HK40006061B - Prl3 antibody - Google Patents

Description

PRL3 antibodies

Technical Field

The present invention relates to humanized antibodies that bind PRL3.

Background

Cancer is fundamentally a disease with disturbed gene expression, leading to the progression of multiple steps into metastasis (1), which is the leading cause of cancer-related death (2). There is increasing evidence that Protein Tyrosine Phosphatases (PTPs) play an important role in driving metastatic progression (3). We identified liver regeneration phosphatase-3 (PRL-3; also known as PTP4A 3) in 1998 as a member of the PRL family of bispecific PTPs (4), which consists of three members: PRL-1, PRL-2, and PRL-3. In 2001, PRL-3 was characterized by the fogerstin group as a metastasis associated phosphatase, specifically highly upregulated in metastatic colorectal cancer samples, but not primary cancer and normal colorectal epithelial cells (5). In recent independent global gene expression studies, PRL-3 was also identified as the most important predictor of metastatic relapse in patients with uveal melanoma (6). Clinically, elevated PRL-3mRNA expression levels are associated with high metastatic potential and poor prognosis for a variety of cancer types, including colorectal, gastric, breast, ovarian, and lung cancers (7).

PRL localizes to the cytoplasmic face of the plasma membrane and endosome via its prenylated C-terminus (8). There is increasing evidence that PRL-3 promotes multiple stages of malignant transformation, including cell proliferation, epithelial-to-mesenchymal transition (EMT), invasion, motility, angiogenesis and survival (9). Molecularly, PRL-3 has been shown to indirectly activate the PI3K/Akt pathway by down-regulating PTEN (10) and to activate oncogenic ERK and SRC signals by constitutive activation of multiple upstream receptor tyrosine kinases (11-13).

PRL-3 was first associated with GC progression in 2004, when higher levels of PRL-3 were found to correlate with increased GC aggressiveness and metastasis (14). Since then, it has been reported that PRL-3 is overexpressed in up to 70% of primary gastric cancers, the higher the level of PRL-3 expression, the shorter the post-operative survival time at all tumor stages in GC patients (15,16). This prognostic potential of PRL-3 is particularly important because GC is the third leading cause of cancer mortality worldwide, with over 700,000 deaths associated with gastric cancer per year (2), largely due to delayed detection and the asymptomatic nature of early disease, coupled with a high rate of relapse after treatment (17). Despite the high failure rate, curative surgery remains the standard treatment modality for GC, and adjuvant chemotherapy is generally considered pre-and/or post-resection (18, 19). Nevertheless, the overall survival rate of chemotherapy remains poor and is associated with adverse side effects due to non-specific targeting to other actively dividing non-cancerous cells (17). For this reason, targeted therapies using tumor-specific biologics have been the focus of anticancer drug development because they have the potential to selectively inhibit specific molecules involved in the growth and survival of cancer cells while retaining normal cells. Current antibody therapies are directed only against extracellular (cell surface or secreted) proteins, as antibodies are generally considered too large to enter cells, leaving large amounts of intracellular therapeutic targets, such as phosphatases, kinases, and transcription factors, unutilized by antibody therapies. For example, in GC, the HER2/neu receptor antagonist trastuzumab (herceptin) has been approved for targeting GCs expressing 13-20% of the cell surface HER2/neu receptor, particularly metastatic gastric or gastroesophageal junction adenocarcinomas (20, 21). However, despite mild responses, patients often develop resistance to trastuzumab (22), thereby impeding their therapeutic efficacy. Alternative targeted therapies for GC are therefore urgently needed and being actively sought.

In 2008, we reported a new approach to antibody therapy, targeting intracellular PRL-1 and PRL-3 anticancer drugs (23). In this report, we found that anti-PRL-3 antibodies inhibit experimental metastasis of PRL-3 (but not PRL-1) expressing cancer cells, while anti-PRL-1 antibodies inhibit PRL-1 (but not PRL-3) expressing cancer cells, thus establishing strict requirements for specific antibody-antigen recognition for therapeutic efficacy when targeting such intracellular oncoproteins. After this, in 2011, we verified the feasibility and effectiveness of this new concept, targeting other endogenous and exogenous intracellular "tumor-specific antigens" by antibody therapy or vaccination in wild-type C57BL/6 and transgenic spontaneous breast tumor MMTV-PyMT mice (24). We and Ferrone propose three possible mechanisms of anti-tumor activity of antibodies specific to intracellular Tumor Antigens (TA), including antibody penetration into cells, antibody binding to an externalized antigen and/or antibody recognition of MHC-bound antigen-derived peptides (25, 26).

Following the success of mice and recent chimeric (27) anti-PRL-3 antibodies targeting PRL-3 expressing tumors, we here transformed our approach into a more clinically relevant four key aspects: 1) Using a PRL-3 humanized antibody (PRL 3-zumab)) instead of a mouse or chimeric antibody; 2) Targeting a human cancer cell line instead of a mouse cancer cell line; 3) Developing more clinically relevant in situ gastric tumor models than the mouse tail vein metastasis model; 4) Identifying potential surrogate biomarkers for monitoring efficacy of PRL 3-mab therapy. We show the first example of a new class of humanized antibodies that block gastric tumorigenesis. Our findings reveal the potential of antibody therapy to target intracellular oncoproteins, opening a new era of cancer therapy.

Summary of The Invention

Off-target effects are a major clinical problem in cancer therapy. We have produced an elegant humanized antibody against tumor specific intracellular PRL-3 (PRL 3-globin), an oncogenic phosphatase that is upregulated in a variety of human cancers. We focused on Gastric Cancer (GC), providing independent evidence that elevated PRL-3mRNA levels were significantly associated with shortened overall survival in GC patients. The PRL-3 protein was overexpressed in 85% of freshly frozen GC tumors, but not in the patient-matched normal stomach tissue examined. Using human GC cell lines, we established a clinically relevant in situ gastric tumor model and demonstrated that PRL 3-globin specifically blocks the growth of PRL-3 positive (PRL-3 +) but does not inhibit the growth of PRL-3 negative (PRL-3) tumors. PRL-3-globin as monotherapy has better therapeutic effect than 5-fluorouracil (5-FU) or 5-FU alone. PRL 3-bead is specifically enriched in PRL-3+ tumor tissue and promotes recruitment of immune cells to the PRL-3+ tumor microenvironment. Unexpectedly, we found secreted PRL-3 oncoprotein in 62% of the urine of various human cancers and 100% of the urine of cancers derived from PRL-3+, but not PRL-3 tumor-bearing mice. In addition, urinary PRL-3 levels were significantly reduced following effective treatment with PRL 3-globin. Urinary PRL-3 can be considered as a potential diagnostic and surrogate biomarker for monitoring of therapeutic response to PRL 3-bead mab therapy in a variety of cancer types in the future.

We also investigated the mechanism of action (MOA) to address how PRL-3 antibodies might bind to their intracellular PRL-3 antigen and concluded that "intracellular oncoproteins" can indeed be relocated to the cell surface as "extracellular oncoproteins" in cancer, thus following the rational basis of tumor elimination through the conventional route of antibodies to extracellular cancer targets.

Consistently, we found that PRL 3-globin blocks tumors expressing PRL-3 'intracellular' antigens, requires host Fc γ II/III receptor interaction, full antibody activity, and increases M1 (but not M2) macrophages, B lymphocytes, natural killer cells to enhance host immunity. These results indicate that the MOA of antibodies targeting "intracellular oncoproteins" do follow a similar principle of targeting "extracellular oncoproteins" via the classical antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis (ADCP) pathways to eliminate tumors.

Finally, using 110 precious freshly frozen human tumors or their matched normal tissues, we further demonstrated that PRL-3 is an excellent tumor-specific cancer target, with an average of >78% being widely overexpressed from 9 different human cancer types: tumor samples from patients with liver, lung, colon, breast, stomach, bladder, prostate, AML and renal disease, but not in matched normal tissues. Thus, PRL-3 may be a useful biomarker for cancer and a very general target for cancer therapy. Thus, PRL-3 may provide a useful biomarker for solid cancers.

The present invention relates to antibodies or antigen-binding fragments that bind to PRL3. Heavy and light chain polypeptides are also disclosed. The antibodies, antigen-binding fragments, and polypeptides may be provided in isolated and/or purified form, and may be formulated into compositions suitable for use in research, therapy, and diagnosis. In particular, the invention relates to humanized antibodies that bind PRL3, in particular PRL3 antagonist antibodies.

In some cases, the antibodies of the invention inhibit the function of PRL3. In some cases, the antibody inhibits Protein Tyrosine Phosphatase (PTP) function of PRL3. In some cases, the antibody induces ADCC and/or ADCP. In some cases, the antibody is capable of binding to an Fc receptor, e.g., fcRII and/or FcRIII. In some cases, binding of the antibody to the cell results in recruitment of immune cells, e.g., B cells, NK cells, or macrophages, preferably M1 macrophages, to the cell.

In one aspect of the invention, there is provided an antibody or antigen-binding fragment, the amino acid sequence of the antibody may comprise amino acid sequences i) to iii), or amino acid sequences iv) to vi), or preferably amino acid sequences i) to vi):

i)KASQSVEDDGENYMN(SEQ ID NO：4)

ii)AASNLES(SEQ ID NO：5)

iii)QQSNEDPFT(SEQ ID NO：6)

iv)GYTFTNYYMH(SEQ ID NO：1)

v)WIYPGNVNTYYNEKFRG(SEQ ID NO：2)

vi)EEKNYPWFAY(SEQ ID NO：3)

or a variant thereof wherein one or two or three amino acids in one or more of sequences (i) to (vi) are substituted with another amino acid.

The antibody or antigen-binding fragment may comprise at least one light chain variable region comprising the following CDRs:

LC-CDR1：KASQSVEDDGENYMN(SEQ ID NO：4)

LC-CDR2：AASNLES(SEQ ID NO：5)

LC-CDR3：QQSNEDPFT(SEQ ID NO：6)

the antibody or antigen-binding fragment may comprise at least one heavy chain variable region comprising the following CDRs:

HC-CDR1：GYTFTNYYMH(SEQ ID NO：1)

HC-CDR2：WIYPGNVNTYYNEKFRG(SEQ ID NO：2)

HC-CDR3：EEKNYPWFAY(SEQ ID NO：3)

the antibody may comprise at least one light chain variable region comprising a CDR as depicted in figure 7. The antibody may comprise at least one heavy chain variable region comprising the CDRs shown in figure 7.

The antibody may comprise at least one light chain variable region (V) comprising one of the amino acid sequences set forth in FIG. 7 _L ) Or V as shown in FIG. 7 _L An amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of the amino acid sequences of the chain amino acid sequence. The antibody may have at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity V to one of the amino acid sequences set forth in figure 7 _L A chain amino acid sequence and comprises the following CDR sequences:

LC-CDR1：KASQSVEDDGENYMN(SEQ ID NO：4)

LC-CDR2：AASNLES(SEQ ID NO：5)

LC-CDR3：QQSNEDPFT(SEQ ID NO：6)

the antibody may comprise at least one heavy chain variable region (V) comprising one of the amino acid sequences set forth in FIG. 7 _H ) Or V as shown in FIG. 7 _H An amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of the amino acid sequences of the chain amino acid sequence. The antibody may have a V with at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of the amino acid sequences set forth in figure 7 _H A chain amino acid sequence and comprising the following CDR sequences:

HC-CDR1：GYTFTNYYMH(SEQ ID NO：1)

HC-CDR2：WIYPGNVNTYYNEKFRG(SEQ ID NO：2)

HC-CDR3：EEKNYPWFAY(SEQ ID NO：3)

the antibody may comprise at least one light chain variable region comprising one of the amino acid sequences set forth in figure 7 (or V as set forth in figure 7) _L An amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of the amino acid sequences of the chain amino acid sequence) and at least one heavy chain variable region comprising one of the amino acid sequences set forth in figure 7 (or a V as set forth in figure 7) _H An amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one of the amino acid sequences of the chain amino acid sequence).

The antibody may bind to PRL3. The antibody may optionally have an amino acid sequence component as described above. The antibody may be IgA, igD, igE, igM or IgM, preferably IgG. In one embodiment, an optionally isolated in vitro complex is provided comprising an antibody or antigen-binding fragment described herein that binds PRL3.

In one aspect of the invention, there is provided an isolated heavy chain variable region polypeptide comprising the following CDRs:

HC-CDR1：GYTFTNYYMH(SEQ ID NO：1)

HC-CDR2：WIYPGNVNTYYNEKFRG(SEQ ID NO：2)

HC-CDR3：EEKNYPWFAY(SEQ ID NO：3)

in one aspect of the invention, there is provided an antibody or antigen-binding fragment comprising heavy and light chain variable region sequences, wherein:

the heavy chain comprises HC-CDR1, HC-CDR2, HC-CDR3 having at least 85% overall sequence identity with HC-CDR1 (SEQ ID NO: 1), HC-CDR2 (SEQ ID NO: 2), HC-CDR3 (SEQ ID NO: 3), respectively, and the light chain comprises LC-CDR1, LC-CDR2, LC-CDR3 having at least 85% overall sequence identity with LC-CDR1 (SEQ ID NO: 4), LC-CDR2 (SEQ ID NO: 5), LC-CDR3 (SEQ ID NO: 6), respectively.

In some embodiments, the degree of sequence identity may be one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

In another aspect of the invention, there is provided an optionally isolated antibody or antigen-binding fragment comprising heavy and light chain variable region sequences, wherein:

the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence set forth in FIG. 7, an

The light chain sequence has at least 85% sequence identity to the light chain sequence set forth in fig. 7.

In some embodiments, the degree of sequence identity may be one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

In some embodiments, the antibody, antigen-binding fragment, or polypeptide is further according to HCFR1: HC-CDR1: HCFR2: HC-CDR2: HCFR3: HC-CDR3: the arrangement of HCFR4 comprises variable region heavy chain framework sequences between the CDRs. The framework sequence may be derived from a human consensus framework sequence.

In some cases, the antibody, antigen-binding fragment, or polypeptide comprises a heavy chain sequence selected from the group consisting of seq id nos:

VQSGAEVKKPGASVKVSCKASGYTFTNYYMHWV(SEQ ID NO：29)；

WIYPGNVNTYYNEKFR(SEQ ID NO：30)；

ASTAYMELSSLRSE (SEQ ID NO: 31); and/or

ASEEKNYPWFAYWGQGTLVT(SEQ ID NO：32)。

In one aspect of the invention, there is provided an isolated light chain variable region polypeptide, optionally in combination with a heavy chain variable region polypeptide as described herein, comprising the following CDRs:

LC-CDR1：KASQSVEDDGENYMN(SEQ ID NO：4)

LC-CDR2：AASNLES(SEQ ID NO：5)

LC-CDR3：QQSNEDPFT(SEQ ID NO：6)

in some embodiments, the antibody, antigen-binding fragment, or polypeptide is further according to LCFR1: LC-CDR1: LCFR2: LC-CDR2: LCFR3: LC-CDR3: the arrangement of LCFR4 comprises variable region light chain framework sequences between the CDRs. The framework sequence may be derived from a human consensus framework sequence.

