Thus, particular assays of the present invention are provided for detection of a microorganism and/or a product of a microorganism. Assays for detection of a microorganism and/or a product of a microorganism are used to assay a sample obtained from a human or a non-human animal to detect infection, for example. In further embodiments, assays for detection of a microorganism and/or a product of a microorganism are used to assay a sample obtained from an environment or object to be tested for contamination with a microorganism and/or a product of a microorganism.

[00208] In embodiments of assays of the present invention, a target analyte is a B-cell receptor, CD20, Her2, ganglioside GM2, glycolic ganglioside GM3, GD3 ganglioside,

caspases, oxidized low density lipoproteins, phosphocholine, EGFR, CD32B, HLADRl,

CD 19, EpCAM, PSA and bacterial antigens such as a staphylococcal antigens.

[00209] Biological Sample

[00210] A biological sample from any source can be assayed for an analyte using compositions and methods of the present invention. A biological sample is typically a fluid, cell, and/or tissue of a mammalian subject, including a primate or human subject. In particular embodiments, a biological sample is obtained from a non-human animal, illustratively including a dog, a cat, a rabbit, a bird, a rodent, a horse, a cow, a goat, a pig, a mini pig and a sheep. A biological sample assayed according to the present invention illustratively includes blood, plasma, serum, cerebrospinal fluid, saliva, ascites, fecal material, a nasopharyngeal secretion, mucus, lymph fluid, urine, tears, semen and milk. A cell or tissue from any source can be a sample assayed according to the present invention. In particular embodiments, a biopsy specimen obtained from a tumor or other abnormal growth is a sample assayed for an analyte.

[00211] A sample obtained from a subject can be processed according to standard methods depending on the particular assay used. For example, a tissue or organ sample can be sectioned or homogenized and a biological fluid can be filtered to remove particulates.

[00212] Environmental Sample

[00213] An environmental sample from any source can be assayed for an analyte using compositions and methods of the present invention. In particular embodiments of the present invention, a sample is obtained by collecting a fluid, solid or combination thereof from a location or object to be assayed for presence of a microorganism and/or a product of a microorganism.

[00214] Immunoassays

[00215] A method according to the present invention for detecting an analyte in a sample using an antibody conjugated to an autophilic peptide can be any of various immunoassays illustratively including ELISA, sandwich immunoassay, immunofluorescence, immunocytochemistry, immunoprecipitation, immunoblot, agglutination assays and biomolecular interaction analysis sensing. Additional suitable immunoassays are listed in T.

Kindt et al., Kuby Immunology, 6th edition, W. H. Freeman, 2006, page 145.

[00216] An antibody conjugated to an autophilic peptide can be attached, covalently or non-covalently, to a solid phase substrate in particular immunoassays if desired. A substrate

in this context can be any suitable solid phase substrate illustratively including a microassay chip, a particle or bead and a multiwell plate.

[00217] In a particular example, an antibody conjugated to an autophilic peptide can be attached to a solid phase substrate such as a latex bead or particle for use in an agglutination immunoassay for an analyte.

[00218] In additional agglutination immunoassays of the present invention, an antibody conjugated to an autophilic peptide is used to detect an analyte, such as a virus, present on a cell or biological particle surface, such as a red blood cell. Aggregation of the cells or biological particles in the presence of an antibody conjugated to an autophilic peptide specific for the analyte is indicative of the presence of the analyte on the cell or biological particle surface.

[00219] Detection of a complex formed by an analyte and an antibody conjugated to an autophilic peptide is achieved by any of various methods.

[00220] In particular embodiments, the complex is detected by detection of a detectable label. Typically a detectable label is joined to the antibody- autophilic peptide conjugate or to a secondary antibody which binds to the antibody- autophilic peptide conjugate. Illustrative examples of a detectable label include a fluorescent label, a radioactive label, a chemiluminescent label, a bioluminescent label, a chromophore, an enzyme and a metal particle. A detectable label can also be a ligand of a ligand binding pair such as avidin-biotin and streptavidin-biotin.

[00221] In further embodiments, no label is used to detect a complex formed by an analyte and antibody-autophilic peptide conjugate. Surface plasmon resonance can be used to detect complex formation, for instance, as described in J. Homola (Ed.), Surface Plasmon

Resonance Based Sensors, Springer, 2006. Turbidimetry and nephalometry are additional examples of detection methods used to detect a complex formed by an analyte and an antibody conjugated to an autophilic peptide.

[00222] In further embodiments, a natural effector mechanism is assayed to detect formation of a complex by an analyte and an antibody-autophilic peptide conjugate. For example, complement activation or cell-mediated cytotoxicity is detected in an appropriate system, indicative of complex formation.

[00223] Any of various modalities for detection of complex formation can be used such as optical detection methods illustratively including flow cytometry or microscopy.

[00224] In preferred embodiments of the present invention, diagnostic and prognostic immunoassays are provided. The term "diagnostic immunoassay" refers to an immunoassay that allows for determination of presence or amount of an analyte indicative of a disease or pathological condition in an animal or human subject. The term "prognostic immunoassay" refers to an immunoassay that allows for determination of presence or amount of an analyte indicative of progression of a disease or pathological condition in an animal or human subject.

[00225] In an example of a diagnostic immunoassay of the present invention, an immunoassay for Her2 is performed using a sample obtained from a human subject. Detection of Her2 in a sample of breast tissue or tumor biopsy is indicative of cancer that tends to be more aggressive than Her2-negative breast tumors. Thus, detection of low levels of Her2 in a patient sample using an anti-Her2 antibody conjugated to an autophilic peptide allows for earlier detection of a tumor and detection of more cancers treatable by immunotherapy compared to diagnostic assays using a non-modified anti-Her2 antibody. In embodiments of the present invention, a trastuzumab-autophilic peptide conjugate is used in an assay to detect Her2 in a patient sample, such as a sample of breast tissue or a tumor biopsy.

[00226] Kits

[00227] In embodiments of the present invention, kits are provided for use in performing an assay using an antibody- autophilic peptide conjugate. In particular embodiments, a kit includes an antibody- autophilic peptide conjugate and instructions for use in detecting an analyte in a sample.

[00228] The following examples are presented to illustrate certain aspects of the invention, and are not intended to limit the scope of the invention.

EXAMPLES

[00229] Example 1: Conjugation of Tl 5 peptide to two Mabs specific for B-cell receptor [00230] Cell Line and Antibodies.

[00231] The human B-cell tumor line (Su-DHL4) and murine B-cell tumor line (38Cl 3) are grown in RPMI 1640 medium (supplemented with 10% fetal bovine serum, 2 μmol/L glutamine, 10 μmol/L HEPES, 50 U/mL penicillin, and 50 μg/mL streptomycin, 50 μmol/L 2- mercaptoethanol) at 37 ⁰ C under 5% carbon dioxide. Two mAbs, 5D10 and S1C5, specific

for the human or murine BCR, respectively, were used in this study. The antibodies are purified from the culture supernatant by protein G and protein A affinity chromatography. [00232] Synthesis of Antibody-Peptide Conjugate.

[00233] T15H peptide ASRNKAND YTTDYSASVKGRFIVSR (SEQ ID NO. 1), a VH- derived peptide from an autophilic antibody-T15, was synthesized by Genemed Synthesis (San Francisco, CA, U.S.A.). Antibodies were dialyzed against PBS (pH 6.0) and 1/10 volume of 200 μmol/L sodium periodate was added and incubated at 4 ⁰ C for 30 minutes in the dark. The reaction was stopped by adding glycerol to a concentration of 30 μmol/L, and the sample was dialyzed at 4 ⁰ C for 30 minutes against PBS (pH 7.0). A one hundred times molar excess of T15H or scrambled T15 peptide (T15scr/T15s) SYSASRFRKNGSIRAVEATTDVNSAYAK (SEQ ID NO: 3) was added to the antibodies and incubated at 37 ⁰ C for 1 hour. L-Lysine was added and incubated at 37 ⁰ C for 30 minutes to block the remaining aldehyde group. The same oxidation reaction (except adding the peptides) was applied to antibodies used as controls. After the blocking step, the antibody conjugates were dialyzed against PBS (pH 7.2) overnight. [00234] Ig Capture ELISA.

[00235] Four μg/mL of murine S1C5-T15H was coated to Costar vinyl assay plates (Costar, Cambridge, MA). After blocking with 3% BSA solution, 8 μg/mL of photobiotinylated S1C5-T15H, S lC5-scrambled peptide conjugate, and control S1C5 were added to the first wells, and 1:1 dilution was performed. The antibodies were incubated for 2 hours at room temperature. After washing with PBS buffer, avidin-HRP (Sigma-Aldrich, St. Louis, MO) was added as a 1:2500 dilution. The binding antibodies were visualized by adding substrate o-phenylenediamϊne. [00236] Size Exclusion Chromatography.

[00237] Antibody conjugate was chromatographed on a 75 mL Sephacryl 300HR column (Pharmacia, Peapack, NJ). 1 :10 diluted PBS (pH 7.2) was chosen as elution buffer. Fractions (0.5 mL/each) were collected and aliquots (100 μL) were assayed on antihuman IgG capture ELISA. The ELISA reading (OD 490 nm) is plotted against elution volume. [00238] Viability Assay for Antibody-Treated Cells.

[00239] Lymphoma cells were grown in 96- well tissue culture wells in 1-mL medium. Two μg of antibodies or antibody-pep tide conjugates were added and incubated for various times as described herein. Ten μL aliquots from the cell suspension were used to determine viability by using trypan blue exclusion.

[00240] FACS Assay of the B-CeIl Lymphoma.

[00241] Human Su-DHL4 and murine 38C 13 cells were fixed with 1 % paraformaldehyde. 1 x 10 ⁶ cells were suspended in 50 μL of staining buffer (Hank's balanced salt solution, containing 0.1% NaN ₃ , 1.0% BSA), then 1.5 μg of photobiotinylated murine

S1C5-T15H conjugates was added and incubated for 30 minutes on ice. Control antibodies and antibody- scrambled T15 peptide conjugates served as controls. The cells were washed twice with staining buffer before avidin-FITC (Sigma-Aldrich) was added to the cells for 30 minutes on ice. Then the cells were washed twice with staining buffer, re-suspended in 200 μL PBS and analyzed by flow cytometry.

[00242] Hoechst-Merocyanin 540 Staining to Detect Apoptosis.

[00243] 1 x 10 ⁶ of lymphoma cells were placed into 24-well tissue culture wells. Four μg of antibodies or antibody-peptide conjugates were added and incubated for various times as described herein. 1 x 10 cells were removed from the culture, re-suspended in 900 μL cold

PBS (pH 7.2). One hundred μL of Hoechst 33342 (50 μg/mL; Molecular Probe, Eugene, OR,

U.S.A.) was added, the cells were incubated at 37 ⁰ C for 30 minutes in the dark. The cells were centrifuged and re-suspended in 100 μL PBS. Then, 4 μL of MC540 solution

(Molecular Probe) was added, and 20-minute incubation was performed at room temperature in the dark. The cells were pelleted, re-suspended in 1 mL cold PBS (pH 7.2), and analyzed by flow cytometry.

[00244] RESULTS

[00245] Characterization of Autophilic Antibodies.

[00246] The T15H (24-mer) peptide was crosslinked to two murine mAb (S1C5 and

5D10), using carbohydrate periodate conjugation. The mAb S1C5 (IgGl) is specific for the tumor idiotype of the mouse 38Cl 3 B -cell line and the 5D10 antibody for the human Su-

DHL4 B-cell tumor. Both mAbs recognize unique idiotypes of the BCR IgM on the B-cell tumors.

[00247] Autophilic Behavior Can Easily be Demonstrated by ELISA.

[00248] The autophilic effect was studied with the S1C5-T15H Mab conjugate. The T15H- crosslinked S1C5 binds to insolubilized S1C5-T15H detected by biotin-avidin ELISA.

Control S1C5 does not bind significantly to S1C5-T15H or S1C5 crosslinked with a scrambled peptide. Similar self-binding of T15H peptide-crosslinked mAb 5D10 to insolubilized T15H- 5D10 was also observed. The specificity of the peptide mediated autophilic effect was tested using the 24-mer peptide T15H itself as an inhibitor. Only the

T15H peptide inhibited S1C5-T15H and 5D10- T15H self-binding while the control- scrambled peptide did not inhibit it. These results are similar to previous inhibition data with the naturally occurring autophilic T15/S107 antibody (Halpern, R., et al., 1991). [00249] Tl 5H- Antibody Conjugates in Monomer - Dimer Equilibrium in Solution. [00250] The non-covalent nature of the self-aggregation of T15H-linked antibodies raises the question of its physical state in solution. To address this issue, the molecular species of T15H-linked monoclonal antibodies were analyzed using gel electrophoresis and sizing gel filtration. The electrophoretic mobility of control and T15H peptide conjugated to S1C5 and 5D10 under reducing and non-reducing conditions show no differences, indicating the absence of chemical bonds between the antibody chains. The molecular species of the peptide-conjugated antibodies (5D10-T15H) was further analyzed by size exclusion chromatography. The elution profile indicated two immunoglobulin species of different sizes. The larger first peak eluted in the position of an antibody dimer. The second smaller peak eluted in the position of non-conjugated 5D10 antibody. The appearance of two peaks resembled monomer and dimer antibodies and could indicate that either a fraction of antibodies was not modified, or that the modification was complete and the antibody establishes an equilibrium of dimers and monomers. To test the latter possibility, material from both peaks were subjected to a second gel filtration on the same column. Reruns of both peaks yielded again two peaks at the same position as in the first chromatography (Zhao and Kohler, 2002). These data show that the T15H peptide-linked antibodies exist in solution as two distinct molecular species in equilibrium as monomer and dimer. [00251] Enhanced Binding of Autophilic Antibodies to Tumors.

[00252] The binding of the peptide-conjugated antibodies against their respective tumor targets was compared with that of the control antibodies in indirect fluorescence activated cell sorting (FACS). As control, antibodies linked with a scrambled peptide were included. The fluorescence intensity of the T15H-S1C5 on 38Cl 3 cells is compared with that of the control S1C5 and the scrambled peptide S1C5. The difference in mean fluorescence channels between S1C5-T15H and controls was greater than 10-fold. Similarly, the FACS analysis of autophilic 5D10-T15H on Su-DHL4 cells shows enhancement of binding over binding of control 5D10 and control peptide-crosslinked 5D10. In both tumor systems, the conjugation of the T15H peptide to tumor- specific antibody enhanced the FACS signals over control antibodies used at the same concentration (Zhao, Lou, et al., 2002). The enhancement of

fluorescence can be explained with the increase of targeting antibodies caused by self- aggregation and lattice formation on the surface of the tumor cells. [00253] Inhibition of Tumor Growth.

[00254] Antibodies binding to the BCR induce crosslinking of the BCR, which, in turn, inhibits cell proliferation and produces a death signal. Furthermore, chemically dimerized antibodies directed against a B-cell tumor induce hyper-crosslinking of the BCR followed by inhibition of cell division and apoptosis of the tumor. To see if similar enhancement of the antitumor effects of dimerizing antibody were induced by noncovalent, dimerizing Tl 5H- linked antibodies, the two B cell tumors were cultured in the absence or presence of control and T15H-linked antibodies. Co-culture of both tumors, 38C13 and Su-DHL4, with their respective T15H-linked antibodies inhibited the cell growth significantly better compared with the control antibodies. To test the tumor target specificity of autophilic antibodies in growth inhibition, criss-cross experiments were performed with the 38Cl 3 and Su-DHL-4 cell lines. Inhibition of murine 38C13 cell growth with S1C5-T15H was statistically greater than mismatched 5D10-T15H. Similar results on the specificity of autophilic antibodies were obtained with the Su-DHL4 cells (Zhao, Y., et al., 2002). [00255] Induction of Apoptosis.

