EP1871810A2 - Anticorps anti-psma conjugués - Google Patents

Anticorps anti-psma conjugués

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Publication number
EP1871810A2
EP1871810A2 EP06749755A EP06749755A EP1871810A2 EP 1871810 A2 EP1871810 A2 EP 1871810A2 EP 06749755 A EP06749755 A EP 06749755A EP 06749755 A EP06749755 A EP 06749755A EP 1871810 A2 EP1871810 A2 EP 1871810A2
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EP
European Patent Office
Prior art keywords
antibody
radioisotope
dota
meo
psma
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP06749755A
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German (de)
English (en)
Inventor
John L. Tedesco
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Cytogen Corp
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Cytogen Corp
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Publication date
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Publication of EP1871810A2 publication Critical patent/EP1871810A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A61K51/1072—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from the reproductive system, e.g. ovaria, uterus, testes or prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to the development of methods and tools effective for treating, preventing, and diagnosing cancer. Specifically, the present invention is directed to methods of treating, preventing, and diagnosing cancer comprising using antibodies which immunospecif ⁇ cally bind to prostate specific membrane antigen and are conjugated to a radioisotope via a MeO-DOTA linkage.
  • Prostate cancer is among the most significant medical problems in the United States, as the disease is now the most common malignancy diagnosed in American males.
  • the American Cancer Society estimates that for the year 2000, 180,400 new cases of prostate cancer were diagnosed with 31 ,900 deaths from the disease.
  • Five year survival rates for patients with prostate cancer range from 88% for those with localized disease to 29% for those with metastatic disease.
  • the rapid increase in the number of cases appears to result in part from an increase in disease awareness as well as the widespread use of clinical markers such as the secreted proteins prostate- specific antigen (PSA) and prostatic acid phosphatase (PAP) (Chiaroda (1991) Cancer Res. 51, 2498-2505).
  • PSA prostate-specific antigen
  • PAP prostatic acid phosphatase
  • the prostate gland is a site of significant pathology affected by conditions such as benign growth (BPH), neoplasia (prostatic cancer) and infection (prostatitis).
  • Prostate cancer represents the second leading cause of death from cancer in man (Chiaroda (1991) Cancer Res. 51, 2498- 2505).
  • the prostate is the leading site for cancer development in men. The difference between these two facts relates to prostatic cancer occurring with increasing frequency as men age, especially in the ages beyond sixty at a time when death from other factors often intervenes.
  • PAP was one of the earliest serum markers for detecting metastatic spread (Nguyen et al.
  • PAP hydrolyses tyrosine phosphate and has a broad substrate specificity. Tyrosine phosphorylation is often increased with oncogenic transformation. It has been hypothesized that during neoplastic transformation there is less phosphatase activity available to inactivate proteins that are activated by phosphorylation on tyrosine residues. In some instances, insertion of phosphatases that have tyrosine phosphatase activity has reversed the malignant phenotype.
  • PSA is a protease and it is not readily appreciated how loss of its activity correlates with cancer development (Henttu et al. (1989) Bioch. Biophys. Res. Comm. 160, 903-908; Yong et al.
  • PSA proteolytic activity of PSA is inhibited by zinc. Zinc concentrations are high in the normal prostate and reduced in prostatic cancer. Possibly the loss of zinc allows for increased proteolytic activity by PSA. As proteases are involved in metastasis and some proteases stimulate mitotic activity, the potentially increased activity of PSA could be hypothesized to play a role in the tumors metastases and spread (Liotta (1986) Cancer Res. 46, 1- 7). Both PSA and PAP are found in prostatic secretions. Both appear to be dependent on the presence of androgens for their production and are substantially reduced following androgen deprivation.
  • PSMA Prostate-specific membrane antigen
  • LNCaP prostatic cancer cell
  • LNCaP is a ' cell line established from the lymph node of a hormone refractory, heavily pretreated patient (Horoszewicz et al. (1983) Cancer Res. 43, 1809-1818). This cell line was found to have an aneuploid human male karyotype. It maintained prostatic differentiation functionality in that it produced both PSA and PAP. It possessed an androgen receptor of high affinity and specificity. Mice were immunized with LNCaP cells and hybridomas were derived from sensitized animals.
  • a monoclonal antibody was derived and was designated 7El 1-C5 (Horoszewicz et al. (1993) Cancer Res. 53, 227-230). The antibody staining was consistent with a membrane location and isolated fractions of LNCaP cell membranes exhibited a strongly positive reaction with immunoblotting and ELISA techniques.
  • This monoclonal antibody was also used for detection of immunoreactive material in serum of prostatic cancer patients (Horoszewicz et al. (1993) Cancer Res. 53, 227-230).
  • the immunoreactivity was detectable in nearly 60% of patients with stage D-2 disease and in a slightly lower percentage of patients with earlier stage disease, but the numbers of patients in the latter group were small.
  • Patients with benign prostatic hyperplasia (BPH) were negative.
  • Patients with no apparent disease were negative, but 50 to 60% of patients in remission, yet with active stable disease or with progression, demonstrated positive serum reactivity.
  • Patients with non prostatic tumors did not show immunoreactivity with 7El 1-C5.
  • the 7El 1-C5 monoclonal antibody is now used as a molecular imaging agent and is the first and currently the only commercial product targeting PSMA.
  • Prostascint® consists of 7El 1- C5 linked to the radioisotope Indium- 111. Due to the selective expression of PSMA by prostate cancer cells, Prostascint® can image the extent and spread of prostate cancer using a common gamma camera.
  • U.S. Patent 5,162,504 discloses and claims the monoclonal antibody 7El 1-C5 and the hyrbirdoma cell line that produces it.
  • Patents 4,671,958; 4,741,900 and 4,867,973 disclose and claim antibody conjugates, methods for preparing such conjugates, methods for using such conjugates for in vivo imaging, testing and therapeutic treatment, and methods for delivering radioisotopes by linking them to such antibodies.
  • J591, J415, and J591 are monoclonal antibody binds with high affinity to an extracellular epitope of PSMA and localizes specifically in PSMA (Smith-Jones et al. (2000) Cancer Res. 60, 5237-5243). These antibodies have been labeled with 131 I and 111 In via a DOTA linkage. The average DOTA to antibody ratio for these antibodies was 5:1, with little apparent loss of immunoreactivity. Conjugation average of eight DOTA molecules to J591 resulted in a 20% reduction in immunoreactivity (Smith- Jones et al. (2000) Cancer Res. 60, 5237-5243). Thus, the art indicates that there is an upper limit of DOTA to antibody ratio without the antibody losing immunoreactivity.
  • the invention encompasses novel compositions which comprise an antibody which immunospecifically binds to prostate specific membrane antigen (PSMA), wherein said antibody is conjugated to a radioisotope via a MeO-DOTA linkage (henceforth known as “conjugated antibody” or “conjugated antibodies”).
  • PSMA prostate specific membrane antigen
  • conjugated antibody or conjugated antibodies
  • conjugated antibodies conjugated to a radioisotope via a MeO-DOTA linkage
  • the ratio of MeO-DOTA to antibody is about 9:1 or greater.
  • the invention also encompasses methods for preventing, treating, or managing cancer in a subject which comprises administering an antibody which immunospecifically binds to prostate specific membrane antigen (PSMA), wherein said antibody is conjugated to a radioisotope via a MeO-DOTA.
  • PSMA prostate specific membrane antigen
  • the MeO-DOTA to antibody ratio is about 9: 1 or greater.
  • the MeO-DOTA to antibody ratio is about 9:1, or greater, with little, if any, loss of immunoreactivity.
  • the conjugated antibodies of the invention can be administered in combination with one or more other cancer therapies.
  • the present invention provides methods of preventing, treating, or managing cancer in a subject comprising administering to said subject a therapeutically or prophylactically effective amount of one or more conjugated antibodies of the invention in combination with the administration of a therapeutically or prophylactically effective amount of one or more chemotherapies, hormonal therapies, biological therapies/immunotherapies and/or radiation therapies, other than the administration of the conjugated antibody of the invention, and/or in combination with surgery.
  • the conjugated antibody of the invention can be administered concurrently to a subject in separate pharmaceutical compositions or in the same composition.