In some cases, the antibody, antigen-binding fragment, or polypeptide comprises a light chain sequence selected from the group consisting of seq id nos:

QSPSSLSASVGDRVT(SEQ ID NO：26)；

KASQSVEDDGENYMNWYQQK (SEQ ID NO: 27); and/or

SGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFT(SEQ ID NO：28)。

In some cases, the antibody, antigen-binding fragment, or polypeptide comprises 2,3, 4,5, 6, or all of the amino acid sequences selected from the group consisting of:

VQSGAEVKKPGASVKVSCKASGYTFTNYYMHWV；

WIYPGNVNTYYNEKFR；

ASTAYMELSSLRSE；

ASEEKNYPWFAYWGQGTLVT；

QSPSSLSASVGDRVT；

KASQSVEDDGENYMNWYQK; and/or

SGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFT。

The antibody may comprise at least one light chain variable region (V) _L ) And/or heavy chain variable region (V) _H ) Comprising one of the amino acid sequences shown in figure 7 or an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of the amino acid sequences shown in figure 7 and comprising the following CDR sequences:

LC-CDR1：KASQSVEDDGENYMN(SEQ ID NO：4)

LC-CDR2：AASNLES(SEQ ID NO：5)

LC-CDR3：QQSNEDPFT(SEQ ID NO：6)

HC-CDR1：GYTFTNYYMH(SEQ ID NO：1)

HC-CDR2：WIYPGNVNTYYNEKFRG(SEQ ID NO：2)

HC-CDR3：EEKNYPWFAY(SEQ ID NO：3)

and comprises at least one of the following sequences:

VQSGAEVKKPGASVKVSCKASGYTFTNYYMHWV；

WIYPGNVNTYYNEKFR；

ASTAYMELSSLRSE；

ASEEKNYPWFAYWGQGTLVT；

QSPSSLSASVGDRVT；

kasqsveddemenynwyqk; and/or

SGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFT。

The antibody may comprise at least one light chain variable region (V) _L ) And/or heavy chain variable region (V) _H ) Comprising one of the amino acid sequences shown in figure 7 or having at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 9% of one of the amino acid sequences shown in figure 73%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

The antibody may comprise at least one light chain variable region (V) selected from _H ): SEQ ID NO: 16. the amino acid sequence of SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. the amino acid sequence of SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. the amino acid sequence of SEQ ID NO:24 or SEQ ID NO: 25. or with the amino acid sequence SEQ ID NO: 16. SEQ ID NO: 17. the amino acid sequence of SEQ ID NO: 18. the amino acid sequence of SEQ ID NO: 19. SEQ ID NO: 20. the amino acid sequence of SEQ ID NO: 21. the amino acid sequence of SEQ ID NO: 22. SEQ ID NO: 23. the amino acid sequence of SEQ ID NO: NO:24 or SEQ ID NO:25, or an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

Preferably, the antibody comprises a light chain variable region (V) selected from the group consisting of _H ): SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. the amino acid sequence of SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO:21 or SEQ ID NO:22, or a sequence identical to the amino acid sequence of SEQ ID NO: 16. the amino acid sequence of SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. the amino acid sequence of SEQ ID NO: 20. SEQ ID NO:21 or SEQ ID NO:22, and more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

The antibody may comprise at least one light chain variable region (V) selected from _L ): SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. the amino acid sequence of SEQ ID NO: 11. the amino acid sequence of SEQ ID NO: 12. the amino acid sequence of SEQ ID NO: 13. SEQ ID NO:14 or SEQ ID NO:15, or a sequence identical to the amino acid sequence SEQ ID NO: 7. the amino acid sequence of SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO:14 or SEQ ID NO:15, or a variant thereof, and more preferably an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

Preferably, the antibody comprises a light chain variable region (V) selected from the group consisting of _L ): SEQ ID NO: 7. SEQ ID NO: 8. the amino acid sequence of SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO:11 or SEQ ID NO:12, or a sequence identical to the amino acid sequence SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO:11 or SEQ ID NO:12, and a polypeptide having at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

The antibody may comprise the following CDR sequences:

LC-CDR1：KASQSVEDDGENYMN(SEQ ID NO：4)

LC-CDR2：AASNLES(SEQ ID NO：5)

LC-CDR3：QQSNEDPFT(SEQ ID NO：6)

HC-CDR1：GYTFTNYYMH(SEQ ID NO：1)

HC-CDR2：WIYPGNVNTYYNEKFRG(SEQ ID NO：2)

HC-CDR3：EEKNYPWFAY(SEQ ID NO：3)

and comprises at least one of the following sequences:

VQSGAEVKKPGASVKVSCKASGYTFTNYYMHWV；

WIYPGNVNTYYNEKFR；

ASTAYMELSSLRSE；

ASEEKNYPWFAYWGQGTLVT；

QSPSSLSASVGDRVT；

kasqsveddemenynwyqk; and/or

SGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFT；

And is capable of binding PRL3 and antagonizing a biological function of PRL3.

In some embodiments, the antibody or antibody binding fragment may further comprise a human constant region. For example one selected from the group consisting of IgG1, igG2, igG3, and IgG 4.

In some embodiments, the antibody or antibody binding fragment may further comprise a murine constant region. For example one selected from the group consisting of IgG1, igG2A, igG2B and IgG 3.

The antibody is preferably a whole antibody, or one comprising an Fc domainAn antibody or antibody fragment. The antibody or antibody fragment may comprise one or both of the CH1 and CH2 domains. Preferably, the antibody comprises a CH2 domain. Antibodies may contain CH1 and CH2 domains. Preferably, the antibody is not Fab ', F (ab)' ₂ Fragments, and/or not scFv and/or not minibody. Preferably, the antibody is an IgG immunoglobulin.

In some aspects, the individual to be treated is immunocompetent. The individual may have been determined to be immunologically active. An individual may have been determined to produce NK cells and/or B cells. The subject can be treated to stimulate production and/or activation of NK cells and/or B cells, for example, by administering a cytokine, or by stopping administration of an agent known to reduce production and/or activation of NK cells and/or B cells.

In another aspect of the invention, a composition, such as a pharmaceutical composition or medicament, is provided. The composition may comprise an antibody, antigen-binding fragment or polypeptide as described herein and at least one pharmaceutically acceptable carrier, excipient, adjuvant or diluent.

In another aspect of the invention, isolated nucleic acids encoding the antibodies, antigen-binding fragments, or polypeptides described herein are provided. The nucleic acid encodes a sequence as set out in figure 7, or is a coding sequence which is degenerate as a result of the genetic code, or may have a nucleotide sequence which is at least 70%, optionally 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto.

The antibody can bind to PRL3. The antibody may bind to an epitope comprising the amino acid sequence KAKFYN and/or HTHKTR. An antibody may be capable of binding both sequences.

In one aspect of the invention, vectors comprising the nucleic acids described herein are provided. In another aspect of the invention, a host cell comprising the vector is provided. For example, the host cell may be a eukaryote, or a mammal, such as the Chinese Hamster Ovary (CHO), or a human cell or may be a prokaryotic cell, such as e. In one aspect of the invention, there is provided a method of making an antibody or antigen-binding fragment or polypeptide described herein, the method comprising culturing a host cell described herein under conditions suitable for expression of a vector encoding the antibody or antigen-binding fragment or polypeptide, and recovering the antibody or antigen-binding fragment or polypeptide.

In another aspect of the invention, an antibody, antigen-binding fragment or polypeptide for use in therapy or in a method of medical treatment is provided. In another aspect of the invention, there is provided an antibody, antigen-binding fragment or polypeptide as described herein for use in the treatment of T cell dysfunction. In another aspect of the invention, there is provided the use of an antibody, antigen-binding fragment or polypeptide as described herein in the manufacture of a medicament or pharmaceutical composition for the treatment of T cell dysfunction.

In another aspect of the invention, a method is provided comprising contacting a sample containing or suspected of containing PRL3 with an antibody or antigen-binding fragment as described herein, and detecting the formation of a complex of the antibody or antigen-binding fragment and PRL3.

In another aspect of the invention, there is provided a method of diagnosing a disease or disorder in a subject, the method comprising contacting a sample from the subject with an antibody or antigen-binding fragment, as described herein, in vitro, and detecting the formation of a complex of the antibody or antigen-binding fragment and PRL3.

In another aspect of the invention, there is provided the use of an antibody or antigen-binding fragment as described herein for the detection of PRL3 in vitro. In another aspect of the invention, there is provided the use of an antibody or antigen-binding fragment as described herein as an in vitro diagnostic agent.

In the methods of the invention, the antibody, antigen-binding fragment, or polypeptide can be provided as a composition as described herein.

In any aspect of the invention, the antibody preferably specifically binds PRL3 but not other PRL phosphatases, e.g. PRL1 or PRL2.

The antibody may be an IgG. It may have a molecular weight of about 140 to 160kDa, preferably about 150kDa.

In some embodiments, the antibody can be PRL 3-mab.

Also disclosed herein is the use of a humanized antibody or antigen binding fragment as disclosed herein in the preparation of a medicament for the treatment of cancer.

In other aspects, humanized antibodies or antibody binding fragments are provided for use in methods of treating cancer. The antibodies are useful for inhibiting tumor formation and/or inhibiting tumor metastasis. The antibodies can be used to reduce the size of a tumor. For example, a treated individual may show a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more reduction in tumor size, or a reduction in the number of tumors, or both, of a particular tumor as compared to an untreated individual, or as compared to the same individual prior to treatment.

Also provided are methods of treating cancer comprising administering a humanized antibody or antibody binding fragment disclosed herein.

The cancer may be a PRL3 expressing or overexpressing cancer. The cancer may be gastric cancer.

The humanized antibody or antibody binding fragment can be administered intravenously. It may be administered at a site remote from the cancer to be treated.

In some methods, the patient has not previously received chemotherapy, particularly anti-metabolic therapy, such as 5-FU. In some cases, patients have not previously received such treatment, or have not received such treatment to treat their cancer, such as gastric cancer. In some cases, the antibody is not co-administered with another agent (i.e., antibody monotherapy). In some cases, the antibody is not co-administered with 5-FU.

In some methods, it has been determined that the patient has not had an impaired immune system. In particular, the patient's white blood cell count may have been determined to be within a normal range. In particular, the patient may have been determined to be free of leukopenia. The patient may have been determined to have a neutrophil, lymphocyte, monocyte, erythrocyte or platelet count within the normal range. The patient's white blood cell count, neutrophil count, lymphocyte count, monocyte count, red blood cell count, or platelet count is not significantly different from a control group, such as counts from individuals known not to have an impaired immune system, or from established normal values. For example, a patient may be determined to have about 4,500 to about 10,000 leukocytes per microliter of blood.

In some aspects, the invention provides methods of selecting a patient for treatment with a humanized anti-PRL 3 antibody or antibody fragment, the method comprising determining the presence of PRL3 in a urine sample from the patient. In some cases, the method involves determining the level of PRL3 in a urine sample from the patient. In some cases, the patient may have gastric cancer, nasopharyngeal cancer, bladder cancer, lung cancer, breast cancer, or prostate cancer.

In some cases, the individual has a family history of PRL3 overexpressing cancer, or has been determined to have a likelihood of developing PRL3 overexpressing cancer. In certain instances, an individual has a cancer that overexpresses PRL3 and is considered to be at risk for metastasis of the cancer.

In another aspect, provided herein are methods involving determining cellular localization of PRL3. An increased proportion of cellular PRL3 on the cell surface may indicate that the individual has cancer. Provided herein is a method comprising determining the cellular location of PRL3 in a cell, wherein expression of PRL3 at the cell surface indicates that the cell is cancerous.

Methods include methods for diagnosing cancer, wherein the presence or increase of PRL3 on the cell surface can be indicative that an individual has cancer. In certain instances, the amount of PRL3 in the cell is the same as the non-cancerous control sample, but the location of PRL3 can be altered compared to the non-cancerous control.

Other methods include methods for determining whether a cell is cancerous comprising determining the presence of PRL3 on the surface of the cell. An increase in the level or proportion of PRL3 compared to control cells may indicate that the individual has become (has) cancer or is about to become (has).

The method can include selecting an individual for anti-cancer therapy based on the cellular localization of PRL3 in the sample. In some cases, the method involves administering an anti-cancer therapy to the individual so selected.

In some cases, the method can include determining the cellular localization of PRL3 in two or more samples from the patient at two or more time points. A change in the amount of PRL3 on the cell surface can indicate an increase or decrease in cancer in the individual. An increase in cell surface PRL3 over time may indicate that the individual has suffered from cancer, or that the cancer has worsened. A decrease in cell surface PRL3 over time may indicate that the cancer has decreased, or that the treatment has led to treatment of the cancer. An increase or an absence of a level of PRL3 on the cell surface may indicate a need for additional or alternative anti-cancer therapies. Thus, the level of PRL3 on the cell surface can be used to select individuals for further or alternative anti-cancer therapy.

A 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold increase in cell surface PRL3 may indicate that the individual has cancer, and/or that the cell is cancerous, or that the individual should be selected for treatment. The level of PRL3 on the cell surface can be compared to a control.

The sample may be a blood sample or a serum sample. The sample may be a urine sample. The cancer may be a sample of a tumor or tissue surrounding a tumor. The method may comprise obtaining a sample, or the method may be performed on a sample previously obtained from the individual.

Diagnostic and detection methods may be performed in vitro or ex vivo, and in some cases do not involve the step of obtaining a sample from an individual.

Description of the invention

Antibodies

The antibodies of the invention preferably bind PRL3 (antigen), optionally with a Kd of 5pM to 8pM, preferably 6-7pM, preferably about 6.3pM. In some cases, the dissociation rate of the antibody is about 7X 10 ^-5 s ^-1 . For example, at about 1X 10 ^-5 s ^-1 And 1X 10 ^-6 s ^-1 In the meantime.

In some embodiments, an antibody of the invention binds PRL3, but not PRL1 or PRL2.

The antibodies of the invention may be provided in isolated form.

By "antibody" we include fragments or derivatives thereof, or synthetic antibodies or fragments of synthetic antibodies.

Given the current technology on monoclonal antibody technology, antibodies can be prepared against most antigens. The antigen-binding portion can be a portion of an antibody (e.g., a Fab fragment) or a synthetic antibody fragment (e.g., a single chain Fv fragment [ ScFv ]). Suitable monoclonal antibodies to the selected antigen can be prepared by known techniques, for example as described in "monoclonal antibodies: technical manual ", H Zola (CRC press, 1988) and" monoclonal hybridoma antibodies: technique and applications ", J G R Hurrell (CRC press, 1982). Neuberger et al (1988, eighth International Biotechnology Symposium) section 2,792-799, discuss chimeric antibodies.

Monoclonal antibodies (mabs) may be used in the methods of the invention and are homogeneous populations of antibodies that specifically target a single epitope on an antigen. Accordingly, mabs that bind PRL3 may be used to treat cancer.

Antigen-binding fragments of antibodies, e.g. Fab and Fab ₂ Fragments may also be provided as genetically engineered antibodies and antibody fragments. Variable weight (V) of antibody _H ) And variable lightness (V) _L ) The fact that the domains are involved in antigen recognition was first identified by early protease digestion experiments. Further confirmation was by "humanization" of rodent antibodies. Variable domains of rodent origin can be fused to constant domains of human origin such that the resulting antibody retains the antigen specificity of the rodent parent antibody (Morrison et al (1984) Proc. Natl. Acad. Sd. USA 81, 6851-6855).

The antibodies and antibody binding fragments of the invention have been humanized. Humanized antibodies are antibodies from non-human species whose protein sequences have been modified to increase their similarity to naturally occurring antibody variants of humans. The "humanization" process is commonly applied to the development of monoclonal antibodies for human administration. When the process of developing specific antibodies involves production in a non-human immune system, such as a mouse, a "humanization" process may be necessary because such antibodies may be immunogenic when administered to a human patient. Humanization may involve the substitution of selective amino acids in the Fab portion of the molecule. Alternatively, humanization may involve insertion of appropriate CDR coding segments into a human antibody scaffold.

Antigen specificity is conferred by variable domains and is independent of constant domains, as is known from experiments involving bacterial expression of antibody fragments, all of which contain one or more variable domains. These include Fab-like molecules (Better et al (1988) Science 240, 1041); fv molecules (Skerra et al (1988) Science 240, 1038); single chain Fv (ScFv) molecules, wherein V _H And V _L The paired domains are linked by a flexible oligopeptide (Bird et al (1988) Science 242,423, huston et al (1988) proc.natl.acad.sd.usa 85, 5879) and a single domain antibody (dAbs) comprising an isolated V domain (Ward et al (1989) Nature 341, 544). A general review of techniques involved in the synthesis of antibody fragments that retain their specific binding sites can be found in Winter and Milstein (1991) Nature 349, 293-299.

"ScFv molecule" means V _H And V _L The pairing domains are covalently linked (e.g., by a flexible oligopeptide) to the molecule.

Fab, fv, scFv and dAb antibody fragments can all be expressed and secreted in E.coli, allowing easy production of large quantities of the fragment.

Whole antibody and F (ab') ₂ Fragments are "bivalent". "bivalent" refers to the antibody and F (ab') ₂ The fragment has two antigen binding sites. In contrast, fab, fv, scFv and dAb fragments are monovalent, having only one antigen binding site. Synthetic antibodies that bind PRL3 can also be prepared using phage display techniques well known in the art.

Antibodies can be produced by the process of affinity maturation, wherein a modified antibody is produced that has improved affinity for an antigen compared to the unmodified parent antibody. Affinity matured antibodies can be produced by methods known in the art, such as Marks et al, rio/Technology 10 (1992); barbas et al Proc nat. Acad. Sci. USA 91 3809-3813 (1994); schier et al Gene 169 (1995); yelton et al J.Immunol.155:1994-2004 (1995); jackson et al, J.Immunol.154 (7): 331-15 (1995); and Hawkins et al J.mol.biol.226:889-896 (1992).

The antibodies of the invention preferably exhibit specific binding to PRL3. Antibodies that specifically bind to a target molecule preferably bind to the target with a higher affinity and/or bind to the target for a longer duration than to other targets. In one embodiment, the degree of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as determined by Radioimmunoassay (RIA).

The dissociation constant (Kd) of the antibody of the present invention is preferably one of 1. Mu.M or less, 100nM or less, 10nM or less, 1nM or less, or 100pM or less. The binding affinity of an antibody for its target is usually described in terms of its dissociation constant (Kd). Binding affinity can be measured by methods known in the art, for example, by a radiolabeled antigen binding assay (RIA) performed with a Fab version of the antibody and an antigenic molecule.

The antibodies of the invention may be "antagonist" antibodies that inhibit or reduce the biological activity of the antigen to which they bind. Blocking PRL3 may inhibit or reduce the phosphatase activity of PRL3. In some cases, the antibody binds to, but does not necessarily affect, the activity of PRL3.

In certain methods, the antibody is PRL 3-mab, or a variant of PRL 3-mab. PRL 3-globin comprises the following CDR sequences:

light chain:

LC-CDR1:(SEQ ID NO：4)

LC-CDR2：(SEQ ID NO：5)

LC-CDR3：(SEQ ID NO：6)

heavy chain:

HC-CDR1:(SEQ ID NO：1)

HC-CDR2：(SEQ ID NO：2)

HC-CDR3：(SEQ ID NO：3)

CDR sequences are determined by Kabat definition.

