WO2018142323A1

WO2018142323A1 - Anti-psma antibodies and uses thereof for diagnostic and therapeutic applications

Info

Publication number: WO2018142323A1
Application number: PCT/IB2018/050644
Authority: WO
Inventors: Serge Moffett; Traian Sulea
Original assignee: Proscan Rx Pharma Inc.
Priority date: 2017-02-02
Filing date: 2018-02-01
Publication date: 2018-08-09

Abstract

Described herein are diagnostic and therapeutic antibodies and antigen binding fragments capable of binding to prostate specific membrane antigen (PSMA). Also described are pharmaceutical compositions and kits comprising these antibodies or antigen binding fragments. These antibodies and antigen binding fragments may be used in the detection and/or treatment of cancer, particularly prostate cancer.

Description

ANTI-PSMA ANTIBODIES AND USES THEREOF FOR DIAGNOSTIC AND

THERAPEUTIC APPLICATIONS

FIELD OF INVENTION

The present invention relates to the field of antibodies (Ab) and to antigen binding fragments thereof. More specifically, the invention relates to diagnostic and therapeutic antibodies and antigen binding fragments capable of binding to prostate specific membrane antigen (PSMA).

BACKGROUND OF INVENTION

Prostate cancer is the most commonly diagnosed nonskin malignancy in males from developed countries. It is estimated that one in six males will be diagnosed with prostate cancer (PCa) in their lifetime. The diagnosis of PCa has greatly improved following the use of serum-based markers such as the prostate-specific antigen (PSA). However, the use of tumor-associated markers offers alternative strategies in disease management and may prove useful for in vivo tumor imaging purposes and further development of targeted therapies.

Identification of the prostate specific membrane antigen (PSMA) marker, a tumor associated marker, has generated interest for both applications. PSMA is a glycoprotein highly restricted to prostate secretory epithelial cell membranes. Its expression has been correlated with tumor aggressiveness. Various immunohistological studies have demonstrated increased PSMA levels in virtually all cases of prostatic carcinoma compared to those levels in benign prostate epithelial cells. Intense PSMA staining is found in all stages of the disease, including prostatic intraepithelial neoplasia, late stage androgen- independent prostate cancer and secondary prostate tumors localized to lymph nodes, bone, soft tissue, and lungs. PSMA was originally identified as the molecule recognized by 7E1 1 , a monoclonal antibody (MAb) reactive to the prostate cancer cell line LNCaP. It was subsequently cloned from these cells as a 2.65 kb cDNA encoding a 750 amino acid cell surface type II integral membrane glycoprotein of 100 kDa. PSMA forms a noncovalent homodimer that possesses glutamate carboxypeptidase activity based on its ability to process the neuropeptide N-acetylaspartylglutamate and glutamate-conjugated folate derivatives. Although the precise biological role played by PSMA in disease pathogenesis remains elusive, its overexpression in PCa might potentially be associated with the growth balance of the prostate gland. Indeed, recent evidence suggests that PSMA performs multiple physiological functions related to cell survival and migration.

Antibody-based therapeutics have emerged as important components of therapies for an increasing number of human malignancies in such fields as oncology, inflammatory and infectious diseases. In most cases, the basis of the therapeutic function is the high degree of specificity and affinity the antibody- based drug has for its target antigen. Arming monoclonal antibodies with drugs, toxins, or radionuclides is yet another strategy by which mAbs may induce therapeutic effect. By combining the exquisite targeting specificity of antibody with the tumor killing power of toxic effector molecules, immunoconjugates permit sensitive discrimination between target and normal tissue thereby resulting in fewer side effects than most conventional chemotherapeutic drugs.

Given the physical properties of PSMA and its expression pattern in relation to disease progression, its large extracellular domain provides an excellent target in the development of ligands for diagnostic and therapeutic intervention. The first PSMA-specific MAb reported, 7E1 1 , was subsequently developed and commercialized as a diagnostic agent for tumor imaging (ProstaScint, Cytogen, Princeton, NJ). However, this antibody recognizes an intracellular epitope of PSMA which limits its usefulness as an imaging agent for the detection of PSMA. More recently, MAbs such as J591 that recognize the extracellular portion of PSMA were developed, however such antibodies have uncharacterized epitope specificities. The development of anti-PSMA antibodies with diagnostic and/or therapeutic activity is needed. The present invention addresses these needs and other needs as it will be apparent from review of the disclosure and description of the features of the invention hereinafter.

BRIEF SUMMARY OF THE INVENTION

The present application relates to anti-PSMA antibodies or antigen binding fragments thereof and their use for the treatment of cancer, for the detection of cancer cells or for the diagnosis or prognosis of cancer.

The present invention also relates to antibodies and antigen binding fragments, cells comprising or expressing these antibodies or antigen binding fragments as well as kits useful for the treatment, detection of tumor cells or tumor neovasculature or in the diagnosis of cancer.

The present application particularly relates to the use of anti-PSMA antibodies for treatment of prostate cancer.

In further aspects, the present invention relates to method of treatment and to method of detection using the antibody or antigen binding fragment described herein.

BRIEF DESCRIPTION OF THE FIGURES

In order that the invention may be readily understood, embodiments of the invention are illustrated by way of example in the accompanying drawings. Figure 1 is a line graph depicting immunoreactivity of humanized antibody variants to prostate cancer cell. Serially diluted cell culture media containing the antibody at the indicated concentrations were tested by ELISA on a cell membrane preparation of the PSMA positive LNCaP, and PSMA negative PC-3 cell line. Figure 2 is a line graph depicting immunoreactivity of purified humanized antibody variants to prostate cancer cell. Serial dilution of the antibody at the concentration indicated were tested by ELISA on a cell membrane preparation of the PSMA positive LNCaP, and PSMA negative PC-3 cell line.

Figure 3 is a bar graph depicting the ratio of immunoreactivity on LNCaP and PC-3 cell. Immunoreativity was measured by ELISA at an antibody concentration of 10 pg/ml.

Figure 4 is a bar graph depicting the ratio of antibody reactivity on PSMA and BSA. Immunoreativity was measured by ELISA at an antibody concentration of 10 pg/ml.

Figure 5 is a panel showing pictures of immunostaining of murine and CH humanized antibodies in normal and prostate cancer tissues in the presence or absence of a competing antigen. Immunostaining is seen in the apical site of epithelial cells within the luminal side of gland (arrow).

Figure 6 is an amino acid alignment of the murine (SEQ ID NO: 6) and humanized (SEQ ID NO: 1 ) light chain variable region (upper alignment) as well as of the murine (SEQ ID NO: 5) and humanized (SEQ ID NO: 2) heavy chain variable region (lower alignment). The complementary determining regions are underlined by a solid line or a dash depending on the definition used. The number of divergent residues in the sequence is indicated. It is noted that residues RADAAP at the end of SEQ ID NO: 6 are not shown in the sequence alignment.

Figure 7 is a line graph depicting immunoreactivity of a humanized monoclonal antibody by ELISA to cell membrane preparation of LNCaP, PC-3 and purified PSMA, BSA.

Figure 8 depicts the results of amino acid alignment one humanized Vlight sequence according to the present invention with the original murine Vlight sequence and with various human Vlight sequences. Figure 9 depicts the results of amino acid alignment one humanized Vheavy sequence according to the present invention with the original murine Vheavy sequence and with various human Vheavy sequences.

Further details of the invention and its advantages will be apparent from the detailed description included below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is concerned with antibodies and antigen binding fragments capable of binding to prostate specific membrane antigen (PSMA).

Anti-PSMA antibodies have been described in International PCT publication WO 2010/1 18522 (PCT/CA2010/000567), International PCT publication WO 2009/127046 (PCT/CA2009/000470), International PCT publication WO 2005/100404 (PCT/CA2005/000601 ), and International PCT publication WO 2004/067570 (PCT/CA2004/000127), the entire content of these patent applications being incorporated herein by reference. According to one particular aspect of the invention, the antibodies of the present invention may be less immunogenic for human than the antibodies described in International PCT publication WO 2010/1 18522 (PCT/CA2010/000567) and/or than the antibodies described in International PCT publication WO 2009/127046 (PCT/CA2009/000470). Indeed, the applicant has attended to the humanization of one of the antibody described in in International PCT publication WO/2010/1 18522 (PCT/CA2010/000567) and has come to the unexpected discovery that an antibody comprising a combination of the variable regions set forth in SEQ ID NO: 1 and SEQ ID NO:5 have advantages over other combinations of variable regions. Without being bound to a theory it appears that this combination of variable regions retains better specificity towards the PSMA antigen, whereas other combination may show depending on the assay used, some level of non- specificity. As used herein, the term "antibody" refers to intact antibody, monoclonal or polyclonal antibodies. The term "antibody" also encompasses multispecific antibodies such as bispecific antibodies. Human antibodies are usually made of two light chains and two heavy chains each comprising variable regions and constant regions. The light chain variable region comprises 3 CDRs, identified herein as CDRL1 , CDRL2 and CDRL3 flanked by framework regions. The heavy chain variable region comprises 3 CDRs, identified herein as CDRH1 , CDRH2 and CDRH3 flanked by framework regions.