  • the conjugated antibody of the invention can be administered prior to the administration of other therapies or after the administration of other therapies.
  • the therapeutic agents may be administered to a subject by the same or different routes of administration.
  • the invention also includes methods and compositions for the treatment of cancer in a mammal, including a human, comprising administering to said mammal an amount of a cytotoxic agent, or a pharmaceutical composition comprising an amount of the cytotoxic agent, that is effective in enhancing the binding of a monoclonal antibody to an epitope on the cytoplasmic domain of PSMA.
  • the cytotoxic agent will induce apoptosis of the malignant cell, and/or increase permeability, and/or otherwise disrupt the cell membrane.
  • the cytotoxic agent is administered to a mammal prior to or simultaneously with the conjugated antibodies of the invention.
  • the cytotoxic agent may disrupt the cancer cell(s), thereby expressing the cytoplasmic domain on the PSMA antigen.
  • the invention further provides diagnostic methods to evaluate or diagnose an individual with a malignant cell expressing PSMA using the conjugated antibodies of the invention.
  • the diagnostic methods of the invention provide methods of imaging and localizing malignant cells expressing PSMA, methods of diagnosis and prognosis using tissues and fluids distal to the primary tumor site (as well as methods using tissues and fluids of the primary tumor and tissues and/or using tissues and fluids surrounding the primary tumor), for example, whole blood, sputum, urine, serum, fine needle aspirates (i.e., biopsies), hi other embodiments, the diagnostic methods of the invention provide methods of imaging and localizing metastases and methods of diagnosis and prognosis in vivo. In such embodiments, primary tumors are detected using the conjugated antibody of the invention.
  • the antibodies of the invention may also be used for immunohistochemical analyses of frozen or fixed cells or tissue assays.
  • the invention further provides a novel, efficient method to conjugate antibodies which immunospecifically bind to prostate specific membrane antigen (PSMA) with MeO-DOTA.
  • PSMA prostate specific membrane antigen
  • MeO-DOTA MeO-DOTA
  • the invention further provides an efficient method to complex the conjugated antibody with a radioisotope.
  • the invention also provides pharmaceutical compositions comprising one or more monoclonal antibodies of the invention either alone or in combination with one or more other agents useful for cancer therapy.
  • kits comprising the pharmaceutical compositions or diagnostic reagents of the invention are provided.
  • Figure 1 is a Coomassie-stained one-dimensional 4 to 20% SDS-PAGE gel of eight lots of 7El 1- C5 conjugates (conjugated to a radioisotope via a MeO-DOTA linkage) prepared under conditions as set forth in Table 1. Arrows indicate heavy chain (50 kDA) and light chain (25 kDA).
  • Figure 2 (A) is size exclusion chromatogram of 7El 1-C5 antibody and (B) a representative chromatogram of its MeO-DOTA conjugate (lot 200402495-5/8). Relative peak areas of unmodified protein and conjugates are summarized in Table 3.
  • Figure 3A-D are size exclusion chromatograms of different complexations of MeO-DOTA- Cyt351, 200402495-29, under the indicated condition. Relative peak areas of unmodified protein and conjugates are summarized in Table 5.
  • Figure 4 is a size exclusion chromatogram of a high specific activity conjugate preparation at 30 minutes. Relative peak areas of unmodified protein and conjugates are summarized in Table 6.
  • Radiotherapy is an effective cancer treatment, but it is difficult to direct and can be devastating to nontargeted parts of the body. Indeed, treatments are often limited by its non- selectivity, resulting in toxicity on the normal tissues. Monoclonal antibodies, or fragments thereof, on the other hand, are adept at selectively targeting diseased cells. The ability of antibodies to exploit antigenic differences between normal and malignant tissues and to exact a variety of antitumor responses offers significant advantages to conventional forms of therapy. However, antibodies alone often have inadequate therapeutic effectiveness.
  • Conjugating a radioisotope to a monoclonal antibody, or fragments thereof, can solve both problems.
  • Monoclonal antibodies are highly specific and can be used as vehicles to deliver substances to specific target sites.
  • the conjugation of a monoclonal antibody to a radioisotope is an effective way to target specific cells, thus reducing the side effects of radiotherapy.
  • compositions comprising an antibody, or fragments thereof, which immunospecifically binds to prostate specific membrane antigen (PSMA), wherein said antibody, or fragment(s) thereof, is conjugated to a radioisotope via a MeO-DOTA linkage.
  • PSMA prostate specific membrane antigen
  • the anti-PSMA monoclonal antibody is 7El 1-C5.
  • the conjugated 7El 1-C5 antibody is complexed with 177 Lu.
  • the conjugated 7El 1-C5 is complexed with 177 Lu at a ratio of isotope to antibody of about 9:1 or greater.
  • the invention also discloses methods of using, formulating and making the antibodies of the invention.
  • the invention comprises antibodies, or fragments thereof, which immunospecifically bind to prostate specific membrane antigen (PSMA), wherein said antibody is conjugated to a radioisotope via a MeO-DOTA linkage.
  • PSMA prostate specific membrane antigen
  • MeO-DOTA ⁇ -(5-isothiocyanato-2-methoxyphenyl)- l,4,7,10-teraazacyclododeczane-l,4,7,10-tretraacetic (Dow Chemical Company) is a bifunctional chelant.
  • a bifunctional chelant is a molecule that has, in addition to chelating functionality, the ability to be conjugated (linked) to a biotargeting molecule ⁇ e.g. monoclonal antibody).
  • MeO- DOTA provides metal complexes with high stability, thereby reducing the incidence of background during imaging procedures or damage to non-targeted tissues in radioimmunotherapy.
  • the antibody is 7El 1-C5.
  • the MeO- DOTA linked antibody is complexed to a radioisotope which is selected from the group consisting Of 3 H, 14 C, 18 F, 19 F, 31 P, 32 P, 35 S 5 131 I 5 125 1, 123 I 5 64 Cu, 187 Re, 111 In, 90 Y, 99 Tc, 177 Lu.
  • the antibody comprises 7El 1-C5 conjugated to 177 Lu via MeO-DOTA.
  • the MeO-DOTA to antibody ratio is about 9:1 or greater.
  • An antibody is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains
  • an IgM antibody consists of five of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contain ten antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising two to five of the basic four chain units along with J chain).
  • the L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains and the methods of the current invention include the use of antibodies with either a kappa or lambda L chain.
  • immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM 5 having heavy chains designated alpha, delta, epsilon, gamma and mu, respectively.
  • the gamma and alpha classes are further divided into subclasses on the basis of relatively minor differences in C H sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3 5 IgG4 5 IgAl and IgA2.
  • the methods of the present invention include the use of antibodies, including monoclonal antibodies, from any of the above classes and/or subclasses.
  • variable refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies.
  • the variable domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed across the 110-amino acid span of the variable domains.
  • the variable regions consist of relatively invariant stretches called framework regions (FR) of about fifteen to thirty amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each about nine to twelve amino acids long.
  • FR framework regions
  • hypervariable regions that are each about nine to twelve amino acids long.
  • the variable domains of native heavy and light chains each comprise four framework regions, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the framework region and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Public Health Service, National Institutes of Health).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
  • hypervariable region when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region generally comprises amino acid residues from a “complementarity determining region” or "CDR" which contributes to the specificity of the antibody.
  • antibodies or fragments thereof as used herein refers to antibodies or fragments thereof that specifically bind to a PSMA polypeptide or a fragment of a PSMA polypeptide and do not specifically bind to other non-PSMA polypeptides.
  • antibodies or fragments that immunospecifically bind to a PSMA polypeptide or fragment thereof do not non-specifically cross-react with other antigens (e.g., binding cannot be competed away with a non-PSMA protein, e.g., BSA in an appropriate immunoassay).
  • Antibodies or fragments that immunospecifically bind to an PSMA polypeptide can be identified, for example, by immunoassays or other techniques known to those of skill in the art.
