Methods and compositions for treating metastatic breast cancer and other cancers in the brain

Abstract

To provide methods for treatment of metastatic dissemination of human breast cancer into the CNS.SOLUTION: A composition comprising at least one AAV vector formulated for central nervous system delivery is described. The composition comprises at least one expression cassette which contains sequences encoding an anti-neoplastic immunoglobulin construct for delivery to the brain operably linked to expression control sequences therefor, and a pharmaceutically acceptable carrier. The anti-neoplastic immunoglobulin construct may be an immunoglobulin modified to have decreased or no measurable affinity for neonatal Fc receptor (FcRn). Also provided are methods of using these constructs in preparing pharmaceutical compositions and uses thereof in anti-neoplastic regimens, particularly for primary and / or metastatic cancers of the brain.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [Incorporation by reference of material submitted in electronic form] Applicants hereby incorporate by reference the sequence listing material submitted in electronic form. This file is designated "14-7028PCT_ST25.txt". [Background technology]

[0002] Brain metastasis is a common and devastating sequela of breast cancer for which treatment options are few and inadequate. Six to 16% of breast cancer patients develop central nervous system (CNS) metastases. These patients have a median 1-year and 5-year survival rate of 20% and 1.3%, respectively, from the time of diagnosis. (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4). Surgical resection of brain metastases is often infeasible, and chemotherapeutic agents are largely excluded from the CNS by the blood-brain barrier (BBB) ​​[Non-Patent Document 5]. Alternative therapies for treating breast cancer brain metastases are needed.

[0003] Breast cancers that overexpress the HER2 receptor tyrosine kinase have a high tendency to metastasize to the CNS and account for 25–30% of all breast cancer cases [Non-Patent Document 6]. Trastuzumab (Herceptin®) is a first-line therapeutic immunoglobulin G (IgG) monoclonal antibody (mAb) directed against HER2; this antibody has been reported to significantly improve survival in patients with HER2-positive disease [Non-Patent Document 7; Non-Patent Document 8]. However, because mAbs do not cross the BBB, patients who benefit from trastuzumab often experience concurrent progression of CNS disease [Non-Patent Document 5, cited above]. Direct infusion of trastuzumab into the CNS has been found to be safe, and intrathecal administration of trastuzumab to patients with meningeal carcinomatosis has been reported to increase overall survival from 2 to 13.5 months [Non-Patent Document 9]. Leptomeningeal carcinomatosis is associated with a compromised rather than intact blood-brain barrier. Non-patent literature 10 reports that focused ultrasound bursts combined with circulating microbubbles can temporarily permeabilize both the blood-brain barrier and the blood-tumor barrier to trastuzumab.

[0004] While current therapies have led to improved control of systemic disease, treatment of metastatic dissemination of human breast cancer to the CNS remains a major therapeutic challenge. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] DiStefano A et al. Cancer.1979;44:1913-1918 [Non-patent document 2] Takakura K et al. Metastatic tumors of the central nervous system. Tokyo: Igaku-Shoin, 1982 [Non-patent document 3] Hall WA et al. Long-term survival with metastatic cancer to the brain.Med Oncol.2000 Nov;17(4):279-86 [Non-patent document 4] Pienkowski T, Zielinski CC.Trastuzumab treatment in patients with breast cancer and metastatic CNS disease.Ann Oncol.2010 May;21(5):917-24 [Non-patent document 5] Nakayama A et al., Antitumor Activity of TAK-285,an Investigational,Non-Pgp Substrate HER2 / EGFR Kinase Inhibitor,in Cultured Tumor Cells,Mouse and Rat Xenograft Tumors,and in an HER2-Positive Brain Metastasis Model, J Cancer.2013 Aug 16;4(7) [Non-patent document 6] Bendell JC et al. Central nervous system metastases in women who receive trastuzumab-based therapy for metastatic breast carcinoma.Cancer.2003 Jun 15;97(12):2972-7 [Non-Patent Document 7] Lin NU et al. Brain metastases:the HER2 paradigm.Clin Cancer Res.2007 Mar 15;13(6):1648-55 [Non-patent document 8] Palmieri D et al. Her-2 overexpression increases the metastatic outgrowth of breast cancer cells in the brain.Cancer Res.2007 May 1;67(9):4190-8 [Non-Patent Document 9] Zagouri F et al., Intrathecal administration of trastuzumab for the treatment of meningeal carcinomatosis in HER2-positive metastatic breast cancer: a systematic review and pooled analysis.Breast Cancer Res Treat.2013 May;139(1):13-22 [Non-Patent Document 10] Park, EJ et al., J Controlled Release, 163(2012), 277-284 Summary of the Invention

[0006] Anti-cancer compositions are provided comprising at least one AAV vector formulated for delivery to the central nervous system, wherein the composition comprises at least one expression cassette containing a sequence encoding an anti-cancer immunoglobulin product for delivery to the CNS operably linked to an expression control sequence therefor, and a pharmaceutically acceptable carrier. In one example, the anti-cancer immunoglobulin construct comprises an immunoglobulin that has been modified to have reduced or no measurable affinity for fetal Fc receptor (FcRn). Suitably, the composition is effective for use in slowing tumor growth in the brain and / or reducing tumor size and / or increasing progression-free survival in subjects.

[0007] In one aspect, the compositions provided herein comprise an AAV viral vector having an AAV9 capsid and having packaged therein an expression cassette encoding an anti-Her2 IgG antibody, or a functional fragment thereof, comprising an anti-Her2 heavy chain with disrupted binding to FcRn.

[0008] In another aspect, provided is a method of slowing the growth of tumors in the brain, which requires administering a composition as described herein to the central nervous system, e.g., intrathecally, hi one aspect, the composition is administered in the absence of chemical or physical disruption of the blood-brain barrier.

[0009] In yet another aspect, the present invention provides a method of treating a tumor in the brain by administering a composition as described herein to a subject in need thereof.

[0010] In yet another aspect, the present invention provides antineoplastic regimens comprising administering a composition as described herein in combination with an antibody or other biologic, a small molecule antineoplastic agent, radiation and / or a chemotherapeutic agent.

[0011] Still other aspects and advantages of the present invention will be readily apparent from the following detailed description of the invention. [Brief explanation of the drawings]

[0012] [Figure 1] The amino acid sequence of the heavy chain of the trastuzumab polypeptide is provided with the sequence listing numbering above the sequence [SEQ ID NO: 25] and conventional Eu(IMGT) numbering below the sequence. [Figure 2]Figure 1 shows survival curves for mice given 1 x 10 GC ICV AAV9.trastuzumab or AAV9.201IA prophylactically and then implanted with BT474-M1.ffluc tumor cells into the brain 21 days after vector administration. The median survival for the 201IA group (mock-treated) was 66 days, while the median survival for the AAV9.trastuzumab-treated group was 99 days, a 33% increase in survival.

[0013] [Detailed Description of the Invention] The compositions and regimens described herein are useful for delivering anti-cancer immunoglobulin constructs to the central nervous system. The compositions described herein comprising AAV-Ig are well suited to cancers (tumors) of the central nervous system (CNS), and specifically those located in the brain.

[0014] As used herein, the term "CNS tumor" includes primary or metastatic cancers that may be located in the brain (intracranial), meninges (the connective tissue layers covering the brain and spinal cord), or spinal cord. Examples of primary CNS cancers may be, among others, gliomas (which may include glioblastomas (also known as glioblastoma multiforme), astrocytomas, oligodendrogliomas, and ependymomas, as well as mixed gliomas), meningiomas, medulloblastomas, neuromas, and primary CNS lymphomas (in the brain, spinal cord, or meninges). Examples of metastatic cancers include those that originate from another tissue or organ, such as the breast, lung, lymphoma, leukemia, melanoma (skin cancer), colon, kidney, prostate, or other types that metastasize to the brain.

[0015] As used herein, an "anti-neoplastic" immunoglobulin construct (including an antibody or antibody fragment, as defined herein) encodes a polypeptide-based moiety that binds to a cell surface antigen or receptor located on cancer cells or solid tumors and inhibits or prevents the growth and spread of malignant cells in tumors or non-solid tumors, and potentially reduces tumor size. Anti-neoplastic immunoglobulin polypeptides can function by multiple mechanisms, such as inhibiting growth factor receptors, cross-linking cell membrane antigens to deliver signals that regulate the cell cycle, inhibiting angiogenesis, inhibiting DNA repair after chemotherapy, or even inhibiting tumor cell growth by inducing cell death. Alternatively, they can indirectly affect tumor growth by activating host immune effector functions, such as antibody-dependent and complement-mediated cytotoxicity. In one embodiment, the anti-neoplastic effects of the compositions and regimens described herein can be assessed by a reduction in tumor size and / or increased progression-free survival compared to subjects not treated or treated with other regimens.

[0016] The term "immunoglobulin" is used herein to encompass antibodies, functional fragments thereof, and immunoadhesins. Antibodies include, for example, polyclonal antibodies, monoclonal antibodies, camelized single domain antibodies, intracellular antibodies ("intrabodies"), and the like. aobodies), recombinant antibodies, multispecific antibodies, Fv, Fab, F(ab)2, F( Antibody fragments such as F(ab')3, Fab', Fab'-SH, F(ab')2, single-chain variable fragment antibodies (scFv), tandem / bis-scFv, Fc, pFc', scFvFc (or scFv-Fc), disulfide Fv (dsfv), bispecific antibodies (bc-scFv) such as BiTE antibodies; camelid antibodies, resurfaced antibodies, humanized antibodies, fully human antibodies, single domain antibodies (sdAb, also known as NANOBORY®), chimeric antibodies, chimeric antibodies comprising at least one human constant region, etc. "Antibody fragment" refers to at least a portion of the variable region of an immunoglobulin that binds to its target, e.g., a tumor cell.

[0017] The term "heterologous" when used with respect to a protein or nucleic acid indicates that the protein or nucleic acid comprises two or more sequences or subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, with two or more sequences from unrelated genes arranged to create a new functional nucleic acid. For example, in one embodiment, a nucleic acid has a promoter from one gene arranged to direct expression of a coding sequence from a different gene. Thus, the promoter is heterologous with respect to the coding sequence.

[0018] As used herein, "expression cassette" refers to a nucleic acid molecule comprising an immunoglobulin gene(s) (e.g., an immunoglobulin variable region, an immunoglobulin constant region, a full-length light chain, a full-length heavy chain, or another fragment of an immunoglobulin construct), a promoter, and possibly other control sequences therefor, which can be delivered to a packaging host cell via a genetic element (e.g., a plasmid) and packaged into a viral vector capsid (e.g., a viral particle). Typically, such expression cassettes for generating viral vectors contain the immunoglobulin sequences described herein flanked by packaging signals of the viral genome and other expression control sequences, such as those described herein.

[0019] As used above, the term "about," when used to modify a numerical value, means a variation of ±10% unless otherwise specified.

[0020] As used throughout this specification and claims, the terms "comprise" and "contain," and variations thereof, including "comprises," "comprising," "contains," and "containing," among others, are inclusive of other components, elements, integers, steps, etc. The terms "consist of" or "consisting of" exclude other components, elements, integers, steps, etc.