The structure of an antibody molecule having a CDR as described herein is typically the heavy or light chain sequence of the antibody molecule, or a substantial portion thereof, in which the CDR is located in a position corresponding to a naturally occurring V encoded by a rearranged immunoglobulin gene _H And V _L The position of the CDRs of the antibody variable domain. The structure and position of immunoglobulin variable domains can be referenced to Kabat, e.a., et al, immunology protein sequences, 4 th edition, U.S. department of health and public services, 1987 and updates thereto. There are manyAcademic and business online resources can be used to query this database. See, for example, martin, a.c.r. computer access to Kabat antibody sequence database proteins: structure, function and genetics, 25 (1996), 130-133 and related online resources, currently at the world wide web address bio in org. Uk/abs/simkab. Html.

The antibody of the invention may comprise the CDRs of PRL 3-globin, or the CDRs of one of SEQ ID NOs 1-6. In the antibodies of the invention, one or two or three or four of the six CDR sequences may vary. A variant may have one or two amino acid substitutions in one or two of the six CDR sequences.

anti-PRL 3-monoclonal antibody cloned V _H And V _L The amino acid sequence of the chain is shown in FIG. 7.

Light and heavy chain CDRs can also be used, inter alia, in conjunction with a number of different framework regions. Thus, light and/or heavy chains with LC-CDR1-3 or HC-CDR1-3 may have alternative framework regions. Suitable framework regions are well known in The art and are described, for example, in M.Lefranc and G.Le: franc (2001) "The immunologlobulin facesbook", academic Press, which is incorporated herein by reference.

In the present specification, the antibody may have V _H And/or V _L A chain comprising an amino acid sequence having a high percentage of sequence identity to one or more of the VH and/or VL amino acid sequences of figure 7.

For example, an antibody of the invention comprises a binding PRL3 and has V _H Antibodies of chain, said V _H The chain has an amino acid sequence that has at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of the amino acid sequences set forth in figure 7.

The antibodies of the invention may be detectably labeled or at least capable of detection. For example, the antibody may be labeled with a radioactive atom or a colored or fluorescent molecule or a molecule that can be easily detected in any other way. Suitable detectable molecules include fluorescent proteins, luciferases, enzyme substrates and radioactive labels. The binding moiety may be directly labeled with a detectable label or may be indirectly labeled. For example, the binding moiety may be an unlabeled antibody, which may be detected by another antibody that is itself labeled. Alternatively, the second antibody may have biotin bound thereto, and labeled streptavidin is bound to the biotin for indirectly labeling the first antibody.

Although various antibody fragments are described herein, the antibody is preferably a whole antibody, which contains an antibody binding fragment (Fab) and a crystallizable fragment (Fc). An antibody may consist of two heavy chains and two light chains. It comprises variable fragments (Fv) and constant domains that provide the antigen specificity of the antibody.

Antibody fragments of the invention preferably comprise a CH2 domain. The CH2 domain of an antibody plays an important role in mediating effector function and maintaining antibody stability. Thus, the antibody fragment of the invention is preferably not a Fab ', F (ab)' 2, scFv or minibody.

The antibodies and fragments of the invention are preferably capable of interacting with Fc γ (Fc- γ) receptors, preferably Fc γ II (CD 32) and Fc γ III (CD 16) receptors.

Detection method

The antibodies or antigen-binding fragments described herein can be used in methods involving binding the antibodies or antigen-binding fragments to PRL3. Such methods may involve detecting a binding complex of an antibody or antigen-binding fragment and PRL3. Thus, in one embodiment, a method is provided that includes contacting a sample containing or suspected of containing PRL3 with an antibody or antigen-binding fragment described herein, and detecting the formation of a complex of the antibody or antigen-binding fragment and PRL3.

Suitable methods formats are well known in the art and include immunoassays, for example sandwich assays such as ELISA. The method may comprise labeling the antibody or antigen-binding fragment or PRL3 or both with a detectable label (e.g., a fluorescent, luminescent, or radioactive label).

Such methods may provide a basis for diagnostic methods for diseases or conditions that require PRL3 detection and quantification. Such methods may be performed in vitro on a patient sample, or after processing of the patient sample. Once the sample is collected, the patient is not required to be present while performing the in vitro method for diagnosis, and thus the method may be a method that is not performed on the human or animal body.

Such methods may involve determining the amount of PRL3 present in a patient sample. The method may further comprise comparing the determined amount to a standard or reference value as part of the process of reaching the diagnosis. Other diagnostic tests may be combined with those described herein to enhance the accuracy of the diagnosis or prognosis, or to confirm the results obtained by using the tests described herein.

Detection in a PRL3 sample can be used for the purpose of diagnosing a cancerous condition in a patient, diagnosing a predisposition to, or providing a prognosis for a cancerous condition. Diagnosis or prognosis may involve an existing (previously diagnosed) cancerous condition, which may be benign or malignant, may involve a suspected cancerous condition, or may involve screening of a patient for a (possibly previously undiagnosed) cancerous condition.

The sample may be removed from any tissue or body fluid. The sample may comprise or may be derived from: a quantity of blood; a quantity of serum from the blood of the individual, possibly comprising a fluid fraction of the blood obtained after removal of fibrin clots and blood cells; a tissue sample or biopsy; or a cell isolated from the individual.

The method of the invention is preferably carried out in vitro. The term "in vitro" is intended to include experiments with cells in culture, while the term "in vivo" is intended to include experiments with intact multicellular organisms.

Therapeutic applications

The antibodies, antigen-binding fragments and polypeptides of the invention, as well as compositions comprising such agents, may be provided for use in methods of medical treatment. Treatment can be provided to a subject having a disease or disorder in need of treatment. The disease or disorder may be cancer, including metastatic cancer.

Administration of the antibody, antigen-binding fragment or polypeptide is preferably in a "therapeutically effective amount", which is sufficient to show benefit to the individual. The actual amount administered, the rate of administration and the time course will depend on the nature and severity of the disease being treated. The treatment regimen, e.g., decisions regarding dosages and the like, is left up to the responsibility of general practitioners and other medical practitioners, and generally takes into account the disease to be treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to practitioners. Examples of the above mentioned techniques and protocols can be found in Remington pharmacy, 20 th edition, 2000 published in Lippincott, williams & Wilkins.

The methods and compositions described herein are suitably capable of improving the measurable criteria of an individual to whom treatment is applied, as compared to a patient who is not receiving treatment.

For this purpose, a number of criteria may be specified that reflect the progression of the cancer or the health of the patient. Useful criteria may include tumor size, tumor maximum size, tumor number, presence of tumor markers (e.g., alpha-fetoprotein), degree or number of metastases, and the like.

Thus, by way of example, treated individuals may show a reduction in tumor size or number as measured by an appropriate assay or test. A treated individual may show, for example, a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more reduction in tumor size, or a reduction in the number of tumors, or both, of a particular tumor as compared to an untreated individual. The term proliferative disease is used herein in a broad sense and includes any disease requiring control of the cell cycle. In particular, proliferative diseases include malignant and pre-neoplastic diseases. The methods and compositions described herein are particularly useful in the treatment or diagnosis of adenocarcinoma, for example: small cell lung cancer, kidney cancer, uterine cancer, prostate cancer, bladder cancer, ovarian cancer, colon cancer, and breast cancer. For example, malignancies that can be treated include acute and chronic leukemias, lymphomas, myelomas, sarcomas such as fibrosarcoma, myxosarcoma, liposarcoma, lymphatic endothelial cell tumor, angiosarcoma, endothelial sarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, lymphatic sarcoma, synovioma, mesothelioma, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, ovarian cancer, prostate cancer, pancreatic cancer, breast cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchopulmonary carcinoma, choriocarcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma seminoma, embryonal carcinoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, ependymoma, pinealoma, 10 hemangioblastoma, acoustic neuroma, medulloblastoma, craniopharyngeal tumor, oligodendroglioma, hemangioma, melanoma, neutroblastoma, and retinoblastoma.

For purposes of this specification, the term "cancer" may include any one or more of: <xnotran> (ALL), (AML), , , , , , , , , , , , , , (CLL), (CML), , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , . </xnotran>

Treatment may result in the alleviation of symptoms of the cancer, or may result in the complete treatment of the cancer. Treatment may slow the progression of the cancer, or may prevent the worsening of the symptoms of the cancer.

Preparation of pharmaceutically useful compositions and medicaments

The antibodies, antigen-binding fragments, and polypeptides of the invention may be formulated into pharmaceutical compositions for clinical use and may include pharmaceutically acceptable carriers, diluents, excipients, or adjuvants.

According to the present invention there is also provided a process for the production of a pharmaceutically useful composition, which process may comprise one or more steps selected from the group consisting of: isolating an antibody, antigen-binding fragment, or polypeptide described herein; and/or mixing the isolated antibody, antigen-binding fragment, or polypeptide described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

For example, another aspect of the invention relates to a method of formulating or manufacturing a medicament or pharmaceutical composition for treating a T cell dysfunctional disease, the method comprising formulating the pharmaceutical composition or medicament by mixing an antibody, antigen-binding fragment, or polypeptide as described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

Cancer treatment

A cancer may be any unwanted cell proliferation (or any disease manifested by unwanted cell proliferation), neoplasm, or tumor, or an increased risk or propensity for unwanted cell proliferation, neoplasm, or tumor. Cancer may be benign or malignant, and may be primary or secondary (metastatic). A neoplasm or tumor can be any abnormal growth or proliferation of a cell and can be located in any tissue. Examples of tissues include adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone marrow, brain, breast, cecum, central nervous system (including or not including brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph node, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsil, trachea, uterus, vulva, leukocytes.

The tumor to be treated may be a nervous system or a non-nervous system tumor. Tumors of the nervous system may originate in the central or peripheral nervous system, such as gliomas, medulloblastomas, meningiomas, neurofibromas, ependymomas, schwannomas, neurofibrosarcomas, astrocytomas and oligodendrogliomas. Non-nervous system cancers/tumors may originate from any other non-nervous tissue, such as melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-hodgkin's lymphoma (NHL), hodgkin's lymphoma, chronic Myelogenous Leukemia (CML), acute Myelogenous Leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic Lymphocytic Leukemia (CLL), liver cancer, epidermoid cancer, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymus cancer, NSCLC, hematologic cancer and sarcoma.

In a particularly preferred aspect, the cancer is a PRL3 expressing cancer. In certain instances, the cancer is a PRL 3-overexpressing cancer. That is, the cancer is associated with or caused by overexpression of PRL 3.PRL3 need not play a role in cancer, but may be a marker or phenomenon of cancer cells. In particularly preferred aspects, the cancer is gastric cancer, nasopharyngeal cancer, bladder cancer, lung cancer, breast cancer, or prostate cancer. The cancer may be acute myeloid leukemia, colon cancer or ovarian cancer. In some cases, the cancer is a metastatic cancer.

Simultaneous or sequential application

Depending on the condition to be treated, the compositions may be administered alone or in combination with other therapies, either simultaneously or sequentially.

In the present specification, the antibody, antigen-binding fragment or polypeptide of the present invention and the anti-infective agent or chemotherapeutic agent (therapeutic agent) may be administered simultaneously or sequentially.

In some embodiments, treatment with an antibody, antigen-binding fragment, or polypeptide of the invention may be accompanied by chemotherapy.

Simultaneous administration refers to administration of the antibody, antigen-binding fragment or polypeptide and the therapeutic agent together, e.g., as a pharmaceutical composition (combined preparation) containing both agents, or in close proximity to each other, and optionally via the same route of administration, e.g., to the same artery, vein, or other blood vessel.

Sequential administration refers to administration of one of the antibody, antigen-binding fragment or polypeptide or therapeutic agent followed by separate administration of the other agent after a given time interval. It is not required that both agents be administered by the same route, although this may be the case in some embodiments. The time interval may be any time interval.

Chemotherapy

Chemotherapy refers to the treatment of cancer with drugs or ionizing radiation (e.g., radiation therapy using X-rays or gamma rays). In a preferred embodiment, chemotherapy refers to treatment with a drug. The drug may be a chemical entity, such as a small molecule drug, an antibiotic, a DNA intercalator, a protein inhibitor (e.g., a kinase inhibitor) or a biological agent, such as an antibody, an antibody fragment, a nucleic acid or peptide aptamer, a nucleic acid (e.g., DNA, RNA), a peptide, a polypeptide, or a protein. The medicament may be formulated as a pharmaceutical composition or medicament. The formulation may comprise one or more drugs (e.g. one or more active agents) together with one or more pharmaceutically acceptable diluents, excipients or carriers.

Treatment may involve the administration of more than one drug. The drugs may be administered alone or in combination with other therapies, either simultaneously or sequentially, depending on the condition to be treated. For example, chemotherapy may be a combination therapy involving the administration of two drugs, one or more of which may be used to treat cancer.

Chemotherapy may be administered by one or more routes of administration, e.g., parenterally, intravenously, orally, or intratumorally.

Chemotherapy may be performed according to a treatment regimen. The treatment regimen may be a predetermined schedule, plan, regimen or chemotherapy regimen that may be prepared by a physician or physician and may be tailored to the patient in need of treatment.

The treatment regimen may indicate one or more of: the type of chemotherapy administered to the patient; the dose of each drug or radiation; the time interval between administrations (dosing); the length of each treatment; the number and nature of any treatment intervals, if any, etc. For combination therapy, a single treatment regimen may be provided that indicates how each drug is to be administered.

The chemotherapeutic agent may be selected from:

alkylating agents such as cisplatin, carboplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide;

purine or pyrimidine antimetabolites such as imidazole azathioprine or mercaptopurine;

alkaloids and terpenoids, such as vinca alkaloids (e.g. vincristine, vinblastine, vinorelbine, vindesine), podophyllotoxin, etoposide, teniposide, taxanes such as Taxol (Taxol) ^TM ) Docetaxel;

topoisomerase inhibitors, such as the topoisomerase inhibitor type I, camptothecin irinotecan and topotecan, or the topoisomerase inhibitor type II, amsacrine (amsacrine), etoposide phosphate, teniposide;

antitumor antibiotics (e.g. anthracyclines) such as dactinomycin, doxorubicin (adriamycin) ^TM ) Epirubicin, bleomycin, rapamycin;

antibody-based agents, such as anti-TIM-3 antibodies, anti-VEGF, anti-TNF α, anti-IL-2, anti-GpIIb/IIIa, anti-CD-52, anti-CD 20, anti-RSV, anti-HER 2/neu (erbB 2), anti-TNF receptor, anti-EGFR antibodies, polyclonal antibodies or antibody fragments, examples include: cetuximab, panitumumab, infliximab, basiliximab, bevacizumabAbciximab, dalizumab, gemtuzumab ozogamicin, alemtuzumab, rituximabPalivizumab, trastuzumab, etanercept, adalimumab, nituzumab;

EGFR inhibitors such as erlotinib, cetuximab and gefitinib;

anti-angiogenic agents such as bevacizumab

The other chemotherapeutic agent may be selected from: 13-cis-retinoic acid, 2-chlorodeoxyadenosine, 5-azacytidine, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, albumin-bound paclitaxel (Abraxane),actinomycin-DAldesleukin, alemtuzumab Ainingda ((ALIMTA)), aliviroc acid,All-trans retinoic acid, alpha interferon, altretamine, methotrexate, amifostine, aminoglutethimide, anagrelide,Anastrozole, arabinocytosine,Arsenic trioxide, asparaginase, ATRAAzacitidine, BCG, BCNU, bendamustine, bevacizumab, bexarotene,Bicalutamide, biCNU,Bleomycin, bortezomib, busulfan,Formyl tetrahydrofolic acid calcium,Kemptol-11, capecitabine, fluorouracil (Carac) ^TM ) Carboplatin, carmustine,CC-5013、CCI-779、CCNU、CDDP、CeeNU、Cetuximab, chlorambucil, cisplatin leucovorin factor, cladribine, cortisone,CPT-11, cyclophosphamide,CytarabineDackinins (Dacogen), actinomycin, daltepatin alpha, dasatinib, daunomycin, daunorubicin hydrochloride, daunorubicin liposome, and pharmaceutically acceptable salts thereof,Dexamethasone, decitabine,Denil interleukin (Denileukin), diphtheria toxin (Diftitox), cytarabine ^TM Dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate dexamethasone sodium phosphate (Dexasone), dexrazoxane, DHAD, DIC, diodex, docetaxel,Adriamycin, adriamycin liposomes, hydroxyurea capsules (Droxina) ^TM )、DTIC、Eligard ^TM 、Ellence ^TM 、Eloxatin ^TM 、Epirubicin, alfa-liprotine, erbitux, erlotinib, erwinia L-asparaginase, estramustine, ethylolEtoposide, etoposide phosphate,Everolimus,Exemestane, exemestane,Filgrastim, floxuridine,Fludarabine,Fluorouracil, fluoxymesterone, flutamide, folinic acid,Fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, gleevec ^TM 、Wafer, goserelin, granulocyte colony stimulating factor granulocyte-macrophage colony stimulating factor,Dexamethasone (Hexadrol),Hexamethamine, HMM,Hydrocort Hydrocortisone, hydrocortisone sodium Phosphate, hydrocortisone sodium succinate, hydrocortisone Phosphate (hydrocodone Phosphate), hydroxyurea, ibritumomab Tiuxetan, ibritumomab tixetan,Idarubicin (Idarubicin) a,Alpha interferon, ifosfamide, IL-11, IL-2, imatinib mesylate, imidazole carboxamide, alpha interferon-2 b (PEG coupling), interleukin-2, interleukin-11, intron(alpha-interferon-2 b),Irinotecan, isotretinoin, ixabepilone, ixempra ^TM Asparaginase, a,Lapatinib, L-asparaginase, LCR, lenalidomide, letrozole, folinic acid, chlorambucil, leukine ^TM Leuprorelin, vincristine, leustatin ^TM Liposome Ara-C and liquidCyclohexylnitrosurea, L-PAM, L-sacolina,LupronDexamethasone, nitrogen mustard, chlormequat hydrochloride,Megestrol, megestrol acetate, melphalan, mercaptopurine, mesna, mesnex ^TM Methotrexate, methotrexate sodium, methylprednisolone,Mitomycin, mitomycin-C mitoxantrone a,MTC、MTX、Mechlorethamine hydrochloride,Mylocel ^TM 、A nelarabine,Neulasta ^TM 、Nilutamide (I) salt a,Nitrogen mustard,Octreotide, octreotide acetate,Onxal ^TM An Oprevelkin (Oprevelkin),Oxaliplatin, paclitaxel, protein-bound paclitaxel, pamidronate disodium, panitumumab,Pegylated interferon, pemetrexed, pegylated filgrastim, PEG-INTRON ^TM PEG-L-asparaginase, pemetrexed, pentostatin, melphalan,Prednisone, prednisone,Methyl benzyl hydrazine,Implant with carmustine20 of pril span (Prolifeprospan), raloxifene,rituximab,(Interferon alpha-2 a),Daunorubicin hydrochloride,"ShangningA sargrastim,Sorafenib, SPRYCEL ^TM STI-571, streptozotocin, SU11248, sunitinib,Tamoxifen,Temozolomide, sirolimus, teniposide, TESPA, thalidomide,Thioguanine, thioguanineSulfur phosphoric acid amide,Thiotepa,Topotecan, toremifene, and,Tositumomab, trastuzumab,Retinoic acid, trexall ^TM 、TSPA、VCR, vickers ratio (Vectibix) ^TM 、Viadur ^TM 、Vinblastine, vinblastine sulfate, vincasarVincristine, vinorelbine tartrate, VLB, VM-26, vorinostat, VP-16,Zevalin ^TM 、Zoledronic acid, vorinostat capsules (Zolinza),