The term "antigen-binding fragment", as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V_L, V_H, C_L and C_Hi domains; (ii) a F(ab')₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_H and Cm domains; (iv) a Fv fragment consisting of the V_L and V_H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a V_H domain; (vi) an isolated complementarity determining region (CDR), e.g., V_H CDR3 comprising or not additional sequence (linker, framework region(s)etc) and (v) a combination of two to six isolated CDRs comprising or not additional sequence (linker, framework region(s) etc.). Furthermore, although the two domains of the Fv fragment, V_L and V_H, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single polypeptide chain in which the V_L and V_H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. Furthermore, the antigen-binding fragments include binding-domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide (such as a heavy chain variable region, a light chain variable region, or a heavy chain variable region fused to a light chain variable region via a linker peptide) that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The hinge region may be modified by replacing one or more cysteine residues with serine residues so as to prevent dimerization. Such binding-domain immunoglobulin fusion proteins are further disclosed for instance in US patent publications US 2003/01 18592 and US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

A typical antigen binding site is comprised of the variable regions formed by the pairing of a light chain immunoglobulin and a heavy chain immunoglobulin. The structure of the antibody variable regions is very consistent and exhibits very similar structures. These variable regions are typically comprised of relatively homologous framework regions (FR) interspaced with three hypervariable regions termed Complementarity Determining Regions (CDRs). The overall binding activity of the antigen binding fragment is often dictated by the sequence of the CDRs. The FRs often play a role in the proper positioning and alignment in three dimensions of the CDRs for optimal antigen binding.

In fact, because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that shows the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., 1998, Nature 332:323-327; Jones, P. et al., 1986, Nature 321 :522-525; and Queen, C. et al., 1989, Proc. Natl. Acad. See. U.S.A. 86: 10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V(D)J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody which contains mutations throughout the variable gene but typically clustered in the CDRs. For example, somatic mutations are relatively infrequent in the amino terminal portion of framework region 1 and in the carboxy- terminal portion of framework region 4. Furthermore, many somatic mutations do not significantly alter the binding properties of the antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody in order to recreate an intact recombinant antibody having binding properties similar to those of the original antibody. Partial heavy and light chain sequence spanning the CDR regions is typically sufficient for this purpose. The partial sequence is used to determine which germline variable and joining gene segments contributed to the recombined antibody variable genes. The germline sequence is then used to fill in missing portions of the variable regions. Heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized to create an entirely synthetic variable region clone. This process has certain advantages such as elimination or inclusion of particular restriction sites, or optimization of particular codons.

Of course, the totality or portions of the framework region of the antibody described herein may be used in conjunction with the CDRs in order to optimize the affinity, specificity or any other desired properties of the antibody.

Antibodies and/or antigen binding fragments of the present invention may originate, for example, from a mouse, a rat or any other mammal or from other sources such as through recombinant DNA technologies.

In accordance with the present invention, the antibody may "naked" or may be conjugated with a therapeutic agent or with a detectable label. The term "naked antibody or antigen binding fragment" refers to an antibody or antigen binding fragment which has the ability to induce cell death in vitro or in vivo, without needed to be conjugated with a toxin, drug or the like. The term "naked", in some instances may also refer to an antibody or antigen binding fragment which is optionally conjugated with a moiety which is considered as being therapeutic. An exemplary embodiment of a therapeutic agent may include for example, a cytotoxic agent (e.g., toxin, anti-mitotics and the like), a chemotherapeutic drug (e.g., taxanes, etc.), a radioisotope. An exemplary embodiment of a detectable moiety comprises for example, a radioisotope, an enzyme, etc. The present invention relates in one particular aspect to isolated antibodies or antigen binding fragments capable of binding to prostate specific membrane antigen (PSMA). More particularly, the present invention relates to diagnostic and/or therapeutic antibodies or antigen binding fragments having specificity for PSMA. The binding site of an antibody has mainly been attributed to the complementarity-determining regions (CDRs). In some instances, a single CDR (e.g., VH CDR3) may be sufficient to provide antigen recognition and specificity of the antibody. The polypeptide, antibody or antigen-binding fragment of the present invention may preferably comprise the heavy and light chain CDR3s of the antibodies listed in Figure 1. The polypeptide, antibody or antigen-binding fragment may further comprise the CDR2s of the antibodies listed in Figure 1. The polypeptide, antibody or antigen-binding fragment may also comprise the CDR1 s of the antibodies listed in Figure 1. The polypeptide, antibody or antigen- binding fragment may further comprise any combinations of the CDRs. CDRs may be identified by analyzing the amino acid sequence and/or structure of the variable domain of an antibody. Computer-implemented analysis and modeling of antigen-binding site are based on homology analysis comparing the target antibody sequence with those of antibodies with known structures or structural motifs in existing data bases. By using such homology-based modeling methods approximate three-dimensional structure of the target antibody is constructed (Kabat and Wu (1972) Proc. Natl. Acad. Sci. USA 69: 960 964). More recently, the canonical loop concept has been incorporated into the computer-implemented structural modeling of an antibody combining site (Chothia et al. (1989) Nature (London) 342:877; Chothia and Lesk JMB 196:901 (1987)). It is also possible to improve the modeling of CDRs of antibody structures by combining the homology-based modeling with conformational search procedures (Martin, A. C. R. (1989) PNAS 86: 9268-72). Antibody modeling software are also available for determining the CDRs (AbM : Accelrys, Cambridge, U.K.) When only one of the light chain variable domain or the heavy chain variable domain is available, an antibody or antigen-binding fragment may be reconstituted by screening a library of complementary variable domains using methods known in the art (Portolano et al. The Journal of Immunology (1993) 150:880-887, Clarkson et al., Nature (1991 ) 352:624-628). Antibodies and antigen binding fragments that binds to PSMA

The variable regions described herein may be fused with constant regions of a desired species thereby allowing recognition of the antibody by effector cells of the desired species. The constant region may originate, for example, from an lgG1 , lgG2, lgG3, or lgG4 subtype. In an embodiment of the invention, the constant region may be of human origin. In another embodiment of the invention, the constant region may be of murine origin. Cloning or synthesizing a constant region in frame with a variable region is well within the scope of a person of skill in the art and may be performed, for example, by recombinant DNA technology.

In certain embodiments of the present invention, antibodies that bind to PSMA may be of the lgG1 , lgG2, lgG3, or lgG4 subtype. More specific embodiments of the invention relates to an antibody of the lgG1 subtype. In another specific embodiment of the invention relates to an antibody of the lgG2 subtype. In yet another specific embodiment of the invention relates to an antibody of the lgG3 subtype. The antibody may be a humanized antibody of the lgG1 subtype that is biologically active in mediating antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CMC), or associated with immune complexes. The typical ADCC involves activation of natural killer (NK) cells and is reliant on the recognition of antibody-coated cells by Fc receptors on the surface of the NK cells. The Fc receptors recognize the Fc domain of antibodies such as is present on lgG1 , which bind to the surface of a target cell, in particular a cancerous cell that expresses an antigen, such as PSMA. Once bound to the Fc receptor of IgG the NK cell releases cytokines and cytotoxic granules that enter the target cell and promote cell death by triggering apoptosis.

An exemplary embodiment of the invention includes, for example an antibody comprising a light chain variable domain as described herein and a heavy chain variable domain described herein. Although the amino acid sequence of some of the antibodies or antigen binding regions have been obtained using recombinant DNA technology, the present invention encompass any isolated or purified antibodies having the same amino acid sequence, whether monoclonal, chimeric, human.

Variant antibody and antigen binding fragments

As indicated above, the present invention also encompasses variants of the antibodies or antigen binding fragments described herein. Variant antibodies or antigen binding fragments included are those having a variation in the amino acid sequence. For example, variant antibodies or antigen binding fragments included are those having at least one variant CDR (two, three, four, five or six variant CDRs), a variant light chain variable domain, a variant heavy chain variable domain, a variant light chain and/or a variant heavy chain. Variant antibodies or antigen binding fragments included in the present invention are those having, for example, similar or improved binding affinity in comparison with the original antibody or antigen binding fragment.

As used herein the term "variant" applies to any of the sequence described herein and includes for example, a variant CDR (either CDRL1 , CDRL2, CDRL3, CDRH1 , CDRH2 and/or CDRH3), a variant light chain variable domain, a variant heavy chain variable domain, a variant light chain, a variant heavy chain, a variant antibody, and a variant antigen binding fragment.

Variant antibodies or antigen binding fragments encompassed by the present invention are those which may comprise an insertion, a deletion or an amino acid substitution (conservative or non-conservative). These variants may have at least one amino acid residue in its amino acid sequence removed and a different residue inserted in its place.

The sites of greatest interest for substitutional mutagenesis include the hypervariable regions (CDRs), but modifications in the framework region or even in the constant region are also contemplated. Conservative substitutions may be made by exchanging an amino acid (of a CDR, variable chain, antibody, etc.) from one of the groups listed below (group 1 to 6) for another amino acid of the same group.

Other exemplary embodiment of conservative substitutions are shown in Table 1A under the heading of "preferred substitutions". If such substitutions result in a undesired property, then more substantial changes, denominated "exemplary substitutions" in Table 1A, or as further described below in reference to amino acid classes, may be introduced and the products screened.

It is known in the art that variants may be generated by substitutional mutagenesis and retain the biological activity of the polypeptides of the present invention. These variants have at least one amino acid residue in the amino acid sequence removed and a different residue inserted in its place. For example, one site of interest for substitutional mutagenesis may include a site in which particular residues obtained from various species are identical. Examples of substitutions identified as "conservative substitutions" are shown in Table 1A. If such substitutions result in a change not desired, then other type of substitutions, denominated "exemplary substitutions" in Table 1A, or as further described herein in reference to amino acid classes, are introduced and the products screened. Substantial modifications in function or immunological identity are accomplished by selecting substitutions that differ significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side chain properties:

(group 1 ) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine

(Val), Leucine (Leu), Isoleucine (He);

(group 2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr); (group 3) acidic: Aspartic acid (Asp), Glutamic acid (Glu);

(group 4) basic: Asparagine (Asn), Glutamine (Gin), Histidine (His), Lysine

(Lys), Arginine (Arg);

(group 5) residues that influence chain orientation: Glycine (Gly), Proline

(Pro); and

(group 6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe).

Non-conservative substitutions will entail exchanging a member of one of these classes for another.