[00256] As suggested by earlier studies, the antitumor effect of antibodies directed against the BCR of B-cell lymphomas in vitro and in vivo might be caused by the induction of apoptosis. Aliquots of tumor cells (38Cl 3 and Su-DHL-4) cultured in the presence of control or T15H-linked antibodies were analyzed for apoptosis using a double stain FACS protocol. 38Cl 3 and Su-DHL4 cells underwent a moderate amount of apoptosis without antibodies over a 6, respectively, 18-hour culture. This apoptosis was enhanced when the respective antibody was added. However, when the T15H-linked antibodies were added, the accumulated number of apoptotic 38Cl 3 cells was almost doubled, and apoptosis of Su- DHL4 cells was more than doubled during the entire culture (Zhao, Y., et al., 2002). [00257] DISCUSSION

[00258] The biologic advantage of the autophilic property is exemplified with the Sl 07/Tl 5 anti-phosphorylcholine antibody. This autophilic antibody is several times more potent in protecting immune-deficient mice against infection with Pneumococci pneumoniae than non-autophilic antibodies with the same antigen specificity and affinity. [00259] As shown here, the autophilic antibody function can be transferred to other antibodies by chemically crosslinking a peptide derived from the Tl 5 VH germline sequence.

The modified antibody mimics the autophilic property of the T15/S107 antibody, producing an autophilic antibody with increased avidity and enhanced targeting. Enhancing the binding of autophilic engineered antibodies to the BCR of B-cell tumor increases the strength of the death signals leading to profound inhibition of cell proliferation in culture. Even though a doubling of apoptosis is demonstrated here, other mechanisms of growth inhibition can be involved.

[00260] Crosslinking the BCR of the mature murine B-cell lymphoma A20 can protect against CD95 mediated apoptosis. This anti-apoptotic activity of engagement of the BCR by crosslinking antibodies is highly restricted to the time window of CD95 stimulation and is not dependent upon protein synthesis. The finding that BCR hypercrosslinking per se is pro- apoptotic is not at variance with reports on the anti-apoptotic activity of the BCR engagement, because it can be due to the use of less mature B-cell lines, to different strength of delivered signals by homodimerizing antibodies, or to Fas -independent apoptosis. [00261] The use of two BCR idio tope- specific antibodies against different tumors offered the opportunity to test the biologic effect of targeting receptors other than the idiotope specific BCR. In criss-cross experiments with autophilic antibodies binding in FACS analysis and inhibition of growth in vitro show a significant enhancement only with the autophilic matched antibody. In this context, it is interesting to speculate whether enhanced tumor targeting would also augment cellular effector functions.

[00262] In an earlier study using chemically homodimerized antibodies, the Fc domain was not involved in the augmentation of growth inhibition and tumor cells lacking Fc receptors were susceptible to the anti growth activity of homodimers. Thus, the anti-tumor effect induced by dimerizing antibodies would not be restricted to lymphoid tumors such as non-Hodgkin's B-cell lymphoma, where anti-tumor effects require the participation of Fc- receptor-bearing effector cells.

[00263] The described approach of transferring the naturally occurring autophilic property to other antibodies thereby enhancing their anti-tumor effect outlines a general method to improve the therapeutic efficacy of antibodies in passive immunotherapy. Such noncovalent antibody complexes offer several advantages over chemically crosslinked antibodies: (i) the equilibrium between monomer and noncovalent homopolymers prevents the formation of precipitating nonphysiologic complexes in solution; (ii) autophilic conversion does not compromise the structural integrity of antibodies; and (iii) the method is simple and efficient and does not require a purification step typically needed for chemically crosslinked

homodimers that reduces the yield of active Ig dimers. One possible limitation of the approach of using dimerizing antibodies might be the ability to penetrate a large tumor mass. Because the homophilic peptide is of murine origin, it might be immunogenic in humans. Thus, it could be necessary to humanize the murine peptide based on sequence and structural homology using computer modeling. The demonstration that adding a single peptide to the structure of antibodies increases the amount of antibody bound to targets and the anti-tumor activity encourages attempts to engineer recombinant antibodies expressing the autophilic activity.

[00264] Example 2: Internalization of Antibodies Conjugated with MTS Peptide [00265] Cell line and antibodies

[00266] Human Jurkat T cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotic (penicillin, streptomycin and amphotericin). Rabbit polyclonal anti-active caspase-3 antibody (#966 IS) and anti cleaved-fodrin, i.e., alpha II spectrins (#2121 S), were purchased from Cell Signaling, Inc (Beverly, MA). Monoclonal (rabbit) anti- active caspase-3 antibody (#C92-605) was purchased from BD PharMingen (San Diego, CA). Mouse monoclonal antibody 3Hl (anti-CEA) was purified from cell-culture supernatant by protein G affinity chromatography. Anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnologies, Inc. ApoAlert Caspase- 3Fluorescent Assay kit was purchased from Clontech Laboratories (Palo Alto, CA). The Cell Death Detection ELISA was purchased from Roche Applied Science (Indianapolis, IN). [00267] Synthesis of MTS peptide-antibody conjugate

[00268] MTS peptide KGEGAA VLLPVLLAAPG (SEQ ID NO. 2) is a signal peptide- based membrane translocation sequence, and was synthesized by Genemed Synthesis (San Francisco, CA). Antibodies were dialyzed against PBS (pH 6.0) buffer, oxidized by adding 1/10 volume of 200 mmol/L NaIO ₄ and incubating at 4 ⁰ C for 30 min in the dark. Adding glycerol to a final concentration of 30 mM terminated the oxidation step. Samples were subsequently dialyzed at 4 ⁰ C for 1 h against Ix PBS (pH 6.0) buffer. The MTS peptide (50X molar excess) was added to couple the antibodies and the samples were incubated at 37 ⁰ C for 1 hour and the resulting antibody-peptide conjugate was dialyzed against Ix PBS (pH 7.4). [00269] Effect of MTS-coniugated antibody on cell growth

[00270] Jurkat cells (2.5 x 10 ⁵ ) were seeded into 96- well culture plate. After incubation with 0.5 μg MTS-antibody conjugates for 6, 12, 18 and 24 hour, aliquots were removed and viability was determined by trypan blue exclusion.

[00271] Study of antibody internalization by ELISA

[00272] Jurkat cells, grown in 1-ml medium in a 6- well culture plate, were incubated with

2 μg of unconjugated or MTS conjugated antibodies for 0, 1, 3, 6, 12 and 18 h. The cells were centrifuged and the culture supernatant was then transferred to a new tube. The cell pellet was washed twice with PBS (pH 7.4) before being homogenized by Pellet Pestle Motor (Kontes,

Vineland, NJ) for 30 sec. All of the cell homogenate and an equal volume of the culture (10 μl) supernatant were added to sheep anti-rabbit IgG coated ELISA plate (Falcon, Oxnard,

CA) and incubated for 2 h at room temperature. After washing, HRP-labeled goat anti-rabbit light chain antibody was added, and visualized using o-phenylenediamine.

[00273] DNA fragmentation

[00274] Jurkat cells were pre-treated with antibodies or a caspase-3 inhibitor (DEVD- fmk) for 1 h, centrifuged, and incubated with fresh medium containing actinomycin D alone

(1 μg/ml) for 4 h. After treatment, Jurkat cells were collected, washed, and resuspended in

700 μl of HL buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.2% Triton X-100, for 15 min at room temperature. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated 24h at -20 ⁰ C with 0.1 volume of 5 M NaCl and 1 volumes of isopropanol.

The DNA was washed, dried, and resuspended in TE pH 8.0. The DNA was resolved by electrophoresis on a 1.5% agarose gel and visualized by UV fluorescence after staining with ethidium bromide. DNA fragmentation was also determined using the Cell Death Detection

ELISA according to the manufacturer's instructions.

[00275] Preparation of total cell lysate

[00276] Jurkat cells were treated as described in the DNA fragmentation section. After treatment, cells were collected and washed with PBS (pH 7.4) twice, then suspended in 300 μl of CHAPS buffer (50 mM PIPES, pH 6.5, 2 mM EDTA, 0.1% CHAPS). The samples were sonicated for 10 sec and centrifuged at 14,000 rpm for 15 min at 4°C. The supernatant was transferred to a new tube and referred as total cell lysate.

[00277] Caspase-3-like cleavage activity assay

[00278] Jurkat cells were treated as described in the DNA fragmentation section. Equal amounts of protein of the total cell lysate were applied for caspase-3 activity assay using

ApoAlert Caspase-3 Fluorescent Assay Kit according to the manufacturer's instruction.

Fluorescence was measured with a Spectra MAX GEMINI Reader (Molecular Devices,

Sunnyvale, CA).

[00279] Western blot analysis

[00280] Jurkat total cell lysates (10 μg) were separated on a 10% SDS-PAGE gel to detect immunoreactive protein against cleaved spectrin. Ponceau staining was used to monitor the uniformity of protein transfer onto the nitrocellulose membrane. The membrane was washed with distilled water to remove excess stain and blocked in Blotto (5% milk, 10 mm Tris- HCl

[pH 8.0], 150 mM NaCl and 0.05% Tween 20) for 2 h at room temperature. Before adding the secondary antibody, the membrane was washed twice with TBST (10 mM Tris-HCl with

150 mM NaCl and 0.05% Tween 20), and then incubated with HRP-conjugated secondary antibodies. The blot was washed extensively and reactivity was visualized by enhanced chemiluminescence (AmershamBiotech, Piscataway, NJ).

[00281] Statistical analysis .

[00282] Statistical analysis was performed using the student Mest (for a pair-wise comparison) and one-way ANOVA followed by Newman-Keuls posttest. Data are reported as means ± SE.

[00283] RESULTS

[00284] As shown in Fig. 1, an MTS conjugated anti-active caspase 3 antibody is internalized more rapidly than unmodified antibody. When cells were exposed to the chemotherapeutic drug, actinomycin D, apoptosis was triggered and the cells died (see Fig.

T). However, if cells were exposed at the same time to the MTS-conjugated antibody

(transMab), most of the toxicity of the chemotherapeutic drug was inhibited.

[00285] Example 3: Enhancing Binding and Apoptosis Using Peptide-Conjugated Anti-

CD20 Antibodies

[00286] Cell Line and Antibodies

[00287] The human B-cell tumor lines SU-DHL-4 and Raj were grown in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 10 μmol/L Hepes,

50 U/mL penicillin, 50 μg/mL streptomycin, and 50 μmol/L 2-mercaptoethanol at 37 ⁰ C under

5% carbon dioxide. Mouse monoclonal antibodies 1F5 IgG2a (ATTC #HB-9645) specific for human B-cell lymphomas 5D10 and 3Hl (Zhao, Lou, et al., 2002.) were purified from cell culture supernatant by protein G or protein A affinity chromatography.

[00288] Synthesis of Antibody-Peptide Conjugate

[00289] Tl 5 peptide ASRNKAND YTTD YS ASVKGRFIVSR (SEQ ID NO. 1), a VH- derived peptide from a self -binding antibody-T15, was synthesized as described in Example

1. 8-azido-adenosine-biotin was synthesized and used to affinity cross-link biotin to

antibodies. The 8-azidoadenosine dialdehyde was prepared as previously described (U.S.

Patent No. 5,800,991, issued to Haley et al., which is incorporated herein by reference).

[00290] Self-Binding Enzyme-Linked Immunosorbent Assay

[00291] Four micrograms per milliliter of 1F5-T15 was used to coat Costar vinyl assay plates (Costar, Cambridge, MA, U.S.A.). After blocking with 1% BSA solution, 8 μg/mL photobiotinylated 1F5-T15 naked 1F5 and control antibody (5D10) were added, diluted to

1 :1, and incubated for 2 hours at room temperature. After washing with PBS buffer, avidin-

HRP (Sigma-Aldrich) was added, and enzyme-linked immunosorbent assay color was developed with o-phenylenediamine.

[00292] FACS Assay of the B-CeIl Lymphoma

[00293] SU-DHL-4 cells were fixed using 1% paraformaldehyde, and 1 x 10 ⁶ cells were suspended in 50 μL staining buffer (Hanks, containing 0.1% NaN3 and 1.0% BSA); 1.5 μg photobiotinylated 1F5-T15 conjugates, naked 1F5, and control antibodies were added and incubated for 30 minutes on ice. The cells were washed twice with staining buffer, and then avidin-FITC was added for 30 minutes on ice. After washing twice with staining buffer, the cells were resuspended in 200 μL PBS for FACS analysis.

[00294] Hoechst-Merocyanin 540 Staining to Detect Apoptosis

[00295] After 1 x 10 ⁶ lymphoma cells were placed into 24-well tissue culture wells, 4 μg antibodies and antibody-peptide conjugates were added. After 24 hours of incubation, 1 x 10 cells were removed from the culture pellet and resuspended in 900 μL cold PBS (pH 7.2), and

100 μL Hoechst (Pierce, Rockford, IL, U.S.A.) 33342 (50 μg/mL) was added and incubated at 37 ⁰ C for 30 minutes in the dark. The cells were centrifuged and resuspended in 100 μL

PBS; 4 μL MC540 dilution solution was added and the cells were incubated for 20 minutes at room temperature in the dark. The cells were pelleted, resuspended in 1 mL PBS, and analyzed by flow cytometry.

[00296] Inhibition of Cell Growth in Culture

[00297] Ix 10 ⁵ tumor cells were seeded in complete culture medium. At days 1, 2, and 3 of culture, aliquots were removed and viable cells were counted (trypan blue).

[00298] RESULTS

[00299] Mouse monoclonal antibodies 1F5 IgG2a were conjugated with self-binding peptide as in Example 1. An average of 1.8 peptides per antibody was found by competitive analysis. The parental antibody was compared to the conjugated form for binding by flow cytometry. As shown in Fig. 3, the binding was increased for the conjugated antibody (Mab-

ap) when assessed with a limiting dilution of antibody. This was characterized by a shift in the binding fluorescence to a higher intensity. When compared over a series of dilutions, conjugated antibody required almost one-tenth the concentration of antibody to achieve the same level of intensity as parental antibody (Fig. 4). As shown in Fig. 5, increasing the amount of conjugated antibody caused a reduction in fluorescence intensity, presumably due to internalization, a property of SAT technology that can be used to enhance potency of immunoconjugates of drugs, toxins and short path length radiotherapeutic isotopes. Furthermore, when tested for the ability to trigger apoptosis, the conjugated form (Sab) was much more active than native antibody, with most cells dead by 3 days, compared to only a small fraction with the native antibody (Fig. 6).