Route of administration

The antibodies, antigen-binding fragments, polypeptides and other therapeutic agents, drugs and pharmaceutical compositions of aspects of the invention may be formulated for administration by a variety of routes including, but not limited to, parenteral, intravenous, intraarterial, intramuscular, intratumoral, and oral. The antibodies, antigen-binding fragments, polypeptides, and other therapeutic agents may be formulated in fluid or solid form. The fluid formulation may be formulated for administration by injection to a selected region of the human or animal body.

In a preferred aspect, the antibody is administered systemically. Intravenous administration is particularly contemplated.

In some cases, the antibody is applied at a site remote from the cancer cell, or remote from a known cancer cell site. In this case, the antibody may migrate in vivo to cancer cells, for example to a tumor.

In some aspects, the antibody is administered at the site of the cancer cell, e.g., directly to the tumor, or to the site of tumor resection. Administration may be performed during the resection procedure or may be performed after the resection procedure. The tumor may be a primary cancer or a metastatic cancer.

Administration may be performed to prevent tumor regeneration at the site of tumor resection, or administration may be performed to prevent formation of cancer cells at sites other than the resected tumor.

Dosage regimen

Multiple doses of the antibody, antigen-binding fragment, or polypeptide may be provided. One or more or each of the doses may be accompanied by the simultaneous or sequential administration of another therapeutic agent.

The multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18,19, 20,21, 22, 23,24, 25,26, 27, 28, 29, 30, or 31 days, or 1,2, 3, 4,5, or 6 months. For example, a dose may be administered once every 7, 14, 21 or 28 days (plus or minus 3,2 or 1 day).

Reagent kit

In some aspects of the invention, kits of parts are provided. In some embodiments, a kit may have at least one container with a predetermined amount of antibody, antigen-binding fragment, or polypeptide. The kit may provide the antibody, antigen-binding fragment, or polypeptide in the form of a medicament or pharmaceutical composition, and may be provided with instructions for administration to a patient for treating a particular disease or disorder. The antibody, antigen-binding fragment or polypeptide may be formulated so as to be suitable for injection or infusion into a tumor or blood.

In some embodiments, the kit may further comprise at least one container having a predetermined amount of another therapeutic agent (e.g., an anti-infective agent or a chemotherapeutic agent). In such embodiments, the kit may further comprise a second drug or pharmaceutical composition such that the two drugs or pharmaceutical compositions may be administered simultaneously or separately such that they provide a combined treatment for a particular disease or condition. The therapeutic agent may also be formulated so as to be suitable for injection or infusion into a tumor or blood.

Subject of the disease

The subject to be treated may be any animal or human. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but more preferably is a human. The subject may be male or female. The subject may be a patient. A subject may be diagnosed as having a disease or condition requiring treatment, or suspected of having such a disease or condition.

Subject or patient selection

In some aspects, the patient is selected for treatment with a humanized anti-PRL 3 antibody or antibody fragment. In some cases, patients have been determined to have a PRL3 expressing cancer. In certain instances, the cancer is a PRL 3-overexpressing cancer. In some cases, the patient is determined to have a functional or active immune system, e.g., as indicated by a patient with a normal white blood cell count. In some methods, it has been determined that the patient's immune system is not compromised. In particular, it may have been determined that the patient's white blood cell count is within a normal range. In particular, the patient may have been determined to be free of leukopenia. The patient may have been determined to have a neutrophil, lymphocyte, monocyte, erythrocyte or platelet count within the normal range. The patient's white blood cell count, neutrophil, lymphocyte, monocyte, red blood cell or platelet count is not significantly different from that of a control group, such as from individuals known not to have an impaired immune system, or established normal values. For example, a patient can be determined to have about 4,500 to about 10,000 leukocytes per microliter of blood.

Some chemotherapeutic agents are associated with a decrease in white blood cell count, so in some cases, a patient is selected for treatment only if the patient has not received chemotherapy or a particular chemotherapeutic agent in the past. In some cases, patients have not received chemotherapy for their cancer in the past. In some cases, the patient is not receiving chemotherapy with an antimetabolite. In certain instances, the patient is not receiving thymidylate synthase inhibitor therapy. In some cases, the patient is not receiving 5-FU treatment.

The data provided herein show that PRL3 found within tumor cells or cancer cells can be present at sufficient levels in the urine of patients to enable detection. Furthermore, the inventors found that PRL3 can be detected in urine at a very early stage of cancer development. Thus, in some cases, a patient is selected for treatment based on the detection or quantification of PRL3 in a sample of bodily fluid obtained from the patient, such as a urine, saliva, blood or plasma sample, or any other bodily fluid, including breast milk. Preferably, the bodily fluid is urine. The presence or absence of oncoprotein may involve an immunoassay, such as an ELISA or western blot based method. In some cases, PRL3 is detected in exosomes in the sample.

Cancers that can be detected by the methods disclosed herein include gastric cancer, bladder cancer, lung cancer, breast cancer, gastric cancer, nasopharyngeal cancer, prostate cancer (e.g., prostate adenocarcinoma or prostate hyperplasia, particularly prostate hyperplasia). The cancer may be distant from the sample source. The cancer may be a poorly accessible and/or invasive cancer, such as an entry sample or biopsy. Thus, in one aspect disclosed herein, a patient can be diagnosed with cancer by detecting PRL3 in a body fluid sample obtained from the patient, and then selecting treatment with a humanized anti-PRL 3 antibody. The cancer may be a solid cancer. As demonstrated herein, PRL3 is associated with a variety of cancers.

As explained herein, detecting can involve determining the cellular location of PRL3, wherein an increase in PRL3 on the cell surface can indicate that the individual has cancer, or that the cell is cancerous.

One skilled in the art will readily understand the methods used to determine the cellular localization of PRL3. In some cases, immunoassays are used to detect a target (e.g., PRL 3) in a sample from an individual. Immunoassays use antibodies with specific affinity for a target molecule in combination with a detectable molecule. In some cases, the antibody is conjugated to a detectable molecule. The detectable molecule may be referred to as a label. When the antibody binds to the target molecule, the detectable molecule produces a detectable signal. The detectable signal may be a quantifiable signal. In some cases, aptamers are used instead of or together with antibodies. Suitable methods include immunohistochemistry, such as in situ hybridization, fluorescence Activated Cell Sorting (FACS) or flow cytometry. The methods may use a binding agent, e.g., an antibody or aptamer that binds PRL3, e.g., PRL3 mab. The method may comprise exposing the sample to a binding agent such that the cell surface PRL3 is bound by the binding agent, whereby the binding agent may be detected.

Protein expression

Molecular biology techniques suitable for producing a polypeptide of the invention in a cell are well known in the art, such as those described in Sambrook et al, molecular Cloning: A Laboratory Manual, new York: cold spring harbor Laboratory Press, 1989 (Molecular Cloning: A Laboratory Manual).

The polypeptide may be expressed from a nucleotide sequence. The nucleotide sequence may be comprised in a vector present in the cell or may be integrated into the genome of the cell.

As used herein, a "vector" is an oligonucleotide molecule (DNA or RNA) that serves as a vector for transferring foreign genetic material into a cell. The vector may be an expression vector for expressing the genetic material in a cell. Such vectors may include a promoter sequence operably linked to a nucleotide sequence encoding the gene sequence to be expressed. The vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer and stop codon known in the art may be used to express the polypeptide from the vector according to the invention. Suitable vectors include plasmids, binary vectors, viral vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

In the present specification, the term "operably linked" may include situations in which a selected nucleotide sequence and a regulatory nucleotide sequence (e.g., a promoter and/or enhancer) are covalently linked in such a way as to place expression of the nucleotide sequence under the influence or control of a regulatory sequence (thereby forming an expression cassette). Thus, a control sequence is operably linked to a selected nucleotide sequence if the control sequence is capable of affecting transcription of the nucleotide sequence. The resulting transcript may then be translated into the desired protein or polypeptide, where appropriate.

Any cell suitable for expression of the polypeptide may be used to prepare the peptides of the invention. The cell may be a prokaryote or a eukaryote. Suitable prokaryotic cells include E.coli. Examples of eukaryotic cells include yeast cells, plant cells, insect cells, or mammalian cells. In some cases, the cell is not prokaryotic, as some prokaryotic cells do not allow the same post-translational modifications as eukaryotic organisms. Furthermore, very high expression levels are possible in eukaryotes and proteins can be more easily purified from eukaryotes using appropriate tags. Specific plasmids can also be used which enhance secretion of the protein into the culture medium.

Methods of producing a polypeptide of interest can involve culture or fermentation of cells modified to express the polypeptide. The cultivation or fermentation may be carried out in a bioreactor with a supply of suitable nutrients, air/oxygen and/or growth factors. Secreted proteins can be collected by separating the cells from the culture medium/fermentation broth, extracting the protein content, and isolating individual proteins to isolate the secreted polypeptide. Culture, fermentation and isolation techniques are well known to those skilled in the art.

The bioreactor comprises one or more vessels in which cells can be cultured. The culture in the bioreactor can occur continuously, with continuous flow of reactants into the reactor and continuous flow of cultured cells therefrom. Alternatively, the culture may be carried out batchwise. The bioreactor monitors and controls environmental conditions such as pH, oxygen, flow rates into and out of the vessel, and agitation within the vessel to provide optimal conditions for the cells being cultured.

After culturing the cells expressing the polypeptide of interest, the polypeptide is preferably isolated. Any suitable method known in the art for isolating polypeptides/proteins from cell cultures may be used. In order to isolate the polypeptide/protein of interest from the culture, it may be necessary to first isolate the cultured cells from the medium containing the polypeptide/protein of interest. If the polypeptide/protein of interest is secreted from the cell, the cell can be isolated from the medium containing the secreted polypeptide/protein by centrifugation. If the polypeptide/protein of interest is collected intracellularly, it may be necessary to disrupt the cells prior to centrifugation, for example using sonication, rapid freeze-thawing or osmotic lysis. Centrifugation will produce a pellet containing the cultured cells or cell debris of the cultured cells, as well as a supernatant containing the culture medium and the polypeptide/protein of interest described above.

It may then be desirable to isolate the polypeptide/protein of interest from the supernatant or culture medium, which may contain other proteins and non-protein components. A common method for isolating polypeptide/protein components from the supernatant or culture medium is by precipitation. Polypeptides/proteins of different solubilities are precipitated in different concentrations of a precipitating agent, such as ammonium sulfate. For example, in low concentrations of precipitant, water soluble proteins are extracted. Thus, by adding increasing concentrations of precipitating agent, proteins of different solubilities can be distinguished. Dialysis can then be used to remove ammonium sulfate from the isolated protein.

Other methods for distinguishing between different polypeptides/proteins are known in the art, such as ion exchange chromatography and size chromatography. These may be used as an alternative to precipitation or may be carried out after precipitation.

Once the polypeptide/protein of interest has been isolated from the culture, it may be necessary to concentrate the protein. Many methods for concentrating the protein of interest are known in the art, such as ultrafiltration or lyophilization.

The medicaments and pharmaceutical compositions of aspects of the invention may be formulated for administration by a variety of routes including, but not limited to, parenteral, intravenous, intraarterial, intramuscular, intratumoral, oral and nasal. The medicaments and compositions may be formulated for injection.

Administration is preferably in a "therapeutically effective amount" sufficient to bring benefit to the individual. The actual amount administered, the rate of administration and the time course will depend on the nature and severity of the disease being treated. Treatment protocols, e.g., decisions regarding dosages, etc., are charged to general practitioners and other physicians, and generally take into account the condition to be treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to practitioners. Examples of the above mentioned techniques and protocols can be found in Remington pharmacy, 20 th edition, 2000 published in Lippincott, williams & Wilkins.

Sequence identity

The alignment used to determine percent amino acid or nucleotide sequence identity can be achieved in a variety of ways known to those skilled in the art, for example using well known computer software, such as ClustalW 1.82.T-coffee or Megalign (DNASTAR) software. When using such software, it is preferred to use default parameters, for example, for gap penalties and extension penalties. The default parameters for ClustalW 1.82 are: protein gap opening penalty =10.0, protein gap extension penalty =0.2, protein matrix = Gonnet, protein/DNA end gap = -1, protein/DNA gap distance =4.

The invention includes combinations of the described aspects and preferred features except where such combinations are expressly not allowed or explicitly avoided.

Section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now be described, by way of example, with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

Throughout the specification, including the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges can be expressed herein as "about" one particular value, and/or "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment.

Control

In some cases, the method involves comparing the cellular localization of the oncoprotein in a sample from the individual to one or more control samples.

The comparison may not require that the analysis of the control sample be performed simultaneously or sequentially with the analysis of the sample from the individual. Instead, a comparison may be made with results previously obtained from a control sample, such as results stored in a database.

The control sample can be a sample obtained from the individual prior to the onset of cancer, or prior to the observation of symptoms associated with cancer, or prior to administration of an anti-cancer therapy.

The control sample can be a sample obtained from another individual, e.g., an individual not suffering from cancer. An individual may be matched to the individual according to one or more characteristics, such as sex, age, medical history, race, weight, or expression of a particular marker. The control sample may be obtained from a body part or may be of the same tissue or sample type as the sample obtained from the individual.

A control sample may be a collection of samples, providing representative values in many different individuals or tissues.

In some cases, the control may be a reference sample or a reference data set. The reference may be a sample previously obtained from a subject with a known fitness for a particular treatment. The reference may be a data set obtained from analyzing a reference sample.

The control may be a positive control in which the target molecule is known to be present or expressed at a high level, or a negative control in which the target molecule is known to be absent or expressed at a low level.

The control may be a tissue sample from a subject known to benefit from treatment. The tissue may be of the same type as the sample being tested. For example, a tumor tissue sample from a subject can be compared to a control sample of tumor tissue from a subject known to be suitable for treatment, e.g., a subject that has previously responded to treatment.

In certain instances, a control can be a sample obtained from the same individual as the test sample, but from a time when the subject is known to be healthy, e.g., a time when the subject is known not to have cancer. Thus, a cancer tissue sample from a subject can be compared to a non-cancer tissue sample.

In some cases, the control is a cell culture sample.

Brief description of the drawings

Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying drawings, in which:

PRL-3 is a novel tumor target highly expressed in gastric tumors. (A) Western blotting of PRV-3 in various normal tissues of FVB/wild type mice (lanes 1 to 15) and spontaneous mammary and metastatic lung tumors from FVB/MMTV-PyMT mice (lanes 16 and 17). The blot was probed with PRL 3-globin antibody. HSP70, loading control. (B) Kaplan-Meier survival analysis of PRL-3mRNA expression in SGset1 GC patient cohorts. n =183; p =0.002, log rank test. (C) Complete western blot analysis of PRL-3 in human primary gastric tumors (T) from GC patients with patient-matched normal tissue (n). Mr, relative molecular weight (kDa).