[0021] For expression from AAV vectors, the amino acid sequence for the antineoplastic immunoglobulin construct is selected from published, commercially available, and coding sequences described herein. The antineoplastic immunoglobulins described herein may target human epidermal growth factor receptors (HERs), such as HER2. One example of trastuzumab is recombinant IgG1κ, a humanized monoclonal antibody that selectively binds with high affinity (Kd=5 nM) to the extracellular domain of the human epidermal growth factor receptor protein in cell-based assays. Commercially available products are produced in CHO cell culture. For example, http: / / www.drugbank.ca / drugs / DB00072 The amino acid sequences of trastuzumab light chains 1 and 2 and heavy chains 1 and 2, as well as the sequence obtained from an X-ray structural study of trastuzumab, are available under the accession number DB 00072, which sequence is incorporated herein by reference. See also 212-Pb-TCMC-trastuzumab [Areva Med, Bethesda, MD]. Other antibodies of interest include, for example, pertuzumab, a recombinant humanized monoclonal antibody targeting the extracellular dimerization domain (subdomain II) of the human epidermal growth factor receptor 2 protein (HER2). It consists of two heavy chains and two light chains, each with 448 and 214 residues. FDA approved June 8, 2012. The amino acid sequences of the heavy and light chains are, for example, www.drugbank.ca / drugs / DB06366 (Synonyms include 2C4, MOAB 2C4, monoclonal antibody 2C4, and rhuMAb-2C4) are provided in this database under accession number DB06366. In addition to HER2, other HER targets may be selected.

[0022] For example, MM-121 / SAR256212 is a fully human monoclonal antibody that targets the HER3 receptor [Merrimack's Network Biology] and has been reported to be useful in the treatment of non-small cell lung cancer (NSCLC), breast cancer, and ovarian cancer. SAR256212 is an investigational fully human monoclonal antibody that targets the HER3 (ErbB3) receptor [Sanofi Oncology]. Another anti-Her3 / EGFR antibody is RG7597 [Genentech], which has been described as useful in head and neck cancer. Another antibody, margetuximab (or MGAH22), a next-generation Fc-optimized monoclonal antibody (mAb) that targets HER [MacroGenics], is also available.

[0023] Alternatively, other human epithelial cell surface markers and / or other tumor receptors or antigens may be targeted. Examples of other cell surface marker targets include, for example, 5T4, CA-125, CEA (e.g., targeted by labetuzumab), CD3, CD19, CD20 (e.g., targeted by rituximab), CD22 (e.g., targeted by epratuzumab or veltuzumab), CD30, CD33, CD40, CD44, CD51 (also integrin α vβ3), CD133 (e.g., glioblastoma cells), CTLA-4 (e.g., ipilimumab, used in the treatment of neuroblastoma), chemokine (C-X-C motif) receptor 2 (CXCR2) (expressed in various regions in the brain; e.g., anti-CXCR2 (extracellular) antibody #ACR-012 (Alomene Labs)); EpCAM, fibroblast activation protein (FAP) [see, e.g., WO2012020006A2, brain cancer], folate receptor alpha (e.g., pediatric ependymal brain tumors, head and neck cancer), fibroblast growth factor receptor 1 (FGFR1) (discussion on cancer treatment with anti-FGFR1 antibodies) For treatment, see WO2012125124A1), FGFR2 (see, e.g., the antibodies described in WO2013076186A and WO2011143318A2), FGFR3 (see, e.g., the antibodies described in US8187601 and WO2010111367A1), FGFR4 (see, e.g., the anti-FGFR4 antibodies described in WO2012138975A1), hepatocyte growth factor (HGF) (see, e.g., the antibodies described in Antibodies include those targeted by the antibody glembatumumab (CR011), integrin α5β1, IGF-1 receptor, ganglioside GD2 (see, for example, the antibodies described in WO2011160119A2), ganglioside GD3, transmembrane glycoprotein NMB (GPNMB) (associated with gliomas, among others, and the target of the antibody glembatumumab (CR011)), mucins, MUC1, phosphatidylserine (targeted, for example, by bavituximab, Peregrine Fc1), and phospholipase A (targeted, for example, by bavituximab). Pharmaceuticals, Inc.], prostate cancer cells, PD-L1 (e.g., nivolumab (BMS-936558, MDX-1106, ONO-4538), fully human gG4, e.g., metastatic melanoma], platelet-derived growth factor receptor alpha (PDGFRα) or CD140, tumor-associated glycoprotein 72 (TAG-72), tenascin-C, These include tumor necrosis factor (TNF) receptor (TRAIL-R2), vascular endothelial growth factor (VEGF) A (e.g., targeted by bevacizumab), and VEGFR2 (e.g., targeted by ramucirumab). Other antibodies and their targets include, for example, monoclonal antibody APN301 (hu14.19-IL2) [pediatric melanoma and neuroblastoma, Apeiron Biologics, Vienna, Austria]. See also, for example, monoclonal antibody 8H9, which has been described as useful for treating solid tumors, including metastatic breast cancer. Monoclonal antibody 8H9 is a murine IgG1 antibody with specificity for the B7H3 antigen [United Therapeutics Corporation]. This murine antibody can be humanized. Still other immunoglobulin constructs targeting the B7-H3 and / or B7-H4 antigens may be used in the present invention. Another antibody is S58 (anti-GD2, neuroblastoma). Cotara TM [Perregrince Pharmaceuticals] is a monoclonal antibody described for the treatment of recurrent glioblastoma. Other antibodies may include, for example, Avastin, ficlatuzumab, medi-575, and olaratumab. Still other immunoglobulin constructs or monoclonal antibodies may be selected for use in the present invention. See, for example, Medicines in Development Biologics, 2013 Report, pp. 1-87, a publication of PhRMA's Communications & Public Affairs Department. (202) 835-3460 (incorporated herein by reference).

[0024] Once a target and immunoglobulin have been selected, the coding sequence of the selected immunoglobulin (e.g., heavy and / or light chain(s)) can be obtained and / or synthesized. Methods for sequencing proteins, peptides, or polypeptides (such as immunoglobulins) are known to those skilled in the art. Once the sequence of a protein is known, there are web-based and commercially available computer programs and service-based companies that will back-translate the amino acid sequence into a nucleic acid coding sequence. For example, backtranseq by EMBOSS, http: / / www.ebi.ac.uk / Tools / st / ;Gene Infinity( http: / / www.geneinfinity.org / sms / sms_backtranslation.html );ExPasy( http: / / www.expasy.org / tools / ) In one embodiment, the RNA and / or cDNA coding sequences are designed for optimal expression in human cells.

[0025] Codon-optimized coding regions can be designed by a variety of different methods. This optimization can be performed using methods available online (e.g., GeneArt), published methods, or companies that provide codon optimization services, such as DNA2.0 (Menlo Park, CA). One codon optimization algorithm is described, for example, in U.S. International Patent Publication No. WO2015 / 012924, which is incorporated herein by reference. See also, for example, U.S. Patent Publication Nos. 2014 / 0032186 and 2006 / 0136184. Suitably, the entire length of the product's open reading frame (ORF) is modified. However, in some embodiments, only a fragment of the ORF may be altered. Using one of these methods, the frequencies can be applied to any given polypeptide sequence and a nucleic acid fragment of a codon-optimized coding region encoding the polypeptide can be produced.

[0026] Numerous options are available for making the actual changes to the codons or for synthesizing the codon-optimized coding regions designed as described herein. Such modifications or synthesis can be carried out using standard and routine molecular biological procedures known to those skilled in the art. In one approach, a series of complementary oligonucleotide pairs, each 80-90 nucleotides in length and spanning the length of the desired sequence, are synthesized by standard methods. These oligonucleotide pairs are synthesized such that, upon annealing, they form 80-90 base pair double-stranded fragments containing cohesive ends, e.g., each oligonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond the region complementary to the other oligonucleotide in the pair. The single-stranded end of each pair of oligonucleotides is designed to anneal with the single-stranded end of another pair of oligonucleotides. The oligonucleotide pairs are annealed, and approximately five to six of these double-stranded fragments are then annealed together via the cohesive single-stranded ends, and then ligated together and cloned into a standard bacterial cloning vector, e.g., the TOPO® vector available from Invitrogen Corporation, Carlsbad, Calif. The constructs are then sequenced by standard methods. Some of these constructs are made up of five or six 80-90 base pair fragments ligated together, i.e., approximately 500 base pair fragments, so that the entire desired sequence is represented in a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and ligated together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector and sequenced. Additional methods will be readily apparent to those skilled in the art. In addition, gene synthesis is readily available commercially.

[0027] The immunoglobulin genes described herein may be used to express "wild-type" i.e., published, commercially available, or other known constant immunoglobulin domains, or may be engineered to reduce or eliminate affinity for binding to Fc binding sites present on immunoglobulins. There are several different types of Fc receptors, classified based on the type of antibody they recognize. As used herein, "FcRn" refers to the fetal Fc receptor that binds IgG. It is similar in structure to MHC class I proteins. In humans, it is encoded by the FCGRT gene. The Fc receptor is located on a variety of cell types, including, for example, epithelial cells of the blood-brain barrier. As used herein, the term "FcRn-binding domain" refers to a protein domain that directly or indirectly binds to FcRn. The FcRn can be a mammalian FcRn. In a further embodiment, the FcRn is a human FcRn. The FcRn-binding domain that directly binds to FcRn is an antibody Fc region. At the same time, a region capable of binding to a polypeptide such as albumin or IgG having human FcRn-binding activity can indirectly bind to human FcRn via albumin, IgG, or the like. Thus, such a human FcRn-binding region can be a region that binds to a polypeptide having human FcRn-binding activity. As used herein, the term "Fc region" refers to an FcRn-binding domain that directly binds to FcRn, i.e., mammalian FcRn or human FcRn. Specifically, the Fc region is the Fc region of an antibody. The Fc region can be a mammalian Fc region or, more specifically, a human Fc region. Specifically, the Fc region can be located within the second and third constant domains (CH2 and CH3) of a human immunoglobulin. Furthermore, the Fc region can be the hinge between CH2 and CH3. In one embodiment, the immunoglobulin construct is an IgG. In a further embodiment, the Fc region is the Fc region of a human IgG1. Other Ig isotypes can be used as well.

[0028] Because these binding domains are located within the constant region of the IgG heavy chain (regions CH2 and CH3), the amino acid positions provided herein for modification in trastuzumab can be readily determined by preparing an alignment with another immunoglobulin heavy chain selected for modification to identify the corresponding amino acid numbers. Methods and computer programs for preparing such alignments are available and known to those skilled in the art. The amino acid positions are based on the numbering system of trastuzumab as provided in SEQ ID NOS: 3 and 25 (heavy chain) and SEQ ID NOS: 4 (light chain). Substitutions can also be written as (amino acid identified by a single-letter code)-position number-(amino acid identified by a single-letter code), whereby the first amino acid is the substituted amino acid and the second amino acid is the substituting amino acid at the specified position. As used herein, the terms "substitution" and "amino acid substitution" refer to the replacement of one amino acid in an amino acid sequence with another, where the latter is different from the replaced amino acid. Methods of amino acid substitution are known to those skilled in the art and include, but are not limited to, mutations in the nucleotide sequence encoding the amino acid sequence. Methods for making amino acid substitutions in IgG are described, for example, in WO2013 / 046704, which is incorporated by reference for its discussion of amino acid modification techniques, although this document describes increasing FcRn affinity rather than reducing or eliminating binding affinity as described herein.