Thus, in some cases, the basic amino acids Lys, Arg and His may be interchangeable; the acidic amino acids Asp and Glu may be interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gin, and Asn may be interchangeable; the non-polar aliphatic amino acids Gly, Ala, Val, He, and Leu are interchangeable but because of size Gly and Ala are more closely related and Val, lie and Leu are more closely related to each other, and the aromatic amino acids Phe, Trp and Tyr may be interchangeable. It should be further noted that if the polypeptides are made synthetically, substitutions by amino acids, which are not naturally encoded by DNA (non-naturally occurring or unnatural amino acid) may also be made. A non-naturally occurring amino acid is to be understood herein as an amino acid which is not naturally produced or found in a mammal. A non-naturally occurring amino acid comprises a D-amino acid, an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, etc. The inclusion of a non-naturally occurring amino acid in a defined polypeptide sequence will therefore generate a derivative of the original polypeptide.

Table 1A. Amino acid substitution

Polypeptides of the present invention may comprise for example, those containing amino acid sequences modified either by natural processes, such as posttranslational processing or by chemical modification techniques which are known in the art. Modifications may occur anywhere in a polypeptide including the polypeptide backbone, the amino acid side chains and the amino- or carboxy- terminus. A given polypeptide may contain many types of modifications. It is to be understood herein that more than one modification to the polypeptides described herein are encompassed by the present invention to the extent that the biological activity is substantially similar to the original polypeptide. Polypeptide modification may comprise, for example, amino acid insertion, deletion and substitution (i.e., replacement), either conservative or non-conservative (e.g., D- amino acids) in the polypeptide sequence where such changes do not substantially alter the overall biological activity of the polypeptide.

Variation in the amino acid sequence of the variant antibody or antigen binding fragment thus may include an amino acid addition, deletion, insertion, substitution etc., one or more modification in the backbone or side-chain of one or more amino acid, or an addition of a group or another molecule to one or more amino acids (side-chains or backbone).

Variant antibody or antigen binding fragment may have substantial sequence similarity and/or sequence identity in its amino acid sequence in comparison with that the original antibody or antigen binding fragment amino acid sequence. The degree of similarity between two sequences is based upon the percentage of identities (identical amino acids) and of conservative substitution.

In addition, a non-naturally occurring amino acid may substitute for a naturally occurring amino acid (i.e., non-naturally occurring conservative amino acid substitution or a non-naturally occurring non-conservative amino acid substitution). Generally, the degree of similarity and identity between variable chains has been determined herein using the Blast2™ sequence program (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250) using default settings, i.e., blastp program, BLOSUM62 matrix (open gap 1 1 and extension gap penalty 1 ; gapx dropoff 50, expect 10.0, word size 3) and activated filters.

Percent identity will therefore be indicative of amino acids which are identical in comparison with the original peptide and which may occupy the same or similar position.

Percent similarity will be indicative of amino acids which are identical and those which are replaced with conservative amino acid substitution in comparison with the original peptide at the same or similar position.

Variants of the present invention therefore comprise those which may have at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an original sequence or a portion of an original sequence.

Exemplary embodiments of variants are those having at least 81 % sequence identity to a sequence described herein and 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity with an original sequence or a portion of an original sequence.

Other exemplary embodiments of variants are those having at least 82% sequence identity to a sequence described herein and 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity with an original sequence or a portion of an original sequence.

Further exemplary embodiments of variants are those having at least 85% sequence identity to a sequence described herein and 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity with an original sequence or a portion of an original sequence.

Other exemplary embodiments of variants are those having at least 90% sequence identity to a sequence described herein and 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity with an original sequence or a portion of an original sequence.

Additional exemplary embodiments of variants are those having at least 95% sequence identity to a sequence described herein and 95%, 96%, 97%, 98%, 99% or 100% sequence similarity with an original sequence or a portion of an original sequence.

Yet additional exemplary embodiments of variants are those having at least 97% sequence identity to a sequence described herein and 97%, 98%, 99% or 100% sequence similarity with an original sequence or a portion of an original sequence.

For a purpose of concision the applicant provides herein a Table 1 B illustrating exemplary embodiments of individual variants encompassed by the present invention and comprising the specified % sequence identity and % sequence similarity. Each "X" is to be construed as defining a given variant.

The present invention encompasses CDRs, light chain variable domains, heavy chain variable domains, light chains, heavy chains, antibodies and/or antigen binding fragments which comprise at least 80% identity with the sequence described herein.

More particularly, the anti-PSMA antibody may have a light chain variable region as set forth in SEQ ID NO:1 and/or a heavy chain variable region as set forth in SEQ ID NO:2.

More particularly, the anti-PSMA antibody may have a light chain variable region as set forth in SEQ ID NO:1 and/or a heavy chain variable region as set forth in SEQ ID NO:5.

Complementarity determining regions (CDRs) are illustrated in bold and underlined:

SEQ ID NO: 1 (humanized light chain variable region):

DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQFLI YKASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQSTHVPYTFGGG

TKVEIK

SEQ ID NO:2 (humanized heavy chain variable region):

EVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYMHWVRQAPGQGLEWMGGI DPADGETKYDPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCVRSFDYW

GQGTLVTVSS

SEQ ID NO:5 (mouse heavy chain variable region)

EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGGIDP ADGETKYDPKFQDKATITTDTSSNTVYLQISSLTSEDTAVYYCVRSFDYWGQG

TTLTVSS

SEQ ID NO:6 (mouse light chain variable region)

DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKFLI YKASNRFSGVPDRFSGRGSGTDFTLKISRVEAEDLGVYFCFQSTHVPYTFGGG

TKLEIKRADAAP

281053.00039/98403226.5 In accordance with the present invention, the antibody may comprise the 3CDRs of the light chain variable region set forth in SEQ ID NO:1 and at least 80 to 1 12 consecutive amino acids of SEQ ID NO:1. The term "at least 80 to 1 12" comprises every possible individual number of amino acids comprised between 80 and 1 12 inclusively, such as 80, 81 , 82, 83, 85, 90, 95, 100, 105, 1 10 and up to 1 12.

In accordance with the present invention, the antibody may comprise the 3CDRs of the heavy chain variable region set forth in SEQ ID NO:2 and at least 80 to 1 13 consecutive amino acids of SEQ ID NO:2. The term "at least 80 to 1 13" comprises every possible individual number of amino acids comprised between 80 and 1 13 inclusively, such as for example, 80, 81 , 82, 83, 85, 90, 95, 100, 105, 1 10 and up to 1 13.

In accordance with the present invention, the antibody may comprise the 3CDRs of the heavy chain variable region set forth in SEQ ID NO:5 and at least 80 to 1 13 consecutive amino acids of SEQ ID NO:5. The term "at least 80 to 1 13" comprises every possible individual number of amino acids comprised between 80 and 1 13 inclusively, such as for example, 80, 81 , 82, 83, 85, 90, 95, 100, 105, 1 10 and up to 1 13.

The present invention relates in a further aspect to an antibody or antigen binding fragment thereof which may have a light chain variable region at least 80% identical (e.g., 85%, 90%, 95%, 99%) to SEQ ID NO:6 and/or a heavy chain variable region at least 80% identical (e.g., 85%, 90%, 95%, 99%) to SEQ ID NO:5 wherein the antibody or antigen binding fragment thereof may comprise, for example, at least one amino acid substitution in comparison with SEQ ID NO:6 or SEQ ID NO:5 and wherein the amino acid substitution may be, for example, outside of a complementarity determining region (CDR)

In accordance with the present invention, the at least one amino acid substitution may be, for example, in the light chain variable region. In accordance with the present invention, the at least one amino acid substitution may be, for example, in the heavy chain variable region.

The amino acid substitution may be conservative or non-conservative. In a more specific embodiment, the amino acid substitution may be conservative.

In accordance with the present invention the antibody or antigen binding fragment may comprise, for example,

a) a light chain variable region which may comprise at least 90 consecutive amino acids of SEQ ID NO:8 and a heavy chain variable region which may comprise at least 90 consecutive amino acids of any of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:1 1 , SEQ ID NO:12 or SEQ ID NO:13;

b) a light chain variable region which may comprise at least 90 consecutive amino acids of SEQ ID NO:9 and a heavy chain variable region which may comprise at least 90 consecutive amino acids of any of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:1 1 , SEQ ID NO:12 or SEQ ID NO:13;

c) a light chain variable region which may comprise amino acids at least 90 consecutive amino acids of SEQ ID NO: 10 and a heavy chain variable region which may comprise at least 90 consecutive amino acids of any of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO: 1 1 , SEQ ID NO:12 or SEQ ID NO:13 or;

d) a light chain variable region which may comprise at least 90 consecutive amino acids of SEQ ID NO:1 and a heavy chain variable region which may comprise at least 90 consecutive amino acids of any of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:1 1 , SEQ ID NO:12 or SEQ ID NO:13.

In accordance with a more specific embodiment of the invention, the light chain variable region may comprise at least 90 consecutive amino acids of SEQ ID NO: 1 and the heavy chain variable region may comprise at least 90 consecutive amino acids of SEQ ID NO:5.

In accordance with an even more specific embodiment of the invention, the light chain variable region may be as set forth in SEQ ID NO:1 and the heavy chain variable region may be as set forth in SEQ ID NO:5.

In accordance with another specific embodiment of the invention, the light chain variable region may comprise at least 90 consecutive amino acids of SEQ ID NO: 1 and the heavy chain variable region may comprise at least 90 consecutive amino acids of SEQ ID NO:2. In accordance with yet another specific embodiment of the invention, the light chain variable region may be as set forth in SEQ ID NO:1 and the heavy chain variable region may be as set forth in SEQ ID NO:2.

The constant region of the antibody may comprise, for example, amino acids of a human constant region. The constant region may comprise amino acids, from an IgG constant region.