FIG. 2PRL 3-globin specific blockade of PRL-3+ in situ gastric tumors. (A) Western blot of endogenous PRL-3 in 22 human GC cell lines. Selection of tumorigenic PRL-3 for subsequent animal models ⁺ And PRL-3 ^- Cell lines are marked with an asterisk in red. Mr, relative molecular weight (kDa). (B) schematic representation of experimental in situ GC model in Balb/C nude mice. (C) PRL 3-bead monoclonal antibody treatment inhibition PRL-3 ⁺ SNU-484 gastric tumor growth in situ. Panels a-b, appearance of mice at the end of experiment (day 28). Arrows highlight abdominal distension in untreated mice. Panels c-d, the stomach was excised and the tumor area was outlined with a black line. Strip, 10mm. (D) Mean gastric tumor volume of untreated and PRL 3-globin treated groups on day 28. N =8 per group; p =0.01, t test; data represent mean ± s.d. (E) Kaplan Meier survival analysis of untreated (red line) and PRL 3-mab treated (black line) groups of mice. N =4 per group; p =0.006, log rank test. p value<0.05 was considered statistically significant.

FIG. 3PRL 3-mAb to PRL-3 ^- In situ gastric tumors had no therapeutic effect. (A) PRL 3-globin treatment failed to block PRL-3-MKN45 in situ gastric tumor growth. Panels a-b, appearance of mice at the end of experiment (day 56). Panels c-d, the stomach was excised and the tumor area was outlined with a black line. Strip, 10mm. (B) Mean gastric tumor volume in untreated and PRL 3-mab treated groups on day 56. n =5 per group; p =0.4,t test; data represent mean ± s.d. (C) Kaplan Meier survival analysis of untreated (red line) and PRL 3-globin treated (black line) groups of mice. N =4 per group; p =0.3, log rank test. (D) Summary of PRL 3-mab treatment results in an in situ model of 4 human GC cell lines. p value<0.05 was considered statistically significant.(E) Mean gastric tumor volume at day 28 from MKN45-PRL3 in situ gastric tumor growth. N =4 (untreated) or 5 (treated) 'p =0.00002, t-test' data represent mean ± SEM.

Figure 4 PRL3-globin was more effective as monotherapy rather than combination therapy with 5-fluorouracil (5-FU) or 5-FU alone. Four treatment groups are used for treating PRL-3+SNU-484 in situ tumors: PBS control (group 1), PRL 3-globin monotherapy (group 2), PRL 3-globin +5-FU combination therapy (group 3) or 5-FU monotherapy (group 4). (A) The stomach of mice was excised from each treatment group on day 28, with the tumor area in situ framed by a black line. Strip, 10mm. Right panel, mean gastric tumor volume for each group on day 28. n =5 per group; p-value for each group when compared to group 1, t-test; data represent mean ± s.d. (B) Representative images of giemsa-stained blood smears from treatment mouse groups before treatment started (day 0) and at the end of the experiment (day 28). White Blood Cells (WBCs) stained blue. Bars, 40 μm. Right panel, mean WBC count of blood smears from each mouse on day 28. n =5 per group; p-value for each group when compared to group 1, t-test; data represent mean ± s.d. A p-value <0.05 is considered statistically significant. (C) Hematological characteristics of the mouse groups at the end of each treatment regimen (day 28). Values highlighted in red represent outliers of the normal reference range for BALB/c nude mice (35).

Figure 5. Intracellular PRL-3 oncoprotein may be secreted into the cell culture medium and is present in 62% of cancer urine, but not in normal urine. (A) Western blot of PRL-3 in matched lysates and conditioned media of indicated GC cell lines. CANX, calnexin. (B) Summary study of% PRL-3 positivity in urine samples from all cancer patients and normal individuals. (C-F) representative Western blots of PRL-3 in the urine of (C) normal individuals and GC patients, (D) nasopharyngeal carcinoma patients, (E) bladder cancer patients, and (F) lung cancer patients. Mr, relative molecular weight (kDa).

Figure 6. Effective PRL 3-bead treatment resulted in the loss of urinary PRL-3, mechanistically involving intratumoral accumulation and recruitment of immune effectors. (A) Tumor-like in matched urine and from untreated or PRL 3-globin treated miceWestern blot of PRL-3 protein in preparations, the mice carrying PRL-3 ⁺ SNU484 or PRL-3 ^- MKN45 gastric tumor in situ. On the upper panel, the stomach was excised on day 28 (SNU-484) or day 56 (MKN 45). (B) In situ SNU-484 and MKN45 tumor tissue cryosections from mice subjected to various treatments were analyzed by immunohistochemistry for PRL 3-mab (panels a-f; bars, 20 μm), or immunofluorescence of B cells (panel e-1) and NK cell markers (panels m-r; bars, 50 μm). B and NK cell markers stained for green, CD45R/B220 and CD335/Nkp46, respectively; blue, DAPI nuclear staining. (C) Description of PRL 3-mAb vs. PRL-3 ⁺ Model of the mechanism of action of cancer cells: 1) PRL-3 antigen, by extravasation of non-conventional secretions (exosomes PRL-3), or from necrotic PRL-3 ⁺ Tumor cells (free PRL-3) leak spontaneously, acting as 2) decoys for PRL 3-globin binding and immune complex accumulation within the tumor niche, subsequently leading to 3) recruitment and activation of effector NK and B cells for anti-tumor effects.

Figure 7 shows the CDR position of the humanized antibody sequence. Heavy chain sequence (A) and light chain sequence (B).

FIG. 8 sequence of murine antibody clone (A) clone #223 and (B) clone #318

FIG. 9 human PRL3 sequences

FIG. 10 PRL-3 sequence analysis of the mAb. (A) Light chain sequence alignments of humanized sequences recognizing the CDR regions (grey boxes) and the important domain sequences (clear boxes). (B) Heavy chain sequence alignments of humanized sequences that identify the CDR regions (grey boxes) and identify the important domain sequences (clear boxes).

FIG. 11 PRL-3 is not expressed in normal adult tissues, but is strongly expressed in human gastric tumors. (A) Immunohistochemistry for PRL-3 expression of (a) multiple normal human tissues from various organs and (b) matched gastric tumor and normal gastric tissues from GC patients. The strips were 50 μm.

FIG. 12 PRL 3-globin specifically binds PRL-3, but is not closely related to PRL-1 or PRL-2. (A-C) human isoforms of PRL-1, PRL-2 and PRL-3 proteins for analysis of PRL 3-globin specificity. (a) Western blots of recombinant GST-PRL1, GST-PRL2, and GST-PRL3 were probed with PRL 3-mAb or anti-GST antibody. (b) PRL 3-mAb was subjected to ELISA using recombinant GST-PRL1, GST-PRL-2, and GST-PRL-3 proteins. (c) Immunofluorescence staining of Chinese Hamster Ovary (CHO) cells overexpressing GFP-PRL1, GFP-PRL2 or GFP-PRL3 cells with PRL 3-globin. Bars, 40 μm.

FIG. 13 inhibition of PRL-3 in mice by PRL 3-mab ⁺ Tumor growth of in situ gastric tumors. Male BALB/C nude mice at 8 weeks of age were implanted with PRL-3 positive NUGC-4 or IM-95 cell lines to induce in situ gastric tumors. At the end of the experiment, visible tumors (black outline) were measured and volumes were compared. (a) Untreated and PRL 3-globin treated mice had an IM-95 tumor in the stomach. The right-most panel shows a graph of the mean tumor volume of IM-95 tumors in untreated and PRL 3-mab treated mice. p =0.008, t-test n =6, data represent mean ± s.d. bars, 10mm. (b) Stomach with NUGC-4 tumor of untreated and PRL 3-globin treated mice. The right-most panel shows the tumor volume of the NUGC-4 tumor in untreated and PRL 3-globin treated mice. P =0.003,t test; n =4, data represent mean ± s.d. Strip, 10mm. (C) PRL 3-mab, but not human IgG isotype control, inhibited PRL 3-positive gastric tumor growth in vivo. In untreated, human IgG-treated (hIgG) and PRL 3-mab treated mice, 8-week-old male BALB/C nude mice were implanted with PRL 3-positive SNU-484 tumors. P<0.001, single factor analysis of variance; n =4 per group and data represent mean ± SEM. * P is<0.001, tukey's post hoc test (untreated and treated).

FIG. 14 post-operative PRL 3-Belizumab treatment inhibition PRL-3 ⁺ Recurrence of the tumor. (A) Xenograft tumors formed by PRL-3+SNU-484 cells grew for 3 weeks prior to tumor resection. Mice were then divided into placebo (untreated) or PRL 3-mab (treated) groups, treated every two weeks for 7 weeks to monitor tumor regrowth. Panel a, appearance of tumor bearing mice at the end of 3 weeks. Panel b, appearance of mice after surgical removal of tumor, lower panel showing dissected tumor. Panels c-d, appearance of mice 7 weeks after resection and treatment. Panel e, dissect tumors that recurred at the site of resection. Panel f, treated mice had no tumor recurrence. Strip, 10mm. (B) Kaplan Meier relapse free survival analysis of untreated (n = 10) and treated (n = 8) groups of mice. P<0.001, log rank test.

FIG. 15 inhibition of PRL-3 by intragastric implantation of PRL 3-globin ⁺ HCT116 colorectal cancer cells form localized and metastatic abdominal tumors. HCT116-luc2 cells were implanted into the submucosa of the stomach of mice to mimic the metastasis of secondary colorectal cancer to the gastric niche. PRL 3-mAb treatment reduced the growth of HCT116-luc2 tumor in the gastric niche. (A) IVIS imaging of whole body in vivo tumor growth within 3 weeks after inoculation. (B) The mice from (a) were analyzed for whole animal IVIS intensity as a function of time. N =4 per group; p is a radical of<0.001, two-way anova. (C) tumor burden in stomach resected at the end of week 3. (D) analyzing the difference in IVIS intensity from the stomach of (C). N =4 per group; p =0.01,t test; data represent mean ± SEM. (E) metastatic tumor burden in the abdominal wall at the end of week 3. (F) analyzing the IVIS intensity differences from the stomach of (E). n =4 per group; p =0.0003,t test; data represent mean ± SEM.

FIG. 16 exosome-associated PRL-3 is present in urine from patients with bladder cancer. Purified exosome fractions from urine samples from bladder cancer patients were analyzed with antibodies against PRL3CD63 exosome markers.

Figure 17 clinical features of sgset1 patient cohort.

Figure 18 univariate and multivariate Cox regression analysis of PRL-3 expression in sgset1 patient cohorts.

Figure 19 PRL3 oncoprotein can be secreted from cancer cells and act as a decoy for PRL 3-mab. (A) Analysis of PRL3 protein expression in intracellular protein pools (cell lysates) and extracellular protein pools (concentrated conditioned medium) after 48 hours of culturing Gastric Cancer (GC) cells in serum-free medium. For extracellular protein analysis, conditioned media (50 mL) from five GC cell culture dishes was first cleared of dead cells and cell debris and then concentrated by centrifugation (final volume 0.2 mL). (B) PRL 3-mab from frozen sections of in situ SNU-484 and MKN45 tumor tissue from mice subjected to various treatments was analyzed by immunohistochemistry using anti-human IgG antibodies. Bars 20 μ < μ M.

FIG. 20 PRL-3 is highly upregulated on the surface of tumor cells in vivo, but not cancer cells cultured in vitro. (a) Experimental outline for cell count analysis of cell surface profiles of in vitro cultured cells and ex vivo tumor cells. (b) Representative histograms of cell surface staining were performed with control (clear), PRL 3-globin (pink) or cetuximab (CTX; grey) antibodies. Positive gates (% pos) were determined after subtracting the background signal inferred from control staining. (c) % cell surface positive population of different antibodies as tested in (b). Data represent mean ± SEM. (d) Western blots of EGFR and PRL-3 showed altered protein levels in cultured cells compared to tumors. GAPDH, loading control.

FIG. 21 PRL 3-mab inhibition of orthotopic liver tumors requires host Fc γ II/III receptor binding. (a) overview of in situ liver tumor model. (b) Western blot of PRL-3 expression in six human cancer cell lines. GAPDH, loading control. (c) Carrying in situ PRL-3 compared to placebo (untreated) ⁺ Mice with MHCC-LM3 liver tumors had a decreased tumor burden after 5 weeks of every two weeks of administration of 100 μ g/dose PRL 3-mab (treatment). Strip, 10mm. (d) Mean liver tumor volume of untreated and treated groups on day 35. P =0.0001,t-test; data represent mean ± SEM. (e) Kaplan Meier survival analysis of untreated (red line) and treated (black line) groups of mice. P =0.002, log rank test. (f) Sketches depict the domain structures of PRL 3-mab and PRL 3-minibody, and their ability to bind Fc receptors (FcR) to host immune cells. 42g2 monoclonal antibodies (mabs) act as FcR blockers, preventing intact IgG from binding to fcrs.

FIG. 22: interaction with host Fc γ II/III receptors is essential for PRL 3-globin-induced recruitment of NK cells, B cells and M1 macrophages into the tumor niche. In situ MHCC-LM3 liver tumor tissue frozen sections from mice subjected to various treatments were analyzed by immunofluorescence against antibodies to (a) F4/80 (pan macrophages), (B) CD206 (M2 macrophages), (c) CD86 (M1 macrophages), (d) CD45/B220 (B cells), or (e) CD335 (NK cells). Tumor infiltration scores were calculated as described in materials and methods. * p <0.05, one-way anova; data represent mean ± SEM. (g) Excised livers of mice treated with placebo (untreated), PRL 3-mab alone, 2.4g2mab, prl3-mab +2.4g2mab combination therapy, human IgG or PRL 3-minibody on day 35 were photographed and tumor volumes were measured. Rectal tumor areas are outlined with black lines. Strip, 10mm. Mean liver tumor volume p =0.003, one-way anova for each group at day 35; data represent mean ± SEM.

FIG. 23: PRL-3 is a general tumor target that is frequently overexpressed in a variety of human tumors. (a-e) PRL-3 in tumor (T) patient-matched normal tissue (n) complete Western blot analysis in (a) liver tumor, (b) lung tumor, (c) colon tumor, (d) breast tumor, and (e) kidney tumor. (f-j) complete Western blot analysis of (f) renal tumor, (g) bladder tumor, (h) Acute Myelogenous Leukemia (AML), (i) gastric tumor, and (j) prostate tumor in other patient samples without matched normal tissue. GAPDH, loading control. The relative molecular masses (in kDa) are shown on the right side of each result set.

FIG. 24: in vitro assays to analyze whether PRL 3-globin can directly inhibit PRL-3+ cancer cells.

FIG. 25: (scFv-CH 3) 2PRL 3-minibody on in situ PRL-3+ SNU-484 gastric tumors.

Examples

Example 1 production of PRL3-Bevacizumab

The PRL 3-globin construct was engineered from a previously characterized murine anti-PRL-3 antibody clone. We hired two independent Contract Research Organizations (CROs) from the united states for humanization or cloning, using a proprietary improvement of the method described by Queen et al (60).

Briefly, complementarity Determining Regions (CDRs) of the heavy (IgG 1) and light (κ) chains of mouse antibodies are grafted onto an "acceptor" human sequence framework, where the framework is defined as a variable segment in addition to the CDRs. Selection of the human acceptor framework is performed by aligning the mouse framework sequence with a database of human framework sequences to find the closest human homolog (typically 65-70% sequence identity) of each strand.

In addition to grafting CDRs from the mouse sequence, approximately three amino acid positions from the mouse sequence (in addition to the CDRs) were grafted into the human acceptor sequence. This retained the CDRs of the original murine anti-PRL-3 antibody, which specifically recognized an epitope within the conserved C-terminal region between mouse and human PRL-3, but did not recognize PRL-1 or PRL-2.

We invited the Sapidyne Instruments Inc. (700 Dr. Simplex, 83705 Edaho) to test the affinity of PRL 3-globin for binding to the PRL-3 antigen. Binding affinity analysis, using a kinetic exclusion assay (Drake et al, 2004), purified PRL 3-globin was characterized as a tight conjugate of purified human PRL-3 with a Kd of 6.29pM, binding Rate (K) _on ) And dissociation rate (K) _off ) Are respectively about 1 × 10 ⁷ M ^-1 s ^-1 And 7X 10 ^-5 s ^-1 (Table 1).

Table 1: summary of KinExA PRL 3-mab binding affinity analysis.

Example 2: use of PRL-globin for treating gastric cancer

Materials and methods

Preparation of tissue and cell lysates

Multiple normal mouse organs were obtained from FVB/wild-type mice, while breast cancer and metastatic lung tumors were dissected from syngeneic FVB/MMTV-PyMT mice-a spontaneous model of mature metastatic breast cancer driven by transgene overexpression of the T oncogene in mammary gland-specific polyomaviruses (28). For tissue, excised samples (5 mm) ³ ) Suspended in RIPA lysis buffer (Sigma) containing protease-phosphatase inhibitor cocktail (Pierce) and disrupted completely with a tissue homogenizer (Polytron). Lysates were clarified by centrifugation at 13,000 × g for 40 min at 4 ℃. For cell lines, 5X 10 lysis in RIPA lysis buffer containing protease-phosphatase inhibitor ⁶ Individual cells, and clarified as described above. Protein concentration was assessed using the bicinchonine assay kit (Pierce). After addition of 2 × Lamelli buffer, the samples were boiled and used immediately for Western blotting or stored at-20 ℃ until use.