[0029] The term "amino acid substitution" and its synonyms, as described above, are intended to encompass the alteration of an amino acid sequence by replacing one amino acid with another substituting amino acid. The substitution may be a conservative substitution. The term "conservative" with respect to two amino acids is intended to mean that the amino acids share a common property recognized by those of skill in the art. The term "non-conservative" with respect to two amino acids is intended to mean that the amino acids differ in at least one property recognized by those of skill in the art. For example, such properties may include amino acids with hydrophobic, non-acidic side chains, amino acids with hydrophobic side chains (which may be further distinguished as acidic or non-acidic), amino acids with aliphatic hydrophobic side chains, amino acids with aromatic hydrophobic side chains, amino acids with polar, neutral side chains, amino acids with charged side chains, amino acids with charged, acidic side chains, and amino acids with charged, basic side chains. Both naturally occurring and non-naturally occurring amino acids are known in the art and may be used as substituting amino acids in the embodiments. Thus, a conservative amino acid substitution may require replacing a first amino acid having a hydrophobic side chain with a different amino acid having a hydrophobic side chain; whereas a non-conservative amino acid substitution may require replacing a first amino acid having an acidic, hydrophobic side chain with a different amino acid having a different side chain, e.g., a basic, hydrophobic side chain or a hydrophilic side chain. Still other conservative or non-conservative changes can be determined by one of skill in the art.

[0030] In still other embodiments, the substitution at a given position can be one amino acid or one of a group of amino acids that would be apparent to one of skill in the art to achieve the goals specified herein.

[0031] In one embodiment, an immunoglobulin construct as defined herein is engineered to remove native sequences located in conserved regions of the immunoglobulin Fc region to eliminate binding to FcRn and minimize or eliminate trafficking of the proteinaceous immunoglobulin construct across the blood-brain barrier (from the CNS region) and into the systemic circulation. In one example, this can be achieved by altering one or more amino acids in the FcRn-binding domain, for example by modifying the codon for selected amino acid(s).

[0032] For example, immunoglobulins may be modified to another suitable amino acid, e.g., alanine (Ala) in one or more of the codons encoding the amino acid residues at positions Y436 (aa 459 of SEQ ID NO:25), S254 (aa 277 of SEQ ID NO:25), I253 (aa 276 of SEQ ID NO:25), and / or H435 (aa 458 of SEQ ID NO:25). However, other suitable amino acids may be modified, e.g., T250 (aa 273 of SEQ ID NO:25), M252 (aa 275 of SEQ ID NO:25), S254 (aa 277 of SEQ ID NO:25), T256 (aa 279 of SEQ ID NO:25), P257 (aa 280 of SEQ ID NO:25), P271 (aa 281 of SEQ ID NO:25), P272 (aa 282 of SEQ ID NO:25), P273 (aa 283 of SEQ ID NO:25), P274 (aa 284 of SEQ ID NO:25), P275 (aa 285 of SEQ ID NO:25), P276 (aa 286 of SEQ ID NO:25), P277 (aa 287 of SEQ ID NO:25), P278 (aa 289 of SEQ ID NO:25), P279 (aa 290 of SEQ ID NO:25), P280 (aa 291 of SEQ ID NO:25), P281 (aa 292 of SEQ ID NO:25), P282 (aa 293 of SEQ ID NO:25), P283 (aa 294 of SEQ ID NO:25), P284 (aa Other positions involved in functional binding to FcRn may be mutated, such as one or more of: T307 (aa 330 of SEQ ID NO:25), Q311 (aa 334 of SEQ ID NO:25), D376 (aa 399 of SEQ ID NO:25), E380 (aa 403 of SEQ ID NO:25), M428 (aa 451 of SEQ ID NO:25), and / or N434 (aa 457 of SEQ ID NO:25), or combinations of these with each other or with other modifications described herein. Other suitable modifications include I253 (aa 276 of SEQ ID NO:25), S254 (aa 277 of SEQ ID NO:25), K288 (aa 311 of SEQ ID NO:25), V305 (aa 328 of SEQ ID NO:25), Q311 (aa 334 of SEQ ID NO:25), D312 (aa 335 of SEQ ID NO:25), K317 (aa 340 of SEQ ID NO:340), K360 (aa 383 of SEQ ID NO:25), Q362 (aa 384 of SEQ ID NO:25), and Q363 (aa 385 of SEQ ID NO:25). The IgG heavy chain CH2 and CH3 positions may be located at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 12 In additional embodiments, the term "one or more" encompasses multiple substitutions in a polypeptide described herein that would result in at least about 85% identity, at least 90% identity, at least about 95% identity, or at least about 99% identity to trastuzumab heavy chain variable region SEQ ID NO:3, light chain variable region SEQ ID NO:4, heavy chain SEQ ID NO:25, or another amino acid sequence identified herein.

[0033] Additionally, mutations that enhance complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC) function may be incorporated into the trastuzumab variants described herein. In further embodiments, such mutations facilitate immune cell killing of tumor cells. Examples of suitable amino acid modifications for enhancing ADCC function are described, for example, in U.S. Patent Publication No. 2008 / 0118501; A. Nasume et al., Drug Des. Devel Ther, 2009, 3;7-16, published online September 21, 2009. Described in GA Lazar et al., Proc Natl Acad Sci, vol. 103, no. 11, pp. 4005-4010 (Mar 14, 2006); and GL Moore et al., MAbs, 2010 Mar-Apr; 2(2):181-189.

[0034] The numbering of heavy chain amino acids used herein to identify the position of variants is based on the EU numbering system [IMGT specific numbering, Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969); http: / / www.imgt.org / IMGTScientificChart / -Numbering / Hu_IGHGnber.html] and refers to positions in the FcRn binding domain, specifically the Fc region. In a similar manner, substitutions are indicated, for example, as "EU387R" or "EU440E," where the number shown after "EU" indicates the position of the substitution according to EU numbering, and the letter following the number is the substituted amino acid, indicated by a one-letter code. Other numbering systems are described, for example, in Kabat, EA, TTWu, HM Perry, KS Gottesman, C. Foeler. (1991) Sequences of Proteins of Immunological Interest. No. 91-3242 US Public Health Services, National Institutes of Health, Bethesda).

[0035] In one embodiment, an anti-Her2 antibody is selected for the compositions and methods described herein. In one embodiment, the antibody selected is trastuzumab. The amino acid sequence of trastuzumab is described, for example, in P. Carter et al., Proc Natl Acad Sci., 89:4285-4289 (May 1982). The amino acid sequence of the trastuzumab heavy chain is provided in Figure 1, showing both the sequence listing [SEQ ID NO:25] and the EU numbering system. The amino acid sequence of the trastuzumab heavy chain variable region is shown in [SEQ ID NO:3], and the trastuzumab light chain variable region is provided in the attached sequence listing [SEQ ID NO:4]. To express trastuzumab, novel nucleic acid molecules were designed containing codons selected for optimal expression of the trastuzumab polypeptide in humans. Furthermore, the novel nucleic acid molecules include leader sequences heterologous to each of the heavy and light chains of trastuzumab, encoding an IL-2 signal leader peptide fused upstream of the heavy and light chain polypeptides composed of the variable and constant regions. However, other heterologous leader sequences may be used in place of one or both of the IL-2 signal / leader peptides. The signal / leader peptides can be the same or different for each heavy and light chain immunoglobulin construct. These may be signal sequences naturally found in immunoglobulins (e.g., IgG) or may be from a heterologous source, such as a cytokine (e.g., IL-2, IL12, IL18, etc.), insulin, albumin, β-glucuronidase, alkaline protease, or fibronectin secretory signal peptide, among others. The promoter(s) may be selected from a variety of sources, such as the human cytomegalovirus (CMV) immediate early enhancer / promoter, the SV40 early enhancer / promoter, the JC polymovirus promoter, the myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoter, the herpes simplex virus (HSV-1) latency-associated promoter (LAP), the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, the neuron-specific promoter (NSE), the platelet-derived growth factor (PDGF) promoter, hSYN, the melanin-concentrating hormone (MCH) promoter, CBA, the matrix metalloprotein promoter (MPP), and the chicken β-actin promoter.

[0036] The expression cassettes described herein may contain at least one internal ribosome entry site (IRES) located between the heavy and light chain coding regions. Alternatively, the heavy and light chains may be separated by a furin 2a self-cleaving peptide linker (see, e.g., Radcliffe and Mitrophanous, Gene Therapy (2004), 11, 1673-1674). The expression cassette may contain at least one enhancer, i.e., a CMV enhancer. Still other enhancer elements may include, for example, an apolipoprotein enhancer, a zebrafish enhancer, a GFAP enhancer element, and a brain-specific enhancer such as those described in WO2013 / 1555222, or a woodchuck post-hepatitis post-transcriptional regulatory element. Additionally or alternatively, other promoter-enhancer elements may be selected, such as the hybrid human cytomegalovirus (HCMV)-immediate early (IE)-PDGR promoter or other promoter-enhancer elements. To enhance expression, other elements may be introns (such as the Promega intron or the chimeric chicken globin-human immunoglobulin intron).

[0037] With reference to the numbering of the engineered nucleic acid molecule of SEQ ID NO: 1, as provided herein, the nucleic acid sequence encoding the heavy chain polypeptide of Trastuzumab is characterized by a leader sequence (1-60 of SEQ ID NO: 1), with nucleic acids 61-423 being the coding region for the immunoglobulin heavy chain (HC) variable sequence, nucleic acids 439-714 being the coding region for HC constant region 1, and nucleic acids 715-1410 being the coding region for HC constant regions 2 and 3. An IRES is located at nucleic acids 1422-2012 of SEQ ID NO: 1 between the leader sequence of the Trastuzumab heavy chain and Trastuzumab light chain coding sequences. The variable region of the Trastuzumab light chain variable sequence is nucleotides 2070-2391 of SEQ ID NO: 1; the light chain constant region is located at nucleic acids 2407-2711 of SEQ ID NO: 1.

[0038] Also encompassed herein are nucleic acid sequences encoding trastuzumab immunoglobulin polypeptides (e.g., heavy chains, light chains, or fragments thereof, which fragments can include, for example, complementarity determining region (CDR) 1, 2, and / or 3, constant region (1, 2, or 3) of SEQ ID NO: 1, or a sequence that is at least about 85% identical thereto, at least about 90%, at least about 95% identical thereto, or at least about 99% identical thereto, or SEQ ID NO: 1, or fragments thereof that encode immunoglobulin polypeptides (e.g., heavy chains, light chains, or fragments thereof (e.g., including variable region (e.g., including complementarity determining region (CDR) 1, 2, and / or 3)), constant region (1, 2, or 3))) having the same amino acid sequence as provided herein for trastuzumab without any FcRN modifications, and fragments thereof).

[0039] The terms "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity) over a specified region (e.g., any one of the modified ORFs provided herein when compared and aligned for maximum correspondence over a comparison window or specified region) as assessed using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters, as described below, or by manual alignment and visual inspection (see, e.g., the NCBI website). As another example, polynucleotide sequences can be compared using the program Fasta in GCG version 6.1. Fasta aligns and calculates the percent sequence identity of the region of the best overlap between the query and search sequences. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta with its default parameters (word size of 6 and NOPAM factor for the scoring matrix) as provided in GCG version 6.1 (incorporated herein by reference). Generally, these programs are used with default settings, although one of skill in the art can modify these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program that provides at least a level of identity or alignment like that provided by the referenced algorithms and programs. This definition can also refer to or apply to the complement of a sequence. The definition also encompasses sequences with deletions and / or additions, as well as those with substitutions. As described below, preferred algorithms can account for gaps, etc.Preferably, identity exists over a region that is at least about 25, 50, 75, 100, 150, 200 amino acids or nucleotides in length, and often over a region that is 225, 250, 300, 350, 400, 450, 500 amino acids or nucleotides in length, or over the entire length of the amino acid or nucleic acid sequence.