  • Antibodies of the invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, diabodies, multispecif ⁇ c antibodies (including bi-specific: antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv) (including bi-specific scfvs), single chain antibodies, Fab' fragments, F(ab') 2 fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • synthetic antibodies monoclonal antibodies, recombinantly produced antibodies, intrabodies, diabodies, multispecif ⁇ c antibodies (including bi-specific: antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv) (including bi-specific scfvs), single chain antibodies, Fab' fragments, F(ab') 2 fragments, disulf
  • antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an PSMA antigen (e.g., one or more complementarity determining regions (CDRs) of an anti-PSMA antibody).
  • an antigen binding site e.g., one or more complementarity determining regions (CDRs) of an anti-PSMA antibody.
  • CDRs complementarity determining regions
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts and includes antibody fragments as defined herein. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies.
  • the modifier "monoclonal” is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al. (1975) Nature 256, 495 or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see U.S. Patent 4,816,567).
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. MoI. Biol. 222, 581-597, for example.
  • an "intact" antibody is one which comprises an antigen-binding site as well as a C L and at least heavy chain constant domains, C H i and C m and C H 3.
  • the constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof.
  • the intact antibody has one or more effector functions.
  • antibody fragment comprises a portion of an intact antibody, preferably the antigen binding CDR or variable region of the intact antibody.
  • antibody fragments include Fab, Fv, Fab' and F(ab') 2 fragments; diabodies; linear antibodies (see U.S. Patent 5,641,870 and Zapata et al. (1995) Protein Eng. 8, 1057-1062); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Fab fragments
  • Fc residual fragment
  • the Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V H ), and the first constant domain of one heavy chain (C H i).
  • V H variable region domain of the H chain
  • C H i first constant domain of one heavy chain
  • Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.
  • Pepsin treatment of an antibody yields a single large F(ab') 2 fragment which roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen.
  • Fab' fragments differ from Fab fragments by having additional few residues at the carboxy te ⁇ ninus of the C ffl domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • the Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides.
  • the effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.
  • Fv is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Single-chain Fv also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the V H and V L antibody domains connected into a single polypeptide chain.
  • the sFv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding (see
  • diabodies refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5 to about 10 residues) between the V H and V L domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites.
  • Bispecif ⁇ c diabodies are heterodimers of two "crossover" sFv fragments in which the V H and V L domains of the two antibodies are present on different polypeptide chains.
  • Diabodies are described more fully in, for example, WO 93/11161 and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90, 6444-6448.
  • an “isolated antibody” is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous components.
  • the antibody will be purified to greater than 95% by weight of antibody, and most preferably more than 99% by weight. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • the conjugated antibody binds to an epitope on the cytoplasmic domain of a protein specific to cancer cells (i.e., a cancer cell marker).
  • the conjugated antibody includes, but is not limited to, an antibody which binds to an epitope on the cytoplasmic domain of PSMA, including but not limited to, the 7El 1-C5 monoclonal antibody as described in U.S. Patent 5,162,504 which is herein incorporated by reference in its entirety.
  • the hybridoma cell line which produces the 7El 1-C5 monoclonal antibody has been deposited with the American Type Culture Collection under Deposit No. HB 10494.
  • the conjugated antibodies used in the methods of the invention include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Patent 4,816,567 and Morrison et a (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).
  • Chimeric antibodies of interest herein include, but are not limited to "humanized" antibodies comprising variable domain antigen-binding sequences derived from a non-human mammal (e.g., murine) and human constant region sequences.
  • Antibodies of the invention may also comprise a fully human antibody sequence. In another embodiment, antibodies of the invention may be a fully human antibody.
  • Antibodies, or fragments thereof, of the invention can be used as diagnostic or detectable agents.
  • the antibody, or fragments thereof immunospecifically bind to prostate specific membrane antigen (PSMA), wherein said antibody is conjugated to a radioisotope via a MeO-DOTA linkage.
  • PSMA prostate specific membrane antigen
  • Antibodies of the invention can be useful for monitoring or prognosing the development or progression of a cancer as part of a clinical testing procedure, such as determining the efficacy of a particular therapy. Additionally, such antibodies can be useful for monitoring or prognosing the development or progression of cancerous conditions.
  • the cancer cells can be imaged and the relative amount of cancerous cells determined by any available means.
  • the invention includes diagnostic methods to detect cancer and/or assess the effect therapeutic agents on cancer cells in an organ or body area of a patient.
  • the present methods include administration of a composition comprising a detectable amount of an anti- PSMA antibody conjugated to a radioisotope via MeO-DOTA to a patient before and after therapy.
  • the MeO-DOTA to antibody ratio is about 9: 1 or greater.
  • the MeO-DOTA to antibody ratio is about 9:1, or greater, with little, if any, loss of immunoreactivity.
  • an additional amount of detectable monoclonal antibody can be administered to determine the relative amount of cancer cells remaining following treatment. Comparison of the before and after treatment images can be used as a means to assess the efficacy of the treatment wherein a decrease in the number of cancer cells imaged following treatment is indicative of an efficacious treatment regimen.
  • the term “detectable amount” refers to the amount of labeled conjugated antibody which binds to PSMA administered to a patient that is sufficient to enable detection of binding of the labeled monoclonal antibody to one or more malignant cancer cells in a tumor.
  • imaging effective amount refers to the amount of the labeled antibody administered to a patient that is sufficient to enable imaging of binding of the antibody to one or more malignant cancer cells in a tumor.
  • the methods of the invention comprise conjugated antibodies of the invention which, in conjunction with non-invasive neuroimaging techniques such as magnetic resonance spectroscopy (MRS) or imaging (MRI), or gamma imaging such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT), are used to identify and quantify abnormal cells in vivo including malignant cells in tumors.
  • non-invasive neuroimaging techniques such as magnetic resonance spectroscopy (MRS) or imaging (MRI), or gamma imaging such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT)
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • the radiation emitted from the tumor or area being examined is measured and expressed either as total binding, or as a ratio in which total binding in one tissue is normalized to (for example, divided by) the total binding in another tissue or the entire body of the same subject during the same in vivo imaging procedure.
  • Total binding in vivo is defined as the entire signal detected in a tumor or tissue by an in vivo imaging technique without the need for correction by a second injection of an identical quantity of labeled compound along with a large excess of unlabeled, but otherwise chemically identical compound.
  • the terms "subject” or "patient” refers to a mammal, preferably a human, and most preferably a human suspected of having abnormal cells, including malignant cells in a tumor.
  • the type of detection instrument available is a major factor in selecting a given label.
  • radioactive isotopes are particularly suitable for in vivo imaging in the methods of the present invention.
  • the type of instrument used will guide the selection of the radioisotope.
  • the radioisotope chosen must have a type of decay detectable by a given type of instrument.
  • Another consideration relates to the half-life of the radioisotope. The half-life should be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that the host does not sustain deleterious radiation.
  • the isotopically-labeled monoclonal antibody can be detected using gamma imaging where emitted gamma irradiation of the appropriate wavelength is detected.
  • gamma imaging include, but are not limited to, positron emission tomography (PET) imaging or for single photon emission computerized tomography (SPECT).
  • PET positron emission tomography
  • SPECT single photon emission computerized tomography
  • the chosen radiolabel will lack a particulate emission, but will produce a large number of photons.
  • the radiolabel will be a positron-emitting radioisotope which will be detected by the PET camera.
  • conjugated antibodies are useful for in vivo detection and imaging of tumors. These compounds are to be used in conjunction with non-invasive neuroimaging techniques such as magnetic resonance spectroscopy (MRS) or imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT).
  • the conjugated antibody may be labeled (complexed) with any acceptable radioisotope. For example, including, but are not limited to, 3 H, 14 C, 18 F, 19 F, 31 P, 32 P, 35 S, 131 1, 125 1, 123 I 3 64 Cu, 187 Re, 111 In, 90 Y, 99 Tc, 177 Lu using techniques described below or known in the art.
  • the diagnostic methods of the present invention may use isotopes detectable by nuclear magnetic resonance spectroscopy for purposes of in vivo imaging and spectroscopy.
  • Elements particularly useful in magnetic resonance spectroscopy include, but are not limited to, 19 F and 13 C.