In accordance with an embodiment of the invention, the antibody may particularly be from an IgGl

Also in accordance with an embodiment of the invention, the antibody may be from an lgG2. Alternatively, the anti-PSMA antibody may have a light chain as set forth in SEQ ID NO:3 and/or a heavy chain as set forth in SEQ ID NO:4.

Preferably, the anti-PSMA antibody may have a light chain as set forth in SEQ ID NO:3 and/or a heavy chain as set forth in SEQ ID NO:7.

SEQ ID NO:3: Humanized Variable Light + human CL

DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQFLI YKASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQSTHVPYTFGGG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

SEQ ID N0:4: Humanized Variable Heavy + human CH1 Fc1

GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS

GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH

EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP

SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGK

SEQ ID N0:7: Murine Variable Heavy + human Constant Heavy Fc1

TTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT

SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK

SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS

NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV

EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA

LHNHYTQKSLSLSPGK

The antibodies of the present invention may be polyclonal, monoclonal, chimeric, hybrid, human or humanized.

The term "humanized" encompassed partially humanized or fully humanized. The antibodies of the present invention may thus comprise a variable region that is partially or fully humanized. The antibody of the present invention may also comprise a variable region that is not humanized.

As used herein the term "hybrid" refers to an antibody comprising a combination including one of a light chain variable region or heavy chain variable region that is partially or fully humanized and the other of the light chain variable region or heavy chain variable region that is not humanized (e.g., from a murine, mouse or chimeric antibody).

In accordance with the present invention, the antibody may comprise one of its light chain or heavy chain variable region having an amino acid sequence identical to that of the corresponding mouse antibody and the other of its light chain or heavy chain variable region having an amino acid sequence that is humanized.

Antibody fragments such as scFV, Fab, Fab', (Fab')₂ etc. are also contemplated. The antibodies of the present invention and especially those comprising a light chain variable region as set forth in SEQ ID NO:1 and a heavy chain variable region as set forth in SEQ ID NO:5 have the ability to bind to prostate specific membrane antigen (PSMA) as demonstrated in ELISA assays, Western blot, Bioacore™ or immunohistochemistry. The antibodies of the present invention have identical CDRs with antibodies that have been found useful in the treatment, detection of cancer. It is expected that the characteristics of the antibody will be preserved but with the added benefit of being humanized and retaining their binding characteristics (e.g., specificity).

Other exemplary embodiments of the invention include for example, antibodies or antigen binding fragments comprising a consensus light chain variable region and/or a consensus heavy chain variable region described herein. SEQ ID NO:8 (consensus light chain variable reqion-1 )

DVVMTQXPLSLPVXXGXXASI SCRSSQSLVHSNGNTYLHWYLQKPGQSPXFLI YKASN RFSGVPDRFSGXGSGTDFTLKISRVEAEDXGVYXCFQSTHVPYTFGGG

TKXEIK

wherein X is any amino acid or wherein at least one of the amino acid identified by X is an amino acid substitution in comparison with a corresponding amino acid in the polypeptide set forth in SEQ ID NO:6 (the murine VL) . The amino acid substitution may be, for example conservative or non-conservative. In accordance with the invention, the amino acid substitution may be conservative.

SEQ ID NO:9 (consensus light chain variable region- 2)

DWMTQXiAPLSLPVXiRXir.GXinXiFASISCRSSQSLVHSNGNTYLHWYLQKPG

QSPXI FFLIYKASN RFSGVPDRFSGXI P;GSGTDFTLKI SRVEAEDXIHGVYXI ICFQ

STHVPYTFGGGTKXHEI K

wherein Xi_A may be, for example, a neutral hydrophilic amino acid (e.g., serine or threonine);

wherein Xi_B may be, for example, a neutral hydrophilic amino acid (e.g., threonine or serine);

wherein Xi_C may be, for example, proline or leucine;

wherein Xi_D may be, for example, an acidic amino acid (e.g. , glutamic acid or aspartic acid):

wherein Xi_E may be, for example, proline or glutamine;

wherein X _F may be, for example, a basic amino acid (e.g., glutamine or lysine);

wherein Xi_G may be, for example, serine or arginine;

wherein Xi_H may be, for example, an hydrophobic amino acid (e.g., valine or leucine);

wherein Xn may be, for example, an aromatic amino acid (e.g. , tyrosine or phenylalanine), and;

wherein X-u may be, for example, an hydrophobic amino acid (e.g. , valine or leucine). In accordance with the present invention, the light chain variable region of SEQ ID NO:9 may have at least one of its amino acid identified by "X" that is different than the corresponding amino acid of SEQ ID NO:6.

SEQ ID NO: 10 (consensus light chain variable reqion-3)

DVVMTQX_{1 a}PLSLPVX_{1 h}X r.GX nX _PASISCRSSQSLVHSNGNTYLHWYLQKPGQ SPX fFLIYKASNRFSGVPDRFSGXmGSGTDFTLKISRVEAEDX hGVYX iCFQST HVPYTFGGGTKXnEIK

wherein Xi_a may be, for example, serine or threonine;

wherein Xi_b may be, for example, threonine or serine;

wherein Xi_c may be, for example, proline or leucine;

wherein Xi_d may be, for example, glutamic acid or aspartic acid;

wherein Xi_e may be, for example, proline or glutamine;

wherein Xi_f may be, for example, glutamine or lysine;

wherein Xi_g may be, for example, serine or arginine;

wherein X-i_h may be, for example, valine or leucine;

wherein X-π may be, for example, tyrosine or phenylalanine, and;

wherein X^ may be, for example, valine or leucine.

In accordance with the present invention, the light chain variable region of SEQ ID NO: 10 may have at least one of its amino acid identified by "X" that is different than the corresponding amino acid of SEQ ID NO:6. SEQ ID NO:1 1 (consensus heavy chain variable region-1 )

EVQLXQSGAEXXKPGASVKXSCXASGFNIKDTYMHWVXQXPXQGLEWXGGID PADGETKYDPKFQXXXTXTXDTSXXTVYXXXSSLXSEDTAVYYCVRSFDYWG

QGTXXTVSS

wherein an amino acid identified by "X" in SEQ ID NO: 1 1 is a) any amino acid, b) an amino acid identical to the corresponding amino acid of SEQ ID NO:5 or, c) wherein at least one of the amino acid identified by X is an amino acid substitution in comparison with a corresponding amino acid in the polypeptide set forth in SEQ ID NO:5 (the murine VH) . The amino acid substitution may be, for example conservative or non-conservative. In accordance with the invention, the amino acid substitution may be conservative. SEQ ID NO: 12 (consensus heavy chain variable reqion-2)

EVQLX_?AQSGAEX_?RX_?r.KPGASVKX_?nSCX_?FASGFNIKDTYMHWVX_?FQX_?nPX_?H QGLE\A^_?iGGIDPADGETKYDPKF(»9jX_?KX9i TX_?MTX_?NDTSX_?nX_?pTVYX_?nX_?RX

2SSSLX2TSEDTAVYYCVRSFDYWGQGTX₂UX2VTVSS

wherein X_2A may be, for example, glutamine or valine;

wherein X₂B may be, for example, an hydrophobic amino acid (e.g., leucine or valine);

wherein X₂c may be, for example, valine or lysine;

wherein X_2D may be, for example, an hydrophobic amino acid (e.g., leucine or valine);

wherein X_2E may be, for example, threonine or lysine;

wherein X_2F may be, for example, a basic amino acid (e.g., lysine or arginine);

wherein X_2G may be, for example, arginine or alanine;

wherein X_2H may be, for example, glutamic acid or glycine;

wherein X₂| may be, for example, an hydrophobic amino acid (e.g. , isoleucine or methionine);

wherein X_2J may be, for example, aspartic acid or glycine;

wherein X_2K may be, for example, a basic amino acid (e.g. , lysine or arginine);

wherein X_2L may be, for example, an hydrophobic amino acid (e.g. , alanine or valine);

wherein X_2M may be, for example, an hydrophobic amino acid (e.g. , isoleucine or methionine);

wherein X_2N may be, for example, threonine or arginine;

wherein X₂₀ may be, for example, a neutral hydrophilic amino acid (e.g., serine or threonine);

wherein X_2P may be, for example, asparagine or serine; wherein X₂Q may be, for example, an hydrophobic amino acid (e.g., leucine or methionine);

wherein X₂R may be, for example, glutamine or glutamic acid;

wherein X₂s may be, for example, an hydrophobic amino acid (e.g., isoleucine or leucine);

wherein Χ₂τ may be, for example, threonine or arginine;

wherein X_2U may be, for example, threonine or leucine;

wherein X_2V may be, for example, an hydrophobic amino acid (e.g., leucine or valine).

Wherein an amino acid identified by "X" in SEQ ID NO: 12 is a) an amino acid identical to the corresponding amino acid of SEQ ID NO:5 or, b) wherein at least one of the amino acid identified by X is an amino acid substitution in comparison with a corresponding amino acid in the polypeptide set forth in SEQ ID NO:5 (the murine VH). The amino acid substitution may be, for example conservative or non-conservative. In accordance with the invention, the amino acid substitution may be conservative.