[0040] Typically, when an alignment is made based on an amino acid sequence, the alignment will contain insertions and deletions identified as such with respect to the reference AAV sequence, and the numbering of the amino acid residues will be based on the reference scale provided for the alignment. However, any given AAV sequence may have fewer amino acid residues than the reference scale. In the present invention, when discussing parent sequences, the terms "same position" or "corresponding position" refer to amino acids located at the same residue number in each of the aligned sequences with respect to the reference scale for the sequences. However, when taken from the alignment, each of the proteins may have these amino acids located at different residue numbers. Alignment can be performed using any of a variety of publicly or commercially available multiple sequence alignment programs. Sequence alignment programs are available for amino acid sequences, such as "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match-Box" programs. Generally, all of these programs are used with default settings, although those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can use another algorithm or computer program that provides at least a level of identity or alignment like that provided by the referenced algorithms and programs. For example, JD Thomson et al. Nucl.Acids.Res. See, "A comprehensive comparison of multiple sequence alignments," 27(13):2682-2690 (1999).

[0041] In another embodiment, a modified anti-Her2 antibody is provided that has altered affinity for FcRn and retains effective anti-cancer activity. One or more amino acid modifications may be selected to eliminate functional binding to FcRn. In one embodiment, the modifications reduce the binding affinity of the trastuzumab immunoglobulin to FcRn to less than 10% of that of the native protein. Suitably, immunoglobulins with these mutations bind substantially normally to all other Fc receptors. For example, the immunoglobulin may have at least one of positions Y436, S254, I253, and / or H435 modified to alanine or another amino acid, or one or more of these modified with each other, or one, two, or more combinations with one or more of the modifications described herein. However, other positions involved in functional binding to FcRn may be mutated, such as, for example, T250 (aa 273 of SEQ ID NO:25), M252 (aa 275 of SEQ ID NO:25), S254 (aa 278 of SEQ ID NO:25), T256 (aa 280 of SEQ ID NO:25), P257 (aa 281 of SEQ ID NO:25), P271 (aa 294 of SEQ ID NO:25), T307 (aa 330 of SEQ ID NO:25), Q311 (aa 334 of SEQ ID NO:25), D376 (aa 399 of SEQ ID NO:25), E380 (aa 403 of SEQ ID NO:25), M428 (aa 451 of SEQ ID NO:25) and / or N434 (aa 457 of SEQ ID NO:25), or a combination of one or more of these with each other or with other modifications described herein.Other suitable modifications include, for example, substitution of I253 (aa 276 of SEQ ID NO:25), S254 (aa 278 of SEQ ID NO:25), K288 (aa 311 of SEQ ID NO:25), V305 (aa 328 of SEQ ID NO:25), Q311 (aa 334 of SEQ ID NO:25), D312 (aa 335 of SEQ ID NO:25), K317 (aa 340 of SEQ ID NO:340), K360 (aa 341 of SEQ ID NO:25), ... a383), Q362 (aa385 of SEQ ID NO:25), E380 (aa403 of SEQ ID NO:25), S415 (aa438 of SEQ ID NO:25), S424 (aa447 of SEQ ID NO:25), H433 (aa456 of SEQ ID NO:25), N434 (aa457 of SEQ ID NO:25), H435 (aa458 of SEQ ID NO:25), and / or Y436 (aa459 of SEQ ID NO:25), or a combination of two or more thereof. Still other mutations may be incorporated. See, e.g., Kuo and Aveson, mAbs, 3:5, 422-430 (Sept / Oct 2011) and Shield, J Biol Chem, 2001, 276:659-6604. Once an amino acid sequence is selected, the nucleic acid sequence can be designed and / or previously described sequences can be manipulated as described above. These modifications are made by manipulating the nucleic acid coding region using site-directed mutagenesis or other genetic engineering techniques known to those skilled in the art.

[0042] Similar modifications may be engineered into another selected anti-HER2 immunoglobulin construct, or alternatively, into another anti-neoplastic immunoglobulin construct as described herein.

[0043] In one embodiment, the immunoglobulin genes described herein are genetic elements (e.g., nucleotides) useful for generating AAV vectors carrying immunoglobulin construct sequences thereon. The selected vector may be delivered to the AAV packaging cell by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Stable packaging cells may also be generated. The methods used to generate such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Molecular Cloning: A Laboratory Manual, edited by Green and Sambrook, Cold Spring Harbor Laboratory Press, 1999. Harbor Press, Cold Spring Harbor, NY (2012).

[0044] AAV vectors As described herein, AAV vectors can each contain one or more nucleic acid sequences encoding one or more heavy and / or light chain polypeptides or other polypeptides of an anti-cancer immunoglobulin construct. Suitably, the composition contains one or more AAV vectors containing all of the polypeptides that form the anti-cancer construct in vivo. For example, a full-length antibody consists of four polypeptides: two identical copies of heavy (H) chain polypeptides and two copies of light (L) chain polypeptides. Each heavy chain contains an N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains an N-terminal variable (VL) region and a C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. In this regard, an AAV vector as described herein can comprise a single nucleic acid sequence encoding two heavy chain polypeptides (e.g., constant, variable) and two light chain polypeptides of an immunoglobulin construct. Alternatively, an AAV vector can comprise a first expression cassette encoding at least one heavy chain constant polypeptide and at least one heavy chain variable polypeptide, and a second expression cassette encoding both light chain polypeptides of an immunoglobulin construct. In yet another embodiment, an AAV vector can comprise a first expression cassette encoding a first heavy chain polypeptide, a second expression cassette encoding a second heavy chain polypeptide, a third expression cassette encoding a first light chain polypeptide, and a fourth expression cassette encoding a second light chain polypeptide.

[0045] Typically, an expression cassette in an AAV vector comprises an AAV 5' inverted repeat translocation (ITR), an immunoglobulin construct coding sequence and any regulatory sequences, and an AAV 3' ITR. However, other configurations of these elements may be suitable. A shortened version of the 5' ITR, termed ΔITR, has been described in which the D sequence and terminal resolution site (trs) are deleted. In other embodiments, the full-length AAV 5' and 3' ITRs are used.

[0046] When pseudotyped AAV is to be produced, the ITRs in expression are selected from a source different from the AAV source of the capsid.For example, AAV2 ITRs can be selected for use with AAV capsids that have a specific efficiency for targeting the CNS or tissues or cells within the CNS.In one embodiment, the ITR sequence from AAV2 or its deleted version (ΔITR) is used for convenience and to facilitate regulatory approval.However, ITRs from other AAV sources can be selected.If the source of ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector can be called pseudotyped.However, AAV Other sources of ITR may be available.

[0047] The abbreviation "sc" refers to self-complementary. "Self-complementary AAV" refers to recombinant AAV nucleic acid We refer to constructs in which the coding region carried by the sequence is designed to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of the scAAV can assemble to form a single double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. See, for example, D. M. McCarty et al., "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis," Gene Therapy, (August 2001), Vol. 8, Number 16, pp. 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683 (each of which is incorporated herein by reference in its entirety).

[0048] Expression cassettes typically contain a promoter sequence as part of the expression control sequence, for example, located between the selected 5' ITR sequence and the immunoglobulin construct coding sequence. Tissue-specific promoters, constitutive promoters, regulatable promoters (see, e.g., WO2011 / 126808 and WO2013 / 04943), or promoters responsive to physiological cues may be utilized in the vectors described herein. In addition to a promoter, the expression cassette and / or vector may contain other appropriate transcription initiation, termination, and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (i.e., Kozak consensus sequences); sequences that increase protein stability; and, if desired, sequences that increase secretion of the encoded product.

[0049] These control sequences are "operably linked" to the immunoglobulin construct gene sequence. As used herein, the term "operably linked" refers to both expression control sequences that are adjacent to a gene of interest and expression control sequences that act in trans or at a distance to regulate the gene of interest.

[0050] In one embodiment, a self-complementary AAV is provided. This viral vector can contain a Δ5' ITR and an AAV 3' ITR. In another embodiment, a single-stranded AAV viral vector is provided. Methods for generating and isolating AAV viral vectors suitable for delivery to a subject are known in the art. See, for example, U.S. Patent No. 7,790,449; U.S. Patent No. 7,282,199; WO2003 / 042397; WO2005 / 033321, WO2006 / 110689; and U.S. Patent No. 7,588,772B2. In one system, a producer cell line is transiently transfected with a construct encoding a transgene flanked by ITRs and a construct(s) encoding rep and cap. In the second system, a packaging cell line stably supplying rep and cap is transiently transfected with a construct encoding a transgene flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpesvirus, necessitating separation of the rAAV from contaminating viruses. More recently, systems have been developed that do not require infection with a helper virus to recover AAV. That is, the required helper functions (i.e., adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also supplied in trans by the system. In these newer systems, helper functions can be supplied by transient transfection of cells with constructs encoding the required helper functions, or cells can be transfected with helper viruses. They can be engineered to stably contain a functionally encoding gene, the expression of which can be controlled at the transcriptional or post-translational level. In yet another system, a transgene flanked by ITRs and rep / cap genes is introduced into insect cells by infection with a baculovirus-based vector. For reviews of these production systems in general, see, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated “viral hybrid for large-scale recombinant adeno-associated virus production”, Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entireties. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of each of which are incorporated herein by reference in their entireties: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065.

[0051] The space available for packaging can be conserved by combining more than one transcription unit into a single expression cassette, thus reducing the amount of regulatory sequences required. For example, a single promoter can direct the expression of a single cDNA or RNA encoding two, three, or more genes, with translation of the downstream gene being driven by an IRES sequence. In another example, a single promoter can direct the expression of a cDNA or RNA containing two, three, or more genes in a single open reading frame (ORF), separated from each other by sequences encoding a self-cleaving peptide (e.g., 2A) and / or a protease recognition site (e.g., furin). The ORF thus encodes a single polyprotein that is cleaved into individual proteins (e.g., heavy and light chains) either during or after translation. However, it should be noted that while these IRES and polyprotein systems can be used to conserve AAV packaging space, they can only be used for the expression of components that can be driven by the same promoter. In another alternative, the transgene capacity of AAV can be increased by providing AAV ITRs from two genomes that can anneal to form head-to-tail concatemers.

[0052] In the following examples, an AAV9 vector is described for expressing trastuzumab directly in the CNS to treat CNS metastases of breast cancer. AAV9 vectors are described, for example, in U.S. Patent No. 7,906,111 (incorporated herein by reference). However, other sources of AAV capsids and other viral elements may be selected, as may other immunoglobulin constructs and other vector elements. Methods for producing AAV vectors have been extensively described in literature and patent documents, including, for example, WO2003 / 042397; WO2005 / 033321; WO2006 / 110689; and U.S. Patent No. 7,588,772B2. The source of AAV capsid may be selected from AAVs that target the CNS, specific cells within the CNS, and / or specific cancer-associated antigens or receptors. Suitable AAVs may include, for example, AAV9 [US 7,906,111; US ​​2011-0236353-A1], rh10 [WO 2003 / 042397], and / or hu37 [see, e.g., US 7,906,111; US ​​2011-0236353-A1]. However, other AAVs may be used herein, including, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 [US 7,790,449; US 7,282,199], and others such as those described in a single word that are considered missing herein. The vectors may be selected to produce the AAV vectors described.