Western blot

200 μ g of lysate was separated in different wells of a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membrane, which was then washed with 1: the indicated primary antibody at1,000 dilution was blocked and probed overnight at 4 ℃. After thorough washing with TBS-T buffer (20mM Tris pH 7.6,140mM NaCl,0.2% Tween-20), the membranes were washed with the corresponding horseradish peroxidase (HRP) -conjugated secondary antibodies in a 1:5,000 dilutions were incubated for 1 hour, washed with TBS-T, and developed using a chemiluminescent substrate (Pierce).

Cell culture

The 22 human GC cell lines studied were from the following sources: MKN7, MKN74, NUGC-3, OCUM-1 (resource base for health scientific research); YCC-1, YCC-3, YCC-7, YCC-17 cells (extending cancer center); AGS, CRL-5822, KATO-III, SNU-1, SNU-5 (American type culture Collection, ATCC); HGC27, NUGC-4, OE19 (Sigma Aldrich); MKN28, MKN45 (japan physical and chemical research institute); IM-95,SCH (Japanese research BioResource cell Bank); SNU-484, SNU-719 (Korean cell line Bank). CHO cells were purchased from ATCC. The production of CHO cells stably expressing GFP-tagged PRL-1, PRL-2 or PRL-3 fusion proteins has been described previously (23). Luciferase-expressing HCT116-luc2 human adenocarcinoma cells (Caliper Life Sciences) were established by stable transduction of lentiviruses containing the luciferase 2 gene into parental HCT116 cells (ATCC) under the control of the human ubiquitin C promoter (pGL 4luc 2). The cell lines were cultured in RMPI-1640 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone) and 1% penicillin-streptomycin (Life Technologies) and stored supplemented with 5% CO ₂ At 37 ℃.

PRL-3mRNA expression analysis

We analyzed a publicly available GC microarray dataset (GSE 15459) from the Gene Expression Omnibus (GEO) database consisting of 200 primary gastric cancer samples analyzed on an Affymetrix Human Genome U133 Plus 2.0 Genechip array. Data pre-processing was performed using the 'affyPLM' R packet (v 2.15). A total of 185 tumor samples were obtained for downstream analysis, excluding outliers (SGset 1; patient characteristics are provided in FIG. 17). Survival analysis was performed using overall survival as a result indicator to compare tumors of "low", "medium" and "high" expression for each gene (n =183 samples with 2 missing survival data), i.e., "low" and "high" expression panels correspond to samples with expression levels below 33.3 percentiles and above 66.7 percentiles, respectively, while the middle percentile was classified as "medium".

Preparation of recombinant GST-tagged protein

The preparation of recombinant GST-PRL-1, GST-PRL-2 and GST-PRL-3 fusion proteins has been previously described (53).

ELISA

ELISA assays were performed as previously described (53). Briefly, 96-well plates coated overnight with GST-PRL-1 (20 ng), GST-PRL-2 (20 ng), or GST-PRL-3 (1 ng, 20ng) were blocked with 3% bovine serum albumin in PBS-0.05-Theeen-20, and then incubated with 200ng PRL3-mab at 37 ℃ for 2 hours. After thorough washing, HRP-conjugated anti-mouse antibody (Pierce) was added for 1 hour at 37 ℃. Colorimetric development Using Turbo-TMB substrate (Pierce) and by Using 2M H ₂ SO ₄ And (5) terminating acidification. The absorbance was measured at 450nm using a plate reader (Dynatech).

Animal models and treatments

Male Balb/C nude mice, 8 weeks old, obtained from a bioresource center (a STAR, singapore) were used in all animal models in this study. Mice were anesthetized with 2.5% avermectin (100 μ l/10g body weight) for intraperitoneal injection. In situ gastric cancer model: the abdomen of the anesthetized mice was opened in layers by a 1cm midline incision starting 0.5cm below the xiphoid process. The stomach was removed through an abdominal incision with surgical forceps and the cancer cells were injected into the serosal layer. Subsequently, the stomach was replaced and the abdomen was sutured layer by layer. The number of cells and duration of the experiment required to induce orthotropic gastric tumors per cell line was confirmed after the preliminary experiment: SNU-484 tumor was 3X 10 ⁶ Individual cell, IM-95, NUGC-4 and MKN-45 tumors 5X 10 ⁶ And (4) cells. The treatment regimen was started on day 2 after inoculation of cancer cells in the submucosa of the gastric serosa. Mice were administered intravenously (iv) with 100 μ g of PRL 3-globin (Wuxi Pharmatech) twice weekly in 100uL PBS for a total of eight times (SNU-484 and NUGC-4 tumors) or ten times (IM-95 and MKN45 tumors). PBS was used as "untreated"control in mouse. Due to the difference in growth rate of individual tumors, the duration of the experiment was as follows: SNU-484 and NUGC-4 tumors were 4 weeks, MKN-45 tumors were 8 weeks, and IM-95 tumors were 12 weeks. Tumor volume was calculated using the formula: volume =0.4 x tumor length x tumor width. Xenograph tumor model: mu.l of 3X 10 in PBS ⁶ Individual SNU-484 cells were injected on both sides of the anesthetized mice. After 3 weeks, the resulting tumors (5-10 mm) were surgically removed under anesthesia, the mice were divided into 2 groups and received PRL 3-mab (100 μ L,100 μ LPBS, i.v.) or PBS (100 μ L) every two weeks after tumor resection. Tumor recurrence was analyzed weekly in untreated and treated groups until 7 weeks after resection. Tumor growth was carefully monitored in both groups. Secondary gastric metastasis model: as stated, 3 × 10 ⁶ HCT116-luc2 cells were implanted directly into the serosa of anesthetized mice. Mice were divided into treated (PRL 3-mab, 100ug in 100uL PBS) or untreated (100 uL PBS) groups, and tumor growth in vivo was monitored by IVIS imaging at1, 2, and 3 weeks post-implantation 15 minutes after intraperitoneal injection of 150mg/kg fluorescein (Caliper Life Sciences) under 2% isoflurane anesthesia.

Analysis of mouse blood samples

WBC staining of mouse blood smears: after a thin layer was applied to a glass slide from a drop of fresh mouse blood, the slide was baked at 37 ℃ for 1 hour, then dipped in a modified Wright Giemsa stain (Sigma) for 1 minute, and then washed with deionized water for 3 minutes. After drying, the stained slides were viewed under a microscope and WBCs stained blue. Total WBCs were estimated under a light microscope (Olympus) by counting in ten fields of view in each slide, calculated with the equation WBCs/μ l = (total number of WBCs counted/number of fields of view) × 2000. Complete blood count: hematological analysis of mouse samples was performed by Quest Laboratories (singapore).

Antibodies

HSP70 (catalog No. EXOAB-HSP 70A) antibody was purchased from System Biosciences, inc. CD63 (catalog No. sc-15363) antibody was purchased from Santa Cruz Biotechnoloy. GAPDH (clone MAB 374) antibody was purchased from Millipore. B cell markers (CD 45/CD220, clone RA3-6B 2) and NK cell markers (CD 335/Nkp46, clone 29A1.4) were purchased from BD Pharmingen.

Immunofluorescence imaging

Preparation of cell slide: cells were directly seeded onto glass coverslips and grown for 48 hours. Using PBSCM (PBS pH 7.0,1mM MgCl) ₂ ,1mM CaCl ₂ ) After two washes, cells were fixed in 3% paraformaldehyde for 20 min at Room Temperature (RT), washed with PBS-0.1% saponin (Sigma) and permeabilized for 15 min. Preparation of tissue section slides: fresh frozen samples of SNU-484 and MKN45 in situ gastric tumors were cut into 10 μm sections using a cryostat (Leica) at-18 ℃. Slides were fixed with 4% paraformaldehyde for 20 minutes, washed with PBS-0.05% Tween-20, and blocked in PBS-FDB (PBS pH 7.0,2% BSA,5% goat serum, 5% fetal bovine serum) for 1 hour at room temperature. The slides were then mixed with the indicated primary antibodies at 1:200 dilution was incubated for 4 hours, washed, and incubated with the corresponding fluorochrome-conjugated secondary antibodies (Life Technologies) for 2 hours. The washed slides were fixed with DAPI-containing fade-resistant mounting reagent (Vector Laboratories) and sealed with nail polish. Confocal imaging was performed with a LSM 510 confocal microscope (Zeiss AG).

Immunohistochemistry for PRL 3-globin

The 10 μm-thick frozen sections were fixed with 4% formalin for 20 minutes and were 1% at1 ℃ by volume H ₂ O ₂ PBS incubation for 5 min. The washed slides were then blocked for 1 hour at room temperature in PBS containing 10% goat serum and 1% BSA (Sigma). Subsequently, the slides were washed four times in PBS-0.05% tween-20 while gently shaking and incubated with goat anti-human labeled polymer-HRP (Dako) for 2 hours, then washed thoroughly and incubated with substrate-chromogen solution (Dako) for 10-20 minutes in the absence of light. The mounted slides were examined using a bright field microscope (Olympus) and representative images were captured.

Statistical analysis

For the human study, kaplan-Meier analysis of significance for overall GC patient survival was evaluated using a log rank test based on PRL-3mRNA expression clustering. Univariate and multivariate analyses were performed using Cox proportional hazards regression. For the mouse study, log rank test was used to assess significant differences in Kaplan-Meier analysis of overall survival between the "untreated" and "treated" groups of mice. The Student's t-test was used to calculate statistically significant differences in tumor volume in situ. Statistical calculations were performed using SPSS software v19.0 (IBM). In all cases, a p-value <0.05 was considered significant.

Results

PRL-3 is a tumor-specific target

A related challenge in the development of anti-cancer targeted therapies is to identify "tumor-specific antigens" that are expressed only in tumors but not in normal tissues to avoid undesirable off-target effects. We first screened normal mouse tissues from all major organs for endogenous PRL-3 by western blotting. In these complete blots, a single-20 kDa endogenous protein corresponding to the predicted molecular weight of PRL-3 was detected (FIG. 1A). We did not observe any non-specific bands, confirming that the PRL-3 antibody did not cross-react with other molecules (27). Although weak PRL-3 protein could be detected in normal colon (fig. 1A, lane 2), it was not detected in 14 other major normal mouse tissues examined (fig. 1A, lanes 1, 3-15), including breast and lung tissues (fig. 1A, lanes 14-15). In contrast, PRL-3 was abundantly expressed in spontaneously occurring breast and lung tumors from MMTV-PyMT mice (FIG. 1A, lanes 16-17) (28). Importantly, PRL-3 protein was also undetectable in 15 major normal human organs examined by immunohistochemistry (fig. S1A). In addition, in patient-matched tissue samples, PRL-3 was not detected in non-cancerous stomach tissue (fig. 11B, panel a), but was highly expressed in gastric tumor sections (fig. 11B, panel B), again showing tumor-specific upregulation. In combination with published literature on the high frequency of overexpression of PRL-3 in cancer (29), and the recent observation that PRL-3 conditional knockout mice show normal (30), PRL-3 is specifically expressed in cancer tissues, but not in normal tissues, confirming that PRL-3 is a suitable tumor-specific target.

PRL-3 oncoprotein is overexpressed in 85% of gastric tumors

In the last decade, many studies have shown that elevated PRL-3 expression is a negative prognostic factor for gastric cancer (14,31,32). We further investigated the clinical significance of elevated PRL-3mRNA levels in an independent cohort of 185 GC patients (clinical features given in figure 17). Kaplan-Meier survival analysis showed that elevated levels of PRL-3mRNA in tumors were associated with a shorter overall survival (p =0.002; FIG. 1B). High PRL-3mRNA expression was also significantly associated with higher tumor grade in multivariate Cox analysis (fig. 18). Next, we examined the level of PRL-3 protein using 20 matched pairs of fresh frozen biopsy tissue samples (tumor versus adjacent normal tissue) from GC patients at the national university hospital of singapore. Western blot clearly showed that endogenous PRL-3 was overexpressed in 17/20 (85%) of the gastric tumors (T; FIG. 1C), but not in any matched normal gastric tissue (n; FIG. 1C), confirming tumor-specific expression of PRL-3. Notably, the PRL-3 protein shows a band of 20 to 25kDa in these blots, indicating a potential post-translational modification of PRL-3 (-20 kDa) in an as yet unidentified human tumor sample. Taken together, our clinical data indicate that PRL-3 oncoprotein overexpression is a common phenomenon in human GC associated with disease severity, again confirming its suitability as a candidate for targeted therapy.

Generation of novel PRL-3 targeting humanized antibodies PRL 3-globin

We previously demonstrated the high efficiency of murine and chimeric PRL-3 antibodies against tumors expressing intracellular PRL-3 in nude and wild type C57BL/6 mice (24,27). In these studies, mice receiving the PRL-3 monoclonal antibody gained weight and showed normal activity, indicating minimal off-target side effects. To translate these early findings into human clinical applications, we produced a humanized monoclonal anti-PRL-3 antibody called 'PRL 3-globin'. Similar to its predecessors, engineered PRL-3-globin specifically recognized PRL-3 and did not cross-react with PRL-3 homologues PRL-1 or PRL-2 as demonstrated by Western blot, ELISA and immunofluorescence analysis (FIGS. 12A-C). Subsequently, we used PRL 3-mab in all further experiments described in this report.

PRL-3-globin specific block PRL-3 positive (PRL-3) ⁺ ) While non-PRL-3 negative (PRL-3) ^- ) Growth of in situ gastric tumors

In mouse tumor modelHuman cancer cells in the form that grow in their natural (in situ) location replicate human disease with high fidelity. More importantly, the response of tumors to treatment has been shown to vary significantly depending on whether the cancer cells are implanted subcutaneously or in situ, (33), highlighting the requirement to select the correct model to test the efficacy of anti-tumor drug treatment. To establish a relevant preclinical in situ mouse model to examine the efficacy of PRL 3-mab against gastric tumors, we first screened a panel of 22 human GC cell lines for PRL-3 protein expression status, and subsequently tested their tumorigenic capacity within the serosal layer of the mouse stomach. PRL-3 protein was detected in 13 of 22 (59%) human GC cell lines analyzed (fig. 2A). However, only a portion of the GC cell lines grew well in culture and within a controlled time frame: (<2 months) to form an in situ tumor. Based on these criteria, three PRL-3 were selected ⁺ Cell lines (SNU-484, NUGC-4 and IM-95) and a PRL-3 ^- Cell lines (MKN 45) were used to develop an in situ GC model to evaluate the anti-tumor efficacy of PRL 3-mab. Cells from these cell lines were seeded into the serosal layer of the stomach and subsequently processed according to the protocol outlined in figure 2B. At the end of the experiment, the stomach was removed from the mice and analyzed for gastric tumor burden.

We first investigated the effect of PRL 3-globin therapy on SNU-484GC cell line, which is superior PRL-3 due to high expression of PRL-3 protein, rapid growth of cultures, reproducible gastric tumor formation within 3-4 weeks ⁺ In situ gastric tumor model (FIG. 2A, lane 1). During the experiment, untreated mice developed a pronounced bloating (fig. 2C, panel a, arrows) and showed a decrease in physical activity and food intake, whereas PRL 3-mab treated mice appeared very normal (fig. 2C, panel b) and maintained normal physical activity by a regular pattern of food intake. After dissection, in situ tumor formation was significantly reduced in the PRL 3-mab treated group compared to the untreated group (fig. 2C, panels C-d). Measurement of tumor volume showed that PRL 3-mab treated group (0.23. + -. 0.25 cm) compared to untreated group ³ ) The tumor burden of (2) is significantly reduced by 20 times (4.08 + -1.52 cm) ³ (ii) a p =0.01; fig. 2D). Consistent with the reduced tumor burden, kaplan-Meier analysis showed PRL 3-globin therapySurvival time was significantly longer in mice than in untreated mice, with median survival times of 7 and 4.5 weeks (p =0.006; fig. 2E), respectively, confirming the carrying of PRL-3 ⁺ Mice with SNU-484 gastric tumors respond effectively to PRL 3-globin anti-tumor therapy. To verify this finding, two additional PRL-3 were used ⁺ The GC cell lines IM-95 and NUGC-4 generated an in situ GC mouse model (FIG. 2A, lanes 2 and 22, respectively). Similar to SNU-484 orthotropic tumors, PRL 3-globin treatment significantly inhibited by PRL-3 ⁺ IM-95 cells (p =0.008; FIG. S13A) or PRL-3 ⁺ Growth of gastric tumors formed by NUGC-4 cells (p =0.03; FIG. S13B).

In sharp contrast, gastric tumors formed by the PRL-3GC cell line MKN45 (FIG. 2A, lane 4) showed no response to PRL 3-globin treatment, with marked bloating (FIG. 3A, panels a-b) and in situ tumor formation (FIG. 3A, panels c-d) in both treated and untreated mice. In the treatment group (0.17. + -. 0.20 cm) ³ ) And untreated group (0.13. + -. 0.19 cm) ³ ) No difference in mean in situ tumor volume was found between (p =0.4; fig. 3B). Kaplan-Meier survival analysis showed no significant difference in overall survival between the untreated and treated groups, with a median survival of 9.25 weeks for the untreated group versus 10 weeks for the PRL 3-globin treated group (p =0.3; fig. 3C). The results of PRL 3-globin treatment of tumors in situ from these four cell lines (summarized in FIG. 3D) underpins our previously proposed rationale for PRL-3 antibody treatment (24) -only PRL-3) ⁺ Tumors responded to PRL 3-globin therapy, whereas tumors lacking PRL-3 oncoprotein expression did not.