  • Suitable radioisotopes for purposes of this invention include beta-emitters, gamma-emitters, positron-emitters and x-ray emitters. These radioisotopes include, but are not limited to, 111 In, 131 1, 123 1, 18 F, 11 Q 75 Br and 76 Br.
  • Suitable stable isotopes for use in Magnetic Resonance Imaging (MRI) or Spectroscopy (MRS), according to this invention include, but are not limited to, 19 F and 13 C.
  • Suitable radioisotopes for in vitro identification and quantification of abnormal cells including tumor cells, in a tissue biopsy or post-mortem tissue include 125 1, 14 C and 3 H. Examples of these radiolabels include, but not limited to, 64 Cu or 18 F for use in PET in vivo imaging, 123 I or 131 I for use in SPECT imaging in vivo, 19 F for MRS and MRI and 3 H or 14 C for in vitro methods.
  • any conventional method for visualizing diagnostic probes can be utilized in accordance with this invention.
  • the dosage of the isotopically-labeled monoclonal antibody will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, contraindications, if any, concomitant therapies and other variables, to be adjusted by the skilled artisan. Dosage can vary from 0.001 mg/kg to 1000 mg/kg, preferably 0.1 mg/kg to 100 mg/kg. Administration to the patient may be local or systemic and accomplished intravenous, intraarterial, intra-thecal (via the spinal fluid), intra-cranial or the like. Administration may also be intra-dermal or intra-cavitary, depending upon the body site under examination.
  • the area of the subject under investigation is examined by routine imaging techniques such as MRS/MRI, SPECT, planar scintillation imaging, PET, and emerging imaging techniques, as well.
  • routine imaging techniques such as MRS/MRI, SPECT, planar scintillation imaging, PET, and emerging imaging techniques, as well.
  • the exact protocol will necessarily vary depending upon factors specific to the patient, as noted above, and depending upon the body site under examination, method of administration and type of label used; the determination of specific procedures would be routine to the skilled artisan.
  • the amount (total or specific binding) of the bound isotopically-labeled monoclonal antibody is measured and compared (as a ratio) with the amount of isotopically-labeled monoclonal antibody bound to the tumor following chemotherapeutic treatment.
  • the conjugated antibodies of the invention can be used for diagnosis and prognosis by using tissues and fluids distal to the primary tumor site (as well as methods using tissues and fluids of the primary tumor and/or tissue and fluids surrounding the tumor).
  • Antibodies of the invention can be used to assay PMSA levels in a biological sample using classical immunohistological methods as known to those of skill in the art (e.g., see Jalkanen et al. (1985) J. Cell. Biol. 101, 976-985; and Jalkanen et al. (1987) J. Cell. Biol. 105, 3087-3096).
  • antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 125 1, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium ( 99 Tc).
  • the present invention provides diagnostic kits, including both immunodetection and imaging kits, for use with the immunodetection and imaging methods described above.
  • the invention encompasses a method for treating cancer which comprises a malignant cell expressing PSMA in a patient in need thereof comprising administering a conjugated antibody which specifically binds to PSMA expressed by a malignant cell.
  • the conjugated antibody binds to a cytoplasmic epitope on the PSMA.
  • the conjugated antibody includes, but is not limited to, the 7El 1-C5 monoclonal antibody as described in U.S. Patent 5,162,504 herein incorporated by reference in its entirety.
  • the hybridoma cell line which produces the 7El 1-C5 monoclonal antibody has been deposited with the American Type Culture Collection under Deposit No. HB 10494.
  • the antibody is conjugated with a radioisotope via a MeO-DOTA linkage.
  • the MeO-DOTA to antibody ratio is about 9:1 or greater.
  • the MeO-DOTA to antibody ratio is about 9:1, or greater, with little, if any, loss of immunoreactivity.
  • the amount of the conjugated antibody composition of the invention which will be effective in the treatment, prevention or management of cancer can be determined by standard research techniques.
  • the dosage of the composition which will be effective in the treatment, prevention or management of cancer can be determined by administering the composition to an animal model such as, e.g., the animal models disclosed herein or known to those skilled in the art.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • Selection of the preferred effective dose can be determined (e.g., via clinical trials) by a skilled artisan based upon the consideration of several factors which will be known to one of ordinary skill in the art. Such factors include the disease to be treated or prevented, the symptoms involved, the patient's body mass, the patient's immune status and other factors known by the skilled artisan to reflect the accuracy of administered pharmaceutical compositions.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
  • the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight.
  • human and humanized antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human or humanized antibodies and less frequent administration is often possible.
  • patients with prostate cancer are administered an effective amount of one or more conjugated antibodies of the invention.
  • the antibodies of the invention can be administered in combination with an effective amount of one or more other agents useful for prostate cancer therapy including but not limited to: external-beam radiation therapy, interstitial implantation of radioisotopes (i.e., 125 I, palladium, iridium), leuprolide or other LHRH agonists, non-steroidal antiandrogens (flutamide, nilutamide, bicalutamide), steroidal antiandrogens (cyproterone acetate), the combination of leuprolide and flutamide, estrogens such as DES, chlorotrianisene, ethinyl estradiol, conjugated estrogens U.S.P., DES-diphosphate, radioisotopes, such as strontium-89, the combination of external-beam radiation therapy and strontium-89, second-line hormonal therapies such as aminoglute
  • certain preferred embodiments will encompass the administration of lower dosages in combination treatment regimens than dosages recommended for the administration of single agents .
  • the invention provides for any method of administrating lower doses of known prophylactic or therapeutic agents than previously thought to be effective for the prevention, treatment, management or amelioration of cancer.
  • lower doses of known anti-cancer therapies are administered in combination with lower doses of conjugated monoclonal antibodies of the invention.
  • the invention also includes methods and compositions for the treatment of cancer in a mammal, including a human, comprising administering to said mammal an amount of a cytotoxic agent, or a pharmaceutical composition comprising an amount of the cytotoxic agent, that is effective in enhancing the binding of an antibody to an epitope of PSMA, for instance a cytoplasmic epitope.
  • Said cytotoxic agent can be administered prior to or simultaneously with said antibody.
  • said antibody binds to an epitope on the cytoplasmic domain of PSMA.
  • said antibody is conjugated with MeO-DOTA.
  • said antibody is 7El 1-C5 and is conjugated to MeO-DOTA.
  • the cytotoxic agent will induce apoptosis of the malignant cell, and/or increase permeability, and/or otherwise disrupt the cell membrane
  • the cytotoxic agent is administered to a mammal prior to or simultaneously with the conjugated antibodies of the invention.
  • the cytotoxic agent disrupts the cancer cell(s), thereby expressing the cytoplasmic domain on the PSMA antigen. Exposure of the cytoplasmic domain allows for targeting of the remaining cancer cells with the conjugated antibody of the invention.
  • chemotherapeutic agents include but are not limited to, BCNU, cisplatin, gemcitabine, hydroxyurea, paclitaxel, temozomide, topotecan, fluorouracil, vincristine, vinblastine, procarbazine, dacarbazine, altretamine, cisplatin, methotrexate, mercaptopurine, thioguanine, fludarabine phosphate, cladribine, pentostatin, fluorouracil, cytarabine, azacitidine, vinblastine, vincristine, etoposide, teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin, dactinomycin, idarubicin, plicamycin, adriamycin, mitomycin, bleomycin, tamoxifen, flutamide, leuprolide, goserelin, aminog
  • the therapeutic method comprises administration of the conjugated antibody of the invention in combination with radiation for the treatment of cancer.
  • the radiation is designed to disrupt the cell membrane of the cancer cell to expose PSMA, for instance the cytoplasmic domain of PSMA. Once the cytoplasmic domain is exposed, an antibody can bind to PSMA. hi a preferred embodiment, said antibody binds to an epitope on the cytoplasmic domain of PSMA.
  • said antibody is conjugated with MeO-DOTA.
  • said antibody is 7El 1-C5 and is conjugated to MeO- DOTA.
  • the methods of the invention are also designed to induce apoptosis (cell death) in cancer cells, reduce the incidence or number of metastases, and reduce tumor size.
  • Tumor cell resistance to radiotherapy agents represents a major problem in clinical oncology.
  • combination therapy with such an antibody could be used on radiation resistant tumors to improve the efficacy of the radiation therapy.