[00300] Example 4: Enhanced Binding and Apoptosis with Anti-GM2 Antibodies [00301] Cell lines and antibody

[00302] Human T-cell leukemia Jurkat cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotic (penicillin, streptomycin and amphotericin). Chimeric hamster anti-GM2 antibody (ch-α-GM2) was obtained from Corixa Corporation (Seattle, WA). After chimerization, the resulting antibody lost its ability to induce apoptosis in ganglioside GM2 expressing target cells. [00303] Synthesis of antibody-peptide conjugate

[00304] Both T15 peptide ASRNKANDYTTEYSASVKGRFIVSR (SEQ ID NO: 1), a VH-derived peptide from a self-binding antibody-T15 (Kaveri et al, 1991), and a scrambled T15 peptide (T15-scr) (SEQ. ID. NO. 3), randomly generated from the T15 amino acid sequence, were synthesized by Genemed Synthesis (South San Francisco, CA). The scrambled peptide was used as a control. Antibodies were dialyzed against PBS (pH 6.0), then 1/10 volume of 200 μM NaIO ₄ was added and incubated at 4 ⁰ C for 30min in the dark. The reaction was stopped by adding glycerol to a final concentration of 30 μM, and the samples were dialyzed at 4 ⁰ C for 30 min against PBS (pH 6.0). Fifty (50) times molecular excess of Tl 5 or scrambled peptide was added to the antibodies and incubated at 37 ⁰ C for 1 h. L-Lysine was added and incubated at 37 ⁰ C for 30min to block the remaining reactive aldehyde group. After the blocking step, the antibody-conjugates were dialyzed against PBS (pH 7.2) at 4 ⁰ C overnight, then stored at 4 ⁰ C until used. [00305] Direct binding ELISA

[00306] GM2 ganglioside was dissolved in methanol and 0.5 μg was coated per well in 96 well polystyrene plates (Costar, Cambridge, MA) and allowed to dry overnight. The wells

were blocked with 1% BSA for 2 h at room temperature and 400 μg of anti-GM2 antibodies, diluted in 1% BSA, were added in the first well and then serially diluted 1 :1. After incubation for 1 h, the wells were washed 5X and HRP-conjugated anti-human IgG (Sigma- Aldrich) was added at a 1 :1000 dilution and incubated for 1.5 h. After washing three times, the bound antibodies were visualized using substrate o-phenylenediamine and read at OD 492 using a spectrophotometer.

[00307] Specific binding ELISA

[00308] Gangliosides GM2, GMl, GM3 were dissolved in DMSO in 0.5 μg and coated in a

96 well polystyrene plate (Costar, Cambridge, MA) dried overnight. The wells were blocked with 1% BSA for 2 h at room temperature, 400 μg of ch-α-GM2 antibodies (anti- GM2-T15) were added in the first well and then serially diluted 1 :1. After incubation for 1 h, the wells were washed 5 times and HRP-conjugated anti-human IgG was added and incubated for 1.5 h. After washing three times, the bound antibodies were visualized using substrate o- phenylenediamine and assayed as described previously.

[00309] Antibody self-binding ELISA

[00310] 2 μg/ml of naked ch-α-GM2 (anti-GM2) or ch-α-GM2-T15 (anti-GM2-T15) were coated onto Costar vinyl assay plates. After blocking with 3% BSA solution, 0.5 μg/well of photobiotinylated anti-GM2-T15 was added. The antibodies were then incubated for 2h at room temperature. After washing three times, avidin-HRP (Sigma-Aldrich) was added at a

1 :1000 dilution and incubated for 1 hour. The bound antibodies were visualized with o- phenylenediamine and assayed as described previously.

[00311] Cell Surface binding detected by FACS

[00312] 2 x 10 ⁵ Jurkat cells per well were seeded in a 6-well plate and incubated overnight, then cells were collected and washed twice with P/B/G/A buffer (0.5% BSA, 5%

Goat Serum in PBS). Cells were then resuspended in 100 μL P/B/G/A buffer containing 5 μg/ml anti-GM2 antibodies for 30 min. After washing with P/B/G/A buffer, FITC-conjugated anti-Human IgG (Sigma-Aldrich, 1 :1000 dilution in 100 μL P/B/G/A) was added and incubated on ice for 30 min. After washing with P/B/G/A buffer, cells were resuspended in

400 μL P/B/G/A containing 10 μg/ml propidium iodide (as viability probe) and analyzed by flow cytometry.

[00313] Apoptosis detected by Annexin V staining

[00314] 2 x 10 Jurkat cells were seeded per well in a 6-well plate. After 6 h, cells were incubated with 20 μg/ml of the anti-GM2 or anti-GM2-T15 antibodies for 12 hr. Following

the incubation, a small portion of cells (50 μL) was saved and assayed for viability, while the remainder of the cells were harvested and washed with cold PBS. Cells were then resuspended in 100 μL annexin staining buffer, 5 μL Alex fluor 488 was added into 95 μL Ix annexin binding buffer, and Sytox was added at a dilution of 1:1000. After incubation at room temperature for 15 min, 400 μL of Ix annexin binding buffer was then added, and samples were analyzed by FACS.

[00315] Viability assay for Antibody-treated cells

[00316] A small portion of the cell samples saved from the annexin experiment was used for viability assay. 10- μL aliquots from the cell suspension were taken to determine viability using trypan blue exclusion assay.

[00317] Statistical analysis.

[00318] Statistical analysis was performed using one-way ANOVA followed by Newman-

Keuls post test. Data are reported as means ± SD.

[00319] RESULTS

[00320] Self -binding peptide enhanced antibody binding to its specific ganglioside.

[00321] Following antibody-peptide conjugation, the binding capacity of the T15- conjugated ch-α-GM2 antibody (anti-GM2-T15) was determined using a direct binding

ELISA. As seen in Fig. 7, both ch-α-GM2 antibody (anti-GM2) and anti-GM2-T15 antibody showed a dose-dependent increase in binding to ganglioside GM2. The anti-GM2-T15 antibody demonstrated a higher binding capacity compared with the naked anti-GM2 at all the doses tested, confirming that the self-binding Tl 5 peptide had increased the antigen binding capacity of the ch-α-GM2 antibody at a given antibody concentration.

[00322] Antibody self-binding behavior demonstrated by ELISA

[00323] Next, it was investigated by ELISA whether the increase in binding to ganglioside

GM2 by the Tl 5 peptide-linked antibody was due to its self -binding feature. As seen in Fig.

8, the anti-GM2-T15 antibody demonstrated a greater dose-dependent increase in binding to the peptide-conjugated anti-GM2-T15 antibody coated on the wells, whereas it did not show significant binding to the non-peptide conjugated anti-GM2 antibody. These data demonstrate that the anti-GM2-T15 antibody can bind to itself or homodimerize through the Fc- conjugated, autophilic peptide moiety.

[00324] Tl 5 conjugation does not change the specificity of the ch-α-GM2 antibody.

[00325] To assess whether conjugation of the Tl 5 peptide might alter the cognate binding specificity of the antibody, a direct antigen-binding ELISA was used to determine the binding specificity of the anti-GM2-T15 conjugated antibody. As shown in Fig. 9, the anti-GM2-T15 antibody demonstrated a specific, dose-dependent increase in binding to ganglioside GM2, whereas no binding above background levels to gangliosides GMl or GM3 was detected. This result confirms that addition of the self-binding Tl 5 peptide did not alter nor reduce the specificity of the ch-α-GM2 antibody.

[00326] Enhanced surface binding of anti-GM2 antibody to target tumor cells [00327] The human T-cell leukemic cell line Jurkat is known to express ganglioside GM2 (Suzuki et al, 1987). The ability of the peptide-conjugated anti-GM2-T15 antibody to bind to native ganglioside GM2 expressed on the surface of Jurkat cells was compared to that of the non-conjugated anti-GM2 antibody by flow cytometry. As shown in Fig. 10, the ch-α- GM2 antibody (anti-GM2) demonstrated a GM2 specific binding signal three times greater than background levels, whereas the binding demonstrated by the T15-conjugated anti-GM2 antibody was 2-fold higher than that of the non-peptide conjugated antibody. This result suggests that the enhanced binding demonstrated by the peptide-conjugated Ab is due to self- aggregation of this antibody. [00328] Inhibition of tumor growth

[00329] Antibodies binding to the B cell receptor have been shown to induce crosslinking of the BCR, which, in turn, inhibits cell proliferation (Ward et al, 1988) and produces a death signal (Hasbold et al, 1990; Wallen-Ohman et al, 1993). Furthermore, chemically dimerized antibodies directed against a B-cell tumor induce hyper-crosslinking of the BCR followed by inhibition of cell division and induction of apoptosis of the tumor cells (Ghetie et al, 1994; Ghetie et al, 1997). To determine whether the T15-conjugated anti-GM2 antibody induced a similar anti-proliferative effect, 2 x 10 ⁵ Jurkat cells were cultured in the presence or absence of anti-GM2 or control antibodies for 12 h, and then the number of viable cells remaining was counted. As summarized in Fig. 11, "no antibody" or control human IgG antibody (HuIgG) treatment had no effect on cell growth or viability, whereas there was some effect with the anti-GM2 antibody. However, the T15-linked antibody demonstrated a marked inhibition of Jurkat cell growth, as cell numbers were reduced > 2-fold compared to naked anti-GM2 antibody treated cells, and more than 4 fold versus the control IgG treatment. As a comparison and positive control, Actinomycin D demonstrated the ability to induce apoptosis, at levels slightly higher than the SuperAntibody.

[00330] Induction of Apoptosis

[00331] In order to determine whether the anti-tumor effect of antibodies directed against cell surface expressed gangliosides might be due to the induction of apoptosis, the cell samples used in the cell growth study were analyzed for apoptosis induction by measuring annexin V staining. The results are summarized in Table 2.

Table 2: Apoptosis analysis using Annexin V staining.

*Data were summarized from four sets of experiments.

[00332] Treatment of Jurkat cells with the ch-α-GM2 antibody (anti-GM2) or the ch-α- GM2 antibody conjugated with a scrambled, control peptide (anti-GM2-T15scr) did not induce apoptosis significantly over levels induced by treatment with control human IgG, as a modest 2-fold increase was observed. However, Jurkat cells treated with the anti-GM2-T15 conjugated underwent a significant amount of apoptosis, nearly 8-fold over background and more than 4-fold higher than that induced by the non-conjugated antibody or the control- conjugated antibody. These results confirmed the activity and specificity of T15-conjugated antibody.

[00333] Example 5: Generation of Autophilic Peptide Sequences T15-scr, T15-scr2, R24. and R24-Charged

[00334] Peptides were synthesized as in Example 1. The sequences are given in Tables 3 and 4.

Table 3: Sequences for Autophilic Binding Peptides

Table 4: Sequences for Membrane Penetrating Peptides

[00335] The peptide derived from R24 is difficult to solubilize except in DMSO or alcohol. Using such solubilizers can not only denature the antibody but also makes it difficult to conjugate to hydrophilic regions of the antibody. To overcome this solubility problem the addition and changes of sequence to charged amino acids, as shown in Table 3, were undertaken. The resultant modified peptide (R24-Charged) was soluble in aqueous buffer, was able to be conjugated to the tryptophan or nucleotide binding sites and preserved self- binding as well as induced apoptosis when conjugated to anti-GM2 antibody. The same amino acids present in the T15 sequence were randomly re-arranged and used to construct a further synthetic peptide; this scrambled sequence (T15scr or Tl 5s), had no self-binding and when conjugated to anti-GM2 antibody did not induce apoptosis (see Example 4, Table 2). In like manner, a second, randomly selected sequence, derived from the amino acids of the Tl 5 sequence, was used to generate a synthetic peptide (T15scr2). Unlike the first scrambled sequence, this peptide demonstrated self-binding and when conjugated to anti-GM2 antibody, induced apoptosis in levels higher than the original Tl 5 sequence. Thus, self -binding behavior can be generated, using the same amino acids from the original Tl 5 sequence but arranged in a different order from the original T 15. A peptide library generated using these same amino acids, combined with a screen for self-binding could be used to identify other self-binding sequences.

[00336] Example 6: Comparison Of Various Immunoglobulin Conjugation Sites [00337] The Tl 5 peptide sequence was conjugated to anti-GM2 antibody via the nucleotide binding site, tryptophan affinity sites, and through periodate oxidation of the carbohydrate on the Fc region. As shown in Fig. 12, when tested for the ability to trigger apoptosis, the nucleotide site conjugation (GM2-N3 -ATP-T 15/biotin) generated a higher

level of apoptosis than the carbohydrate linkage (Anti-GM2-T15). This was in spite of the fact that carbohydrate linkage installed 8-10 peptides per antibody and nucleotide linkage only 2 peptides per antibody. Hence, affinity site conjugation was the best method of conjugation of peptides. Conjugation to epsilon-amino acids of antibody, via hetero- bifunctional cross-linking agents, gave an inactive conjugate (not shown). [00338] Example 7: Restoration of Apoptotic Activity

[00339] A parental antibody to GM2 glycolipid, derived from a non-human hybridoma, was tested for the ability to trigger apoptosis against human cancers including non-small cell lung cancer (Fig. 13). The parental antibody demonstrated a high level of apoptosis and killing of cancer cells. The antibody was also effective in inhibiting growth of cancers in nude mouse models (not shown). To remove the potential for immunogenicity in humans, the antibody was "humanized" via cloning the heavy and light chain CDR's into the context of a human IgGl. Despite retention of affinity and specificity (not shown), the humanized antibody demonstrated much reduced ability to trigger apoptosis. In contrast, the humanized antibody, conjugated to a self-binding peptide (Sab), demonstrated high levels of apoptosis, similar to that of the parental antibody.

[00340] A further example is of a murine antibody, R24, which targets the GD3 ganglioside on human melanoma cells. When naturally expressed, this antibody has self- binding and therapeutic activity in patients, but as a humanized antibody it loses avidity, self- binding and therapeutic activity (Chapman et al., 1994). Restoration of therapeutic activity of the humanized R24 antibody can also be achieved by conjugation of a self -binding peptide to the antibody.

[00341] The humanized versions of antibody TEPC- 15 and T15/S107 can also benefit from conjugation with a self-binding peptide to restore or enhance self-binding and therapeutic activity.

[00342] Example 8: Enhanced binding and tumor recognition by Herceptin® SuperAntibody.

[00343] Herceptin® (monoclonal antibody to HER2/neu), has been approved by the FDA for treatment of breast cancer. The antigen is expressed in approximately 30% of breast cancers but in only about half of those patients is the level of expression sufficient to trigger therapeutic effects. In fact, patients are normally pre-screened in a diagnostic test to determine their suitability for treatment. HER2/neu is also expressed on other cancers, such as non-small cell lung cancer but typically in only low levels, making this type of cancer

unsuitable for treatment. An autophilic peptide was conjugated to Herceptin and tested for ability to bind non-small cell lung cancer. As shown in Fig. 14 (top panel), Herceptin reacts very weakly to this cancer; only 0.5% of cells are positive compared to an irrelevant antibody. In contrast, the same cancer can be better detected with the autophilic peptide conjugated form (i.e., SuperAntibody form) of Herceptin; over 57% are positive compared to irrelevant antibody (bottom panel). In separate tests, a SuperAntibody form of Herceptin also inhibited growth better than the parent antibody and could trigger apoptosis unlike the parent.

[00344] Example 9: Photo-crosslinking of tryptophan peptides to antibodies.

[00345] Antibodies and Reagents

[00346] Anti-human IgG (whole molecule) -peroxidase-conjugated secondary antibody, avidin-conjugated peroxidase, anti-human IgG (whole molecule) antibody, monoganglioside

GM2 were purchased from Sigma-Aldrich. Anti-GM2 antibody, Herceptin and anti-GM3 were obtained from Corixa (Seattle, WA), Genentech (San Francisco, CA) and CMI (Havana,

Cuba), respectively.

[00347] Two kinds of Trp-biotin peptides were designed: KAAGW (SEQ ID NO: 8) containing a biotin molecule on the alpha amino group [single biotin-peptide] , and

KAAKGEAKAAGW (SEQ ID NO: 9) containing biotin molecules on the alpha and epsilon amino groups of lysine [Multiple biotin-peptide]. These peptides were synthesized by .

Genemed Synthesis, Inc. (San Francisco, CA).