SEQ ID NO: 13 (consensus heavy chain variable region-3) EVQLX_?aQSGAEX_?hX_?r.KPGASVKX_?riSCX_?PASGFNIKDTYMHWVX_?fQX_?nPX_?hQ GLEWX_?iGGIDPADGETKYDPKFQX_?jX_?kX_?lTX_?mTX_?nDTSX_?nX_?nTVYX_?nX_?rX_?.SS LX_2tSEDTAVYYCVRSFDYWGQGTX_2uX_2vTVSS

wherein X_2a may be, for example, glutamine or valine;

wherein X_2b may be, for example, leucine or valine;

wherein X_2c may be, for example, valine or lysine;

wherein X_2d may be, for example, leucine or valine;

wherein X_2e may be, for example, threonine or lysine;

wherein X_2f may be, for example, lysine or arginine;

wherein X_2g may be, for example, arginine or alanine;

wherein X_2h may be, for example, glutamic acid or glycine;

wherein X_2i may be, for example, isoleucine or methionine;

wherein X_2j may be, for example, aspartic acid or glycine; wherein X₂k may be, for example, lysine or arginine;

wherein X₂i may be, for example, alanine or valine;

wherein X_2m may be, for example, isoleucine or methionine;

wherein X_2n may be, for example, threonine or arginine;

wherein X_2o may be, for example, serine or threonine;

wherein X_2p may be, for example, asparagines or serine;

wherein X_2q may be, for example, leucine or methionine;

wherein X_2r may be, for example, glutamine or glutamic acid;

wherein X_2s may be, for example, isoleucine or leucine;

wherein X_2t may be, for example, threonine or arginine;

wherein X_2u may be, for example, threonine or leucine, and;

wherein X_2v may be, for example, leucine or valine.

Wherein an amino acid identified by "X" in SEQ ID NO: 13 is a) an amino acid identical to the corresponding amino acid of SEQ ID NO:5 or, b) wherein at least one of the amino acid identified by X is an amino acid substitution in comparison with a corresponding amino acid in the polypeptide set forth in SEQ ID NO:5 (the murine VH). The amino acid substitution may be, for example conservative or non-conservative. In accordance with the invention, the amino acid substitution may be conservative. Nucleic acids, vectors and cells

Antibodies are usually made in suitable host cells allowing expression of the light chain and heavy chain expressed from a vector(s) comprising a nucleic acid sequence encoding the light chain and heavy chain.

The antibodies that are disclosed herein can be made by a variety of methods familiar to those skilled in the art, such as hybridoma methodology or by recombinant DNA methods.

Considering degeneracy of the genetic code, many different nucleic acid sequences and nucleic acid molecules can be devised for encoding the antibodies described herein. Nucleotide sequences derived from the amino acid sequences described herein may also be selected in order to optimize the gene expression in a particular host. Devising, obtaining and using such nucleic acid sequences for expression of antibodies is within the skills of those in the art.

Particular examples of nucleic acids capable of encoding the CDRs, light chain variable domains, heavy chain variable domains, constant light chains, constant heavy chains described herein include, but are not limited to SEQ ID NO: 15 (sequence encoding a humanized light chain variable region and a human light chain constant region as defined in SEQ ID NO: 16) and SEQ ID NO: 17 (sequence encoding a murine heavy chain variable region and a human heavy chain constant region as defined in SEQ ID NO: 18). The present therefore encompasses the above nucleic acid sequences and any additional nucleic acid molecule capable of encoding any of the CDRs, light chain variable domains, heavy chain variable domains, light chains, heavy chains described herein.

The present invention further encompasses nucleic acid molecules encoding any of the amino acid sequence SEQ ID NOs: 1 , 2, 3, 4, 5, 6 7, 8, 9, 10, 1 1 , 12, 13, 16 and 18 and hybridizing specifically to any of the above nucleic acid molecules and/or to a nucleic acid molecule comprising SEQ ID NO: 15 or NO:17.

In some embodiments, nucleic acid molecule molecules of the invention has (i) a sequence which hybridizes under stringent conditions to at least 10, 15, 25, 50, 100, 250 or more contiguous nucleotides of any of SEQ ID NO: 15. Yet, in other embodiments the nucleic acid molecule of the invention is (ii) a fragment comprising at least 10, 15, 25, 50, 100, 250 or more contiguous nucleotides of any of SEQ ID NO:17.

The term "specifically hybridizing" or "hybridizing specifically" refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed "substantially complementary"). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non- complementary sequence. Appropriate conditions enabling specific hybridization of single-stranded nucleic acid molecules of varying complementarity are well known in the art. For instance, one common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is set forth below (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press):

T_m=81.5 °C +16.6 Log [Na+]+0.41(% G+Q-0.63 (% formamide)-600/#bp in duplex

As an illustration of the above formula, using [Na+]=[0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T_m is 57°C. The T_m of a DNA duplex decreases by 1 -1.5 with every 1 % decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.

The stringency of the hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe with its target, the hybridization is usually carried out at salt and temperature conditions that are 20-25°C below the calculated T_m of the hybrid. Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In general, wash conditions are selected to be approximately 12-20°C below the T_m of the hybrid. With regard to the nucleic acids of the current invention, a moderate stringency hybridization is defined as hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS and 100 g/ml denatured salmon sperm DNA at 42°C and washed in 2xSSC and 0.5% SDS at 55°C for 15 minutes. A high stringency hybridization is defined as hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS and 100 pg/ml denatured salmon sperm DNA at 42°C, and washed in 1xSSC and 0.5% SDS at 65°C for 15 minutes. A very high stringency hybridization is defined as hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS and 100 g/ml denatured salmon sperm DNA at 42°C, and washed in O.lxSSC and 0.5% SDS at 65°C for 15 minutes.

In yet another aspect, the present invention relates to a vector comprising any of the nucleic acid described herein. In accordance with one embodiment, the vector may be an expression vector.

The term "vector" encompasses, without being limited to, autonomously replicating DNA or RNA molecule into which foreign DNA or RNA fragments may be inserted and then propagated in a host cell for expression and/or amplification of the foreign DNA or RNA molecule. A vector may comprise, without limitation, a linear plasmid and/or circular plasmid.

Vector that contains the elements for transcriptional and translational control of the inserted coding sequence in a particular host are known in the art. These elements may include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' un-translated regions. Methods that are well known to those skilled in the art may be used to construct such expression vectors. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

In order to express the antibodies, nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein may be inserted into an expression vector, i.e., a vector that contains the elements for transcriptional and translational control of the inserted coding sequence in a particular host. These elements may include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' un-translated regions. Methods that are well known to those skilled in the art may be used to construct such expression vectors. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

A variety of expression vector/host cell systems known to those of skill in the art may be utilized to express a polypeptide or RNA derived from nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with baculovirus vectors; plant cell systems transformed with viral or bacterial expression vectors; or animal cell systems. For long-term production of recombinant proteins in mammalian systems, stable expression in cell lines may be effected. For example, nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein may be transformed into cell lines using expression vectors that may contain viral origins of replication and/or endogenous expression elements and a selectable or visible marker gene on the same or on a separate vector. The invention is not to be limited by the vector or host cell employed. In certain embodiments of the present invention, the nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein may each be ligated into a separate expression vector and each chain expressed separately. In another embodiment, both the light and heavy chains able to encode any one of a light and heavy immunoglobulin chains described herein may be ligated into a single expression vector and expressed simultaneously. Alternatively, RNA and/or polypeptide may be expressed from a vector comprising nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein using an in vitro transcription system or a coupled in vitro transcription/translation system respectively.

In an additional exemplary embodiment of the invention, the antibodies may be produced by recombinant DNA methods.

In another aspect, the present invention relates to a host cell which comprises and/or expresses an antibody and/or an antigen binding fragment as described herein. The cell may comprise a nucleic acid encoding a light chain variable domain and a nucleic acid encoding a heavy chain variable domain as described herein. Preferably, the cell is capable of expressing, assembling and/or secreting an antibody or antigen binding fragment thereof.

In another aspect the present invention relates to an isolated cell which comprises and/or expresses the antibody or antigen binding fragment of the present invention, and/or comprises any of the nucleic acid molecules or vector described herein. In another aspect, the present invention relates to an isolated cell that may produce the antibody or antigen binding fragment described herein.

The isolated cell may comprise a nucleic acid encoding a light chain variable domain and a nucleic acid encoding a heavy chain variable domain either on separate vectors or on the same vector. The isolated cell may also comprise a nucleic acid encoding a light chain and a nucleic acid encoding a heavy chain either on separate vectors or on the same vector.

The isolated cell may be a hybridoma cell producing an antibody described herein. Alternatively, the isolated cell may be a hybridoma cell producing an antibody having the same epitope specificity as the antibody or antigen binding fragment described herein.

The present invention, therefore encompasses a cell (an isolated cell) which comprises and/or expresses an antibody or antigen binding fragment of the present invention or a portion thereof (e.g., such as during cloning procedures etc.). Although conventional hybridoma cells are contemplated, a person of skill in the art will readily know that other cells are suitable for expressing antibodies or antigen binding fragments, such as bacterial cells, yeast cells, mammalian expression system (e.g., CHO, 293 etc.). Cells that are particularly useful for expression of antibodies, are those which are able to suitably express the antibody (complete antibody, antibody chain(s) or fragments), suitably glycosylate it and/or suitably secrete it.

In an exemplary embodiment of the invention, the antibodies may be produced by the conventional hybridoma technology, where a mouse is immunized with an antigen, spleen cells isolated and fused with myeloma cells lacking HGPRT expression and hybrid cells selected by hypoxanthine, aminopterin and thymine (HAT) containing media.

In general, host cells that contain nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein and/or that express a polypeptide encoded by the nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein, or a portion thereof, may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA/DNA or DNA/RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques that include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or amino acid sequences. Immunological methods for detecting and measuring the expression of polypeptides using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). Those of skill in the art may readily adapt these methodologies to the present invention.

Host cells comprising nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein may thus be cultured under conditions for the transcription of the corresponding RNA (mRNA, siRNA, shRNA etc.) and/or the expression of the polypeptide from cell culture. The polypeptide produced by a cell may be secreted or may be retained intracellular^ depending on the sequence and/or the vector used. In an exemplary embodiment, expression vectors containing nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein may be designed to contain signal sequences that direct secretion of the polypeptide through a prokaryotic or eukaryotic cell membrane.

Due to the inherent degeneracy of the genetic code, other DNA sequences that encode the same, substantially the same or a functionally equivalent amino acid sequence may be produced and used, for example, to express a polypeptide encoded by nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein. The nucleotide sequences of the present invention may be engineered using methods generally known in the art in order to alter the nucleotide sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.