[0053] Uses and Regimen Suitably, the compositions of the invention are designed such that the AAV vector carries an immunoglobulin construct and a nucleic acid expression cassette encoding a regulatory sequence that directs expression of the immunoglobulin in selected cells. After administration of the vector into the CNS, the vector delivers the expression cassette to the CNS and expresses the proteinaceous immunoglobulin construct in vivo. Use of the compositions described herein in anti-neoplastic methods is also described, as is use of these compositions in anti-neoplastic regimens that may optionally require the delivery of one or more other anti-neoplastic or other effective agents.

[0054] As mentioned above, the composition can contain a single type of AAV vector, as described herein, containing an expression cassette for delivering an anti-cancer immunoglobulin construct in vivo. Alternatively, the composition can contain two or more different AAV vectors, each of which packages a different expression cassette therein. For example, the two or more different AAVs can have various expression cassettes expressing immunoglobulin polypeptides that assemble in vivo to form a single functional immunoglobulin construct. In another example, the two or more AAVs can have different expression cassettes expressing immunoglobulin polypeptides for different targets, for example, two of which provide two functional immunoglobulin constructs (e.g., one anti-Her2 immunoglobulin construct and a second anti-cancer immunoglobulin construct). In yet another alternative, the two or more different AAVs can express immunoglobulin constructs directed to the same target, where one of the immunoglobulin constructs has been modified to ablate FcRn binding and a second immunoglobulin construct that retains that ability or has an enhanced ability to bind FcRn. Such compositions can be useful for simultaneously providing antibodies with increased retention in brain regions and for systemic delivery of the immunoglobulin construct.

[0055] In some cases, one or both of these immunoglobulin constructs described herein have enhanced ADCC activity. The regimens described herein can include one or more of the combinations described herein, as well as further combinations with one or more antineoplastic biologics, antineoplastic small molecule drugs, chemotherapeutic agents, immune enhancers, radiation, surgery, etc. The biologics described herein are based on peptides, polypeptides, proteins, enzymes, nucleic acid molecules, vectors (including viral vectors), etc.

[0056] Suitably, the compositions described herein comprise an antineoplastically effective amount of one or more AAVs suspended in a pharmaceutically suitable carrier designed for delivery to a subject via infusion, osmotic pump, intrathecal catheter, or another device or route. In one example, the composition is formulated for intrathecal delivery. As used herein, intrathecal delivery encompasses injection into the spinal canal, more specifically the subarachnoid space. However, other delivery routes and pharmaceutically acceptable carriers for AAV compositions may be selected, including, for example, intracranial, intranasal, intracisternal, intracerebrospinal fluid delivery, and particularly suitable direct or systemic routes, i.e., via an Ommaya reservoir.

[0057] The composition contains about 1×10 9 Genome copies (GC) or approximately 5 x 10 13 GC (weight 70 In one embodiment, about 15 mL (or less) to about 40 mL of CSF is removed therein, and the vector is mixed and / or compatible with the CSF. In one example, the vector concentration is about 3×10 13 GC, but about 1 × 10 9 GC, approx. 5×10 9 GC, approx. 1×10 10GC, approx. 5×10 10 GC, approx. 1×10 11 GC, approx. 5×10 11 GC, approx. 1×10 12 GC, approx. 5×10 12 GC or approximately 1.0 x 10 13 Other quantities such as GC.

[0058] rAAV, preferably suspended in a physiologically compatible carrier, can be administered to a human or non-human mammalian patient. Suitable carriers can be readily selected by those skilled in the art based on the indication for which the imported virus is intended. For example, one suitable carrier includes saline (e.g., phosphate-buffered saline), which can be combined with various buffer solutions. Other exemplary carriers include sterile saline, lactose, sucrose, maltose, and water. The selection of the carrier is not a limitation of the present invention. Optionally, the compositions of the present invention may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to the rAAV and carrier(s).

[0059] In one embodiment, the compositions described herein are used in methods for slowing tumor growth. In yet another embodiment, the compositions described herein are useful for reducing tumor size in a subject. In a further embodiment, the compositions described herein are useful in reducing the number of cancer cells in non-solid tumor cancer. In another embodiment, the compositions provided herein are used in methods for increasing overall survival and / or progression-free survival in a patient. For example, the data in the Examples below show a 33% increase in survival rate for metastatic breast cancer in the brain as a monotherapy over the period tested. However, an even more modest increase in survival rate may be desirable. The anti-cancer immunoglobulin construct is selected with an eye toward the tumor to be treated. For example, for the treatment of metastatic breast cancer in the brain, an expression cassette for an anti-HER antibody may be engineered into a recombinant AAV as described herein. Optionally, the AAV compositions described herein are administered in the absence of additional exogenous pharmacological or chemical agents or other physical disruption of the blood-brain barrier.

[0060] In combination therapy, the AAV-delivered immunoglobulin constructs described herein are administered before, during, or after initiating treatment with another agent, as well as any combination thereof, i.e., before and during, before and after, during and after, or before, during and after initiating anti-cancer therapy. For example, the AAV may be administered between 1 and 30 days, preferably between 3 and 20 days, and more preferably between 5 and 12 days, before initiating radiation therapy. In another embodiment of the invention, chemotherapy is administered simultaneously with or, more preferably, after AAV-mediated immunoglobulin (antibody) therapy. In yet other embodiments, the compositions of the invention may be combined with other biologics, such as recombinant monoclonal antibody drugs, antibody-drug conjugates, and the like. Furthermore, combinations of various AAV-delivered immunoglobulin constructs, as discussed above, may be used in such regimens.

[0061] Any suitable method or route can be used to administer the AAV-containing compositions described herein, and optionally co-administer antineoplastic agents and / or antagonists of other receptors. The antineoplastic agent regimens utilized by the present invention include any regimen believed to be optimally suitable for treating the patient's tumor condition. Various malignancies may require the use of specific anti-tumor antibodies and specific antineoplastic agents, which can be determined on a patient-by-patient basis. Routes of administration include, for example, systemic, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. The dose of the antagonist administered depends on numerous factors, including, for example, the type of antagonist, the type and severity of the tumor being treated, and the route of administration of the antagonist.

[0062] The following examples are illustrative only and not limiting of the invention described herein.

[0063] [Example] Example 1

[0064] CNS expression of AAV9-mediated delivery of GFP Both GFP and mAb were 5 × 10 12 AAV9 vectors containing a GFP transgene under either the CMV or CB7 promoter were expressed in the CNS of cynomolgus monkeys after intracisternal injection of 1 genome copy (gc) / kg. Fourteen days later, the macaques were necropsied and histology and biodistribution studies were performed. Post-necropsy histological analysis showed widespread CSN expression of GFP in the cerebrum, cerebellum, choroid plexus, meninges, and spinal cord anterior horn.

[0065] In addition, 3 × 10 cells containing the 201 anti-SIV immunoadhesin (201IA) transgene under the control of the CB7 promoter 12 AAV9 vector was injected intracisternally at gc / kg, and CSF samples were collected at regular intervals to measure immunoadhesin concentrations. The resulting levels of 201IA expressed in the CSF peaked at approximately 600 ng / mL, plateaued at approximately 250 ng / mL, and remained stable for 198 days after injection.

[0066] A.201IA expression construct Rhesus monkey anti-SIV mac251 gp120 IgG-201 (Glamann et al. J Virol. 1998;74(15):7158-7163. doi:10.1128 / JVI.74.15.7158-7163.2000. Updated.) The codon-optimized nucleotide sequence of the immunoadhesin (201IA) was cloned into an AAV expression construct flanked by AAV2 inverted terminal sequences and containing the CB7 promoter, chimeric intron, and rabbit globin polyadenylation sequence (pAAV.CB7.CI.201IA.rBG).

[0067] B. I253A mutation of 201IA to abolish FcRn binding A 768-bp-long nucleotide sequence complementary to the 201IA gene but containing a mutation corresponding to I253A (SEQ ID NO:24 provides the CH2.CH3 fragment with this mutation) or H453A (SEQ ID NO:23 provides the CH2.CH3 fragment with this mutation) in the heavy chain amino acid sequence (Kabat numbering) was obtained from GeneArt (Life Technologies). The sequence was flanked by Pst1 and BstZ17I restriction sites matching those in pAAV.CB7.CI.201IA.rBG. The mutated sequences were cloned separately into pAAV.CB7.CI.201IA.rBG by restriction digestion and ligation (TaKaRa Inc.) using the indicated enzymes (NEB) as described by the manufacturer. Sanger sequencing (GeneWiz) was used to confirm the complementarity of pAAV.CB7.CI.201IA.rBG [SEQ ID NO:5 (SEQ ID NO:6 corresponds to the encoded 201IA sequence)], pAAV.CB7.CI.201IA(I253A).rBG [SEQ ID NO:7 (encoding SEQ ID NO:8)] and pAAV.CB7.CI.201IA(H435A).rBG [SEQ ID NO:9 (encoding SEQ ID NO:10)] on either side of the desired mutation.

[0068] B. IA expression in HEK293 cells and purification with Protein A 3×10 8 HEK293 cells (293 cells) were cultured in 10% FBS and 1% penicillin / The cells were seeded in 10-stack CellSTACK® (Corning) in Dulbecco's Modified Eagle's Medium (DMEM, Corning CellGro) supplemented with streptomycin (DMEM complete) and incubated at 37°C, 5% CO2 for 48 hours. 1 mg of pAAV.CB7.CI.201IA.rBG or pAAV.CB7.CI.201IA(I253A).rBG in TE buffer (Qiagen) was diluted with 42 mL of room temperature antibiotic- and serum-free DMEM. Two mL of PEI-Max 40 kDa, linear (Polysciences), pH 7.1, was separately diluted in 42 mL of room temperature antibiotic- and serum-free DMEM. The diluted DNA and diluted PEI were combined and incubated at room temperature for 15 minutes. The DNA-PEI mixture was added to a final volume of 1 L of antibiotic- and serum-free DMEM. 293T cells were washed twice with sterile PBS. The DNA-PEI DMEM mixture was added and incubated with the cells at 37°C, 5% CO2 for 72 hours. The supernatant was collected and centrifuged at 3000 × g for 10 minutes to pellet cell debris. The supernatant was then concentrated using Centrikon® Plus-70 centrifugal filter units (EMD Millipore) according to the manufacturer's instructions. 201IA or 201IA (I253A) was then purified using a Protein A antibody purification kit (Sigma) and quantified using a NanoDrop 2000 (Thermo Scientific). The purified IA was then diluted to 1 mg / mL using glycerol and stored at −20°C.