[00348] GMl, 2 and 3 were obtained from Sigma-Aldrich, glycolylic GM3 was obtained from Alexis USA (San Diego, CA).

[00349] Photobiotinylation using the tryptophan site.

[00350] All antibodies were incubated with the tryptophan-containing peptides for 1 hr at room temperature. The antibodies were photo-biotinylated at 200, 100, 50, 25, 10 and 1 μM concentrations of biotin-peptide. Photo-crosslinking was done using UV crosslinker FP-

UVXL-1000 (Fisher Scientific) on the optimum setting at 100 μj/cm ² . The samples were dialyzed against PBS (pH 7.4) buffer. The antibody concentration was determined using

Comassie Plus Protein Assay (Pierce). Chemical biotinylation was performed with NHS- biotin (Pierce Chemical, Rockford, IL). Chimeric anti-GM3 glycolylic (CIMAB, Havana,

Cuba) was biotinylated with 15 molar excess of NHS-biotin according to the manufacturer's protocol.

[00351] Direct Antibody Binding ELISA

[00352] Photobiotinylated antibody was coated by adding 2 μg to the first well and serially diluted and incubated overnight at 4 ⁰ C. The wells are washed 3X and blocked with 3% BSA dissolved in PBS, pH 7.4 for 2 hours. The plate was washed 3X and 100 μL of a 1/1000 dilution of avidin peroxidase conjugate was added per well. After incubating for 1 hour at room temperature, the wells were washed 3X with washing solution. 100 μL of OPD solution (OPD buffer, o-phenylenediamine and 1 μL of 30% hydrogen peroxide per ml) were added to each well. The color development was stopped by adding 30 μL of 4N H ₂ SO ₄ and the optical density is determined by scanning each well at 492 nm with a Fisher Scientific Multiskan RC plate reader.

[00353] Antibody Capture ELISA

[00354] Goat anti -human IgG whole molecule was coated at a 1/100 dilution per well, overnight at 4 ⁰ C. The plate was washed 3X and blocked 2 hours at room temperature with 3% BSA in PBS, pH 7.4. The plate was washed 3X and 2μg of the photobiotinylated antibody was added to the first well, serially diluted and incubated for 2 hours at room temperature or 4 ⁰ C, overnight. The plate was washed 3X and 100 μL of a 1/1000 dilution of avidin peroxidase conjugate was added per well. After incubating for 1 hour at room temperature, the wells were washed 3X with washing solution. 100 μL of OPD solution (OPD buffer, o-phenylenediamine and 1 μL of 30% hydrogen peroxide per ml) were added to each well. The color development was stopped by adding 30 μL of 4N H ₂ SO ₄ and the optical density was determined by scanning each well at 492 nm with a Fisher Scientific Multiskan RC plate reader.

[00355] Monoganglioside ELISA

[00356] GMl, GM2, GM3 and glycolylic GM3 monoganglioside were dissolved in methanol and coated overnight by drying on polystyrene microtiter plates at 0.5 μg per well. The wells were blocked with 1% BSA for 2 hours. GM2 tryptophan Tl 5 conjugate was added to 1 % BSA to a concentration of 2μg/μl and 200 μL was added to the first row of wells and serially diluted. After incubation at room temperature for 1 hr, the wells were washed 5X with washing solution. The plate was washed 3X and 100 μL of a 1/1000 dilution of avidin peroxidase conjugate was added per well. After incubating for 1 hr at room temperature, the wells were washed 3X with washing solution. 100 μL of OPD solution (OPD buffer, o- phenylenediamine and 1 μL of 30% hydrogen peroxide/ml) were added to each well. The color development was stopped by adding 30 μL of 4N H ₂ SO ₄ and the optical density was determined by scanning each well at 492 nm (Fisher Scientific Multiskan RC plate reader).

[00357] Photobiotinylation at different pH

[00358] The antibodies were incubated with 100 μM biotin peptide at pHs 5, 6, 7, 8, 9, 10 for 1 hour at room temperature and UV-crosslinked. The samples were dialyzed against PBS pH 7.4 and analyzed by capture ELISA.

[00359] RESULTS

[00360] Screening of biotin amino acids for photo-biotinylation.

[00361] Several biotinylated amino acids were mixed with a monoclonal antibody, OKT3, and exposed to UV. The mixture was then dot-blotted and developed with avidin-HRP. The dots were scanned and the relative color intensity was recorded. As shown in Fig. 15, OKT3 photolyzed with biotinylated tryptophan yielded the strongest reaction with avidin followed by biotin-tyrosine. OKT3 photolyzed with other biotin amino acid gave only background reaction with avidin.

[00362] Titrating Trp-biotin photolysis.

[00363] To obtain data on the affinity of biotin- Trp the monoclonal chimeric anti- ganglioside (anti-GM2) antibody was photolyzed at increasing concentrations of biotin- Trp.

The results shown in Fig. 16A indicate a saturating plateau of biotinylation of the antibody at the 100 μM level. Similar results were obtained with the titration of another monoclonal chimeric antibody against ganglioside (data not shown).

[00364] The dependence of affinity Trp photobiotinylation on pH was probed. The humanized antibody Herceptin® was photolyzed at different pH. As seen in Fig. 16B, the highest biotinylation was at pH 9. Similar pH dependence on biotinylation was observed with other monoclonal antibodies (data not shown).

[00365] Testing the covalent attachment of the biotin-Trp-peptides.

[00366] To prove that the photobiotinylation creates covalent bonds between the biotin peptide and the antibody, the biotinylated chimeric anti-ganglioside antibody was exposed to

6M guanidine HCL, then dialyzed against PBS and tested in direct avidin-HRP ELISA. Fig.

17 shows the ELISA reading of the native biotinylated anti-GM2 antibody and the de/re- natured antibody. Both preparations gave identical ELISA colors. Anti- GM2 not exposed to

UV did not react with avidin in the ELISA. These results provide evidence that the photobiotinylation using a Trp-biotin peptide attaches the biotin-peptide covalently to the antibody.

[00367] Antigen binding of single and multiple biotinylated antibodies.

[00368] Next, the use of biotin-peptides that contain terminal Trp was examined. Two kinds of Trp-biotin peptides were synthesized: 1) KAAGW containing a biotin molecule on the alpha amino group [single biotin-peptide] and 2) KAAKGEAKAAGW containing biotin molecules on the alpha and epsilon amino groups of lysine [multiple biotin-peptide] . [00369] In Fig. 18 A, the single biotin-peptide humanized anti-GM3 was compared to insolubilized ganglioside with the multiple biotin-peptide anti-GM3. The multiple biotin antibody produced stronger ELSIA signals with avidin-HRP. Similar differences (Fig. 18B) between a single and the multiple biotinylated antibody were seen with the chimeric anti- GM2.

[00370] Comparing the efficiency of photo-biotinylation with chemical biotinylation. [00371] Chemical biotinylation techniques are based on the variable availability of reactive amino acid side chains to produce mixtures of biotin proteins. For antibodies the number of biotins attached is 8-12 per molecule. In contrast, affinity-based biotinylation is limited by the number of affinity sites per antibody. In targeting the nucleotide site two affinity sites are available per Ig molecule. The number of Trp sites is variable in antibodies between 3 and 5 per molecule as estimated by a commercial biotin determination assay (data not shown). In Fig. 19, the reaction of avidin-HRP with insolubilized antibodies is shown. As expected, the chemically biotinylated antibodies produce stronger ELISA readings than the photo-biotinylated antibodies.

[00372] To compare the, detection sensitivity in an antigen- specific ELISA, photo- and chemical biotinylation of the chimeric anti-glycolylic GM3 antibody was performed. As shown in Fig. 20, the chemically biotinylated antibody produces a stronger signal than the photo- biotinylated antibody due to the greater number of biotin molecules on the antibody with chemical method.

[00373] To demonstrate the antigen specificity of affinity-photobiotinylated antibody, the chimeric anti-glycolylic GM3 antibody in ELISA was used. As seen in Fig. 21, the photo- biotin antibody recognizes its target antigen, not control ganglioside GMl, GM2 and GM2. [00374] DISCUSSION

[00375] Conjugating peptides with biological or chemical properties is an attractive method to enhance the potency of antibodies or endow antibodies with diagnostic and therapeutic utility [Zhao, et al (2001); Zhao, et al (2002)a; Zhao, et al (2002)b]. For example, the targeting of antibodies has been increased by conjugating autophilic peptides to produce dimerizing antibodies with enhanced targeting and induction of apoptosis. In another study,

membrane transporting sequence (MTS) was conjugated to antibodies and demonstrated that such MTS-antibodies penetrate the cellular membranes of living cells without harming the cells [Zhao, et al (2001)]. MTS antibodies against caspase-3 enzyme can inhibit induction of apoptosis in tumor cells. Attaching a peptide from the C3d complement fragment enhances the immune response to antibody vaccines creating a molecular adjuvant vaccine [Lou (1998)].

[00376] In all of these conjugations the invariant carbohydrate or the invariant nucleotide binding site were used. Both methods have drawbacks involving complex chemical reactions. The carbohydrate method requires oxidation of the antibody to create a reactive aldehyde and the nucleotide affinity photocrosslinking involves the synthesis of an azido- adenosine peptide [Lou and Kohler (1998)].

[00377] Here is presented a simple one-step affinity crosslinking technique for peptides based on the discovery that antibodies can be photo-crosslinked to aromatic hydrocarbon moieties (AHMs), including heterocyclic amino acids, such as tryptophan. Thus, peptides that contain terminal tryptophan are affinity photo-crosslinking reagents for antibodies. [00378] These new affinity conjugation methods have been demonstrated using biotinylated peptides. Exposing UV energy to a mixture of antibody and Trp-biotin peptides produces a biotin antibody that can be used in ELISA and other biotin-based detection methods. Such affinity-biotinylated antibodies have a defined number of biotins attached that are less than conventional biotinylation chemistries, but sufficient to produce useful signals in ELISA. Currently, the Trp-affinity photo-crosslinking method is used to attach peptides with biological and chemical properties similar to those previously published [Lou et al. (1998); Zhao, et al (2001); Zhao, et al (2002)a; Zhao, et al (2002)b].

[00379] Advantages of the tryptophan affinity- site based biotinylation are: (i) gentle one- step procedure without modifying amino acid side chains, and (ii) generates a reproducible antibody product labeled with defined number of biotin molecules. [00380] Example 10. Detection of circulating ox-LDL with super-antibodies [00381] The ability of autophilic antibodies, prepared according to the principles of the present invention, to recognize epitopes of circulating ox-LDL can be determined by conducting a sandwich assay. First, goat anti-mouse IgG-Fc antiserum is coated on microtiter wells, to which mouse mAbs having specific binding affinity for LDL particles, such as for apoB, are added. Next, plasma is contacted with the coated microtiter wells, followed by extensive washing. Then, a super-antibody, comprising a mAb specific for ox-LDL

conjugated to an autophilic peptide is added to top the sandwich. The completed sandwich can be visualized by a labeled secondary antibody specific for the autophilic peptide. Super- antibodies having specific binding affinity for ox-LDL should show at least a several-fold increase in detection over analogous super-antibodies nonspecific for ox-LDL. Controls for ox-LDL can be provided by Cu ⁺² -oxidized LDL (see U.S. Patent No. 6,225,070 to Witztum et al.).

[00382] Example 11. Inhibition of chronic inflammation in atherosclerosis. [00383] Chronic inflammation leading to atherosclerosis can be inhibited by the capacity of super- antibodies to bind avidly to ox-LDL, thereby blocking or reducing uptake of ox- LDL by macrophages. Humanized autophilic antibodies having specificity for ox-LDL are administered to a patient according to the regimen described hereinabove. The self-binding property of the autophilic antibodies increases their affinity for ox-LDL over that of unconjugated antibodies, and reduces recognition of the LDL particles by macrophages. Macrophage binding to ox-LDL should be effectively inhibited greater than 50% in the presence of the immunoconjugate. [00384] Example 12. Cell Lines

[00385] SV-DHL-4 (DHL-4) cells were a kind gift of Dr. Ron Levy, JOK-I cells were a gift of Affimed Inc. DHL-4 and JOK-I cells are grown in RPMI1640 with Glutamax (Gibco), supplemented with 10% FBS-Premium-HI (Aleken Biologicals), and 1% Penicillin/Streptomycin (Gibco). 1F5 hybridoma, Raji, and Ramos, cells are obtained from the American Type Culture Collection (ATCC), numbers HB-9645, CCL-86, CRL- 1596, and TIB-152, respectively. Raji and Ramos cells are maintained in RPMI-1640 Medium with HEPES (ATCC), supplemented with 10% FBS-Premium-HI (Aleken Biologicals), and 1% Penicillin/Streptomycin (Gibco). 1F5 cells are maintained in RPMI-1640 Medium with HEPES (ATCC), supplemented with 10% FBS-low-IgG (Gibco), 1% Penicillin/Streptomycin (Gibco), and 0.5% Glutamax (Gibco). CHO-S cells are purchased from Invitrogen, and are grown in CD CHO medium, supplemented with 1% HT supplement (Gibco), 2% Glutamax (Gibco), and 100 U/ml pen/strep (Gibco). After introduction of vector DNA, CHO-S cells are grown as above with the addition of 1.2 mg/ml G418 (Invivogen) for selection. All cells are maintained at 37 ⁰ C and 5% CO ₂ .

[00386] Example 13. Construction of chimeric antibody genes

[00387] Total RNA is isolated from about 7x10 ⁶ 1F5 hybridoma cells using an RNeasy kit (Qiagen) according to the manufacturer's instructions. First strand cDNA synthesis, cDNA

amplification by Long-Distance PCR (LD-PCR), and Proteinase K digestion are carried out using the materials and protocol of the Creator SMART cDNA library kit (Clontech). The 1F5 heavy chain variable regions are amplified from the cDNA pool by PCR using primers modVHlF5fwd (SEQ ID No. 15) and modVHlF5rev (SEQ ID No. 16). The 1F5 light chain variable regions are amplified from the cDNA pool by PCR using primers modVLlF5fwd (SEQ ID No. 17) and modVLlF5rev (SEQ ID No. 18). The heavy chain and light chain PCR products are cloned into the Xhol-Nhel and Sacl-Hindlll sites, respectively, of vector pAc-k- CH3 (Progen Biotechnik GmbH), to form pAc-k-lF5H and pAc-k-lF5K. Clones are verified by sequencing in both directions. All restriction enzymes are purchased from Takara or New England Biolabs. Taq polymerase (Promega) is used for all PCR. All enzymatic reactions are carried out using manufacturers' protocols.

[00388] Example 14. Construction of antibody expression vectors

[00389] Oligos LongT15fwd (SEQ ID No. 19), LongT15rev (SEQ ID No. 20), and PrimerB (SEQ ID No. 21) are used in a nested PCR similar to Horton, R. M., 1995, MoI Biotechnol 3: 93-99, to construct a DNA sequence that encodes the T15E peptide. The resulting PCR product is cloned into the Sall-Notl sites in MCS B of pIRES (Clontech) to form pDXL. The complete heavy and light chains of pAc-k-lF5H and pAc-k-lF5K are PCR amplified using primers modVHXfwd (SEQ ID No. 22) and modVHXrev (SEQ ID No. 23), or VKXfwd (SEQ ID No. 24) and VKXrev (SEQ ID No. 25), respectively. The light chain is cloned into the Nhel-Xhol sites of MCS A of vector pDXL, and the heavy chain is cloned into the Sall-Notl sites of the resulting vector to form pchlF5-DXL. Clones are verified by sequencing in both directions. To produce pchlF5 (anti-CD20 without the Tl 5 peptide), pchlF5-DXL and pIRES are digested with Notl and CIaI. Resulting DNA fragments of ~6 Kb from pchlF5-DXL, and -2.2 Kb from pIRES are each gel purified from a 1% agarose gel using a Qiaquick kit (Qiagen), and ligated together to form pchlF5. Clones are verified by sequencing in both directions. Oligo DNA sequences are provided in Table 5. All oligos are purchased from Operon.