PRL 3-globin as monotherapy is more effective than 5-fluorouracil (5 FU) alone or 5-FU in combination therapy

Since 5-FU is a chemotherapeutic drug used as first line therapy for gastric cancer (17), we investigated whether PRL 3-globin could be used more effectively in combination with 5-FU in inhibiting in situ tumor growth. We tested four treatment regimens: PBS control (group 1), PRL 3-globin monotherapy (group 2), PRL 3-globin +5-FU combination therapy (group 3) or 5-FU monotherapy (group 4). According to the treatment regimen, a biweekly dose of PRL 3-globin (100. Mu.g/dose) or5-FU (30 mg/kg/dose) alone or in combination for intravenous administration carrying PRL-3 in situ ⁺ Tumors in the nude mouse group of SNU-484 stomach. During the experiment we observed a decrease in overall animal activity in 5-FU treated mice (groups 3 and 4). Tumor volume analysis showed that the single drug treatment of PRL 3-globin (group 2) had the highest therapeutic effect, and the mean tumor volume was the lowest at 0.67. + -. 0.59cm ³ Followed by PRL 3-globin +5-FU combination therapy (group 3; 1.49. + -. 0.27 cm) ³ ) 5-FU monotherapy (group 4; 1.76 +/-0.52 cm ³ ) And finally a PBS control (group 1; 3.98 +/-0.60 cm ³ (ii) a Fig. 4A). These results indicate that PRL 3-globin is more effective in reducing gastric tumors when the chemotherapeutic agent 5-FU is not used.

Previously, we highlighted a key role of the host immune system in the efficacy of PRL-3 antibody therapy (24). In view of the known side effects (34) of 5-FU treatment causing a non-specific reduction in the white blood cell count (WBC), we investigated whether the observed reduction in therapeutic effect might be due to this phenomenon. In whole blood smears we found a 5-fold decrease in peripheral WBC counts after 5-FU treatment (groups 3 and 4) compared to control (group 1) or PRL 3-globin monotherapy (group 2; FIG. 4B). To validate these results, we performed a complete blood count of mouse samples to analyze the hematological impact of different treatment regimens at the end of the experiment (day 28). While mice receiving PRL 3-globin had general hematological characteristics in the normal range of BALB/C nude strains (35), those receiving 5-FU in combination with PRL 3-globin or 5-FU alone showed a reduction in neutrophil, lymphocyte and monocyte counts, with a significant reduction in erythrocyte and platelet counts (fig. 4C). In summary, our results indicate that the reduced immune function resulting from 5-FU therapy may be responsible for the reduced efficacy of PRL 3-globin when used in combination with 5-FU, and support the finding that our previous PRL-3 antibody therapy requires a stronger immune system.

Post-operative PRL 3-bead monoclonal antibody treatment inhibits PRL-3 ⁺ Recurrence of tumor

Although surgery is the cornerstone of treatment for GC, nearly 80% of patients die in a short time mainly due to local recurrence and/or a lower degree of distant metastasis (36). IdentificationIn vivo inhibition of PRL-3 in PRL 3-bead monoclonal antibodies ⁺ Ability of GC to grow, we investigated whether PRL 3-globin also has efficacy in post-operative adjuvant therapy to inhibit tumor recurrence. Using PRL-3 ⁺ SNU-484GC cells, we first established xenograft tumors (5-10 mm wide) in both flanks of nude mice over a period of 3 weeks (FIG. 19, panel a). The resulting solid tumors were then completely removed by careful surgery (fig. 19, panel b) and the mice were divided into 2 groups and injected twice weekly with either control antibody (untreated) or PRL 3-globin (treated). Local tumor recurrence was then monitored weekly. At 7 weeks post-surgery, the untreated group developed large local tumors at the site of resection (fig. 19, panel c). In contrast, no visible tumor growth was observed at the resection sites of PRL 3-globin treated mice at the same time period (fig. 19, panel d). This was confirmed after dissection-whereas large solid tumors could be obtained from the untreated group (fig. 19, panel e) and no solid tumor was found at the resection site in the PRL 3-mab treated group (fig. 19, panel f). Taken together, these results indicate that PRL 3-mab has efficacy in inhibiting postoperative local tumor recurrence, suggesting a possible approach for clinical transformation of this drug as an adjuvant therapy.

PRL 3-globin inhibition of gastric secondary PRL-3 ⁺ Growth of tumor metastasis

The presence of metastasis in the stomach is a rare condition (37-39), almost always associated with poor prognosis (40, 41). To address whether PRL 3-globin can block metastatic tumor formation, we used PRL-3 surgically injected into the gastric serosal layer of mice ⁺ HCT116-luc2 colorectal cancer cells developed an experimental model for colorectal cancer metastasis to the stomach. There are two main reasons we have used HCT116-luc2 cells: 1) Gastric metastasis from colon cancer has been described in humans (38, 39, 42), and 2) HCT116-Luc2 constitutively expresses firefly luciferase, allowing tumor growth to be monitored using an In Vivo Imaging System (IVIS). PRL-3 in 2 separate experimental replicates ⁺ HCT116-luc2 tumors grew rapidly in untreated mice, and PRL 3-globin treated mice were PRL-3 in the same period ⁺ HCT116-luc2 tumor growth was greatly reduced (FIG. 14A). After dissection, a heavy swelling was observed in the stomach of untreated miceTumor burden (fig. 14B, panels a, a'). In contrast, PRL 3-globin treated mice had much lower gastric tumor burden (fig. 14B, panel B, B'). In addition, extensive metastatic spread to the abdominal wall was observed in untreated mice (fig. 14B, panels c, c ') was also greatly reduced in treated mice (fig. 14B, panels d, d'). Taken together, these results indicate that PRL 3-globin can reduce PRL-3 in and around the gastric niche ⁺ Growth and metastasis of HCT116-luc2 colorectal carcinoma tumors.

Intracellular PRL-3 oncoprotein can be secreted and is present in 62% of the cancer urine, but not in normal urine

Having demonstrated the anti-tumor efficacy of PRL 3-globin in various cancer models, we next sought a simple method to identify PRL3 ⁺ Cancer patients are treated with PRL 3-mab. We have previously reported that anti-PRL-3 antibodies can be PRL-3 in vitro ⁺ Tumor cells internalize (23). However, it is not clear how and where antibody recognition of the "intracellular" PRL-3 antigen occurs. Here, we report a previously unrecognized natural phenomenon that PRL-3 protein can be expressed in vitro in the corresponding PRL-3 ⁺ Instead of PRL-3 ^- Cancer cell line secretion and detection in concentrated medium (FIG. 5A, lanes 1-4). To exclude non-specific contamination of dead cells or cell debris, we detected ER-localized Calnexin (CANX) as a control, only in the lysate (FIG. 5A, lanes 5-8), but not in the conditioned medium (FIG. 5A, lanes 1-4).

Since PRL-3 has promising cancer biomarker potential based on microarray and histological studies (7), we began to investigate whether "secreted" PRL-3 might have clinical relevance as a biomarker by analyzing urine samples from healthy individuals and cancer patients. A total of 15 urine samples from healthy individuals and 199 urine samples from cancer patients were analyzed by western blot to detect PRL-3 protein. Encouraging, PRL-3 was readily detectable in urine samples from different types of cancer patients, averaging 62% (123 out of 199) (FIG. 5B), but was completely absent from normal urine samples (FIG. 5C, lanes 1-7). Specifically, urinary PRL-3 protein was detected in up to 14/16 (88%) of gastric cancer patients, 12/17 (70%) of nasopharyngeal cancer patients (fig. 5D), 30/67 (45%) of bladder cancer patients (fig. 5E), 56/85 (66%) of lung cancer patients (fig. 5F), 8/10 (80%) of breast cancer patients, and 3/4 (75%) of prostate cancer patients (data not shown) (fig. 5C, lanes 8-23). Our results from these 214 urine samples indicate that PRL-3 is a common cancer-specific urine protein.

Since the PRL-3 protein does not have a sequence peptide for classical secretion via the ER/golgi pathway, we considered whether secretion is possible by non-classical exosome secretion. Exosomes are cell membrane and/or endosome-derived vesicles (43) of 50-150nm present in many biological fluids and cell culture media. We used transmembrane tetrameric protein CD63 as a control exosome marker for exosome fractionation of urine samples from different types of cancer patients (44). Surprisingly, we detected exosome PRL-3 only from urine of bladder cancer patients (fig. 16), but not from other types of cancer (data not shown). Thus, urinary PRL-3 exists as a cancer specific marker comprising at least two forms-soluble, "free" form (urine from multiple cancer patients) and exosome-associated form (urine from bladder cancer patients only).

Urinary PRL-3 may be a potential surrogate biomarker for monitoring of treatment response to PRL 3-bead mab therapy

Since PRL-3 may often be detected in urine samples from cancer patients, we asked whether the urine PRL-3 reflects the true PRL-3 in vivo ⁺ The presence of a tumor. Since it is difficult to obtain a clinically matched tumor-urine sample to verify this relationship, we instead used PRL-3 ⁺ SNU-484 and PRL-3 ^- MKN45 orthotopic gastric mouse model to compare the expression of PRL-3 in matched tumor-urine pairings. Furthermore, each in situ model was subdivided into 2 groups-untreated or PRL 3-globin (treatment) -to elucidate the relationship between PRL 3-globin treatment and urinary PRL-3 expression. In untreated PRL-3+SNU-484 tumor-bearing mice, PRL-3 protein was highly abundant in urine samples (FIG. 6A, odd lanes 1-9). However, after PRL 3-globin treatment, it was no longer possible to treat all miceUrinary PRL-3 was detected, consistent with reduced intratumoral expression of PRL-3 (fig. 6A, even lanes 2-10). Importantly, the loss of PRL-3 signaling in PRL 3-mab treated mice in urine corresponded to gastric tumor reduction in each case (fig. 6A, upper panel), suggesting that urinary PRL-3 could be used as a surrogate biomarker for PRL 3-mab treatment efficacy. In contrast, we did not detect PRL-3 in urine from mice bearing PRL-3MKN45 in situ tumors, regardless of PRL 3-globin treatment (FIG. 6A, lanes 11-12). Thus, carrying PRL-3 ⁺ But does not carry PRL-3 ^- Urinary PRL-3 was specifically detected in cancer mice, and reduced tumor burden and decreased urinary PRL-3 when treated with PRL 3-globin.

PRL-3-globin post-treatment PRL-3 ⁺ Increased B cell and NK cell infiltration in tumors

An important consideration in clinical antibody development is the biodistribution of antibodies between tumors and normal (or non-antigen expressing) tissues in vivo (46). In view of this, we explored the distribution of PRL 3-mab at the tumor site in our in situ model. After PRL 3-globin treatment, we detected an enrichment of PRL 3-globin within the PRL-3+ SNU484 tumor (FIG. 6B, panels B-c), but not in the PRL-3-MKN45 tumor (FIG. 6B, panel f). As a control, no signal was observed in untreated mice (fig. 6B, panel a) or 5-FU alone (fig. 6B, panel d). These results indicate the specific accumulation of PRL 3-mab in the microenvironment of PRL-3 expressing tumors. Immune effector cells recognize antibodies through immunoglobulin receptors (fcrs), which bind the Fc portion of the antibody, resulting in recruitment and activation of these effector cells (47). To determine whether accumulation of PRL 3-globin in tumor tissue results in infiltration of immune cells, PRL-3 was paired with specific antibodies against B cells and NK cells ⁺ NU-484 gastric tumor sections were immunofluorescent, and two FcR-bearing immune cell types were thought to be critical for the efficacy of intrabody antibody therapy (26). At PRL-3 ⁺ In SNU-484 in situ tumor sections, the number of infiltrating B cells and NK cells was significantly higher in PRL 3-globin treated tumors compared to untreated tumor sections (FIG. 6B, panels g and m) (FIG. 6B, panels h and n). Remarkably, we have an overviewThe lack of B or NK cell infiltration in mice treated with PRL 3-globin and 5-FU (FIG. 6B, panels i and o) and 5-FU (FIG. 6B, panels j and p) was observed, possibly due to a decrease in lymphocyte populations following 5-FU administration (FIG. 4C). No difference in B cell or NK cell infiltration was observed in PRL-3-MKN45 tumor sections regardless of PRL 3-mab treatment (FIG. 6B, panels k-1 and q-r). Based on these findings, we propose a new mechanism of how PRL 3-globin and PRL-3 antigen interact to elicit a therapeutic effect in vivo (fig. 6C): 1) PRL-3 antigen, by non-conventional secretion (exosome PRL-3), or from necrotic or apoptotic PRL-3 ⁺ Tumor cells (free PRL-3) leak spontaneously, acting as decoys for 2) PRL 3-globin binding and immune complex accumulation within the tumor niche, subsequently leading to 3) recruitment and activation of effector NK and B cells for anti-tumor effects.

PRL 3-bead monoclonal antibody inhibiting PRL-3 ⁺ Possible mechanism of action of tumors

Studies of autoimmune pathology show that autoantibodies can bind to specific intracellular antigens and accumulate in the cytoplasmic and nuclear compartments of antigen expressing cells (47). Similarly, we have observed that anti-PRL-3 antibodies can be expressed by PRL-3 in vitro ⁺ Tumor cells internalize (4). However, the manner of antibody uptake has not yet been clearly established. Here we found two new findings of antibodies in which the intracellular PRL-3 antigen may be involved in specific binding and tumor suppression: 1) The intracellular PRL-3 oncoprotein may be secreted. In tumor cells, it has been reported that several classical "intracellular" proteins are externalized by secretion and/or cell surface relocation, making them available for therapeutic intervention using antibodies (48, 49). We compared three PRL-3 ⁺ Cell lines SNU-484, NUGC-4, IM-95 and a PRL-3 ^- Expression of PRL-3 intracellularly and extracellularly in cell line MKN45 PRL-3 was investigated to see if PRL-3 could be externalized as a PRL 3-globin-bound target antigen as well. PRL-3 expression was compared to non-secreted ER dockerin calnexin as a control. PRL-3 protein was detected in the intracellular protein fraction (cell lysate) of PRL-3+ GC cells (FIG. 6A, lanes 1-3), as well as PRL-3 ⁺ Extracellular protein pool (concentrated conditioned medium) of GC cells (FIG. 6A, lanes 5-7), but without PRL-3-GC cell line (FIG. 6A, lanes 4, 8). In contrast, calnexin is only present in PRL-3 ⁺ And PRL-3 ^- PRL GC cells in the intracellular pool (fig. 6A, lanes 1-4), but not in the extracellular pool (fig. 6A, lanes 5-8). This observation ruled out non-specific contamination of dead cells or cell debris and characterized PRL-3 as a novel secreted protein. 2) The externalized PRL-3 can act as a decoy to which PRL 3-mAb binds. We next studied tumor sections from treated orthotopic GC mice and analyzed the distribution of PRL 3-mab in the tumor niche. As a control, no signal was observed in untreated mice (fig. 6B, left-most panel). After treatment, PRL 3-bead monoclonal antibody was administered at PRL-3 ⁺ Enrichment in the microenvironment of SNU-484 tumors, but not receiving 5-FU monotherapy or PRL-3 ^- Those of MKN45 tumors (fig. 6B). These results indicate that PRL 3-globin is present in PRL-3 ⁺ Specific accumulation in the tumor microenvironment, but not in PRL-3 ^- Accumulated in the tumor.

Discussion of the related Art

This study further demonstrates the previously unrecognized potential of tumor-specific intracellular oncoproteins as viable molecular targets for cancer-targeted immunotherapy with minimal side effects. Using a human gastric cancer cell line to produce an in situ tumor model, our results characterize PRL-3 as an excellent tumor specific target and demonstrate specific anti-tumor efficacy of PRL 3-mab in clinically relevant environments. PRL 3-monoclonal antibody specificity inhibition in situ PRL-3 ⁺ (but not PRL-3) ^- ) Growth of gastric tumors, determination of PRL 3-Belizumab for treatment of PRL-3 ⁺ Suitability for gastric cancer. In addition, secreted urinary PRL-3 can be used as a biomarker for diagnosis and monitoring of therapeutic response.

To create a clinically relevant in situ GC model using human GC cell lines, we used immunodeficient nude mice but not severely immunocompromised mouse strains such as NOD/SCID, BALB/c-RAG2null or their derivatives (48). These latter strains have little or no intact endogenous immune system, and thus are converting the results of the study into positive immune functionThe usual human patient aspect creates a gap. The use of more clinically relevant mouse models also overcomes the limitations of in vitro drug screening in culture dishes, they fail to generalize complex interactions in vivo, and predict poor in vivo toxicity (49). In fact, anti-PRL-3 antibodies have been demonstrated in immunocompromised SCID mice (24), or PRL-3 was added directly in vitro ⁺ Lack of anticancer efficacy in cancer cells (27), indicates the importance of therapeutic interaction with immune effectors for successful treatment. Here, we demonstrate the therapeutic efficacy of PRL 3-globin in inhibiting the growth of primary and metastatic gastric tumors, as well as its value for postoperative adjuvant therapy to prevent cancer recurrence. Furthermore, we extended these findings by demonstrating accumulation of PRL 3-mab and increased infiltration of B and NK cells in PRL-3+ tumor cells following PRL 3-mab treatment, thereby potentiating the antitumor activity of these key immune effectors involved in PRL 3-mab. It has recently been shown that PRL-3 promotes secretion of ULBP2 (NKG 2D ligand), leading to decreased tumor recognition and cytolysis of NK cells (50). This finding indicates that the increased infiltration of NK cells to PRL-3 was observed in PRL 3-globin treated mice ⁺ Possibly synergistic in tumor niches with an increase in NK cell lytic activity leading to PRL-3 ⁺ Immune targeting of tumors is more effective.