  • Types of cancer that can be treated the methods of the invention include solid tumors.
  • solid tumors include, but are not limited to, endothelial cell carcinoma.
  • endothelial cell carcinoma include, but are not limited to, renal cell carcinoma, colon carcinoma, transitional cell carcinoma, lung carcinoma, breast carcinoma and prostatic adenocarcinoma.
  • renal cell carcinoma examples include, but are not limited to, clear cell carcinoma, papillary carcinoma, chromophobe carcinoma, collecting duct carcinoma and unclassified carcinoma.
  • lung carcinoma examples include, but are not limited to, adenocarcinoma, alveolar cell carcinoma, squamous cell carcinoma, large cell and small cell carcinoma.
  • breast carcinoma examples include, but are not limited to, adenocarcinoma, ductal carcinoma in situ, lobular carcinoma in situ, invasive ductal carcinoma, medullary carcinoma and mucinous carcinoma.
  • Another example of solid tumor treatable by the methods of the invention includes endothelial cell sarcoma. In one embodiment, the sarcoma is a soft tissue sarcoma. Metatstatic tumors are also treatable.
  • U.S. patents 5,435,990 and 5,652,361 disclose methods of making and using MeO-DOTA and other bifunctional chelators.
  • U.S. patents 5,435,990 and 5,652,361 are herein incorporated by reference in their entireties for all purposes.
  • the modification of antibodies for the addition of MeO-DOTA may be accomplished by formation of a covalent linkage with an amino acid residue of the protein and a functional group of the bifunctional chelator which is capable of binding proteins.
  • MeO-DOTA could also be attached to the antibody via a linker molecule.
  • linker molecules useful for conjugating MeO-DOTA to a polypeptide are disclosed in, for example, DeNardo et al. (1998) Clin Cancer Res. 4, 2483-2490; Peterson et al. (1999) Bioconjug. Chem. 10, 553-557; and Zimmerman et al. (1999) Nucl. Med. Biol. 26, 943-950, which are hereby incorporated by reference in their entirety.
  • U.S. Patents 5,652,361 and 5,756,065 disclose chelating agents that may be conjugated to antibodies.
  • the method by which the disclosed chelating agents are conjugated are also disclosed in U.S. Patents 5,652,361 and 5,756,065. Any suitable process that results in the formation of the conjugates of this invention is within the scope of this invention.
  • one important aspect for an efficient reaction is the antibody to conjugate ratio. Once the conjugate and the antibody are purified and ready for conjugating both the antibody and are added to a reaction mixture.
  • the conjugate to antibody ratio should be approximately 5:1, 10:1, 30:1, 40:1, 50: 1, 60:1, 70:1, 80:1, 90:1, 100:1, 150:1, 200:1, 250:1, respectively.
  • the pH of the reaction mixture should be about 10 to about 5, preferably about 9 to about 8. More preferably the pH should be about 8.5.
  • the conjugation reaction should be carried out at a temperature range from about 2O 0 C to about 37°C. Up to 5, 6, 7, 8, 9, 10, 11, 12 or more MeO-DOTA chelates could be bound to the antibody following the described protocol.
  • the MeO-DOTA to antibody ratio is about 9:1 or greater. In another embodiment, the MeO-DOTA to antibody ratio is about 9:1, or greater, with little loss, if any, of immunoreactivity.
  • Binding of a therapeutic radioactive isotope to the bifunctional chelator may be accomplished either with the bifuntional chelator, MeO-DOTA, that is not complex to an antibody or, alternatively, a composition consisting of MeO-DOTA conjugated to an antibody.
  • the complexing reaction should be accomplished at a pH of about 7 to about 4, preferably about 5 to about 6. More preferably the pH should be about 5.5.
  • the complexing reaction may be accomplished in an acetate buffer (e.g. sodium acetate, potassium acetate) at a molar concentration of at least about 50 mM, 100 mM, 150 mM, 200 mM, or 250 mM or more.
  • the molar concentration should be about 100 mM.
  • the complexing reaction should carried out at a temperature range from about 20 0 C to about 37°C.
  • the percent of radionucleotide complexed with DOTA should be between about 80% and about 100%, preferably, between about 87% to about 95%.
  • the radioisotope that can be complexed with MeO-DOTA include, but are not limited to,
  • compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient) which can be used in the preparation of unit dosage forms.
  • Such compositions comprise a prophylactically or therapeutically effective amount of a prophylactic and/or therapeutic agent disclosed herein or a combination of those agents and a pharmaceutically acceptable carrier.
  • compositions of the invention comprise a prophylactically or therapeutically effective amount of one or more antibodies of the invention and a pharmaceutically acceptable carrier or an agent that reduces expression (e.g. , antisense oligonucleotides) and a pharmaceutically acceptable carrier.
  • the composition of the invention further comprises an additional therapeutic, e.g., anti-cancer, agent.
  • the term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • compositions of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • Various delivery systems are known and can be used to administer the antibody of the invention or the combination of a conjugated antibody of the invention and a prophylactic agent or therapeutic agent useful for preventing or treating cancer, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or antibody fragment, receptor-mediated endocytosis (see for example, Wu and Wu (1987) J. Biol. Chem. 262, 4429-4432).
  • Methods of administering a prophylactic or therapeutic agent of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal, inhaled, and oral routes).
  • parenteral administration e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous
  • epidural e.g., intranasal, inhaled, and oral routes
  • mucosal e.g., intranasal, inhaled, and oral routes.
  • prophylactic or therapeutic agents of the invention are administered intramuscularly, intravenously, or subcutaneously.
  • the prophylactic or therapeutic agents may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g. , oral mucosa, rectal and intestinal mucos
  • the prophylactic or therapeutic agents of the invention may be desirable to administer locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion (such as limb perfusion), by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Kits
  • the invention provides a pharmaceutical pack or kit comprising one or more containers filled with an monoclonal antibody of the invention.
  • the kit comprises an anti-PSMA antibody conjugated to a radioisotope via MeO-DOTA to a patient before and after therapy.
  • the MeO-DOTA to antibody ratio is about 9: 1 or greater.
  • the MeO-DOTA to antibody ratio is about 9:1, or greater, with little, if any, loss of immunoreactivity.
  • one or more other prophylactic or therapeutic agents useful for the treatment of a cancer can also be included in the pharmaceutical pack or kit.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • kits that can be used in the above methods.
  • a kit comprises one or more a monoclonal antibodies of the invention.
  • a kit further comprises one or more other prophylactic or therapeutic agents useful for the treatment of cancer, in one or more containers.
  • the antibody of the invention is an antibody which immunospecifically binds to prostate specific membrane antigen (PSMA), wherein said antibody is conjugated to a radioisotope via a MeO-DOTA linkage.
  • PSMA prostate specific membrane antigen
  • said antibody is 7El 1-C5 conjugated to 177 Lu via MeO-DOTA.
  • the conjugated 7El 1-C5 is complexed with 177 Lu at a ratio of isotope to antibody of about 9: 1 or greater.
  • the other prophylactic or therapeutic agent is a chemotherapeutic.
  • the prophylactic or therapeutic agent is a biological or hormonal therapeutic.
  • the LNCaP cell line was established from a metastatic lesion of human prostatic carcinoma.
  • the LNCaP cells grow readily in vitro (up to 8 xlO 5 cells/cm 2 ; doubling time, 60 hours), form clones in semisolid media, and show an aneuploid (modal number, 76 to 91) human male karyotype with several marker chromosomes.
  • the malignant properties of LNCaP cells are maintained.
  • Athymic nude mice develop tumors at the injection site (volume-doubling time, 86 hours). Functional differentiation is preserved; both cultures and tumor produce a prostate acid phosphatase (PAP) and prostate specific antigen (PSA).
  • PAP prostate acid phosphatase
  • PSA prostate specific antigen
  • High-affinity specific androgen receptors are present in the cytosol and nuclear fractions of cells in culture and in tumors. Estrogen receptors are demonstrable in the cytosol.