TABLE 5. Primers Used

[00390] All vector constructs are introduced into E. coli (XL-10 cells, from Stratagene) using the provided heat shock protocols. Plasmids are purified from 3 ml of overnight bacterial culture using a Qiagen mini-prep kit. Vectors pchlF5 and pchlF5-DXL are electroporated into CHO-S cells using a 4 mm gap cuvette in an Eppendorf Multiporator set to 580 V and 40 μs. Two days of recovery are allowed before the start of selection. [00391] Example 15. Purification of recombinant antibodies

[00392] Cell culture supernatant is harvested every 3-5 days, depending on cell density. Cell suspensions are centrifuged at low speed (480-740 x g) for 7 to 10 minutes, and the supernatant is held at -2O ⁰ C prior to additional processing. After rapid thawing at 37 ⁰ C, supernatant is passed through a 0.2 filter (Corning) by vacuum filtration to remove cell debris, and filtered supernatant is then passed over HiTrap Protein G HP column (GE Healthcare). Bound antibodies are eluted with 0.1 M glycine buffer pH 2.7, collected in ImL fractions, and the pH is neutralized with 50 μL IM Tris pH 9. Elution profile is determined by reading UV absorbance at 280. Fractions with significant protein content are then pooled and concentrated using Amicon Ultra centrifugal filtration device 50,000 MW cutoff (Millipore) according to the manufacturer's instructions. [00393] Example 16. Cell Surface Binding

[00394] 3xlO ⁵ per well of Raji, Ramos, DHL-4, JOK-I, or Jurkat cells are seeded in a 24 well plate and incubated overnight at 37 ⁰ C and 5% CO ₂ . Cells are then harvested and washed twice with PBS. Cells are resuspended in ImL PBS and are incubated with either chlF5 or chlF5-DXL at increasing concentrations (1 μg, 5 μg, 10 μg/mL, 20 μg/mL) and incubated at 4 ⁰ C for 30 minutes. Excess antibody is removed by washing cells twice with PBS, and then cells are resuspended in a ImL solution of FITC conjugated goat anti-Human (Sigma, 1 :1000)

and incubated at 4 ⁰ C for 30 minutes. After washing twice, cells are resuspended in 200 μL PBS and analyzed by flow cytometry (BD FACSCalibur Instrument, BD Bioscience). Specific mean fluorescence intensity is determined by using the formula: specific MFI= MFI (primary Ab + goat anti-Human FITC) - MFI (goat anti-Human FITC). [00395] Figure 24 shows the ability of the recombinant chlF5 and chIF5-DXL antibodies to bind to cells from the human B -cell JOK-I line using fluorescence activated cell sorting (FACS). The dotted line shows the mean fluorescence intensity (MFI) of staining with the chlF5-DXL antibody, while the solid line represents the staining using the chlF5, non-DXL antibody. Binding of the chlF5-DXL antibody is approximately four-fold higher than binding of chlF5.

[00396] Example 17. Apoptosis Assay

[00397] The induction of apoptosis by the chlF5 and chlF5-DXL antibodies is tested in various cell lines. 2xlO ⁵ per well of Raji, Ramos, DHL-4, JOK-I, or Jurkat cells are seeded in a 24 well plate and incubated overnight at 37 ⁰ C and 5% CO ₂ . Cells are then treated with increasing concentrations of Abs for 20 hours at 37 ⁰ C. Cells are harvested, washed once with PBS, and resuspended with 100 μL IX annexin binding buffer containing 3 μL annexin V Alexa Fluor 488 conjugate (Invitrogen) and propidium iodide (Sigma) at a final concentration of 4 μg/mL to detect apoptosis and cell death, respectively. After 20 minutes incubation at 37 ⁰ C, cells are diluted with 150 μL of IX annexin binding buffer and analyzed by flow cytometry (BD FACSCalibur Instrument, BD Bioscience). Percent apoptotic cells is determined by gating the healthy population in the untreated control samples and using the formula: Percent Apoptotic Cells = (1 - (Live Treated Target Cells/Live Untreated Target Cells)) * 100.

[00398] Results are consistence with dependence of induction of apoptosis by DXL antibodies on receptor cross-linking. Figure 25 shows a comparison of induction of apoptosis by treatment with chlF5 or chlF5-DXL on Raji (panels A-C) and Ramos (panels D-F) cells. Results of analysis of untreated cells is shown in panels A and D, cells treated with chlF5 in panels B and E, and cells treated with chlF5-DXL in panels C and F.

[00399] In each panel of Figure 25, the x-axis of the graph (FL-I) shows the intensity of annexin-V binding, while the y-axis (FL-2) refers to the intensity of propidium iodide staining. Addition of 20 μg of chlF5 induces apoptosis in approximately 30% of the cells (Figure 25B versus Figure 25A). The DXL chimeric antibody induces significantly more

apoptosis than the non-DXL chimeric antibody (compare Figure 25C to Figure 25B). Similarly, the DXL antibody is a more potent inducer of apoptosis in Ramos cells at a concentration of 10 μg (compare Figure 25F to 25D and 25E).

[00400] In Table 6 the apoptotic effect of the two antibodies over a range of concentrations is shown.

TABLE 6. Induction of Apoptosis

Differing amounts of antibodies are added for 20 hours to each cell line

² Percent apoptotic cells induced by chlF5

³ Percent apoptotic cells induced by DXL-chl F5

[00401] It is interesting to note, at lower concentration of Abs the enhancing effect is much more pronounced. For example after treatment of Raji cells with of either antibody, the percent of apoptotic cells is 2.5 fold higher after DXL treatment, but it is slightly less than 2- fold higher after treatment with 20 μg/mL. JOKl cells showed little or no difference between chlF5 and DXL-chl F5.

[00402] Example 18. Complement Dependent Cytotoxicity (CDC) Assay [00403] The CDC activity of the chlF5 and chlF5-DXL is compared in this example. 2x10 cells are seeded into a 24 well plate and incubated overnight at 37 ⁰ C and 5% CO ₂ . Cells are then treated with increasing concentrations of Abs for 2 hours at 37 ⁰ C in the presence of 5% rabbit HLA-ABC complement enriched sera (Sigma). Cells are harvested and washed once with PBS, resuspended in 200 μL of PBS containing 50 nM calcein-AM (Biochemica) and 4 μg/mL propidium iodide (Sigma). After incubation for 20 minutes at 37 ⁰ C cell viability is analyzed by flow cytometry (BD FACSCalibur Instrument, BD

Bioscience). Percent killing is determined by the formula: Percent Dead Cells = (1 - (Live Treated Target Cells/Live Untreated Target Cells)) * 100.

[00404] CDC is induced after binding of complement components to the Fc region of an antibody, and is potent in the IgGl isotype, which is the isotype of the DXL construct. An enhancing effect is observed in all cell lines. Figure 26 shows graphs relating number of apoptotic cells to antibody concentration. Error bars show the standard deviation of the mean of two or more experiments. Student's t-test (two-tail) is used to test for statistical significance, *, P<0.05; **, P<0.01. As seen in Figure 26A, for example, at 5 μg/mL there is virtually no CDC activity in Raji cells with the non-DXL chimeric antibody. However, 35% of cells are killed with the DXL chimeric antibody. This correlates to the highest improvement of effectiveness in apoptosis. It is interesting to note that the potency of the DXL antibody plateaus at 5 μg/ml in Ramos cells (see Figure 26B). The chlF5 appears to plateau at 10 μg/ml, but does not reach the potency of DXL Ab at any level tested, suggesting that even higher doses would not reach the killing capacity of 5 μg/ml DXL Ab. [00405] Example 19. PBMC Separation

[00406] Peripheral blood mononuclear cells (PBMC) are prepared from healthy donors' buffy coat (Kentucky Blood Center, Lexington KY) by Ficoll-Hypaque density gradient centrifugation. PBMC are diluted to 6xlO ⁶ cells/mL in hRPMI (10% FBS, low IgG) culture media and maintained for a maximum of three days. PBMC viability and day-to-day cell population variation is analyzed by flow cytometry (BD FACSCalibur Instrument, BD Bioscience) before experimentation.

[00407] Example 20. Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) Assay [00408] Target cells (Raji, Ramos, DHL-4, or JOK-I) are harvested from T75 flasks and resuspended in ImL of media containing 400 nM calcein-AM (Biochemica) and 8 μL of TFL2 dye (Oncolmmunin), used according to manufacturer's instructions. Target cells are labeled for 45 minutes at 37 ⁰ C, washed twice in media, and resuspended to a density of 6xlO ⁵ cells/mL. Effector cells (PBMC) are then harvested from T75 flasks and resuspended to a density of 1.2xlO ⁷ cells/mL. Target cells (T) and effector cells (E) are mixed at an E:T ratio of 20:1. Then, 250 μL of the cell mixture is aliquoted into individual 5mL round bottom tubes and incubated with increasing concentrations of Abs for 2 hours at 37 ⁰ C. After incubation, target cell viability is analyzed by flow cytometry (BD FACSCalibur Instrument, BD Bioscience). Percent killing is determined by the formula: Percent Dead Cells = (1 - (Live Treated Target Cells/Live Untreated Target Cells)) * 100.

[00409] CDC can be used as a criterion to divide different anti-CD20 antibodies into two types, as described in Cragg, M.S. et al., Blood, 103:2738-2743, 2004. Type I anti-CD20 activates complement efficiently, while type II mediates ADCC not CDC. The 1F5 anti- CD20 belongs together with Rituxan to the type I class. Even though the parental 1F5 anti- CD20 belongs to the type I class, the DXL version shows a significant increase of ADCC activity, therefore gaining type II properties. This creates a new class of therapeutic antibodies, designated here as type III. Figure 27 shows graphs relating number of apoptotic cells to antibody concentration. Error bars show the standard deviation of the mean of two or more experiments. Student's t-test (two-tail) is used to test for statistical significance, *, P<0.05; **, P<0.01. As shown in Figures 27A and 27B, the DXL antibody induces significantly more ADCC than chlF5 in Raji and Ramos cells at 1 μg/ml and 3 μg/ml, but the increase in potency is not significant at 7.5 μg/ml. [00410] Example 21. Inhibition of Lymphoma Growth In Vitro

[00411] The anti-proliferative effects of the chlF5 and chlF5-DXL antibodies is determined in Raji and Ramos cell lines to approximate the in vivo killing potential of these anti-CD20 antibodies on tumor cells. The assay measures the level of fluorescence dye binding to nucleic acid. 5x10 ³ cells per well of Raji or Ramos cells are seeded into a 96 well plate and treated with decreasing concentrations of Abs. Cells are incubated for 6 days at 37 ⁰ C and 5 % CO ₂ . At the end of six days cells are centrifuged at low speed (450 x g) for seven minutes. Supernatant is removed and cells are resuspended with 100 μL Cyquant NF DNA binding dye reagent (Invitrogen) for 45 minutes at 37°C. Fluorescence is measured using a Synergy 2 microplate reader (Biotek), emission 485 nm and excitation 530 nm. Higher fluorescence is indicative of cell proliferation.

[00412] As shown in Figure 28A and Figure 28B, the DXL antibody inhibited proliferation to a greater extent than the non-DXL antibody in both cell lines at all concentrations tested.

[00413] Example 22. Construction of antibody expression vectors

[00414] DNA encoding the rituximab heavy chain is synthesized by PCR using overlapping primers to produce SEQ ID No. 31.

SEQ ID No. 31 DNA encoding Rituximab heavy chain 5' to 3' ATGGGATGGTCTTGTATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCAGGCCTAC CTGCAG

GGCCATCTACCCAGGCAACGGCGATACCTCTTACAATCAGAAGTTCAAGGGAAAGGC CACACTGA CAGTGGACAAGTCTTCTAGCACCGCCTACATGCAGCTGAGCAGCCTGACCTCCGAGGATT CCGCCG

TGTACTTTTGCGCCAGAGTGGTGTATTATTCCAATTCCTACTGGTACTTCGATGTGT GGGGGACCGG

CACAACCGTGACCGTGTCCGGCCCAAGCGTGTTCCCACTGGCCCCTTCCTCTAAATC TACCTCTGG

CGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTTCCAGAGCCAGTGACCGT GTCCTGGAA

TTCCGGCGCCCTGACATCTGGAGTGCACACATTCCCTGCCGTGCTGCAGTCCTCCGG CCTGTATTCT

CTGTCCAGCGTGGTGACCGTGCCTTCTAGCAGCCTGGGCACACAGACCTACATCTGC AATGTGAAT

CCTGCCCTCCCTGTCCTGCCCCAGAGCTGCTGGGCGGGCCCAGCGTGTTTCTGTTCC CTCCCAAGCC

TAAAGACACACTGATGATCAGCAGAACCCCAGAGGTGACCTGTGTGGTGGTGGATGT GTCTCACG

AGGACCCCGAGGTGAAGTTCAACTGGTACGTGGATGGGGTGGAGGTGCACAATGCCA AAACCAAA

CTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGAGCAATAAAGCCCTGCCTGCCCC AATCGAAA

AGACAATCAGCAAGGCCAAAGGCCAGCCTAGGGAACCCCAGGTGTACACACTGCCTC CCTCTCGG

GACGAGCTGACAAAGAATCAGGTGAGCCTGACCTGCCTGGTGAAAGGCTTCTACCCC AGCGATAT

CGCCGTGGAGTGGGAGTCCAACGGCCAGCCAGAGAATAACTATAAGACCACCCCTCC CGTGCTGG

ACTCCGACGGCAGCTTTTTCCTGTACTCCAAGCTGACCGTGGACAAAAGCCGGTGGC AGCAGGGA

AATGTGTTCAGCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAA TCCCTGTCT

CGCCAGCGTGAAGGGGAGGTTCATTGTGAGCAGATGA

[00415] DNA encoding the rituximab light chain is synthesized by PCR using overlapping primers to produce SEQ ID No. 32.

[00416] SEQ ID No. 32 DNA encoding Rituximab light chain 5' to 3'

ATGGGCTGGTCTTGTATCATTCTGTTTCTGGTGGCCACAGCCACCGGGGTGCAG

ACCTGCAGGGCCTCCTCTTCCGTGTCCTACATGCACTGGTACCAGCAGAAGCCCGGC TCT AGCCCAAAACCCTGGATCTACGCCCCCTCTAACCTGGCCTCCGGCGTGCCAGCCAGATTC TCTGGCTCCGGAAGCGGCACCTCCTACAGCCTGACCATCTCCAGAGTGGAAGCCGAAGAC GCCGCCACCTACTACTGCCAGCAGTGGTCTTTCAATCCTCCCACCTTCGGGGCCGGGACA AAACTGGAGCTGAAGCGGACCGTGGCCGCCCCCTCCGTGTTCATCTTCCCTCCTTCCGAC GAGCAGCTGAAGTCCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCACGC GAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGAGCGGCAATAGCCAGGAATCT GTGACCGAGCAGGACAGCAAGGATTCTACCTACAGCCTGTCCAGCACCCTGACCCTGAGC AAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACACACCAGGGCCTGAGC AGCCCTGTGACCAAGTCTTTCAACAGATGA

[00417] The light chain is cloned into the Xhol-EcoRI sites of Multiple Cloning Site (MCS) A of vector pDXL, and the heavy chain is cloned into the Xbal-Sall sites of MCS B the same vector to form the bicistronic plasmid pRituximab-DXL having DNA sequences encoding the chimeric heavy chain and light chain separated by the IRES. [00418] pRituximab-DXL is introduced into E. coli (XL-10 cells, from Stratagene) using the provided heat shock protocols. Plasmids are purified from 3 ml of overnight bacterial culture using a Qiagen mini-prep kit. Vector pRituximab-DXL is electroporated into CHO-S cells using a 4 mm gap cuvette in an Eppendorf Multiporator set to 580 V and 40 μs. Two days of recovery are allowed before the start of selection. Recombinant autophilic antibodies which include the rituximab heavy chain fused to the T15E autophilic peptide are purified and tested as described herein.