[0069] C. SDS-PAGE / Western blot analysis of IAs SDS-PAGE using NuPage reagent (Life Technologies) was performed according to the manufacturer's instructions. Briefly, 1 μg of 201IA, 201IA(I253A), or 201IA(H453A) purified from 293 supernatant, or 201IA previously purified in-house, was mixed with NuPage sample buffer and NuPage reducing agent and heated to 70°C for 10 minutes. Samples and MagicMark XP Western protein standards (Life Technologies) were loaded onto a precast NuPage 4-12% Bis-Tris 1 mm acrylamide gel, and 1× NuPage Electrophoresis was carried out in MOPS SDS running buffer at 200 V for 1 hour. Trans-Blot® Turbo TMProteins were transferred to LF PVDF membranes using a 1x transfer apparatus (BioRad). The ion reservoir stack was transferred to a 1x Trans-Blot® Turbo TM The membrane was wetted with TBT transfer buffer for 2-3 minutes. Precut LF PVDF membranes were soaked in 100% ethanol until clear, then transferred to 1x TBT buffer for 2-3 minutes. The transfer stack was assembled and run at 1.3 A and 25 V for 7 minutes. The LF PVDF membrane was blocked overnight in 1x NET buffer + 2% gelatin (50 mM Tris-HCl, pH 7, 125 mM NaCl, 5 mM EDTA pH 8, 0.05% Triton X-100, 2% gelatin in double-distilled H2O) with gentle shaking. Biotin-conjugated goat anti-human IgG polyclonal antibody (Abcam) was diluted in 1x NET + 2% gelatin and incubated with the membrane at room temperature, washed with 1x NET, incubated with streptavidin-horseradish peroxidase (Abcam) diluted in 1x NET + 2% gelatin, and washed with 1x NET. Western blots were detected using SuperSignal® West Pico chemiluminescent substrate (Thermo Scientific) according to the manufacturer's protocol. Images were captured using a BioRad with high-resolution chemiluminescence auto setting. ChemiDoc TM Images were taken using an MP imager (Thermo Scientific).

[0070] D.201IA ELISA All procedures were performed at room temperature unless otherwise indicated. Plates were washed with a BioTek 405TS microplate washer using PBS + 0.05% Tween-20. mac251 gp120 (Immune) diluted to 2 μg / mL in PBS was used. Technology Corp.) was used with a Costar® 96-well EasyWash TMThe plates were then incubated overnight at 4°C on ELISA assay plates (Corning). The plates were then blocked with 201IA ELISA blocking buffer (PBS + 5% heat-inactivated fetal bovine serum + 1 mM EDTA + 0.07% Tween-20). The diluted samples were added to the plates and diluted two-fold a minimum of four times. The plates were incubated at 37°C for 1 h and blocked again in 201IA ELISA blocking buffer. The plates were then incubated with AffiniPure polyclonal goat anti-human IgG-biotin (Jackson ImmunoResearch Labs) diluted in PBS, followed by streptavidin-horseradish peroxidase (Abcam) diluted in PBS. The plates were developed using 3,3',5,5'-tetramethylbenzidine (TMB) substrate. After stopping the color reaction with H2SO4, the plates were transferred to a SpectraMax M3 (Molecular The plate was read at 450 nm using a (Devices) plate reader.

[0071] The equivalent performance of 201IA or 201IA(I253A), as well as that of 201(H453) purified from 293 cells and the in-house produced 201IA standard protein as described in these Examples, was confirmed by 201IA ELISA. Each IA was diluted to 50 ng / mL and assayed as described above. The 201IA used as a standard was produced as follows: RAG KO mice were injected with 3x10 AAV8.TBG.201IA vectors. 11 GC / mouse were injected intravenously, and orbital bleeds were collected weekly for 8 weeks, after which the mice were terminated by cardiac bleeding. There were generally 5 mice per group. All collected sera were pooled together and loaded onto a Protein A affinity column (Sigma) as described above. Purified 201IA was generally diluted to 1 mg / ml, and glycerol was added so that the final glycerol was approximately 20% for better storage.

[0072] E. AAV9 Vector Production pAAV.CB7.CI.201IA.rBG and pAAV.CB7.CI.201IA(I253A).rBG were packaged into AAV9 capsids by triple transfection of 293 cells and purified as previously described in M. Lock et al., Hum Gene Ther. 2010 Oct;21(1);1259-1271, published online September 24, 2010.

[0073] F. Expression of 201IA and 201IA(I253A) in mouse brain and serum All animals were maintained in accordance with NIH and USDA guidelines for the care and use of animals in research. Six- to eight-week-old female Rag1- / - (Jackson Labs #002216), FcRn- / - Rag1- / - (Jackson Labs #017700), or human FcRn transgenic mice (mFcRn- / - hFcRn+ / +, Jackson Labs #016919) on a C57BL / 6 background were obtained and housed at the University of Pennsylvania.

[0074] For vector administration, AAV9.CB7.CI.201IA.rBG or AAV9.CB7.CI.201IA(I253A).rBG was diluted in sterile PBS. For intravenous (IV) administration, the vector was diluted to 1 x 10 per 100 μL. 10 Or 1 x 10 11 For intracerebroventricular (ICV) administration, the vector was diluted to 1 × 10 genome copies (GC) per 10 μL. 10 GC or 1×10 11IV injections were performed via tail vein injection, and ICV injections were performed freehand after isofluorane induction of anesthesia as previously described (Glascock et al. J Vis Exp. 2011 Oct 3; (56)). Blood was collected on days 3, 7, 14, 21, 28, 42, 56, and at the final time point on day 60 for 201IA (I253A) or day 76 for 201IA, using Z-Gel TM Blood was collected by retro-orbital bleeding into a microtube serum separator (Sarstedt). Blood was incubated at room temperature for 20 minutes and then centrifuged at 5000 x g for 5 minutes. Serum was kept at -80°C and analyzed by 201IA as described above in Part D to measure serum 201IA concentrations. Used in IA ELISA.

[0075] At necropsy, mice were deeply anesthetized with 100 mg / kg ketamine and 10 mg / kg xylazine in sterile PBS to the spinal plane of anesthesia. The thoracic cavity was exposed. A 20-gauge angiocath was inserted. TM Autoguard TM An IV catheter (Becton Dickenson) was inserted into the left ventricle of the heart, and the right atrium was nicked with scissors. 50 mL of PBS containing heparin (10 U / mL, Sigma) was slowly administered through the IV catheter into the left ventricle using a 30 mL hand syringe. The fluid exiting the right atrium was clear at the end of the perfusion procedure. The brain, liver, and spleen were removed and immediately frozen on dry ice. Brain tissue extracts were prepared by quartering frozen mouse brains (approximately 100 mg brain per quarter) and dissolving them in 1 mL of tissue lysis buffer (25 mM Tris-HCl, 5 mM EDTA, 1% Triton X-10). TM The samples were prepared by immersion in a TissueLyzer (TissueLyzer-X, 150 mM NaCl, pH 7.6). TMThe brain extracts were homogenized using a stainless steel bead mixer (Qiagen) at 30 Hz for 2 minutes, frozen overnight at -80°C, thawed in a room temperature water bath, and centrifuged at 10K x g for 10 minutes at 4°C. The supernatants from each of the four sections of individual mouse brains were combined. After gentle vortexing, the brain extracts were aliquoted and frozen at -80°C until use. The diluted brain extracts were used in the 201IA ELISA described above to confirm 201IA expression in the brain.

[0076] Serum expression of each I253A and H435A 201IA mutant was significantly higher at both tested doses (1 × 10 10 GC / mouse or 1×10 11 GC / mouse) were significantly lower than wild-type 201IA (standard) after both iv and icv administration. 11 Brain extracts tested after GC / mouse showed expression of the I253A mutant in the brain at levels exceeding that of the wild-type (standard). H435A (1 × 10 11 GC / mouse) expression was observed.

[0077] G. Expression of 201IA and 201IA(I253A) in CSF and serum of cynomolgus monkeys Four 3-4 year old female cynomolgus monkeys weighing between 3 and 5 kg were housed in stainless steel cages with a 12-h light / dark cycle at the University of Pennsylvania in accordance with NIH and USDA guidelines for the care and use of animals in research. Animals were allowed to acclimate for 7 weeks before the start of the study. Monkeys were fed Primate Diet 5049 (PMI Feeds Inc.). Water was provided ad libitum from an automatic watering system.

[0078] On the day of vector administration, animals were anesthetized using ketamine (10–15 mg / kg) and dexmedetomidine (0.05–0.10 mg / kg) given intramuscularly (IM). Animals were weighed, and vital signs were recorded. The hair on the head and dorsum of the cervical spine was shaved. The skin was aseptically prepped with Betadine. The neck was flexed so that the chin was almost touching the chest (care was taken not to obstruct the animal's airway). The dorsal occipital protuberance and the wings of the atlas (C1) vertebra were palpated, and a spinal or standard needle (20–24 gauge) was inserted midway between them. If bone was encountered, the needle was redirected anteriorly or posteriorly. Once in the subarachnoid space, CSF was collected (up to 2 mL) into a syringe or other sterile container via gravity flow as it rose into the hub of the needle. No suction was applied to the needle. Up to 2 mL of vector solution was infused at a slow, steady pace using a syringe pump at 0.5 mL / min or manually. The needle was removed and direct pressure was applied to the puncture site. Two monkeys received AAV9.CB7.CI.201IA.rBG.N401 and two received AAV9.CB7.CI.201IA(I253A).rBG. The dose was 1.00 x 10 per kilogram of body weight. 12 It was VG.

[0079] Animals were monitored for vital signs, clinical pathology, and immunology at least every two weeks. Blood and lumbar CSF were collected on days 8 and 15 after treatment and monthly thereafter. Serum and CSF were stored at approximately -65 to -80°C. 201IA expression was assessed by 201IA ELISA as described above. Animal blood chemistry and changes in blood profile were monitored by a contract facility, Antech Diagnostics, Inc.

[0080] Monkeys can be euthanized at the end of their study period. Animals are initially sedated with ketamine (10–15 mg / kg) and dexmedetomidine (0.05–0.10 mg / kg) IM. They are euthanized using pentobarbital (80 mg / kg IV). Death is confirmed by the absence of heartbeat and respiration. Animals may also be exsanguinated to ensure death. Collected tissues can be placed in 10% neutral-buffered formalin for histopathology. For genome copy analysis, tissue samples can be immediately frozen on dry ice and maintained at <-60°C. Samples can be directly frozen in OCT embedding medium for cryosectioning. Slides can be prepared by the Cellular and Morphology Core of the Gene Therapy Program at the University of Pennsylvania. Other appropriate stains may be used at the discretion of the study pathologist. Example 2

[0081] Production of AAV9 expressing trastuzumab A well-published mouse xenograft model of breast cancer brain metastasis is used to determine whether CNS-expressed trastuzumab prolongs survival or reduces tumor burden [Martinez-Aranda A et al., Development of a Preclinical Therapeutic Model of Human Brain Metastasis with Chemoradiotherapy. Int J Mol Sci. 2013;14:8306-8327]. HER2-positive human BT474 breast ductal carcinoma cells are transfected with luciferase and stereotactically injected into the brain parenchyma of nude mice. Tumor size can be monitored by luminescence intensity. Tumors exceeding 10 mm are considered to be resistant to trastuzumab. 2 When multiplying to Next, mice can be intracerebroventricularly injected with varying concentrations of AAV9 vectors carrying the trastuzumab transgene.

[0082] The transgene is created by cloning the codon-optimized nucleic acid sequence, now provided in SEQ ID NO: 1, encoding the published sequences of the light and heavy variable chains of trastuzumab, into an IgG expression cassette. The constant region amino acid sequences described in WO 2015 / 012924 (incorporated herein by reference) may be used. See, for example, Carter P et al., Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci USA. 1992 May 15;89(10):4285-9, which describes the humanization of the murine mAb precursor of trastuzumab. These amino acid sequences are those of the clinical product sequenced by mass spectrometry in 2013 [Gahoual R et al., Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry. MAbs. 2013 Apr 5;5(3)]. [Epub ahead of print]. Following vector injection, tumor size and mouse survival can be monitored for 30 days. At necropsy, pathological examination of tumors can be performed, and levels of trastuzumab expression by ELISA of brain extracts can be measured. The vectors and methods described herein can be used to improve tumor burden and / or tumor size, including prolonged survival, progression-free survival, and / or reduction and / or stable tumor burden. It should provide a

[0083] A. Trastuzumab Expression Construct Sequences consistent with the WHO-published nucleotide sequences of the heavy and light chains of trastuzumab were obtained from GeneArt (Life Technologies). The light chain sequence is flanked by EcoRV and BsiW1 restriction sites, and the heavy chain sequence is flanked by Xba1 and Sal1 restriction sites (the nucleic acid sequences are provided in SEQ ID NO: 11, which encode the trastuzumab variable heavy chain (SEQ ID NO: 12), constant heavy chain (SEQ ID NO: 13), variable light chain (SEQ ID NO: 14), kappa chain (SEQ ID NO: 15), and Amp-R (SEQ ID NO: 16)), thus providing the sequences of the plasmids containing the trastuzumab heavy and light chains.