In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. In an exemplary embodiment, antibodies that contain particular glycosylation structures or patterns may be desired. Post-translational processing, which cleaves a "prepro" form of the polypeptide, may also be used to specify protein targeting, folding, and/or activity. Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and W138) are available commercially and from the American Type Culture Collection (ATCC) and may be chosen to ensure the correct modification and processing of the expressed polypeptide. Those of skill in the art will readily appreciate that natural, modified, or recombinant nucleic acid sequences may be ligated to a heterologous sequence resulting in translation of a fusion polypeptide containing heterologous polypeptide moieties in any of the aforementioned host systems. Such heterologous polypeptide moieties may facilitate purification of fusion polypeptides using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein, thioredoxin, calmodulin binding peptide, 6-His (His), FLAG, c-myc, hemagglutinin (HA), and antibody epitopes such as monoclonal antibody epitopes.

In yet a further aspect, the present invention relates to a polynucleotide which may comprise a nucleotide sequence encoding a fusion protein. The fusion protein may comprise a fusion partner (e.g., HA, Fc, etc.) fused to the polypeptide (e.g., complete light chain, complete heavy chain, variable regions, CDRs etc.) described herein.

Those of skill in the art will also readily recognize that the nucleic acid and polypeptide sequences may be synthesized, in whole or in part, using chemical or enzymatic methods well known in the art. For example, peptide synthesis may be performed using various solid-phase techniques and machines such as the ABI 431 A Peptide synthesizer (PE Biosystems) may be used to automate synthesis. If desired, the amino acid sequence may be altered during synthesis and/or combined with sequences from other proteins to produce a variant protein.

Antibody conjugates

Although not necessary for therapeutic purposes, if desired, the antibody or antigen binding fragment of the present invention may nevertheless be conjugated with a therapeutic moiety. For detection purposes it may be particularly useful to conjugate the antibody or antigen binding fragment with a detectable moiety.

A "detectable moiety" is a moiety detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical and/or other physical means. A detectable moiety may be coupled either directly and/or indirectly (for example via a linkage, such as, without limitation, linked with DOTA) to antibodies and antigen binding fragments thereof of the present invention using methods well known in the art. A wide variety of detectable moieties may be used, with the choice depending on the sensitivity required, ease of conjugation, stability requirements and available instrumentation. A suitable detectable moiety include, but is not limited to, a fluorescent label, a radioactive label (for example, without limitation, ¹²⁵l, In¹¹¹, Tc", I¹³¹ and including positron emitting isotopes for PET scanner etc), a nuclear magnetic resonance active label, a luminescent label, a chemiluminescent label, a chromophore label, an enzyme label (for example and without limitation horseradish peroxidase, alkaline phosphatase, etc.), quantum dots and/or a nanoparticle. Detectable moiety may cause and/or produce a detectable signal thereby allowing for a signal from the detectable moiety to be detected.

In another exemplary embodiment of the invention, the antibody or antigen binding fragment thereof may be coupled (modified) with a therapeutic moiety (e.g., drug, cytotoxic moiety).

In an exemplary embodiment, the antibodies and antigen binding fragments may comprise a chemotherapeutic or cytotoxic agent. For example, the antibody and antigen binding fragments may be conjugated to the chemotherapeutic or cytotoxic agent. Such chemotherapeutic or cytotoxic agents include, but are not limited to, Yttrium-90, Scandium-47, Rhenium-186, lodine-131 , lodine-125, and many others recognized by those skilled in the art (e.g., lutetium (e.g., Lu¹⁷⁷), bismuth (e.g., Bi²¹³), copper (e.g., Cu⁶⁷)). In other instances, the chemotherapeutic or cytotoxic agent may be comprised of, among others known to those skilled in the art, 5-fluorouracil, adriamycin, irinotecan, taxanes, pseudomonas endotoxin, ricin and other toxins.

Alternatively, in order to carry out the methods of the present invention and as known in the art, the antibody or antigen binding fragment of the present invention (conjugated or not) may be used in combination with a second molecule (e.g., a secondary antibody, etc.) which is able to specifically bind to the antibody or antigen binding fragment of the present invention and which may carry a desirable detectable, diagnostic or therapeutic moiety.

Pharmaceutical compositions of the antibodies and their use

The present invention also relates to pharmaceutical composition comprising the anti-PSMA antibodies and/or antigen binding fragment described herein and a pharmaceutically acceptable carrier. In order to inhibit the growth of a tumor cell or in order to promote tumor cell death, the pharmaceutical composition may comprise a naked antibody or an antigen binding fragment and may also contain a pharmaceutically acceptable carrier. Of course, as indicated herein, it may be useful to also add a therapeutic moiety to the pharmaceutical composition (e.g., as a drug combination or conjugated to the antibody or antigen binding fragment described herein).

Yet other aspects of the invention relate to the use of the isolated antibody or antigen binding fragment described herein in the detection of tumor cells or in the diagnosis of cancer. Tumors cells which may be particularly detected are those which expresses PSMA, especially if PSMA is located at the cell surface. The antibody or antigen binding fragment of the present invention are particularly useful for the detection of prostate tumor cells or of other PSMA-expressing cells such as neovasculature (in the case of psoriasis) including tumor neovasculature.

In addition to the active ingredients, a pharmaceutical composition may contain pharmaceutically acceptable carriers comprising water, PBS, salt solutions, gelatins, oils, alcohols, and other excipients and auxiliaries that facilitate processing of the active compounds into preparations that may be used pharmaceutically. In other instances, such preparations may be sterilized.

As used herein, "pharmaceutical composition" means therapeutically effective amounts of the agent together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers. A "therapeutically effective amount" as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen. Such compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCI, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20™, Tween 80™, Pluronic F68™, bile acid salts). Solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc., or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines). Other embodiments of the compositions of the invention incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, oral, vaginal, rectal routes. In one embodiment the pharmaceutical composition is administered parenterally, paracancerally, transmucosally, transdermal^, intramuscularly, intravenously, intradermally, subcutaneously, intraperitonealy, intraventricularly, intracranially and intratumorally.

Further, as used herein "pharmaceutically acceptable carrier" or "pharmaceutical carrier" are known in the art and include, but are not limited to, 0.01 -0.1 M or 0.05 M phosphate buffer or 0.8 % saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's orfixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like. "Pharmaceutically acceptable carriers" thus may include, without limitation, diluents (such as phosphate buffered saline buffers, glycine buffer, water, saline), preservatives, solubilizers, emulsifiers, adjuvant and/or carriers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents is well known in the art. Except insofar as any conventional media or agent is incompatible with antibodies of the present invention, its use in pharmaceutical compositions is contemplated.

For any compound, the therapeutically effective dose may be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the concentration range and route of administration. Such information may then be used to determine useful doses and routes for administration in humans. These techniques are well known to one skilled in the art and a therapeutically effective dose refers to that amount of active ingredient that ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating and contrasting the ED₅₀ (the dose therapeutically effective in 50% of the population) and LD₅₀ (the dose lethal to 50% of the population) statistics. Any of the therapeutic compositions described above may be applied to any subject in need of such therapy, including, but not limited to, mammals such as dogs, cats, cows, horses, rabbits, monkeys, rats, mouse and humans.

The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

The present invention also relates to non-pharmaceutical composition which may contain the antibody or antigen binding fragment in aqueous solution or in other forms (e.g., freeze-dried, etc.). These non-pharmaceutical compositions may have utility in in vitro assays or the like. In a further aspect the present invention relates to a formulation comprising the antibody or antigen binding fragment described herein and a glycine buffer. The pH of the formulation may be between 7.0 to 8.0. In accordance with a more specific embodiment of the invention, the pH of the formulation may be around 7.4.

The term "treatment" for purposes of this disclosure refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

The antibodies and antigen binding fragments may have therapeutic uses in the treatment of various diseases involving PSMA, such as prostate cancer. In an exemplary embodiment, the antibodies or antigen binding fragments may interact with cancer cells that express PSMA and induce an immunological reaction by mediating cellular immunity, humoral immunity or complement-mediated immunity. In other instances, the antibodies and fragments may block the interaction of PSMA with its protein partners.

In certain instances, the antibodies and antigen binding fragments therein may be administered concurrently in combination with other treatments given for the same condition. As such, the antibodies may be administered with anti-mitotics (e.g., taxanes), platinum-based agents (e.g., cisplatin), DNA damaging agents (e.g. doxorubicin), and other anti-cancer therapies that are known to those skilled in the art. In other instances, the antibodies and antigen binding fragments therein may be administered with other therapeutic antibodies.

The present invention relates in a further aspect thereof to a method for inhibiting the growth of a PSMA-expressing cell, the method may comprise contacting the cell with an effective amount of the antibody or antigen binding fragment described herein. The use of a naked anti-PSMA antibody is especially contemplated herein. The present invention also encompasses method of treating cancer or inhibiting the growth of a PSMA expressing cells in a mammal, the method may comprise administering the antibody or antigen binding fragment described herein to a mammal in need. The use of a naked anti-PSMA antibody is also especially contemplated herein.

It is to be understood herein that by "inhibiting" it is meant a process by which the growth of a PSMA-expressing cell may be reduced, delayed, prevented and/or impaired. The term "inhibiting" may also encompass cell death.

As it will become apparent from the method described herein and in accordance with the present invention, the method may be performed using a naked antibody or antigen binding fragment described herein. The method may also be performed using the naked antibody either alone or in combination with a second therapeutic molecule. Furthermore, the method of the present invention may be carried out by using an antibody or antigen binding fragment which carries a diagnostic or therapeutic moiety.