  • the model is hormonally responsive. In vitro, ⁇ - dihydro-testosterone modulates cell growth and stimulates acid phosphatase production. In vivo, the frequency of tumor development and the mean time of tumor appearance are significantly different for either sex. LNCaP cells, therefore, meet criteria of a versatile model for immunological studies of human prostatic cancer in the laboratory.
  • Fresh normal, benign and malignant prostate cancer tissues were obtained either from the Department of Surgery or the Department of Pathology at Roswell Park Memorial Institute. The tissues were quick frozen in M-I embedding matrix (Lipshaw Corp) and stored at -80 0 C.
  • mice West Seneca Laboratoryreceived intraperitoneal injections (2 x 10 7 cells/0.2ml) of washed (three times in RPMI medium 1640) live LNCaP cells suspended in RPMI medium 1640, at monthly intervals for 3 months.
  • mice received an intraperitoneal challenge of 2 x 10 7 cells in RPMI medium 1640 and an intravenous injection of the plasma membrane isolated from 1 x 10 8 LNCaP cells.
  • Cell fusion was carried out using a modification of the procedure developed by Kohler and Milstein (1975) Nature 256, 495-497.
  • Mouse splenocytes (1 x 10 8 cells) were fused in HyBRL-Prep 50% polyethylene glycol 1450 (Bethesda Research Laboratories) with 5 x 10 7 mouse myeloma cells (P3 x 63Ag8.653). Fused cells were distributed to ten 96-well culture plates (Falcon, Oxnard, CA) and grown in hypoxanthine/aminopterin/thymidine (HAT) medium at 37°C with 7.5% CO 2 in a humid atmosphere.
  • HAT hypoxanthine/aminopterin/thymidine
  • Plasma membrane-enriched fractions were obtained from LNCaP cells and normal human diploid fibroblast strain MLD by modification of published methods (Kartner et al. (1977) J. Membrane Biol. 36, 191-211). Briefly, MLD cells in roller bottles or LNCaP cells in plastic culture flasks were gently rinsed 4 times with phosphate buffered saline (PBS). The cells were then rinsed once with hypotonic lysing buffer (3 mM Hepes [hydroxyethylpiperazine- ethanesulfonic acid] (pH 7.0), 0.3 mM MgCl 2 , 0.5 mM CaCl 2 ) and the buffer discarded.
  • hypotonic lysing buffer 3 mM Hepes [hydroxyethylpiperazine- ethanesulfonic acid] (pH 7.0), 0.3 mM MgCl 2 , 0.5 mM CaCl 2
  • Fresh lysing buffer (5-25 ml) was added to each bottle or flask and the cells allowed to swell for 30 minutes at room temperature. The swollen cells were removed from the surface and disrupted by manual shaking. The progress of disruption was monitored by phase microscopy of a sample droplet. Gentle trituration (8-10 times) with a 10 ml pipette was used to complete disruption of the LNCaP cells. Vigorous shaking and pipetting were necessary to completely break-up the MLD cells. Phenyl-methylsulfonyl fluoride (PMSF, 0.5 mM) (Calbiochem) was added to minimize proteolysis.
  • PMSF Phenyl-methylsulfonyl fluoride
  • the disrupted cell suspensions were centrifuged at 100 x g to remove nuclei and incompletely disrupted cell clumps.
  • the nuclei pellet was washed once with the lysing buffer and after centrifugation the supernatant was combined with the first supernatant and centrifuged at 3,000 x g for 10 minutes, at 4°C.
  • the pellet consisting of mitochondria and debris was discarded and the supernatant designated as membrane lysate was layered over a discontinuous density gradient composed of 15 ml each of 10, 30 and 38% sucrose (w/v) and centrifuged at 60,000 x g for 2-1/2 hours in an SW 25.2 rotor (Beckman).
  • the dot-immunobinding assay was used to screen large numbers of supernatants of hybridomas producing monoclonal antibodies (Hawkes et al. (1982) Anal. Biochem. 119, 142- 147).
  • the crude plasma membrane isolate, the 10/30 plasma membrane isolate and/or the cytosol fractions containing the cellular antigen were dotted (1-3 ⁇ l) on a washed nitrocellulose filter paper grid (Bio-Rad).
  • the protein concentration of the "antigen" ranged from between 0.1 to 0.1 mg/ml. After thorough drying, the filter was washed in Tris Buffered Saline (TBS, 50 mM Tris- HCl, 200 mM NaCl, pH 7.4).
  • the enzyme linked immunosorbent assay was used for general enzyme immunoassay of antigen and screening for monoclonal antibody production.
  • Target cells were seeded (2-30 x 10 4 cells/ml) on microtiter plates (Falcon) 4-7 days before assay. Nonspecific binding sites on the plates were blocked with 1% (w/v) swine gelatin in a special media (FL), formulated to keep the cells viable.
  • FL media consisted of Dulbecco's Modified Eagle Medium (Gibco) supplemented with 15 mM Hepes, 0.3% NaCl, 10 mM NaN 3 and swine gelatin (1% for blocking or 0.3% for washing) pH 7.2-7.4.
  • Hybridoma culture fluids 50 ⁇ l per well were added and incubation was carried out at 37°C. for 60 minutes.
  • the plates were washed 4 times with FL media and F(ab') 2 goat anti-mouse IgG conjugated to horseradish peroxidase (1:1250 in 0.3% swine gelatin, 0.01M PBS) was used in place of the ⁇ -galactoxidase conjugate.
  • 100 ⁇ l of substrate 25 ml of 0.1 M citrate buffer pH 5.0, 10 ⁇ l of 30% H 2 O 2 and 10 mg of ⁇ -phenylenediamine (Sigma) was added to each well.
  • the plate was incubated for 30 minutes in the dark, and the reaction stopped with 50 ⁇ l of 2 N H 2 S(VWeIl.
  • the absorbance was determined at 490 nm using the Bio-Tek EIA reader.
  • Dried crude plasma membrane isolates from LNCaP cells or dried cells were used initially in the primary screening procedure of hybridoma culture fluids.
  • Approximately 400 ng of membrane protein in 50 ⁇ l of buffer (S3 mM Hepes, 0.3 mM MgCl 2 , 0.5 mM CaCl 2 ) was dried (35°C overnight) in 96 well flat bottom microtiter plates (Falcon).
  • Nonspecific binding sites on the plates were blocked with 1% swine gelatin in PBS containing 0.1% NaN 3 .
  • the wash buffer consisting of 0.01M Hepes and 0.2 ⁇ M of PMSF in saline (pH 7.6), all other reagents used were as described above for cell surface enzyme immunoassay.
  • LNCaP cells attach poorly to plastic wells and detach from the plastic surface during the ELISA procedure.
  • immunof ⁇ ltration on a disposable microfold system V&P Scientific
  • whole LNCaP cells were employed as a confirmatory assay for the dried membrane assay.
  • HSA human serum albumin
  • 2.5 x 10 4 LNCaP cells in 100 ⁇ l of 5% HSA buffer was deposited on the filter discs with vacuum. After washing the filters with 0.3% gelatin in 0.01M phosphate buffer, the plates were processed as described above.
  • Hybridoma cultures showing specificity restricted to the LNCaP cells and membranes were cloned by limiting dilution and subcloned in agarose (see e.g., Schreier et ⁇ /.(1980) Hybridoma Techniques pp. 11-15, Cold Spring Harbor Laboratory Press).
  • Stable cultures of antibody-producing hybridomas were expanded in complete media (RPMI 1640 media supplemented with 10% (w/v) heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 10 ⁇ g/ml insulin) and cryopreserved. After cloning, two stable monoclonal hybridoma cell lines were obtained and designated as 7El 1-C5 and 9H10-A4 respectively.
  • Monoclonals 7El 1-C5 and 9H10-A4 are of the IgGl subclass, as determined by double diffusion gel precipitation with isotype specific antisera (Miles). Consistent with this finding were observations that Protein A conjugated with either fluorescein or horseradish peroxidase (BIO-RAD) failed to react with smears of LNCaP cells following incubation with either monoclonal.
  • Conjugates were prepared at pH 8 and 9 in 0.1 M HEPES buffer, at chelant to protein ratios of 50 and 200, at 20 and 37°C for four hours. Antibody 7El 1-C5 concentration was 0.022 iiiM. Conjugates were purified by gel filtration chromatography. Table 1 summarizes the lot numbers and reaction conditions. As Table 2 indicates average loading values from 0.2 to 5.3 were observed. Increasing molar ratios, temperature and pH facilitated the conjugation reaction.