[0084] The heavy and light chain sequences were cloned into an AAV expression construct using restriction digestion (NEB) and ligation (TaKaRa Inc.) using known cloning techniques. The heavy and light chain sequences were separated from each other by an F2A self-cleaving peptide. The construct was flanked by AAV2 inverted terminal repeats and contained a CMV immediate-early promoter, a chimeric intron, and an SV40 polyadenylation signal, and was designated pAAV.CMV.CI.trastuzumab.SV40 [SEQ ID NO: 17, encoding the trastuzumab variable heavy chain, constant heavy chain, variable light chain, and kappa chain (SEQ ID NOs: 18-21, respectively)].

[0085] B. Trastuzumab expression in HEK293 cells and purification with Protein A 3×10 8 HEK293 cells [obtained from ATCC] were cultured in 10 stacks of CellST Plate in ACK® DMEM complete at 37°C and 5% CO for 48 hours. The cells were seeded with 1 mg of pAAV.CMV.CI.trastuzumab.SV40 (described in Part A) in 42 mL of room temperature antibiotic- and serum-free DMEM. 2 mL of PEI-Max 40 KDa, linear (Polysciences) at 1 mg / mL and pH 7.1 was separately diluted in 42 mL of room temperature antibiotic- and serum-free DMEM. The diluted DNA and diluted PEI were combined and incubated at room temperature for 15 minutes. The DNA-PEI mixture was added to a final volume of 1 L of antibiotic- and serum-free DMEM. 293 cells were washed twice with sterile PBS, and the cells were then incubated with the DNA-PEI DMEM mixture for 72 hours. The supernatant was collected and centrifuged at 3000 × g for 10 minutes to pellet cell debris and filtered using Centricon® Plus-70 centrifugal filter units (EMD) according to the manufacturer's instructions. The trastuzumab was concentrated using a Protein A antibody purification kit (Sigma) and quantified using a NanoDrop 2000 (Thermo Scientific). Purified trastuzumab was diluted to 1 mg / mL using glycerol and stored at -20°C.

[0086] C. SDS-PAGE / Western blot analysis of IAs SDS-PAGE using NuPage reagent (Life Technologies) was performed according to the manufacturer's instructions. Briefly, 1 μg of trastuzumab purified from 293 supernatant as described in Part B of this Example, or trastuzumab clinical product (Hoffmann-La Roche, HUP Pharmacy) resuspended in PBS, was mixed with NuPage sample buffer and NuPage reducing agent and heated at 70°C for 10 minutes. Precast NuPAGE® 4-12% gradient Bis-Tris (neutral pH) 1 mm acrylamide gels were loaded with the sample and MagicMark TMXP Western Protein Standards (Life Technologies) were loaded and run at 200 V for 1 hour in 1x NuPage® 3-morpholinopropane-1-sulfonic acid (MOPS) SDS running buffer. TM Proteins were transferred to a low-fluorescence (LF) polyvinylidene fluoride (PVDF) membrane using a 1x transfer system (BioRad). The ion reservoir stack was placed in a 1x Trans-B lot® Turbo TM The LF PVDF membrane was wetted with 1x NET buffer (TBT) transfer buffer for 2-3 minutes. A pre-cut LF PVDF membrane was soaked in 100% ethanol until clear, then transferred to 1x TBT buffer for 2-3 minutes. The transfer stack was assembled and run at 1.3 A and 25 V for 7 minutes. The LF PVDF membrane was then resuspended in 1x NET buffer + 2% gelatin (50 mM Tris-HCl pH 7, 125 mM NaCl, 5 mM ethylenediaminetetraacetic acid in double-distilled HO). Aminotetraacetic acid (EDTA) pH 8, 0.05% Triton TM X-100 (Triton TM The membranes were blocked overnight with gentle shaking in 2% gelatin (X-100 is a nonionic surfactant with hydrophilic polyethylene oxide chains and aromatic hydrocarbon lipophilic, i.e., hydrophobic, groups). Biotin-conjugated goat anti-human IgG polyclonal antibody (Abcam) was diluted in 1x NET + 2% gelatin and incubated with the membrane at room temperature, washed with 1x NET, incubated with streptavidin-horseradish peroxidase (Abcam) diluted in 1x NET + 2% gelatin, and washed with 1x NET. Western blots were detected using SuperSignal® West Pico chemiluminescent substrate (Thermo Scientific) according to the manufacturer's protocol. Images were captured using a BioRad ChemiDoc with high-resolution chemiluminescence automatic settings. TM Images were taken using an MP imaging system (Thermo Scientific).

[0087] D. Vector Production pAAV.CMV.CI.trastuzumab.SV40 was packaged into AAV9 capsids by triple transfection of 293 cells and purified as previously described (Lock et al., 2010, cited above).

[0088] E. Trastuzumab ELISA An ELISA for the trastuzumab mimotope was developed as previously described (Jiang et al. J Biol Chem. 2005 Feb 11;280(6):4656-62. Epub 9 Nov 2004). All steps were performed at room temperature unless otherwise stated. Plates were washed with a BioTek 405TS microplate washer. The peptide mimotope LLGPYELWELSH [SEQ ID NO: 22] of the HER2 epitope to which trastuzumab binds was obtained from the mimotope, resuspended in DMSO, and stored at -80°C. Costar® 96-well EasyWash TM ELISA assay plates (Corning) were coated with 1 μg / mL LLGPYELWELSH [SEQ ID NO: 22] in 100 mM bicarbonate solution (pH 9.6), incubated overnight at 4°C, and blocked with trastuzumab ELISA blocking buffer (TEB, PBS + 5% bovine serum albumin + 1 mM EDTA + 0.07% Tween-20). Samples were diluted in TEB and plated, followed by two-fold dilutions of the ELISA plate in TEB and incubation. The plate was then incubated with AffiniPure polyclonal goat anti-human IgG-biotin (Jackson ImmunoResearch Labs) diluted in TEB, followed by streptavidin-horseradish peroxidase (Abcam) diluted in TEB. Plates were developed with TMB substrate, stopped with 2N H2SO4, and then read at 450 nm using a SpectraMax M3 (Molecular Devices) plate reader.

[0089] F. Expression of 201IA and 201IA(I253A) in mouse brain and serum Six- to eight-week-old female Rag1 − / − mice (Jackson Labs #002216) on a C57BL / 6 background were obtained and housed at the University of Pennsylvania and maintained in accordance with NIH and USDA guidelines for the care and use of animals in research.

[0090] For vector administration, pAAV.CMV.CI.trastuzumab.SV40 was used. The vector was diluted in sterile PBS at 1 × 10 per 100 μL for intravenous (IV) administration. 10 Or 1 x 10 11 For intracerebroventricular (ICV) administration, the vector was diluted to 1 × 10 per 10 μL. 10 Or 1 x 10 11 The vector was diluted in GC. IV injections were performed via tail vein injection, and ICV injections were performed freehand after isofluorane induction of anesthesia as previously described (Glascock et al.). Blood was collected by retro-orbital bleeding into Z-Gel microtube serum separators (Sarstedt) on days 3, 7, 14, 21, 28, 42, 56, and 60 after vector administration. Blood was incubated at room temperature for 20 minutes and then centrifuged at 5000 × g for 5 minutes. Serum was kept at -80°C and used in the trastuzumab ELISA described above to measure serum trastuzumab concentrations.

[0091] At necropsy, mice were deeply anesthetized with 100 mg / kg ketamine and 10 mg / kg xylazine in sterile PBS to the spinal plane of anesthesia. The thoracic cavity was exposed. A 20-gauge angiocath was inserted. TM Autoguard TM An IV catheter (Becton Dickenson) was inserted into the left ventricle of the heart, and the right atrium was scored with scissors. 50 mL of PBS containing heparin (10 U / mL, Sigma) was slowly administered through the IV catheter into the left ventricle using a 30 mL hand syringe. The fluid exiting the right atrium was clear at the end of the perfusion procedure. The brain, liver, and spleen were removed and immediately frozen on dry ice.

[0092] Brain tissue extracts were prepared by quartering frozen mouse brains (approximately 100 mg brain per quarter) and immersing them in 1 mL of tissue lysis buffer (25 mM Tris-HCl, 5 mM EDTA, 1% Triton-X, 150 mM NaCl, pH 7.6). Samples were homogenized using a stainless steel bead on a TissueLyzer (Qiagen) at 30 Hz for 2 minutes, frozen at -80°C overnight, thawed in a room temperature water bath, and centrifuged at 10K × g for 10 minutes at 4°C. Supernatants from each of the four sections of a single mouse brain were combined. After gentle vortexing, the brain extract was aliquoted and frozen at -80°C until use. The diluted brain extract was used in the trastuzumab ELISA described above to measure brain trastuzumab concentrations.

[0093] These data demonstrate steady-state expression levels of greater than 1000 μg / mL in the serum of Rag1− / − mice after intravenous vector delivery for the duration of the experiment (60 days). For mice receiving ICV vector administration, steady-state expression levels of greater than 750 μg / mL are observed for the duration of the experiment (60 days). These amounts are considered to represent levels of expression capable of providing a therapeutic effect.

[0094] Brain research is 1×10 10 and 1 × 10 11 Those receiving intravenous vectors demonstrated concentrations of between about 1200 and about 1800 μg of trastuzumab in the test mice. There did not appear to be any significant differences between these dosage levels for iv delivery. At the same dose, greater variation was observed for vectors delivered at these concentrations via ICV. The concentrations ranged from about 1000 μg to about 2500 μg.