In exemplary embodiment of the invention the method may be carried out using antibodies which may comprise a portion capable of attracting immune effector cells (e.g. natural killer cells, macrophages, etc.). Such portion may be a Fc region derived from the same species or from another species, e.g. a mice antibody Fc region, a human antibody Fc region, etc.

The present invention relates in an additional aspect thereof to a method for treating cancer, which may comprise administering to a subject in need an effective amount of a pharmaceutical composition that may comprise the antibody or antigen binding fragment described herein. According to the present invention, a "subject" may be a mammal. In accordance with the present invention, the mammal may be a human being. A subject in need thereof encompasses a subject that may need PSMA expressing-cell detection and/or a subject that may need cancer treatment (such as prostate cancer). The term "cancer" is intended to mean any cellular malignancy whose unique trait is the loss of normal controls which may result in unregulated growth, lack of differentiation and/or ability to invade local tissues and metastasize. Cancer may develop in any tissue of any organ. In a non-limitative embodiment of the present invention, cancer is intended to include prostate cancer.

The present invention also encompasses method of detecting cancer or detecting a PSMA-expressing cells in a mammal, the method may comprise administering the antibody or antigen binding fragment described herein to a mammal in need.

According to the present invention, contacting and/or detecting may occur in vivo, ex vivo or in vitro. In vivo contacting involves administering to a subject an antibody (effective amount thereof) of the invention, for example in a composition and/or pharmaceutical composition. Ex vivo contact and/or in vitro contact involves contact with a biological sample obtained from a subject. A biological sample may comprise a sample of blood, serum and/or tissue biopsies. It is to be understood herein that the PSMA expressing cell may be a normal cell or a cell which aberrantly expresses PSMA (e.g., a tumor cell). A PSMA- expressing cell may also include neovasculature (non-tumor, e.g., psoriasis) and including tumor neovasculature. Such tumor neovasculature is not only found in prostatic cancer but also in bladder and lung tumors and also in breast tumor, colon tumor and pancreatic tumor.

According to the present invention, a cell which aberrantly expresses PSMA may be a cell that simply overexpresses PSMA without being tumoral. Alternatively and in accordance with the present invention, cell which aberrantly expresses PSMA may be a tumor cell. In accordance with the present invention, a tumor cell may be a prostate cancer cell, an astrocytoma cell, a breast carcinoma cell, a carcinoid cell, a gastric carcinoma cell, a hepatocarcinoma cell, a Hodgkin's lymphoma cell, a leiomyoma cell, a lung adenocarcinoma cell, a lymphoma cell, a melanoma cell, an ovarian carcinoma cell, a rhabdosarcoma cell and/or a thyroid carcinoma cell. In an embodiment of the present invention, a tumor cell is a prostate cancer cell. In another embodiment, the prostate cancer cell may be a metastatic prostate cancer cell.

The present invention relates in another aspect thereof to a method for detecting a PSMA -expressing cell, the method may comprise contacting the cell with an antibody or antigen binding fragment described herein and detecting a complex formed by the antibody and the PSMA-expressing cell.

Another aspect of the invention relates to a method for detecting PSMA, or a variant having at least 80% sequence identity with PSMA, the method may comprise contacting a cell or a sample (biopsy, serum, plasma, urine etc.) comprising or suspected of comprising PSMA or the PSMA variant with the antibody or antigen binding fragments described herein and measuring binding.

The sample may originate from a mammal (e.g., a human) which may have cancer (e.g., prostate cancer) or may be suspected of having cancer (e.g., prostate cancer). The sample may be a tissue sample obtained from the mammal or a cell culture supernatant.

In accordance with the invention the sample may be a biopsy, a serum sample, a plasma sample, a blood sample or ascitic fluid obtained from the mammal. The antibody or antigen binding fragment described herein may advantageously detect PSMA.

The method may comprise quantifying the complex formed by the antibody or antigen binding fragment bound to PSMA or to the PSMA variant.

The antibody or antigen binding fragment of the present invention may more particularly be used in the detection, diagnosis or treatment of prostate cancer. Additional aspects of the invention relates to kits which may include one or more container containing one or more antibodies or antigen binding fragments described herein. Kits of the present invention may additionally include, if desired, one or many conventional components, for example, containers that may comprise one or many excipients and/or pharmaceutically acceptable vehicles, or any other additional containers that may be evident to a person skilled in the art. A kit according to the present invention may also advantageously include instructions in the form of a pamphlet or of any other support, indicating the quantities to be used and/or administered and/or the instructions to mix given components.

The following examples are presented to illustrate the invention but it is not to be considered as limited thereto. EXAMPLES

A murine lgG1 antibody was transformed so that its protein sequence contains a majority of human sequence in such a way that it's binding reactivity and specificity is not significantly altered from the parental murine Ab. The portion of an antibody that is responsible for its ability to bind an antigen is located in the variable region of the antibody, and is formed by 6 loops collectively called the complementarity determining region (CDR). A humanized version of antibody can be accomplished by inserting the murine CDR segments into an appropriate human Ig in place of the original human CDR segments. The methodology to design the humanized antibody involves a combination of In Silico analysis of protein structure, cDNA synthesis, and recombinant DNA techniques for cloning of the genes into appropriate expression vector. The recombinant genes for the heavy and light chain are then expressed either transiently or stably in mammalian cells such as CHO cells, for the production of the recombinant protein. The antibody harvested from the cell culture media or following purification by affinity chromatography is tested by ELISA or other analytical methods to assess its reactivity and specificity.

Modification in the natural sequence of amino acid of a protein may affect diverse properties of the protein. For instance, taking the CDR loops of a murine antibody out of its natural murine context and grafting them into a human variable backbone domain may significantly alter the antigen specificity and/or the reactivity for which the antibody was created. Therefore, as a general strategy to test the impact of humanization content on the overall binding properties of the antibody, we created humanized antibodies of gradual human content. A mouse- human chimeric antibody was created in which the intact murine variable region of the light and the heavy chains was linked to the respective human lgG1 Fc domain (CC; chimeric heavy chain and chimeric light chain). Further humanization was achieved with an antibody combining a humanized heavy chain variable domain with an intact murine light chain variable domain linked to a human lgG1 Fc domain (HC). The reverse antibody design was also made (CH). The strategy of testing one humanized chain at a time in a chimeric antibody backbone can bring useful information to further improve the antibody design. Another antibody combined both the humanized light and heavy variable regions linked to the human lgG1 Fc (HH). The four antibody variants; CC, HC, CH, HH were produced by transient co-transfection of CHO-3E7 cells a ratio of 1 :1 of DNA of expression vector for the appropriate heavy chain (either chimeric or humanized) and light chain (either chimeric or humanized). The transfected cells were maintained in culture for a minimum of 3 days after which the cell culture media containing the secreted antibody was harvested. The concentration of antibody in the media was measured by ELISA using a standard curve of human IgG antibody. The harvested antibody was also purified by affinity chromatography using a protein A matrix and concentrated to 2-5 mg/ml by ultrafiltration. The concentration of the purified antibody was measured by a Bradford assay or by absorbance at 280 nm and calculated using the 0.1 % solution extinction coefficient of 1.348. An ELISA assay was used to assess and compare the reactivity profile of the humanized antibodies produced. Briefly, 96-well plates (Maxi-Sorp™, Nalgene Nunc, Rochester, NY) were coated overnight at 4°C or for 2 h at 37°C with 100 μΙ of PBS containing 5 ug of LNCaP or PC-3 cell membrane preparation, or 5 ng of purified recombinant human PSMA or BSA. Plates were washed four times with 200 μΙ of 10 mM Tris-HCI, 150 mM NaCI, and 0.05% Tween-20™ (TBST, pH 7.5), and blocked for a minimum of 30 min with 200 μΙ of TBST containing 3% casein. Plates were then washed and incubated for 1 h at room temperature with gentle agitation with 100 μΙ of diluted cell culture media containing the antibody or the purified antibody in TBST at the indicated concentration. Antibody binding was detected by the sequential addition, followed by washing, of 100 μΙ of horseradish peroxidase (HRP) conjugated goat anti-human IgG whole molecule (Jackson Immunoresearch, West Grove, PA) secondary antibody diluted 1 :10000 in TBST, for 1 h at room temperature, and 100 μΙ of HRP colorimetric substrate solution 3,3,5,5-tetramethylbenzidine (BioFX Laboratories, Owings Mills, MD). The reaction was stopped with the addition of 100 μΙ of 0.5 M sulphuric acid and the absorbance was read at 450 nm in a microplate reader (Bio-Tek Instruments, Highland Park, VT).

The graph in Figure 1 presents the reactivity profile of the unpurified CC, CH, HC, CC antibodies for the PSMA expressing LNCaP cell extract and for the PSMA negative PC-3 prostate cancer cell lines. All four antibodies recognize membrane extracts prepared from LNCaP cells in a dose-dependent manner that plateau at approximately 1 ug/ml, but lacked reactivity with extracts derived from the PC-3 cell line deficient in PSMA expression. Those results indicate that the four antibodies constructs still recognize PSMA and that the parental murine antibody specificity was maintained, despite the introduction of a minimum of 70% human amino acids content in their sequence. The graph also indicates that the humanized antibodies CH and CC react more strongly to LNCaP than the two other variants, HC and HH. Figure 2 compares the ratio of LNCaP to PC-3 reactivity of the unpurified antibodies at a concentration of 10ug/ml. The HH and HC generate a 4 fold signal to noise versus more than the double, 9.7 and 9.3, for the CH and CC respectively.