[00419] Example 23

[00420] Preparation of anti-Her2 antibody-autophilic peptide conjugate [00421] An anti-Her2 antibody is compared to an anti-Her2 antibody-autophilic peptide conjugate in this example. The Tl 5 autophilic peptide of SEQ ID No. 14 is used in this example with a four amino acid spacer, H-Trp-Gly-Ala-Ala-OH (SEQ ID No. 52). The autophilic peptide and spacer: H-Trp-Gly-Ala-Ala-Ala-Ser-Arg-Asn-Lys-Ala-Asn-Asp-Tyr- Thr-Thr-Glu-Tyr-Ser-Ala-Ser-Val-Lys-Gly-Arg-Phe-Ile-Val-Ser- Arg-OH (SEQ ID No. 53) (MW 3207.5 g/mole) is conjugated to Herceptin (trastuzumab, Genentech). The resulting anti-Her2 antibody-autophilic peptide conjugate is referred to as DXL702. [00422] Herceptin (Genentech; NDC 50242-134-68) is reconstituted per package insert and dialysis is performed against sodium citrate saline (25 mM sodium citrate, pH 6.5/150 mM NaCl). Dialysis is achieved in three steps against a minimum of 50 volumes for at least four hours each utilizing dialysis tubing with a 25,000 MWCO (Spectrum; 132-126). The dialyzed Herceptin is then sterile filtered through a 0.2 μ syringe filter (Fisherbrand; 09- 719C) and a protein concentration is determined utilizing the absorbance at 280 nm. Based upon the protein concentration, the Herceptin is then diluted to 4.5 mg per mL in sterile sodium citrate saline. A sterile solution of 20 mg/mL Tl 5 peptide is added aseptically to the diluted Herceptin at a molar ratio of two moles of Tl 5 peptide to every one mole of Herceptin and gently mixed.

[00423] A sterile quartz tube (Technical Glass Products, Inc.; 8x12, 30 cm in total length) is sealed on one end with parafilm (Pechiney Plastic Packaging; PM996). The diluted Herceptin/T15 mixture is transferred aseptically using a sterile syringe to slowly fill the quartz tube to avoid the introduction of air bubbles. The open end of the quartz tube is also sealed with parafilm and is place within the UV crosslinker (Spectroline; Spectrolinker XL- 1500 UV Crosslinker with UV lamps at 254 nm). The Herceptin/T15 mixture in the quartz tube is exposed to a UV dose equivalent to 500 mJ/cm ² . After exposure to the UV, the mixture is transferred to a container with an equal volume of quench buffer (25 mM sodium citrate, pH 6.5/150 mM NaCl/100 mM L-arginine/100 mM L-glutamic acid/2% ethanolamine). The quenched mixture is initially concentrated two-fold in a Millipore Stir Cell using a membrane with a 30,000 MWCO (Millipore, YM30 Regenerated Cellulose) at room temperature. Then a diafiltration step is performed by adding two volumes of dialysis buffer A (25 mM sodium citrate, pH 6.5/150 mM NaCl/50 mM L-arginine/50 mM L- glutamic acid) to the concentrated mixture, and concentrating back to the original volume

(i.e., post initial concentration volume). A repeat of the diafiltration with dialysis buffer A is performed. A second diafiltration step is performed by adding two volumes of dialysis buffer B (25 mM sodium citrate, pH 6.5/150 mM NaCl) to the concentrated mixture, and again concentrating back to the original volume (i.e., post initial concentration volume). This diafiltration step with dialysis buffer B is repeated five more times. After the final diafiltration, the antibody is concentrated to approximately 30 mg/mL. The concentrated antibody is stored at 4 ⁰ C for a minimum of 16 hours. A centrifugation is performed in a Fisher accuSpin 3R centrifuge with swinging bucket rotor at 200Og for 10 minutes at 4 ⁰ C. The supernatant is removed and sterile filtered through a 0.2 μ nylon syringe filter into a sterile container. The antibody-autophilic peptide conjugate can be stored indefinitely at 4 ⁰ C. [00424] ELISA - Using anti-Her2 antibody-autophilic peptide conjugate [00425] One volume of 702 Bt-Peptide, Biotin-Ala-Ala-Leu-Leu-Gly-Pro-Tyr-Glu-Leu- Trp-Glu-Leu-Ser-His-OH (SEQ ID No. 54) (MW 1826.1 g/mole), is combined with 41 volumes Avidin and mixed thoroughly.

[00426] The mixture is diluted in ELISA Coating Buffer to a final peptide concentration of 0.075 ug/ml. The diluted mixture is used to coat wells of an assay plate using 100 ul per well. The mixture is incubated in the wells at room temperature (RT) for four hours or at 2-8 deg C overnight. The plate is then washed 2X with PBS Tween. Non-specific binding in the wells is blocked by addition of 275 ul 1% BSA in PBS. The plate is incubated at RT for 60 minutes and then washed with 3X with PBS Tween. One hundred microliters of primary antibody is added per well at desired concentration(s). The plate is then incubated at RT for 60 minutes followed by three washes with PBS Tween. One hundred microliters of 1 :25K diluted HRP-labeled anti- Human IgG is added per well and incubated at RT for 60 minutes followed by three washes with PBS Tween. One hundred microliters of TMB Substrate is added per well and incubated at RT for 15 minutes.

[00427] Fifty microliters of 1.0 N Sulfuric Acid is added to each well to stop the reaction. Results are then read immediately on a plate reader at 450 nm. [00428] Figure 29 shows a graph of the results comparing ELISA using an unmodified anti- Her2 antibody and an anti-Her2 antibody-autophilic peptide conjugate (DXL702). More sensitive detection of Her2 is observed at all concentrations of the antibodies used. [00429] Example 24

[00430] Biomolecular interaction sensing - anti-Her2 antibody-autophilic peptide conjugate

[00431] In this example, a Biacore TlOO system is used to compare binding avidity of an unmodified anti-Her2 and an anti-Her2 antibody-autophilic peptide conjugate, DXL702. A biotinylated Her2 peptide is immobilized to a strepavidin sensor chip. A sample of each antibody is injected into the system and passed over the sensor chip surface. Interaction of each antibody with the immobilized analyte is detected according to system manufacturer's directions.

[00432] Figure 30 shows a resulting sensorgram which illustrates an increasing response as Anti-Her2 (left) and DXL702 (right) bind to the Her2 peptide on the sensor chip surface. This interaction levels off as equilibrium is reached. When the sample is replaced by the buffer, the response decreases as the Anti-Her2 and DXL702 dissociate from the Her2 peptide. Biacore detection methods illustrate increased avidity of DXL702 compared to an unmodified anti-Her2 antibody. [00433] Example 25

[00434] Immunofluorescence Assay- anti-Her2 antibody-autophilic peptide conjugate [00435] MDA MB 231 cells (ATCC, HTB-26) are grown in T150 Canted Neck Tissue Culture Flasks (Becton-Dickinson, 355001) in a growth media of RPMI + GlutaMAX (Gibco, 61870) with 10% Fetal Bovine Serum (Gibco, 26140) and 1% Penicillin/Streptomycin (Gibco, 15140) at 37°C/5% CO ₂ to confluence. After removal of the media the cells are rinsed with Phosphate Buffered Saline (Fisher, BP3994, diluted to IX). The cells from each T 150 flask are then harvested through trypsinization by the addition of 2.0 mL of 0.25% Trypsin-EDTA (Gibco, 25200) and incubation for 4 minutes at 37°C/5% CO ₂ . The trypsinization is stopped by the addition of 10 mL of the aforementioned growth media to each flask. The trypsinized cells are combined, and then centrifuged at 70Og for 7 minutes. To wash the cells, the supernatant is removed and an equal volume of IX PBS is added. The resuspended cells are centrifuged at 70Og for 7 minutes. The supernatant is again removed, and a second wash of the cells is performed. The washed cells are resuspended in PBS + 2.5% Fetal Bovine Serum to a cell density of approximately 5 million cells per mL. [00436] An 8% Formaldehyde solution is prepared by the diluting an equal volume of 16% Formaldehyde (Ted Pell Inc., 18505) and MiIIiQ water. While mixing the cells at a low speed on a vortex, an equal volume of 8% Formaldehyde slowly is added. The cells are allowed to fix overnight at 4 ⁰ C. Immediately prior to use, the fixed cells are centrifuged at 800g for 10 min. The supernatant is carefully removed. The excess Formaldehyde is removed by washing the cell pellet with an equal volume of IX PBS and centrifuging at 800g

for 10 min. The supernatant is again carefully removed. The cell pellet is resuspended to a concentration of approximately 2 million cells per mL in PBS + 2.5% Fetal Bovine Serum. [00437] Samples of unmodified anti-Her2 (Genentech, Herceptin) and anti-Her2 antibody- autophilic peptide conjugate are diluted in IX PBS so that addition of 10 μL of sample to 200 μL cells creates test concentrations of 0.1 to 10 μg of antibody per mL. Replicates of the 10 μL test samples are aliquoted to the wells of a U-bottom 96-well plate (Becton-Dickinson, 353910). In addition to the test samples, replicate wells with only 10 μL of IX PBS are aliquoted for use as setup and negative controls. After placement of all samples (i.e., primary antibody or PBS) in the wells, 200 μL of Fixed Cells are added to each well. The cells are incubated with the primary antibodies for 1 hour at room temperature with gentle shaking. The plate is then centrifuged at 800g for 10 min and the supernatant is removed by a quick inversion of the plate. The cells are washed by the addition of 200 μL of IX PBS to each well and subsequent centrifugation at 800g for 10 min. Again the supernatant is removed by quick inversion of the plate.

[00438] As a secondary antibody goat anti-Human IgG (Fab specific)-FTTC (Sigma, F5512) is diluted 1:10,000 in IX PBS + 2.5% Fetal Bovine Serum and 200 μL of this is added to each test well. As a negative control, half of the wells without any primary antibody also have the diluted secondary antibody added to them. The remaining wells without any primary antibody have IX PBS + 2.5% Fetal Bovine Serum added to them for use in setting up the flow cytometer parameters. The plate is incubated with the secondary antibody for 1 hour at room temperature with gentle shaking. The plate is then centrifuged at 800g for 10 min and the supernatant is removed by a quick inversion of the plate. The cells are washed by the addition of 200 μL of IX PBS to each well and subsequent centrifugation at 800g for 10 min. Again the supernatant is removed by quick inversion of the plate. A repeat of the cell wash is performed. Each well then has 200 μL of IX PBS added to it. The plate contains the following samples: (1) No Primary, No Secondary - used for flow cytometry setup, (2) No Primary, With Secondary - to show the level of non-specific binding that the secondary antibody has with the fixed cells and (3) Test Samples - tested each antibody at specified concentrations. The 96-well plate is placed into a FACS Canto II Flow Cytometer (Becton-Dickinson, 338960) with a High Throughput Sampler (Becton-Dickinson, 640009). The no primary, no secondary sample is used to setup the following parameters (FSC, SSC and FITC fluorescent channel). A minimum of 20,000 events are recorded for each of the

remaining samples and the Mean Fluorescent Intensity (MFI) is calculated using FACSDiva software.

[00439] Figure 31 shows results of this assay graphically. The results show that the anti-

Her2 antibody conjugated to an autophilic peptide, DXL702, produces more signal than the unmodified anti-Her2, allowing for more sensitive detection of the Her2 analyte.

[00440] Example 26

[00441] Anti-Her2 antibody- autophilic peptide conjugate detection of Her2 in high- and

Iow-Her2 expressing cells

[00442] Quantitiative reverse transcription polymerase chain reaction (RT-PCR) is performed using nucleic acid samples isolated from cell lines BT-20 (ATCC No. HTB-19),

BT-474 (ATCC No. HTB-20), H1650 (ATCC No. CRL-5883), HCC1419 (ATCC No. CRL-

2326), MCF7 (ATCC No. HTB-22), MDA-231 (ATCC No. HTB-26), MDA-453 (ATCC

No. HTB-131), MDA-468 (ATCC No. HTB-132), SKBR3 (ATCC No. HTB-30), ZR-75-1

(ATCC No. CRL-1500), and Ramos (ATCC No. CRL-1596), to detect and quantify Her2.

Cells are classified as "high" Her2 expressers or "low" Her2 expressers as indicated in graphs shown in Figure 32.

[00443] High and low Her2 expressers are incubated with fluorescently labeled non- autophilic Herceptin antibody or Herceptin antibody conjugated to an autophilic peptide and bound antibody is measured using flow cytometry. Figure 33 is a series of graphs showing that autophilic peptide conjugated Herceptin provides improved signal strength compared to non-autophilic Herceptin antibody and allows for more sensitive detection of low Her2 expressing cells.

[00444] Example 27

[00445] Preparation of anti-CD20 antibody- autophilic peptide conjugate

[00446] Rituxan (Genentech; NDC 50242-053-06) is diluted to 4.0 mg per mL (based upon package insert concentration) in sterile sodium citrate saline (25 mM sodium citrate, pH

6.5/150 mM NaCl). A sterile solution of 20 mg/mL T15 peptide + four amino acid spacer

(SEQ ID No. 53) is added aseptically to the diluted Rituxan at a molar ratio of two moles of

Tl 5 peptide to every one mole of Rituxan and gently mixed. A sterile quartz tube (Technical

Glass Products, Inc.; 8x12, 30 cm in total length) is sealed on one end with parafilm

(Pechiney Plastic Packaging; PM996). The diluted Rituxan/T15 mixture is transferred aseptically using a sterile syringe to slowly fill the quartz tube to avoid the introduction of air bubbles. The open end of the quartz tube is also sealed with parafilm and is place within the

UV crosslinker (Spectroline; Spectrolinker XL- 1500 UV Crosslinker with UV lamps at 254 nm). The Rituxan/T15 mixture in the quartz tube is exposed to a UV dose equivalent to 600 mJ/cm ² . After exposure to the UV, the mixture is transferred to a container with an equal volume of quench buffer (25 mM sodium citrate, pH 6.5/150 mM NaCl/100 mM L- arginine/100 mM L-glutamic acid/2% ethanolamine). The quenched mixture is initially concentrated two-fold in a Millipore Stir Cell using a membrane with a 30,000 MWCO (Millipore, YM30 Regenerated Cellulose) at room temperature. Then a diafiltration step is performed by adding two volumes of dialysis buffer A (25 mM sodium citrate, pH 6.5/150 mM NaCl/50 mM L-arginine/50 mM L-glutamic acid) to the concentrated mixture, and concentrating back to the original volume (i.e., post initial concentration volume). A repeat of the diafiltration with dialysis buffer A is performed. A second diafiltration step is performed by adding two volumes of dialysis buffer B (25 mM sodium citrate, pH 6.5/150 mM NaCl) to the concentrated mixture, and again concentrating back to the original volume (i.e., post initial concentration volume). This diafiltration step with dialysis buffer B is repeated five more times. After the final diafiltration, the antibody is concentrated to approximately 30 mg/mL. The concentrated antibody is stored at 4 ⁰ C for a minimum of 16 hours. A centrifugation is performed in a Fisher accuSpin 3R centrifuge with swinging bucket rotor at 200Og for 10 minutes at 4 ⁰ C. The supernatant is removed and sterile filtered through a 0.2 μ nylon syringe filter into a sterile container. The rituximab-T15 autophilic peptide conjugate (DXL625) can be stored indefinitely at 4 ⁰ C.