[0095] G. Generation of a HER2+ BT474-M1 breast cancer cell line expressing firefly luciferase Passage 27 HER2+ BT474.M1 human breast ductal carcinoma cells were kindly provided by Louis Chodosh and Jason Ruth. [BT474.MI cells are a subclone of BT474 available from California Pacific Medical Center; Si Tuen Lee-Hoeflich et al., Cancer Res July 15, 2008 68:5878.] Cells were grown in T75 tissue culture flasks (Corning) in DMEM / F12 medium (Corning Cellgro) supplemented with 10% FBS and 1% penicillin / streptomycin (DMEM / F12 complete). VSVG, HIV, SIN, cPPT, CMV, ff-luciferase, WPRE lentivirus. The vector was obtained from the Penn Vector Core [E. Coprini et al., Viruses, Aug 2010, 2(8):1577-1588.] When BTB474-M1 cells were 60-70% confluent, the medium was aspirated, the cells were washed with sterile PBS, trypsinized, and counted. 2.5 x 10 cells were cultured in 2 mL of DMEM / F12 complete. 5 Cells were plated onto wells of 6-well tissue culture-treated plates (Falcon). The vector was added to the tube and incubated overnight at 37°C, 5% CO2. 3.5 × 10 cells in serum-free DMEM / F12 8 Dilute to VG / mL, and Five two-fold serial dilutions were prepared. 1 mL of each of the six vector dilutions was added to corresponding wells of a 6-well plate containing PBS-washed BT474-M1 cells and 1 mL of antibiotic- and serum-free DMEM / F12. The plate was incubated at 37°C, 5% CO2 for 4 h. The cells were incubated for 8 hours. The medium was aspirated and replaced with DMEM / F12 complete. Microscopic examination revealed no cytopathology. After another 72 hours, the cells in the 3 wells that received the highest concentration of vector were washed with sterile PBS, trypsinized, mixed, and cultured in a T75 flask in DMEM / F12 complete. After 72 hours at 37°C, 5% CO2, the cells were trypsinized and replaced with 200 μL of DMEM / F12 complete. The cells were diluted to a concentration of 1 cell per 12 complete wells. 200 μL of the cell suspension was plated per well in a 96-well tissue culture-treated plate (Falcon) and incubated at 37°C with 5% CO2 for 6 weeks. The medium was changed every 2 weeks. After 2 weeks of incubation, microscopic examination revealed wells containing single colonies of clonal cells. When wells were 70% confluent with single clusters of clonal cells, 15 clones were selected for further expansion to 70-80% confluency in 24-well plates, then 6-well plates, and then T25 tissue culture flasks (Corning). Cell morphology was compared to the parental BT474.M1 cell line and shown to be equivalent.

[0096] Luciferase activity of the cells was measured using the Dual Luciferase® Reporter Assay System (Promega) according to the manufacturer's instructions. DNA was isolated from the cells using the DNeasy kit (Qiagen). The copy number of luciferase per cell in the five clones with the highest luminescence was measured by TaqMan real-time PCR (Life Technologies) using the lentivirus packaging signal as a probe [A. Hachiya et al., Gene Ther, Apr 2007;14(8):648-656, Epub 2007 Feb 1 (E ... Clones selected for xenograft experiments were expanded to passage number 52 and cryopreserved in liquid nitrogen in DMEM / F12 containing 5% DMSO and 20% FBS.

[0097] H. HER2+ breast cancer brain metastasis xenograft model in Rag1- / - mice BT474-M1.ffluc cells, prepared as described in part G of this example, were thawed, washed with DMEM / F12 complete, and grown in T175 flasks (Corning) in DMEM / F12 complete at 37°C, 5% CO2; and Tumor cells were passaged once at least one week before implantation. On the day of implantation, cells at passage 53 were trypsinized at 70%-80% confluency and counted using a hemocytometer. Cells were centrifuged at 1000 × g for 3 minutes and washed with sterile PBS. After centrifugation again, cells were collected at 1 × 10 5 The cells were resuspended in sterile PBS at 1 μL / well and stored on ice until injection. For the tumor cell injection procedure, mice were anesthetized with an intraperitoneal (IP) injection of 100 mg / kg ketamine and 10 mg / kg xylazine in sterile PBS to induce a spinal plane of anesthesia. Ophthalmic ointment was applied to the eyes of the mice appropriately. Hair was clipped from the top of the mouse's head using electric clippers. Estrogen pellets (1.6 mg, 60-day release) were implanted subcutaneously by disinfecting the exposed skin first with povidone-iodine and then with 70% ethanol. A small incision was made in the skin overlying the thoracic spine, and the skin and underlying fascia were bluntly cut. The incision was made. An estrogen pellet was implanted subcutaneously, and the incision was sutured with 4.0 vicryl. The mouse was then secured in a stereotaxic apparatus. The exposed skin on the skull was cleaned with povidone-iodine and then 70% ethanol. An approximately 1 cm long anteroposterior incision was made on the top of the skull with a 22-blade scalpel. The bregma was identified. The position of the air drill was determined at the bregma, and the coordinates were noted. The drill point was moved -0.8 mm anterior-posterior and +2.2 mm medial-lateral to the bregma, and the burr hole was penetrated until the brain parenchyma was reached. The drill was removed from the stereotaxic apparatus, and a 10 μL Hamilton syringe was loaded with 1 μL of cell suspension. The needle was positioned on the apparatus, brought to the bregma, and moved to the coordinates indicated above. The needle was checked for accurate positioning over the burr hole, and the coordinates were adjusted accordingly before penetrating -4.0 mm DV of the bregma, then +1.0 mm. One μL of the cell suspension was injected over 5 minutes. The needle was left in place for 5 minutes after injection and then slowly removed. The mouse was removed from the stereotaxic device, and the incision on the skull was sutured using 4.0 vicryl. The mouse was placed in a clean cage on a heating pad set at 37°C. After recovery from anesthesia, the mouse received 100 μL of 15 mg / kg enrofloxacin (Bayer) subcutaneously in sterile PBS along with 0.3 mg / kg buprenorphine in sterile PBS. The mouse received subcutaneous enrofloxacin for 2 days after the procedure.

[0098] Tumor growth was monitored every 3–4 days using bioluminescence imaging (BLI). Mice were first injected IP with 150 mg / kg luciferin in sterile PBS, followed 5 minutes later by 100 mg / kg ketamine and 10 mg / kg xylazine in PBS. Five to 10 minutes after anesthesia administration, mice were imaged using an IVIS Xenogen imager. Bioluminescence was measured for at least 5 seconds. Regions of interest (ROIs) corresponding to luminescent tumors were measured by drawing a gate around the ROI. Luminescence was reported in photons / second. At necropsy, mice were euthanized by CO2 overexposure followed by cervical dislocation. Brains were removed and preserved in formalin, then 70% ethanol, and embedded in paraffin for sectioning. Hematoxylin and eosin and luciferin immunostaining were performed. Liver and spleen were also collected at necropsy for analysis of vector genome biodistribution.

[0099] I. Prophylactic treatment of a xenograft model of breast cancer brain metastasis Six to eight-week-old female Rag1 mice (Jackson Labs #002216) were cultured at 1 × 10 11 Mice were treated 21 days prior to tumor implantation with ICV injection of AAV9.CMV.CI.trastuzumab.SV40 (n=9), AAV9.CB7.CI.201IA.rBG (n=10), or no treatment (n=5). Tumors were implanted and bioluminescence measured as described above. Blood was collected retroorbitally from mice at D20, 36, 62, and 72 after vector injection to measure serum trastuzumab as a surrogate for CNS trastuzumab expression. 1x10 8 After reaching a tumor BLI of 10 photons / second, mice were monitored daily and Mice are sacrificed at a clinical endpoint defined as neurological impairment or significant morbidity, including lethargy, hunching, paralysis, neurological deficits, or convulsions.

[0100] Figure 2 shows the 1×10 11This figure shows survival curves for mice given prophylactic GC ICV AAV9.trastuzumab or AAV9.201IA and then implanted with BT474-M1.ffluc tumor cells into the brain 21 days after vector administration. The curves reflect results 99 days after tumor implantation. The median survival for the 201IA group (mock-treated) was shown to be 66 days, while the median survival for the AAV9.trastuzumab-treated group was 99 days, a 33% increase in survival.

[0101] The biodistribution of AAV9.trastuzumab delivered iv and icv in Rag1 mice was evaluated. 1 x 10 10 At a dose of GC, relatively low levels of vector genome were observed in either the liver or brain. 11 GC), significantly higher levels of vector While trastuzumab was found in the liver with both delivery methods, significantly higher levels in the brain were found only in animals receiving intravenous administration. It is noteworthy that this vector contained a non-tissue-specific promoter. Safety concerns may be reduced through the use of a tissue-specific promoter that specifically targets brain cells and possibly other neural or central nervous system cells to minimize liver expression. Alternatively, liver expression may be beneficial for systemic delivery of trastuzumab to prevent or control breast cancer metastasis to other organs.

[0102] An alternative mouse model suitable for studying BT474-M1 breast cancer brain metastasis using J.NSG mice Six- to eight-week-old female NSG mice (Jackson Labs #005557) were maintained at the University of Pennsylvania. Tumors were implanted as described above with the following modifications to the tumor cell preparation and injection technique: Tumor cells were implanted at 1 x 10 cells per 5 μL in 50% MatriGel® (Corning) / 50% sterile PBS. 5 Cells were resuspended in The volume was increased from 1 μL to 5 μL. After positioning the needle in the brain parenchyma, 5 minutes elapsed before the injection began. The injection was performed slowly over 10 minutes, and the syringe was left in place for 5 minutes before being removed. Mice were monitored, and bioluminescence was measured as described elsewhere in this document. The data indicated successful engraftment of tumor cells into the brain. The rate of tumor growth can be assessed to determine whether this model is desirable for studying breast cancer metastasis to the brain. [Sequence List Free Text]

[0103] The following information is under numerical headings: <223> is provided for sequences containing free text.

[0104] [Table 1]

[0105] [Table 2]

[0106] [Table 3]

[0107] [Table 4]

[0108] [Table 5]

[0109] [Table 6]

[0110] [Table 7]

[0111] [Table 8]

[0112] This application contains sequences and a sequence listing which are incorporated herein by reference. U.S. Provisional Application No. 61 / 984,646, filed April 25, 20145, and all publications, patents, and patent applications cited in this application are hereby incorporated by reference in their entireties as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

1. (a) adeno-associated virus (AAV) capsid; (b) an expression cassette comprising a nucleic acid sequence encoding an anti-human epidermal growth factor receptor 2 (anti-Her2) immunoglobulin construct and a nucleic acid sequence comprising an expression control sequence operably linked to the anti-Her2 encoding sequence, wherein the expression cassette is packaged in the AAV capsid; 1. A pharmaceutical composition comprising a recombinant adeno-associated virus (AAV) comprising: The nucleic acid sequence (i) a nucleic acid sequence encoding an anti-Her2 immunoglobulin heavy chain comprising an interleukin (IL)-2 leader sequence and a heavy chain variable region encoded by nucleotides 1314 to 1673 of SEQ ID NO: 17 and one or more heavy chain constant regions; (ii) a linker sequence, and (iii) a nucleic acid sequence encoding an anti-Her2 immunoglobulin light chain comprising an interleukin (IL)-2 leader sequence, and a light chain variable region and a light chain constant region encoded by nucleotides 2805 to 3110 of SEQ ID NO:

17. The pharmaceutical composition comprising:

2. The pharmaceutical composition described in claim 1, wherein the expression cassette includes an internal ribosome entry site (IRES).

3. The pharmaceutical composition described in claim 1, wherein the nucleic acid sequence comprises the sequence of nucleotides 1254 to 2660 of SEQ ID NO:

17.

4. The pharmaceutical composition described in claim 1, wherein the nucleic acid sequence comprises the sequence of nucleotides 1314 to 2660 of SEQ ID NO:

17.

5. The pharmaceutical composition described in claim 1, wherein the nucleic acid sequence comprises the sequence of nucleotides 2805 to 3452 of SEQ ID NO:

17.

6. The pharmaceutical composition described in claim 1, wherein the nucleic acid sequence comprises the sequence of nucleotides 2745 to 3452 of SEQ ID NO:

17.

7. The pharmaceutical composition of claim 1, wherein the expression control sequence comprises a human cytomegalovirus (CMV) immediate-early (IE) enhancer / promoter and an SV40 polyadenylation (poly A) signal.

8. The pharmaceutical composition of claim 1, wherein the nucleic acid sequence comprises nucleotides 1254 to 3452 of SEQ ID NO: 17.

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