  • b ALV is the average loading value
  • MeO-DOTA-7El 1-C5 was complexed with 177 Lu at 2.25 mg/mL protein concentration at pH 5.2 at 20 0 C in sodium acetate buffer. Buffer concentrations of 50, 100 and 250 mM were studied. Complexation was monitored using size exclusion chromatography with radiometric and UV detection. Samples were analyzed after 30 min reaction time. Results are summarized in Table 4.
  • an antibody of the invention preferably 7El 1-C5
  • 7El 1-C5 is incubating said antibody in 0.1 M HEPES buffer at a pH of 8.5 with molar ratio of linker to antibody of approximately 200: 1 at 37°C for approximately 4 hours with slight agitation.
  • the resulting antibody will be conjugated with molar ratio of MeO-DOTA to antibody of approximately 9:1 or greater.
  • Optimal complexing reactions occur in 10OmM Na-Actetate, pH 5.5 at 20°C for 0.5 hours.
  • the resulting antibody is 1.2mCi/mg of 7El 1-C5 at 90% complexation.
  • Example 6 Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500)
  • Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) is comprised of 7El 1C5-3 monoclonal antibody (CYT-351) that is currently used in the manufacture of its commercial product ProstaScint®.
  • ProstaScint® is comprised of CYT-351 conjugated via periodate oxidation of the carbohydrate groups located on the heavy chains to the linker-chelator GYK-DTPA HCl [glycyl-tyrosyl-(N-C-diethylenetriaminepentaacetic acid)-lysine hydrochloride] which is complexed with the gamma emiting radioistope 111 In.
  • Anti-PSMA-meO-DOTA Immunoconjugate is comprised of CYT-351 covalently conjugated to the linker-chelator meO- DOTA [ ⁇ -(5-isothiocyanato-2-methoxyphenyl) ⁇ 1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7, 10- tetraacetic acid].
  • the CYT-351-meO-DOTA immunoconjugate has been shown to be stable in human serum thereby decreasing the chance for secondary toxicities as a result of shed linker and/or radioisotopes.
  • CYT-351 is a murine IgGl monoclonal antibody secreted by a murine/murine hybridoma cell line, which was produced by immunizing BALB/c mice with live LNCaP human prostatic adenocarcinoma cells and partially purified LNCaP plasma membranes.
  • the LNCaP cell line used to immunize the mice is a well characterized continuous cell line which was established from a needle biopsy taken from a lymph node metastasis of human prostatic adenocarcinoma.
  • LNCaP cells grow readily in vitro, form clones in semisolid media, show an aneuploid (modal number 76- 91) human male karyotype with several marker chromosomes and maintain the malignant properties of an adenocarcinoma.
  • the CYT-351 hybridoma was established and originally described by Horoszewicez et al. (1987) Anticancer Res. 7, 927-936 and U.S. Patents 5,162,504 and 5,578,484). Spleen cells from mice immunized with live LNCaP cells were fused with P3X63Ag8.653 murine myeloma cells. The cells were cloned twice by limited dilution cloning and a stable hybridoma, designated hybridoma 7El 1-C5, was expanded and cryopreserved. This clone secreted a prostate-specific monoclonal antibody of the IgGl subclass which was originally designated monoclonal antibody 7E11-C5.
  • a culture of the CYT-351 seed stock was used to establish a 100 vial Master Cell Bank
  • MCB MCB
  • FBS fetal bovine serum
  • the cells were subsequently expanded into 75 cm 2 flasks, 150 cm 2 flasks, a 500 ml spinner flask, and finally on into a three liter spinner.
  • the cells were harvested and placed into freezing medium (basal medium supplemented with 20% FBS and 10% DMSO).
  • the cells were then aliquoted into 100 vials, each containing approximately 9 x 10 6 cells and labeled with the designation 2MM0180- M001-9M, and subsequently stored in the vapor phase of liquid nitrogen.
  • Ten vials from the serum-grown MCB were used for tests to determine if the preparation was sterile and free of infectious adventitious agents. The results of these tests demonstrated that the CYT-351 MCB was sterile and free of infectious adventitious agents
  • Methoxy-DOTA ( ⁇ -(5-isothiocyanato-2-methoxyphenyl)-l,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid) is prepared from a purely synthetic process.
  • meDOTA and its methods of use and manufacture is disclosed in U.S. Patents 5,435,990 and 5,652,361 both of which are herein incorporated by reference in their entirety.
  • CYT-351 intermediate antibody for use in producing Anti-PSMA-meO-DOTA Immunoconjugate CYT-500
  • the cell banks, components, raw materials and manufacturing process used to produce CYT-351 intermediate antibody for use in producing Anti-PSMA-meO-DOTA Immunoconjugate are done so in accordance with the GMP manufacturing process.
  • the growth/production medium for the CYT-351 hybridoma is a defined, serum-free media available from HyClone Laboratory (HyQ-CCMTM) and is comprised of 925 basal medium.
  • Cell culture is performed in an AcuSyst-Xcell hollow fiber bioreactor and pH, temperature and oxygen levels monitored throughout the run. Samples are removed to monitor glucose, lactate and CYT-351 levels. Media feed is achieved via peristaltic pump. Medium is perfused through the bioreactor and the conditioned medium containing CYT-351 is harvested, clarified by filtration and stored at 2 to 8 0 C. The production run typically lasts for 60 to 70 days.
  • Each CYT-351 harvest is sampled and tested for CYT-351 titer, immunoreactivity, endotoxin and bioburden. Prior to purification, pooled harvest samples are tested minimally for: CYT-351 concentration, Mycoplasma, sterility and virus by reverse transcriptase, XC Plaque, S+L- Focus and in vitro viral testing.
  • CYT-351 Harvest and purification of CYT-351 are performed in classified rooms with appropriate environmental monitoring to allow for aseptic processing.
  • the CYT-351 harvest is filtered through a 0.45 ⁇ m filter, concentrated to approximately 6 to 12 mg/ml CYT-351 using a Pellicon tangential-flow ultrafiltration device fitted with a 30 kDa cutoff membrane.
  • the concentrated crude CYT-351 product is passed over a Sephadex G-25 column to remove low molecular weight moieties.
  • the G-25 column is equilibrated and eluted with 0.7 M ammonium sulfate (pH 8.0 to 8.4).
  • the eluted protein (CYT-351) peak is loaded onto a Protein A affinity column equilibrated with 0.7 M ammonium sulfate.
  • the loaded Protein A column is washed with thirty (30) column volumes of 0.7 M ammonium sulfate followed by a short wash with 55 mM sodium acetate (pH 7.0 to 8.5).
  • Bound CYT-351 is eluted from the Protein A column with 55 mM sodium acetate (pH 4.0 to 4.5) and the pH of the eluted product adjusted to 5.1 to 5.3 with 55 mM sodium acetate (pH 7.0 to 8.5).
  • the Protein A purified material is passed over a DEAE Sepharose column equilibrated in 55 mM sodium acetate (pH 5.1 to 5.3). This is a passive purification step in that the CYT-351 passes over the column whereas DNA, albumin and other acidic components bind to the support.
  • the CYT-351 peak is then loaded onto a S-Sepharose column equilibrated with 55 mM sodium acetate (pH 5.1 to 5.3). The column is washed with 10 mM sodium phosphate buffer (pH 5.9 to 6.1). The bound CYT-351 is eluted with 10 mM phosphate buffered saline (pH 5.9 to 6.1). Purified CYT-351 is filtered through a sterile 0.22 ⁇ m, sampled for Quality Control testing and stored at 2 to 8°C until needed for conjugation. Sterile filtered bulk CYT-351 has an approved shelf life of three years at 2 to 8°C.
  • Example 10 Manufacturing Process for the Immunoconjugate CYT-500
  • the purified CYT-351 Prior to conjugation, the purified CYT-351 is passed through a DV-20 (PALL) virus removal filter.