[00447] Binding Kinetics of rituximab compared to rituximab-autophilic peptide conjugate.

[00448] The binding interaction between rituximab-autophilic peptide conjugate DXL625 and CD20 is evaluated by surface Plasmon resonance (SPR) and Scatchard analyses of flow cytometry data.

[00449] To determine the real time interaction of DXL625 with CD20, a biotinylated- CD20 mimetic cyclized peptide, Biotin-AHTPYINIYNCEPANPSEKNSPSTOYCY-OH (s-s bonded, MW 3430.8 g/mole) (SEQ ID No. 51), is immobilized to the surface of a neutravidin-coated SPR chip (-200 RU immobilized) and various concentrations of antibody are flowed over (at room temperature) and the resonance-change or response unit (RU) is recorded. The RU changes reflect the amount of antibody binding to the CD20 mimetic peptide coated on the chip over time and at saturation, estimate the binding maximum (Bmax). At saturation, antibody flow ceases and buffer is flowed through. The reduction of

RU during this phase is a measure of dissociation. Modeling of the shape of the resultant tracing is only validated for mono- or bi-valent binding interactions and therefore do not provide for the polyvalent-interactions observed with autophilic (self interacting) antibodies such as DXL625. For a given antibody-antigen interaction, taking the RU value at the shallowest point of the dissociation phase (determined by the instrument) divided by the maximum RU value at saturation (Bmax) results in a measure of avidity (strength of binding). DXL625 has a significantly greater avidity (0.66 ± 0.023) than Rituxan (0.08 ± 0.002) Data represents assays performed in triplicate and compared by pair- wise t-test. Measurement of avidity of DXL625 is compared to Rituxan by chaotropic avidity ELISA using the mimetic peptide of the CD20 antigen. Antibody bound to the CD20 peptide is challenged with varying concentrations of the chaotropic agent potassium thiocyanate (0- 3M). The avidity index is determined by the concentration of chaotropic agent needed to reduce antibody binding by 50%. The Al for Rituxan is 2.25M, whereas the Al for DXL625 is 3.0M.

[00450] The mimetic peptide is selected for the SPR method with a reduced affinity to ensure appropriate visualization of the binding kinetics of DXL625. Thus, the kinetics of the control anti-CD20 monoclonal antibody may be lower than that reported in Rituxan® product literature (~1 μM vs. ~8 nM). A second method used to investigate DXL625 binding utilized flow cytometry. To determine the binding characteristics to native antigen-expressing cells (human peripheral blood lymphocytes), are incubated with 8-fold dilutions of antibody at 4°C for 20 minutes and the amount antibody bound to cells is detected using a fluorescent secondary (FITC goat anti-mouse IgG). The binding maxima (Bmax) are illustrated in the graph shown in Figure 34. Thus, on a concentration basis, DXL625 detectable on the surface of human B cells is much greater than Rituxan and represents a 5.4 fold increase. [00451] Example 28

[00452] ELISA - Using anti-CD20 antibody-autophilic peptide conjugate [00453] DXL625 is compared to an anti-CD20 antibody-autophilic peptide conjugate in this example. One volume of CD20 Bt-Peptide Biotin-

AHTPYINIYNCEPANPSEKNSPSTOYCY (SEQ ID NO. 51), is combined with 41 volumes Avidin and mixed thoroughly.

[00454] The mixture is diluted in ELISA Coating Buffer to a final peptide concentration of 0.075 ug/ml. The diluted mixture is used to coat wells of an assay plate using 100 ul per well. The mixture is incubated in the wells at room temperature (RT) for four hours or at 2-8 deg C

overnight. The plate is then washed 2X with PBS Tween. Non-specific binding in the wells is blocked by addition of 275 ul 1% BSA in PBS. The plate is incubated at RT for 60 minutes and then washed with 3X with PBS Tween. One hundred microliters of primary antibody is added per well at desired concentration(s). The plate is then incubated at RT for 60 minutes followed by three washes with PBS Tween. One hundred microliters of 1 :25K diluted HRP-labeled anti- Human IgG is added per well and incubated at RT for 60 minutes followed by three washes with PBS Tween. One hundred microliters of TMB Substrate is added per well and incubated at RT for 15 minutes. Fifty microliters of 1.0 N Sulfuric Acid is added to each well to stop the reaction. Results are then read immediately on a plate reader at 450 nm. More sensitive detection of CD20 peptide analyte is observed using DXL625. [00455] Example 29

[00456] Immunofluorescence Assay - using rituximab-T15 autophilic peptide conjugate DXL625

[00457] Ramos cells (ATCC, CRL-1596) are grown in T150 Canted Neck Tissue Culture Flasks (Becton-Dickinson, 355001) in a growth media of RPMI + GlutaMAX (Gibco, 61870) with 10% Fetal Bovine Serum (Gibco, 26140) and 1% Penicillin/Streptomycin (Gibco, 15140) at 37°C/5% CO ₂ to a cell density of 1 to 2 million cells per mL. The cells are combined and then centrifuged at 70Og for 7 minutes. To wash the cells, the supernatant is removed and an equal volume of Phosphate Buffered Saline (Fisher, BP3994, diluted to IX) is added. The resuspended cells are centrifuged at 70Og for 7 minutes. The supernatant is again removed, and a second wash of the cells is performed. The washed cells are resuspended in PBS + 2.5% Fetal Bovine Serum to a cell density of approximately 5 million cells per mL.

[00458] An 8% Formaldehyde solution is prepared by the diluting an equal volume of 16% Formaldehyde (Ted Pell Inc., 18505) and MiIIiQ water. While mixing the cells at a low speed on a vortex, an equal volume of 8% Formaldehyde slowly is added. The cells are allowed to fix overnight at 4 ⁰ C. Immediately prior to use, the fixed cells are centrifuged at 800g for 10 min. The supernatant is carefully removed. The excess Formaldehyde is removed by washing the cell pellet with an equal volume of IX PBS and centrifuging at 800g for 10 min. The supernatant is again carefully removed. The cell pellet is resuspended to a concentration of approximately 2 million cells per mL in PBS + 2.5% Fetal Bovine Serum. [00459] Samples of unmodified anti-CD20 (Genentech, Rituxan) and anti-CD20 autophilic antibody (InNexxus, DXL625) are diluted in IX PBS so that addition of 10 μL of sample to

200 μL cells created test concentrations of 0.1 to 10 μg per mL. Replicates of the 10 μL test samples are aliquoted to the wells of a U-bottom 96-well plate (Becton-Dickinson, 353910). In addition to the test samples, replicate wells with only 10 μL of IX PBS are aliquoted for use as setup and negative controls. After placement of all samples (i.e., primary antibody or PBS) in the wells, 200 μL of Fixed Cells is added to each well. The cells are incubated with the primary antibodies for 1 hour at room temperature with gentle shaking. The plate is then centrifuged at 800g for 10 min and the supernatant is removed by a quick inversion of the plate. The cells are washed by the addition of 200 μL of IX PBS to each well and subsequent centrifugation at 800g for 10 min. Again the supernatant is removed by quick inversion of the plate.

[00460] As a secondary antibody goat anti-Human IgG (Fab specific)-FTTC (Sigma, F5512) is diluted 1:10,000 in IX PBS + 2.5% Fetal Bovine Serum and 200 μL of this is added to each test well. As a negative control, half of the wells without any primary antibody also have the diluted secondary antibody added to them. The remaining wells without any primary antibody have IX PBS + 2.5% Fetal Bovine Serum added to them for use in setting up the flow cytometer parameters. The plate is incubated with the secondary antibody for 1 hour at room temperature with gentle shaking. The plate is then centrifuged at 800g for 10 min and the supernatant is removed by a quick inversion of the plate. The cells are washed by the addition of 200 μL of IX PBS to each well and subsequent centrifugation at 800g for 10 min. Again the supernatant is removed by quick inversion of the plate. A repeat of the cell wash is performed. Each well then has 200 μL of IX PBS added to it. The plate contained the following samples: (1) No Primary, No Secondary - as use for flow cytometry setup, (2) No Primary, With Secondary - to show the level of non-specific binding that the secondary antibody has with the fixed cells and (3) Test Samples - tests each antibody at specified concentrations.

[00461] The 96-well plate is placed into a FACS Canto II Flow Cytometer (Becton- Dickinson, 338960) with a High Throughput Sampler (Becton-Dickinson, 640009). The no primary, no secondary sample is used to setup the following parameters (FSC, SSC and FITC fluorescent channel). A minimum of 20,000 events is recorded for each of the remaining samples and the Mean Fluorescent Intensity (MFI) is calculated using FACSDiva software (Becton-Dickinson, version 6.0).

[00462] More sensitive detection of CD20 analyte is observed using DXL625. [00463] Example 30 - Assays anti-EGFR antibody- autophilic peptide conjugate

[00464] Lyophilized Erbitux (Bristol-Meyer Squibb; NDC 66733-948-23) is reconstituted with sodium citrate saline (25 mM sodium citrate, pH 6.5/150 mM NaCl) to a concentration of approximately 2 mg per mL. Dialysis is performed against sodium citrate saline (25 mM sodium citrate, pH 6.5/150 mM NaCl) in one step against a minimum of 100 volumes for at least 16 hours utilizing dialysis tubing with a 25,000 MWCO (Spectrum; 132-126). The dialyzed Erbitux is then clarified by filtering through a 0.45 μ syringe filter (Fisherbrand; 09- 719D) and a protein concentration is determined utilizing the absorbance at 280 nm. Based upon the protein concentration, the Erbitux is then diluted to 1.75 mg per mL in sterile sodium citrate saline. A sterile solution of 20 mg/mL Tl 5 peptide + spacer of SEQ ID No. 53 is added aseptically to the diluted Herceptin at a molar ratio of two moles of Tl 5 peptide to every one mole of Erbitux and gently mixed. A sterile quartz tube (Technical Glass Products, Inc.; 8x12, 30 cm in total length) is sealed on one end with parafilm (Pechiney Plastic Packaging; PM996). The diluted Erbitux/T15 mixture is transferred aseptically using a sterile syringe to slowly fill the quartz tube to avoid the introduction of air bubbles. The open end of the quartz tube is also sealed with parafilm and is place within the UV crosslinker (Spectroline; Spectrolinker XL-1500 UV Crosslinker with UV lamps at 254 nm). The Erbitux/T15 mixture in the quartz tube is exposed to a UV dose equivalent to 350 mJ/cm . After exposure to the UV, the mixture is transferred to a container with an equal volume of quench buffer (25 mM sodium citrate, pH 6.5/150 mM NaCl/100 mM L-arginine/100 mM L- glutamic acid/2% ethanolamine). A diafiltration step is performed in a Millipore Stir Cell using a membrane with a 30,000 MWCO (Millipore, YM30 Regenerated Cellulose) at room temperature. Three volumes of dialysis buffer A (25 mM sodium citrate, pH 6.5/150 mM NaCl/50 mM L-arginine/50 mM L-glutamic acid) is added to the quenched mixture, and the mixture is concentrated back to the original volume (i.e., original volume of quenched mixture). A repeat of the diafiltration with dialysis buffer A is performed. A second diafiltration step is performed by adding three volumes of dialysis buffer B (25 mM sodium citrate, pH 6.5/150 mM NaCl) to the mixture, and concentrating back to the original volume (i.e., original volume of quenched mixture). This diafiltration step with dialysis buffer B is repeated four more times. After the final diafiltration, the antibody is concentrated to approximately 1.35 mg/mL. The concentrated antibody is then further concentrated by use of Amicon Ultra-4 Centrifugal Filter Devices (Millipore; Ultracel-30K, UFC803024) in a Fisher accuSpin 3R centrifuge with swinging bucket rotor at 120Og for 5-7 minutes at 15 ⁰ C

(multiple runs) until the concentration is at least 15 mg/mL. The antibody-autophilic peptide conjugate can be stored indefinitely at 4 ⁰ C.

[00465] ELISA - Using anti-epidermal growth factor receptor (EGFR) antibody-autophilic peptide conjugate

[00466] An anti- EGFR antibody is compared to an anti- EGFR antibody-autophilic peptide conjugate in this example. One volume of EGFR Bt-Peptide: Biotin-Gly-Gly-Ala-Ala-Cys- Val-Tφ-Gln-Arg-Trp-Gln-Lys-Ser-Tyr-Val-Cys-Ala-OH (s-s bonded, MW2138.5 g/mole, SEQ ID No. 55) is combined with 41 volumes Avidin and mixed thoroughly. [00467] The mixture is diluted in ELISA Coating Buffer to a final peptide concentration of 0.075 ug/ml. The diluted mixture is used to coat wells of an assay plate using 100 ul per well. The mixture is incubated in the wells at room temperature (RT) for four hours or at 2-8 deg C overnight. The plate is then washed 2X with PBS Tween. Non-specific binding in the wells is blocked by addition of 275 ul 1% BSA in PBS. The plate is incubated at RT for 60 minutes and then washed with 3X with PBS Tween. One hundred microliters of primary antibody is added per well at desired concentration(s). The plate is then incubated at RT for 60 minutes followed by three washes with PBS Tween. One hundred microliters of 1 :25K diluted HRP-labeled anti- Human IgG is added per well and incubated at RT for 60 minutes followed by three washes with PBS Tween. One hundred microliters of TMB Substrate is added per well and incubated at RT for 15 minutes. Fifty microliters of 1.0 N Sulfuric Acid is added to each well to stop the reaction. Results are then read immediately on a plate reader at 450 nm. More sensitive detection of analyte is observed using the anti-EGFR antibody-autophilic peptide conjugate. [00468] Example 31

[00469] Immunofluorescence Assay- Using anti-EGFR antibody-autophilic peptide conjugate

[00470] MDA MB 231 cells (ATCC, HTB-26) were grown in T150 Canted Neck Tissue Culture Flasks (Becton-Dickinson, 355001) in a growth media of RPMI + GlutaMAX (Gibco, 61870) with 10% Fetal Bovine Serum (Gibco, 26140) and 1% Penicillin/Streptomycin (Gibco, 15140) at 37°C/5% CO ₂ to confluence. After removal of the media the cells were rinsed with Phosphate Buffered Saline (Fisher, BP3994, diluted to IX). The cells from each Tl 50 flask were then harvested through trypsinization by the addition of 2.0 mL of 0.25% Trypsin-EDTA (Gibco, 25200) and incubation for 4 minutes at 37°C/5% CO ₂ . The trypsinization was stopped by the addition of 10 mL of the aforementioned growth media to each flask. The trypsinized cells were combined, and then centrifuged at 70Og for 7

minutes. To wash the cells, the supernatant was removed and an equal volume of IX PBS was added. The resuspended cells were centrifuged at 70Og for 7 minutes. The supernatant was again removed, and a second wash of the cells was performed. The washed cells were resuspended in PBS + 2.5% Fetal Bovine Serum to a cell density of approximately 5 million cells per mL.