The purification of the antibodies did not change the differential pattern reactivity (Figure 3), suggesting that the lower reactivity observed for the HH and HC does not results from the presence of a contaminant in the cell culture supernatant that cross-reacts with those two antibodies and partially prevent them to bind the LNCaP. Moreover, a similar pattern of reactivity with the purified antibody was also observed on PSMA vs BSA (Figure 4). Altogether, these results suggest that the CC and CH humanized antibody variants (i.e. comprising a heavy chain variable region set forth in SEQ ID NO:5 and a light chain variable region set forth in SEQ ID NO: 1 ) are more reactive to PSMA than the other two sister molecules, HC and HH, while exhibiting the same background reactivity. The CH humanized antibody was selected for further characterization based on its stronger reactivity than the HC and HH, and its higher degree of humanization than CC.

The interaction kinetic between the CH and the PSMA antigen was measured by surface plasmon resonance at room temperature and compared to the parental murine antibody. The affinity measurement assay was design to measure the monovalent interaction of the antibody with the antigen. The epitope of the antibody, peptide PSMA^490"500 (GKSLYESWTKK; SEQ ID NO: 14), was used as a surrogate antigen in place of the whole protein in the binding experiment. The peptide was coupled to human serum albumin at a stoechiometric ratio (Ag- HSA). Briefly, a proteon chip was first prepared with bound anti-mouse Fc or anti- human Fc antibodies. The immunoreactive chip was then used to capture the murine antibody and humanized CH variant on the appropriate channel. Following the preparation of the sensor chip, various dilutions of the Ag-HSA in running buffer (1.23-100 nM) were then injected. Each injection was followed by injection of buffer in order to record the dissociation of the antibody: Ag-HSA complexes. Both complex formation and dissociation were recorded in real time and a sensorgram was generated. The curves from the phase of association and the entire dissociation phase were then analysed and fitted to derive the kinetics of association (k_a) and dissociation (k_d) of the complex. The affinity (K_D) of the antibody for the antigen is calculated as the ratio of k_d/k_a.

The results presented in Table 2 show that the measured association and dissociation constant for the humanized and the parental murine antibody are similar, and their respective K_D are calculated at 1.71x10^"9 and 1.6x10^"9M. These results indicate that the humanisation of the murine antibody into the CH variant did not results in a loss of antigen recognition and neither in a change in the binding affinity. Table 2: Antigen binding kinetics and calculated affinity for the murine antibody and humanized CH variant

K_a (1/Ms) K_d (1/s) K_D (M)

Murine _{e t} 6.72x10^"4 ± 4.55x10^" 1.60x10^"9 ± 1.59x10^"

4.27x10⁺⁵ ± 7.1 1x10⁺⁴

antibody ^{5 10}

CH _{K A} 7.41x10^"4 ± 6.79x10^" 1.71x10^"9 ± 3.10x10^"

4.45x10⁺⁵ ± 8.56x10⁺⁴

antibody ^{5 10}

The immunocytochemical patterns of localization of PSMA by the humanized CH antibody and the parental murine antibody in human prostate gland and prostate cancer tissue were compared. Paraffin sections of tissue (Folio) were cleared with 3 xylene baths, rehydrated in a series of alcohol (100% to 50%) and washed in PBS. Sections were then pre-treated in 3% H₂0₂ in water for 10 minutes, washed in PBS and treated with an antigen retrieval solution (citrate buffer 10 mM, pH 6.2), before heating in a microwave oven for 5 minutes, and allowed to slowly return to room temperature, thereafter. Non-specific sites were blocked by incubating the sections in PBS containing 1 % BSA for 1 hour. Tissues were then incubated overnight at 4°C with the PSMA-specific antibodies diluted in the same buffer (Murine; 1 ;100 to 1 :200, CH; 1 : 1000 to 1 :2000). In some conditions, a blocking control was performed in which the primary antibody was incubated in the presence of 1 pg of peptide epitope (GKSLYESWTKK; SEQ ID NO: 14). After incubation with the primary antibody, sections were washed 3 times within 15 minutes in PBS, and incubated 30 minutes with either a goat anti-mouse- biotinylated antibody (Vector™ #BA-9200, 1 :300), or goat anti-human IgG (1 :300), washed and followed by a rabbit anti-goat-biotinylated antibody (Vector™ #BA-5000). After washing 15 minutes in PBS, the immunoreactivity was revealed using Vectastain Elite ABC Kit™ (Vector™ #PH-6200). The immunoreaction product appeared as brown precipitate on brightfield.

Figure 5 compares the prostate tissue immunostaining by the murine and the CH antibodies. The murine and CH humanized antibodies generate specific immunostaining in normal prostate epithelial cells and in prostate cancer acini with Gleason 2 to 4 grade. The immunostaining density is similar for both antibodies under the conditions tested and the distribution pattern is in agreement with the known localization of PSMA in prostate tissue. The displacement of the CH immunoreactivity by the peptide epitope indicates that the antibody is specific.

As used herein the term CH designate an antibody having a chimeric heavy chain (comprising the variable region set forth in SEQ ID NO:5) and a humanized light chain (comprising the variable region set forth in SEQ ID NO: 1 ). As used herein the term HC designate an antibody having a humanized heavy chain (comprising the variable region set forth in SEQ ID NO:2) and a chimeric light chain (comprising the variable region set forth in SEQ ID NO:6).

As used herein the term CC designate an antibody having a chimeric heavy chain (comprising the variable region set forth in SEQ ID NO:5) and a chimeric light chain (comprising the variable region set forth in SEQ ID NO:6).

As used herein the term HH designate an antibody having a humanized heavy chain (comprising the variable region set forth in SEQ ID NO:2) and a humanized light chain (comprising the variable region set forth in SEQ ID NO: 1 ).

As shown in Figure 6 the humanized light chain and heavy chain variable regions described herein have a reduced number of divergent residues compared to the mouse regions.

Various combinations of the intact murine or humanized variable regions linked to respective human constant regions were tested as CH (i.e., chimeric heavy chain-humanized light chain), HC, HH and CC combinations (Heavy Light) for reactivity with PSMA. As shown in Figures 3 and 4 the CH gave the best reactivity profile.

One particular humanized monoclonal antibody named "PSC1700" was selected for further analysis and experimentation. This antibody is composed of an intact murine V-heavy chain and a humanized V-light chain linked to the respective human lgG1 constant regions. Its percentage of humanization is 96.1 % (hC_L=107/107; hC_H=330/330; muV_H=60/82; hV_L= 78/79).

Figure 7 depicts immunoreactivity of the humanized monoclonal antibody PSC1700 according to the present invention. As shown, immunoreactivity was twice higher for purified PSMA than for the LNCaP cell membrane preparation, and substantially non-existent for the PC-3 cell membrane preparation (PSMA negative prostate cancer cells) or BSA.

As shown in Figure 8 and 9, the humanized Vlight sequence in this figure, obtained according to the present invention, is in conformity with most common human residues.

Claims

CLAIMS:

1. An antibody or antigen binding fragment thereof, comprising a light chain variable region as set forth in SEQ ID NO:8 and a heavy chain variable region as set forth in SEQ ID NO: 1 1.

2. An antibody or antigen binding fragment thereof, comprising a light chain variable region as set forth in SEQ ID NO:1 and a heavy chain variable region as set forth in SEQ ID NO:2.

3. An antibody or antigen binding fragment thereof, comprising a light chain variable region as set forth in SEQ ID NO:8 and a heavy chain variable region as set forth in SEQ ID NO:5.

4. An antibody or antigen binding fragment thereof, comprising a light chain variable region as set forth in SEQ ID NO:1 and a heavy chain variable region as set forth in SEQ ID NO:5.

5. An antibody or antigen binding fragment thereof, comprising a light chain as set forth in SEQ ID NO:3 and a heavy chain as set forth in SEQ ID NO:4.

6. An antibody or antigen binding fragment thereof, comprising a light chain as set forth in SEQ ID NO:3 and a heavy chain as set forth in SEQ ID NO:7.

7. The antibody or antigen binding fragment thereof of any one of claims 1 to 6 comprising a constant region of a human lgG1.

8. The antibody or antigen binding fragment thereof of any one of claims 1 to 7, conjugated to a therapeutic moiety.

281053.00039/98403226.5

9. The antibody or antigen binding fragment thereof of claim 8, wherein the therapeutic moiety is a chemotherapeutic agent or a cytotoxic agent.

10. The antibody or antigen binding fragment thereof of any one of claims 1 to 7, conjugated to a detectable moiety.

11. A pharmaceutical composition comprising the antibody or antigen binding fragment of any one of claims 1 to 10, and a pharmaceutically acceptable carrier.

12. Use of the antibody or antigen binding fragment of any one of claims 1 to 10, for the treatment of cancer.

13. The use of claim 12, wherein the cancer is prostate cancer.

14. Use of the antibody or antigen binding fragment of any one of claims 1 to 10, in the manufacture of a medicament for the treatment of cancer.

15. Use of the antibody or antigen binding fragment of any one of claims 1 to 10, in the detection of a cancer or the detection of cancer cells.

16. The use of claim 14 or 15, wherein the cancer is prostate cancer.

17. A method for the treatment of cancer, comprising administering to a subject in need thereof the antibody or antigen binding fragment of any one of claims 1 to 10.

18. The method of claim 17, wherein the cancer is prostate cancer.

19. A method for detecting PSMA, or a variant having at least 80% sequence identity with PSMA, the method comprising contacting a cell or a sample comprising or suspected of comprising PSMA or the PSMA variant with the antibody or antigen binding fragments any one of claims 1 to 10.

20. A kit containing one or more antibodies or antigen binding fragments of any one of claims 1 to 10, and at least one of a container and instructions of use.

21. An isolated nucleic acid molecule encoding for one or more antibodies or antigen binding fragments of any one of claims 1 to 10.

22. The isolated nucleic acid molecule of claim 21 , comprising a nucleic acid sequence as set forth in any one of SEQ ID NO:15 and SEQ ID NO:17.

23. An expression vector comprising a nucleic acid molecule as defined in claim 21 or 22.

24. An isolated cell expressing one or more antibodies or antigen binding fragments of any one of claims 1 to 10.