  • a DV-20 (PALL) virus removal filter Prior to conjugation, the purified CYT-351 is passed through a DV-20 (PALL) virus removal filter.
  • An additional 5 to 6 log viral removal is obtained using the DV-20 filter, resulting in approximately 14 log removal.
  • Purified monoclonal antibody CYT-351 is combined with 0.22 ⁇ m filtered (cellulose acetate) meO-DOTA in 0.5 M HEPES (pH 8.85).
  • the linker to CYT-351 ratio is 70: 1 with a total of 6 grams CYT-351 used for the toxicology lot (clinical lots also are 6 gram CYT-351 scale).
  • the reaction mixture is incubated for three hours at 35 to 37°C with gentle stirring. Following three hours, the reaction mixture is adjusted to 7.0 with 1 M acetic acid to slow the reaction.
  • the resultant product was concentrated from approximately 1900 to 300 ml using a Millipore Labscale TFF system with one Pelicon XL Biomax 50 filter. The concentrate was stored overnight at 2-8°C.
  • the concentrate was chromatographed with 0.1M sodium acetate (pH 5.5) on a 9 x 90 cm Superose 12 column.
  • the main (product) fraction was collected and concentrated to approximately 21 mg/ml using a Millipore Labscale TFF system with one Pelicon XL Biomax 50 filter.
  • This material (CYT-500) was filtered through a 0.22 ⁇ m filter and stored at 2 to 8°C.
  • the bulk CYT-500 is stored at 2 to 8 0 C and tested for contaminants before being released for use.
  • Released CYT-351 is filtered through a sterile 0.22 ⁇ m filter and filled into 10 ml Type 1 borosilicate glass vials and stoppered with presterilized 20 mm stoppers.
  • the filled, unlabeled vials are sealed with 20 mm flip-off crimp, visually inspected and sampled for Quality Control testing. Vials are placed in trays marked "quarantine" pending release.
  • antibody-chelating agent immunoconjugates form kinetically inert complexes with metals of interest, in this case, 177 Lu. These complexes must be stable following conjugating to a protein and should stay intact in vivo to avoid secondary toxicities. Similarly, loss of lanthanide metals can result in toxic effects, such as radioactive doses to the liver and bone. Accordingly, we tested serum stability of 177 Lu labeled CYT-500 and compared it to in In-labeled ProstaScint.
  • the purpose of this study was to determine the potential toxicity (including neurotoxicity) of Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) when administered once by intravenous injection to male Sprague Dawley rats.
  • Eighty male rats were randomly assigned to one of four groups and administered 100 mM sodium acetate buffer (control article) or Anti- PSMA-meO-DOTA Immunoconjugate (CYT-500) at 3, 15 or 30 mg/kg once on Study Day (SD).
  • Forty rats (10/group) were subjected to a full gross necropsy on SD 4; the remaining rats were necropsied on SD 15.
  • An additional 27 rats were assigned to one of the three treated groups (9/group) and blood was collected at selected timepoints for future toxicokinetic profiling.
  • the purpose of this study was to determine the potential toxicity of Anti-PSMA-meO- DOTA Immunoconjugate (CYT-500) when administered once by intravenous injection to male beagle dogs.
  • Twenty four male dogs were randomly assigned to one of four groups and administered 100 mM sodium acetate buffer (control article) or Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) at 0.6, 3 or 6 mg/kg once on SD 1. Twelve dogs (three per group) were subjected to a full gross necropsy on SD 4; the remaining 12 dogs were necropsied on SD 15.
  • Parameters evaluated included mortality, clinical observations, body weights, food consumption, ophthalmology, cardiology, clinical pathology, gross pathology, absolute and relative organ weights, and histopathology.
  • Treatment with Anti-PSMA-meO-DOTA Immunoconjugate had no effect on mortality, clinical observations, body weights, food consumption, ophthalmology, cardiology, clinical pathology, gross pathology or absolute and relative organ weights.
  • Test article related findings consisted of vasculitis of the central veins of the liver in treated animals. Lesions were more pronounced in SD 4 animals and, although present, appeared to be resolving in SD 15 animals. The most severe lesions in SD 4 animals were seen in animals treated at 3 or 6 mg/kg (10 and 2Ox the anticipated human dose, respectively). By SD 15, the lesions were milder overall, suggesting that with additional time resolution may be possible.
  • intravenous injections of Anti-PSMA-meO-DOTA Immunoconjugate were generally well tolerated.
  • the purpose of this study was to evaluate cardiovascular safety following intravenous administration of Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) in male Beagle dogs. Seven male dogs were given an intravenous injection of 100 mM sodium acetate buffer on SD 1, and Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) at 0.6 mg/kg on SD 8, 3 mg/kg on SD 15, and 6 mg/kg on SD 22 and 29. Each dose administration was followed by at least a one- week wash-out period. Cardiovascular profiling and body temperature data were collected via telemetry following doses on SD 1, 8, 15 and 22. Other parameters evaluated included mortality, clinical observations, and body weights.
  • the purpose of this study was to evaluate respiratory function following intravenous administration of Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) in male Beagle dogs.
  • Six male dogs were given an intravenous injection of 100 mM sodium acetate buffer on SD 1, and Anti-PSMA-meO-DOTA Immunoconjugate (CYT-500) at 6 mg/kg on SD 4.
  • Parameters evaluated included mortality, clinical observations, body weights and respiratory function assessment.
  • Respiratory function assessment included respiratory rate, saturated blood oxygen levels (Sp ⁇ 2 ) and end-tidal pressures (ETCO 2 ).

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Abstract

L'invention concerne de nouvelles compositions, des méthodes de traitement du cancer, des méthodes de diagnostic du cancer, des compositions pharmaceutiques et des méthodes pour fabriquer des anticorps conjugués comprenant un anticorps qui se lie immunospécifiquement à l'antigène de la membrane spécifique à la prostate et est conjugué à un radio-isotope par une liaison MeO-DOTA.
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EP3184539A3 (fr) 2001-10-23 2017-09-13 PSMA Development Company L.L.C. Anticorps contre le psma
US20050215472A1 (en) 2001-10-23 2005-09-29 Psma Development Company, Llc PSMA formulations and uses thereof
EP1726650A1 (fr) * 2005-05-27 2006-11-29 Universitätsklinikum Freiburg Anticorps monoclonaux et fragments d'anticorps monocaténaires contre l'antigène spécifique de surface membranaire de la prostate
GB0624587D0 (en) * 2006-12-08 2007-01-17 Univ Muenchen Tech Chelating agent
CN103372221B (zh) * 2007-01-11 2016-08-24 免疫医学股份有限公司 用于蛋白质、肽和其他分子的改进的f-18标记的方法和组合物
US8889100B2 (en) 2007-01-11 2014-11-18 Immunomedics, Inc. Methods and compositions for improved F-18 labeling of proteins, peptides and other molecules
US8545809B2 (en) 2007-01-11 2013-10-01 Immunomedics, Inc. Methods and compositions for improved 18F labeling of proteins, peptides and other molecules
EP2326350B1 (fr) 2008-09-08 2013-09-04 Psma Development Company, L.L.C. Composés pour tuer des cellules cancéreuses exprimant psma et résistantes au taxane
EP3495000A1 (fr) * 2009-02-17 2019-06-12 Cornell Research Foundation, Inc. Procédés et kits pour le diagnostic du cancer et la prédiction d'une valeur thérapeutique
WO2016130819A2 (fr) * 2015-02-11 2016-08-18 Emergent Product Development Seattle Llc Compositions et méthodes de polythérapie combinées à des protéines se liant à l'antigène membranaire spécifique de la prostate
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CA3050085A1 (fr) 2017-01-20 2018-07-26 Juno Therapeutics Gmbh Conjugues de surface cellulaire et compositions cellulaires et methodes associees
JP7284707B2 (ja) 2017-04-07 2023-05-31 ジュノー セラピューティクス インコーポレイテッド 前立腺特異的膜抗原（ｐｓｍａ）またはその改変型を発現する操作された細胞および関連方法

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WO2004058309A1 (fr) * 2002-12-23 2004-07-15 Human Genome Sciences, Inc. Conjugue neutrokine-alpha, complexe neutrokine-alpha, et leurs utilisations
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