[00471] An 8% Formaldehyde solution was prepared by the diluting an equal volume of 16% Formaldehyde (Ted Pell Inc., 18505) and MiIIiQ water. While mixing the cells at a low speed on a vortex, an equal volume of 8% Formaldehyde slowly was added. The cells were allowed to fix overnight at 4 ⁰ C. Immediately prior to use, the fixed cells were centrifuged at 800g for 10 min. The supernatant was carefully removed. The excess Formaldehyde was removed by washing the cell pellet with an equal volume of IX PBS and centrifuging at 800g for 10 min. The supernatant was again carefully removed. The cell pellet was resuspended to a concentration of approximately 2 million cells per mL in PBS + 2.5% Fetal Bovine Serum.

[00472] Samples of unmodified anti-EGFR (Bristol-Meyer Squibb, Erbitux) and anti- EGFR autophilic antibody (InNexxus, DXL1218) were diluted in IX PBS so that addition of 10 μL of sample to 200 μL cells created test concentrations of 0.1 to 10 μg per mL. Replicates of the 10 μL test samples were aliquoted to the wells of a U-bottom 96- well plate (Becton-Dickinson, 353910). In addition to the test samples, replicate wells with only 10 μL of IX PBS were aliquoted for use as setup and negative controls. After placement of all samples (i.e., primary antibody or PBS) in the wells, 200 μL of Fixed Cells were added to each well. The cells were incubated with the primary antibodies for 1 hour at room temperature with gentle shaking. The plate was then centrifuged at 800g for 10 min and the supernatant was removed by a quick inversion of the plate. The cells were washed by the addition of 200 μL of IX PBS to each well and subsequent centrifugation at 800g for 10 min. Again the supernatant was removed by quick inversion of the plate.

[00473] As a secondary antibody goat anti-Human IgG (Fab specific) -FITC (Sigma, F5512) was diluted 1 :10,000 in IX PBS + 2.5% Fetal Bovine Serum and 200 μL of this was added to each test well. As a negative control, half of the wells without any primary antibody also had the diluted secondary antibody added to them. The remaining wells without any primary antibody had IX PBS + 2.5% Fetal Bovine Serum added to them for use in setting up the flow cytometer parameters. The plate was incubated with the secondary antibody for 1 hour at room temperature with gentle shaking. The plate was then centrifuged at 800g for 10

min and the supernatant was removed by a quick inversion of the plate. The cells were washed by the addition of 200 μL of IX PBS to each well and subsequent centrifugation at 800g for 10 min. Again the supernatant was removed by quick inversion of the plate. A repeat of the cell wash was performed. Each well then had 200 μL of IX PBS added to it. The plate contained the following samples: (1) No Primary, No Secondary - used for flow cytometry setup, (2) No Primary, With Secondary - to show the level of non-specific binding that the secondary antibody has with the fixed cells and (3) Test Samples - tested each antibody at specified concentrations.

[00474] The 96-well plate was placed into a FACS Canto II Flow Cytometer (Becton- Dickinson, 338960) with a High Throughput Sampler (Becton-Dickinson, 640009). The no primary, no secondary sample was used to setup the following parameters (FSC, SSC and FITC fluorescent channel). A minimum of 20,000 events were recorded for each of the remaining samples and the Mean Fluorescent Intensity (MFI) was calculated using FACSDiva software. More sensitive detection of analyte is observed using the anti-EGFR antibody-autophilic peptide conjugate.

[00475] A similar immunofluorescence assay is performed using SW-480 colon cancer cells to compare anti-EGFR antibody and anti-EGFR antibody-autophilic peptide conjugate (also called DXL1218) Figure 39 is a graph showing more sensitive detection of EGFR analyte using the anti-EGFR antibody-autophilic peptide conjugate. [00476] Example 32

[00477] ELISA - Using anti-Staphlococcal clumping factor protein antibody-autophilic peptide conjugate

[00478] An anti-Staphlococcal clumping factor protein antibody is compared to an anti- Staphlococcal clumping factor protein antibody-autophilic peptide conjugate in this example. The Tl 5 peptide of SEQ ID No. 14 is crosslinked to the antibody by UV irradiation for a total energy of 1200 microjoules per centimeter squared at 254nm. The autophilic anti-Staphlococcal clumping protein antibody is by expressing in CHO cells. The recombinant clumping factor protein, expressed in E. coli, is diluted to one microgram per milliliter. The diluted mixture is used to coat wells of an assay plate using 100 ul per well. The mixture is incubated in the wells at room temperature (RT) for four hours or at 2-8 deg C overnight. The plate is then washed 2X with PBS Tween. Non-specific binding in the wells is blocked by addition of 275 ul 1% BSA in PBS. The plate is incubated at RT for 60 minutes and then washed with 3X with PBS Tween. One hundred microliters of primary antibody is added per well at desired

concentration(s). The plate is then incubated at RT for 60 minutes followed by three washes with PBS Tween. One hundred microliters of 1:25 K diluted HRP-labeled anti-Human IgG is added per well and incubated at RT for 60 minutes followed by three washes with PBS Tween. One hundred microliters of TMB Substrate is added per well and incubated at RT for 15 minutes.

[00479] Fifty microliters of 1.0 N Sulfuric Acid is added to each well to stop the reaction. Results are then read immediately on a plate reader at 450 nm. Figure 35 is a graph showing increased sensitivity of an assay using autophilic peptide conjugated anti-Staphlococcal clumping factor protein antibody in contrast to the same antibody without the autophilic peptide.

[00480] Example 33

[00481] ELISA - Using anti-CD32B antibody- autophilic peptide conjugate [00482] Anti-CD32B antibodies are compared to anti-CD32B antibody-autophilic peptide conjugates in this example. The Tl 5 peptide + spacer of SEQ ID No. 53 is crosslinked to recombinantly produced chimeric anti-CD32B antibody or recombinantly produced humanized anti-CD32B antibody by UV irradiation for a total energy of 1200 micojoules per centimeter squared at 254nm. Recombinant 32B protein, expressed in E. coli, is diluted to one microgram per milliliter. The mixture is diluted in ELISA Coating Buffer to a final peptide concentration of 0.075 ug/ml. The diluted mixture is used to coat wells of an assay plate using 100 ul per well. The mixture is incubated in the wells at room temperature (RT) for four hours or at 2-8 deg C overnight. The plate is then washed 2X with PBS Tween. Non-specific binding in the wells is blocked by addition of 275 ul 1% BSA in PBS. The plate is incubated at RT for 60 minutes and then washed with 3X with PBS Tween. One hundred microliters of primary antibody is added per well at desired concentration(s). The plate is then incubated at RT for 60 minutes followed by three washes with PBS Tween. One hundred microliters of 1:25K diluted HRP-labeled anti-Human IgG is added per well and incubated at RT for 60 minutes followed by three washes with PBS Tween. One hundred microliters of TMB Substrate is added per well and incubated at RT for 15 minutes. Fifty microliters of 1.0 N Sulfuric Acid is added to each well to stop the reaction. Results are then read immediately on a plate reader at 450 nm. Sensitivity of an assay using autophilic peptide conjugated anti-CD32B antibody is increased in contrast to the same antibody without the autophilic peptide. Figure 38 is a graph showing results of an ELISA for analyte CD32B binding assay using A, chimeric 2B6 antibody and autophilic peptide conjugate; B, humanized 2B6 antibody and autophilic peptide conjugate.

[00483] Example 34

[00484] Immunofluorescence Assay- Using anti-CD32B antibody-autophilic peptide conjugate

[00485] Ramos cells (ATCC) are washed with IX PBS. To 300,000 cells, varying amounts of primary antibody are incubated on ice for 30 minutes. After washing the cells, one hundred microliters of 1:5000 dilution of goat anti-human IgG labeled with FITC are incubated for thirty minutes on ice. After washing, the cells are analyzed by FACS and the mean fluorescence determined. Sensitivity of the assay using autophilic peptide conjugated anti-

CD32B antibody is increased in contrast to the same antibody without the autophilic peptide.

[00486] Example 35

[00487] ELISA - Using anti-GM2 antibody-autophilic peptide conjugate

[00488] An anti-GM2 antibody is compared to an anti-GM2 antibody-autophilic peptide conjugate in this example. The Tl 5 peptide + spacer of SEQ ID No. 53 is crosslinked to the antibody by UV irradiation for a total energy of 1200 micojoules per centimeter squared at

254nm. GM2 gangliosides (Sigma Chemical) are diluted in methanol to 5 ng/ml. The diluted mixture is used to coat assay plate using lOOul per well allowed to dry over night.

Nonspecific binding is blocked by adding 250μl of 1% BSA into each well for 2 hours at room temperature. The contents are decanted and one hundred microliters of the primary antibody diluted in 1% BSA, are added and at the desired concentration (s). The plate is incubated for 1 hour followed by three washes of PBST. One hundred microliters of 1 :1000 diluted HRP labeled anti-human IgG is added per well and incubated at room temperature for

60 minutes. After washing three times, one hundred microliters per well of TMB substrate is added per well and incubated for 15 minutes. Fifty microliters of IN sulfuric acid is added to each well to stop the reaction. Results are read immediately on a plate reader at 450nm.

Sensitivity of the assay using autophilic peptide conjugated anti-GM2 antibody is increased in contrast to the same antibody without the autophilic peptide.

[00489] Example 37

[00490] ELISA - Using anti-glycolic GM3 antibody-autophilic peptide conjugate

[00491] An anti-glycolic GM3 is compared to an anti-glycolyl GM3 antibody-autophilic peptide conjugate in this example. The Tl 5 peptide + spacer of SEQ ID No. 53 is crosslinked to the antibody by UV irradiation for a total energy of 1200 micojoules per centimeter squared at 254nm. The glycolyl GM3 gangliosides were purchased from Sigma. Glycolic GM3 gangliosides are diluted in methanol to 5 ng/ml. The glycolyl GM3 gangliosides were

purchased from Sigma. The diluted mixture is used to coat assay plate using lOOul per well allowed to dry over night. Nonspecific binding is blocked by adding 250μl of 1% BSA into each well for 2 hours at room temperature. The contents are decanted and one hundred microliters of the primary antibody diluted in 1% BSA, are added and at the desired concentration (s). The plate is incubated for 1 hour followed by three washes of PBST. One hundred microliters of 1: 1000 diluted HRP labeled anti-human IgG is added per well and incubated at room temperature for 60 minutes. After washing three times, one hundred microliters per well of TMB substrate is added per well and incubated for 15 minutes. Fifty microliters of IN sulfuric acid is added to each well to stop the reaction. Results are read immediately on a plate reader at 450nm. Figure 36 is a graph showing increased sensitivity of an assay using autophilic peptide conjugated anti-glycolic GM3 antibody in contrast to the same antibody without the autophilic peptide. [00492] Example 38

[00493] Immunofluorescence Assay- Using anti-glycolic GM3 antibody-autophilic peptide conjugate

[00494] P3 X63 myeloma cells are washed with IX PBS. To 300,000 cells, varying amounts of primary antibody, anti-glycolic GM3 or anti-glycolyl GM3 antibody-autophilic peptide conjugate, are incubated on ice for 30 minutes. After washing the cells, one hundred microliters of 1:5000 dilution of goat anti-human IgG labeled with FTTC are incubated for thirty minutes on ice. After washing, the cells are analyzed by FACS and the mean fluorescence determined. Sensitivity of the assay using autophilic peptide conjugated anti- glycolyl GM3 antibody is increased in contrast to the same antibody without the autophilic peptide.

[00495] Example 39

[00496] Immunofluorescence Assay- Using anti-HLADRl antibody-autophilic peptide conjugate

[00497] Raji cells are washed with IX PBS. To 300,000 cells, varying amounts of primary antibody, anti-HLADRl or anti-HLADRl antibody-autophilic peptide conjugate, are incubated on ice for 30 minutes. After washing the cells, one hundred microliters of 1:5000 dilution of goat anti-human IgG labeled with FITC are incubated for thirty minutes on ice. After washing, the cells are analyzed by FACS and the mean fluorescence determined. Sensitivity of the assay using autophilic peptide conjugated anti-HLADRl antibody is increased in contrast to the same antibody without the autophilic peptide.

[00498] Example 40

[00499] Immunofluorescence Assay- Using anti-CD19 antibody-autophilic peptide conjugate

[00500] Raji or Jokl cells are washed with IX PBS. To 300,000 cells, varying amounts of primary antibody, anti-CD19 or anti-CD19 antibody-autophilic peptide conjugate, are incubated on ice for 30 minutes. After washing the cells, one hundred microliters of 1:5000 dilution of goat anti-human IgG labeled with FITC are incubated for thirty minutes on ice.

After washing, the cells are analyzed by FACS and the mean fluorescence determined.

Sensitivity of the assay using autophilic peptide conjugated anti-CD19 antibody is increased in contrast to the same antibody without the autophilic peptide.

[00501] Example 41

[00502] Immunofluorescence Assay- Using anti-EpCAM antibody-autophilic peptide conjugate

[00503] Jokl cells are washed with IX PBS. To 300,000 cells, varying amounts of primary antibody, anti-EpCAM or anti-EpCAM antibody-autophilic peptide conjugate, are incubated on ice for 30 minutes. After washing the cells, one hundred microliters of 1:5000 dilution of goat anti-human IgG labeled with FTTC are incubated for thirty minutes on ice. After washing, the cells are analyzed by FACS and the mean fluorescence determined. Sensitivity of the assay using autophilic peptide conjugated anti-EpCAM antibody is increased in contrast to the same antibody without the autophilic peptide.

[00504] Example 42

[00505] Electrochemical Antigen Detection Assay - anti-prostate specific antigen (PSA) antibody-autophilic peptide conjugate

[00506] The Tl 5 peptide + the four amino acid spacer of SEQ ID No. 53 is crosslinked to the anti-PSA antibody by UV irradiation for a total energy of 1200 micojoules per centimeter squared at 254nm.

[00507] A microtiter plate is coated with prostate specific antigen beads for 1 hour at room temperature. The beads are washed with BV clean (BioVeris). The beads are rehydrated in

BV diluent (BioVeris). A volume of 50-10OuI of anti-prostate specific antigen antibody or autophilic anti-prostate specific antigen antibody is added to test wells as primary antibody

(1-lOug/ml). The mixture is incubated for 1 hour. The plate is washed with BV diluent

(BioVeris) and then detection antibody is added diluted in BV-diluent (BioVeris). The mixture is incubated for 1 hour to overnight and then BV-GIo buffer(BioVeris) is added to

wells and the plate is read on fluidics plate reader (BioVeris). Figure 37 is a graph showing increased sensitivity of an assay using autophilic peptide conjugated anti-prostate specific antigen antibody in contrast to the same antibody without the autophilic peptide. Any patents or publications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication is specifically and individually indicated to be incorporated by reference. U.S. Patent Application No. 60/937,023; U.S. Provisional Patent Application Serial No. 61/031,545, filed February 26, 2008; U.S. Patent Application Serial No. 11/912,992, filed October 29, 2007, Patent Cooperation Treaty No. PCT/US2006/016844, filed April 29, 2006, U.S. Patent Application Serial No. 09/865,281, filed May 29, 2001, U.S. Patent No. 6,238,667; U.S. Patent Application Serial No. 11/119,404, filed April 29, 2005; U.S. Patent Application Serial No. 10/652,864, filed August 29, 2003; U.S. Provisional Patent Application Serial No. 60/407,421, filed August 30, 2002; U.S. Patent Application Serial No. 12/144,361, filed June 23, 2008, are all incorporated herein by reference in their entirety.

[00508] The compositions and methods described herein are presently representative of preferred embodiments, exemplary, and not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art. Such changes and other uses can be made without departing from the scope of the invention as set forth in the claims.

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