Discovery of a secreted form of PRL-3 increases recruitment of immune cells to PRL-3 ⁺ Specific antibody-antigen interactions required at the tumor site and anti-tumor efficacy of PRL 3-mab. Interestingly, although soluble PRL-3 was detected in the urine of multiple cancer patients, we only detected exosome-associated PRL-3 in the urine of bladder cancer patients, but not in the urine of other malignant patients. A possible explanation for this is the physical exclusion limit imposed by glomerular filtration, which only allows proteins of less than 70kDa from plasma to enter Bowman's capsule for urinary excretion (45). Our results indirectly suggest that PRL-3 can be secreted from at least two forms of tumor cells in vivo: 1) First, it is in a soluble, filterable form that is present in many types of cancer urine. This "free PRL-3" may be in tumor necrosis, apoptosis or tumor cell divisionDuring the lysis process, it leaks into body fluids, has a low molecular weight of 20-25kDa, and may pass through the glomerulus and be excreted in the urine. 2) Second, as the 'exosome PRL-3', it is found only in the urine of patients with bladder cancer, as bladder cancer cells entering the bladder urinary system unimpeded can excrete this PRL-3 containing exosome directly into the urinary pool. However, circulating exosomes from other cancerous tissues (e.g. stomach, liver, lung) cannot be filtered through the glomerulus, but rather from PRL-3 ⁺ Budding exosomes of cancer cells could be used as anchor points within the tumor region for PRL 3-mab recognition in vivo to initiate a cascade immune response (fig. 6C).

Recently, a large number of FDA-approved anti-cancer drugs have been demonstrated to have poor target selectivity (51). In our study, over 400 clinical cancer samples were studied for expression of PRL-3 at mRNA or protein levels in tumor tissues and/or cancer urine. On average, PRL-3 oncoprotein is overexpressed in 62% of the various types of cancer examined (stomach, liver, lung, nasopharynx, kidney, breast, colon, bladder). With such high PRL-3 tumor positivity, the development of PRL 3-globin targeted therapy against tumor specific PRL-3 is an exciting step towards personalized medicine. By maximizing therapeutic efficacy while minimizing off-target side effects (PRL-3 is not expressed at detectable levels in most normal adult tissues), PRL 3-mab demonstrates clinical validation and development as an accurate anti-cancer drug.

We summarize here five main findings on PRL 3-mab cancer treatment: 1) PRL 3-mAb specifically recognizes PRL-3 tumor specific antigen. PRL 3-mAb is highly specific-it does not cross-react with its two homologues (PRL-1 or PRL-2) which have>75% amino acid sequence identity. Furthermore, PRL 3-globin specifically recognizes PRL-3 antigen in tumor tissue but not in normal tissue, indicating low toxicity and minimal off-target side effects. 2) PRL-3-monoclonal antibody specific inhibition PRL-3 ⁺ Growth of gastric tumors in situ and prevention of post-operative PRL-3 ⁺ The tumor recurs. The expression of the PRL-3 protein in tumors is an absolute prerequisite for a therapeutic response, indicating that specificity is requiredSex antigen-antibody recognition of tumor inhibition. 3) PRL 3-globin is more effective as monotherapy than in combination with chemotherapy. Overall, our results indicate that PRL 3-mab treatment outcome is dependent on the host immune system, as chemotherapy-induced immunosuppression (34) reduces the therapeutic efficacy of PRL 3-mab. 4) PRL 3-globin should have broad utility in a variety of PRL-3 positive cancers. Although our findings focused on several GC models as case studies of efficacy of PRL 3-globin, PRL-3 was also widely associated with tumor metastasis and poor prognosis for various cancer types, with higher PRL-3 expression associated with shorter overall survival (7). Based on the principle that PRL 3-mab only exerts its effect when it recognizes the PRL-3 antigen, targeting most, if not all, PRL-3 positive cancers in immunocompromised patients is envisioned to be effective, opening up a new therapeutic approach in the treatment of general cancers. 5) Urinary PRL-3 may be a potential novel biomarker for cancer diagnosis and monitoring of therapeutic response. We averaged 62% of the urinary PRL-3 detected in multiple human cancer patients. The close correlation between tumor and urinary PRL-3 expression observed in mouse models suggests that urinary PRL-3 expression can be used as a prospective diagnostic biomarker for PRL-3 targeted cancer therapies, including PRL 3-globin, in various human malignancies. Furthermore, our data suggest that urinary PRL-3 may serve as a surrogate biomarker, providing clinicians with a rapid and simple qualitative approach to infer PRL 3-mab therapeutic efficacy. Although the biomarker values of urinary PRL-3 require further validation, the potential to develop this "companion diagnosis" of PRL 3-mab would accelerate its drug development process by performing robust hypothesis testing in early clinical trials (52).

Herein, we demonstrate that PRL 3-globin is the first anti-intracellular tumor target to block PRL-3 ⁺ Humanized antibodies to human cancers. Overall, our results here and elsewhere (23, 24, 27) challenge the textbook of intracellular anti-cancer drugs where therapeutic antibodies failed to achieve anti-cancer effects. We suggest that other intracellular oncoproteins may also have great potential as targeted immunotherapies. Countless candidate tumor-specific intracellular anticancer drugs should now be reconsideredPotential as a viable molecular target for future clinical trials.

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Example 3: study of the mechanism of action of PRL3 mAb

In this study we mainly used a 'seed and soil' liver in situ tumor model, focusing on the molecular mechanism of action (MOA), to address how PRL-3 antibodies bind to their intracellular PRL-3 antigen. We have studied from different aspects and come to the important conclusion that indeed the proportion of "intracellular oncoproteins" in vivo is higher than in vitro, and that they relocate to the cell surface in the form of "extracellular oncoproteins", thus following a reasonable basis for tumor elimination by the conventional route of antibodies against extracellular tumor targets. Consistently, we have mechanistically found that PRL 3-globin blocks tumor requirements for expression of PRL-3 'intracellular' antigen: 1. the host Fc γ II/III receptor interacts because both Fc γ II/III blockers abolish therapeutic efficacy. 2. Full antibody activity, small bodies (CH 1 and CH2 domains) lacking the Fc fragment abrogated therapeutic efficacy, 3. Increase M1 (but not M2) macrophages, B lymphocytes, natural killer cells to enhance host immunity. These results indicate that the MOA of antibodies directed against "intracellular oncoproteins" do follow a similar principle of targeting "extracellular oncoproteins" via the classical antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis (ADCP) pathways to eliminate tumors. Finally, using 110 precious fresh-frozen human tumors or their matched normal tissues, we further showed that PRL-3 is an excellent tumor-specific target, broadly overexpressed by an average of >78% of 9 different human cancer types: tumor samples from patients with liver, lung, colon, breast, stomach, bladder, prostate, AML and renal disease, but not from normal tissues. These findings make the PRL 3-bead clinical trial the next frontier for targeted immunotherapy of most "refractory" cancers.

Materials and methods

A cell line. Human HCC Cell lines Hep3B2.1, hepG2, huh-7, PLC, SNU449 were purchased from American Type Cell Culture (Marnsas, virginia, USA). The murine HCC Cell line Hep53.4 was purchased from CLS Cell Lines Service GmbH (Epeheim, germany). All cell lines were cultured in their recommended medium. MHCC-LM3 human HCC cancer cell line was routinely stored in the Kam-Man doctor's laboratory (national cancer center, singapore).

Western blotting. For patient tissues, 3 pieces of 5mm tissue were suspended in RIPA lysis buffer (Sigma) containing a protease-phosphatase inhibitor cocktail (Pierce) and completely disrupted with a tissue homogenizer (Polytron). Lysates were clarified by centrifugation at 13,000 × g for 40 min at 4 ℃. For cell lines, 5X 10 lysis in RIPA lysis buffer containing protease-phosphatase inhibitor ⁶ Individual cells, and clarified as described above. Tissue lysates (40 μ g) or cell lysates (200 μ g) were separated in a 12% SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and then blocked and probed with murine anti-PRL-3 34 or anti-GAPDH antibody (clone MAB374, milipore) at 4 ℃. After thorough washing with TBS-T buffer (20mM Tris pH 7.6,140mM NaCl,0.2% Tween-20), the membranes were washed with corresponding horseradish peroxidase (HRP) -conjugated secondary antibodies in a 1: the 5,000 dilutions were incubated for 1 hour, washed with TBS-T buffer, and visualized using a chemiluminescent substrate (Pierce).

Animal models and treatments. Male BALB/c nude mice at 8 weeks of age obtained from the center of biological resources (a STAR, singapore) were used in all animal models in this study. Mice were anesthetized with 2.5% averin (100 μ l per 10g body weight). The abdomen of the anesthetized mice was opened by stratification through a 1cm midline incision starting from below the xiphoid process. The left lobe of the liver was removed by abdominal incision and 3X 10 ⁶ And (3) inoculating MHCC-LM3 liver cancer cells to the lower layer of the envelope. The liver was placed back into the abdominal cavity and the abdominal wall was sutured layer by layer. The treatment regimen was started on day 5 after inoculation of the cancer cells. For tumor growth/volumeExperimental, mice were treated with 100 μ g of each of PRL 3-mab, human IgG isotype control (catalog BE0092; bio X Cell), or PRL 3-minibody intravenously, biweekly for 5 weeks. Co-treatment was performed by co-administration of 100. Mu.g of anti-CD 16/32 antibody (clone 2.4 G2. All antibodies were diluted into 100 μ L (final) PBS for injection. The final tumor volume was calculated using the formula: volume =0.4 x tumor length x tumor width. For survival studies, treated mice were given 100 μ l of PRL 3-mab diluted in pbs intravenously, twice weekly, 10 times total. Untreated mice were given an equal amount of placebo (buffer only) intravenously as a control. When mice had reduced physical activity and became sick, they were euthanized and recorded as "death" events in the survival analysis.

And (5) separating the cells. A tumor cell. In situ MHCC-LM3 liver tumors were harvested and gently dissociated using a MACS tissue dissociation kit (130-095-929, miltenyi Biotec) according to the manufacturer's instructions. The kit is optimized for high-yield tumor cells, and important cell surface epitopes are reserved. Isolated tumor cells were then counted, resuspended in RPMI, and placed on ice until analyzed. The cells are cultured. MHCC-LM3PRL-3 ⁺ Hepatoma cells were grown at 80% confluence index in complete RPMI medium (RPMI supplemented with 10% FBS and 1% antibiotics) in T-75 flasks, washed once with PBS, and incubated for 5 minutes with non-enzymatic cell dissociation buffer (C5914; sigma) to engraft adherent cells into suspension. Cells were washed once with PBS, counted, resuspended in complete RPMI medium, and placed on ice until analysis.

Cell surface labeling and flow cytometry analysis. Will be 4X 10 ⁵ The cells were incubated with 2. Mu.g cetuximab (anti-EGFR, chimeric antibody), herceptin (anti-HER 2, humanized antibody), PRL 3-globin (anti-PRL-3, humanized antibody) in a total volume of 100. Mu.l for 30 minutes at 4 ℃. A separate tube without any added primary antibody served as a negative control. After incubation, 1mL PBS was added to each sample, centrifuged, and the cell pellet was resuspended in 100 μ LPBS containing 1.5 μ L of anti-human FITC antibody. After incubation at 4 ℃ for 15 minutesCells were washed with PBS as before and finally resuspended in 200 μ LPBS. Cells were passed through a cell filter to obtain a single cell suspension and immediately obtained on a BD facscan II flow cytometer equipped with 2 lasers (488 nm and 633 nm) using FACS Diva software (data). The data was stored as FCS3 files and analyzed using the floating Software version 2.5.1. Live cells were gated on the basis of FSC and SSC. Single cells were gated using FSC and SSC widths. Single antibody stained cells (secondary antibody only) and unstained control cells were used for compensation.

Preparation of recombinant GST-tagged protein and ELISA. The preparation and ELISA assay of recombinant GST-PRL-1, GST-PRL-2 and GST-PRL-3 fusion proteins have been previously described (59). Briefly, 96-well ELISA plates coated with the indicated antigen amounts were blocked with 3% bovine serum albumin and then incubated with 0.5ng or 1ng of PRL-3 miniantibody for 2 hours at 37 ℃. After extensive washing and incubation with secondary antibody, colorimetric development was carried out using Turbo-TMB substrate (Pierce) and by using 2M H ₂ SO ₄ And (5) terminating acidification. The absorbance was measured at 450nm using a plate reader (Dynatech).

Immunofluorescence imaging. Freshly frozen MHCC orthohepatic tumor specimens were cut into 10 μm sections at 16 ℃ using a cryostat (Leica). Slides were fixed with 4% paraformaldehyde for 20 minutes, washed with PBS-0.05% Tween-20, and blocked in PBS-FDB (PBS pH 7.0,2% BSA,5% goat serum, 5% fetal bovine serum) for 1h at room temperature. The slides were then incubated with the indicated primary antibodies at 4 ℃ in a 1:200 dilutions were incubated overnight, washed, and incubated with the corresponding fluorochrome-conjugated secondary antibodies (Life Technologies) for 2 hours. The washed slides were fixed with DAPI-containing fade-resistant mounting reagent (Vector Laboratories) and sealed with nail polish. Confocal imaging was performed with an LSM 800 confocal microscope (Zeiss AG). A representative image of Tumor Infiltrating Lymphocytes (TILs) in the tumor region near the tumor pouch (border of normal and tumor tissue) was taken (n = 3). The total number of immune cells (green) and DAPI positive cells (blue) was analyzed by Image J software and the percentage of TIL was determined by the ratio of immune cells to DAPI. The results of averaging 3 images represent data for 1 sample.

Results

PRL-3 'intracellular oncoprotein' can be identified in vivo as an "extracellular oncoprotein".

The PRL-3 antibody has been shown to have efficacy against PRL-3 expressing xenograft tumors, metastatic lung tumors, and orthotopic gastric tumors. To understand these unconventional antibody therapies for intracellular oncoproteins, how do PRL3 antibodies bridge intracellular PRL-3 and Fc γ R on immune cells? One possible hypothesis is that certain portions of PRL-3 itself may be inverted to be exposed to the cell surface in vivo to trigger a circulatory effect, allowing direct PRL 3-mab binding, as with other cell surface (extracellular) antigens. To test this, we prepared single cell suspensions from solid liver tumors using a mild enzymatic method and compared the cell surface expression of these ex vivo tumor cells to cells cultured in vitro using a flow cytometry method (fig. 20 a). Cytometric analysis of these unpermeated cell banks revealed major antigen-specific differences between them. Cetuximab is an anti-Epidermal Growth Factor Receptor (EGFR) chimeric antibody, with significantly reduced EGFR surface expression in tumors in vitro, as compared to cultured cells artificially supplemented with large amounts of growth factor, as opposed to PRL-3 (representative flow histogram in fig. 20 b). Quantitative assays showed a 3-fold reduction in surface EGFR staining in ex vivo tumor cells (T) relative to cultured cells (CC; fig. 20c, columns 3 and 4). In contrast, PRL-3 expression was increased about 7-fold in ex vivo tumor cells as analyzed by PRL 3-globin staining, where in vivo cancer cells were under hypoxic stress and serum deprivation, conditions likely to enhance the ability of the cancer cells to externalize intracellular PRL-3 protein compared to cultured cells (CC; FIG. 20c, columns 5 and 6). To verify whether these changes seen in flow cytometry are likely due to changes in the total cellular level of these antigens, we performed western blot analysis of lysates in parallel. Consistent with the early cell count observations, the total levels of EGFR were significantly down-regulated in ex vivo tumor cells compared to cultured cells (fig. 20 d). In contrast, PRL-3 expression was significantly increased in tumors relative to the primary cells (fig. 20 d). However, since the increase in total levels of PRL-3 is much less than the increase in surface levels of PRL-3, we believe that the latter observation may be due primarily to the relocation of PRL-3 within the cell. To verify this, transmission Electron Microscopy (TEM) of cultured MHCC cells and MHCC tumors was performed, in which a significant increase in anti-PRL-3 immunogold staining on the extracellular lobules of the cell membrane in MHCC tumors was observed compared to MHCC cells, while no significant difference was observed in the intracellular lobules of the cell membrane.

PRL 3-mAb shows therapeutic efficacy in an Fc-dependent manner in a mouse 'seed and soil' orthotopic liver tumor model

These higher levels of "extracellular" PRL-3 antigen in vivo can then be recognized by the PRL-3 antibody to recruit immune cells and follow classical ADCC and ADCP of antibody therapeutics against traditional extracellular oncoproteins. In situ tumor models, in which human cancer cells ("seeds") are allowed to grow in their natural location ("soil") to replicate human disease with high fidelity. We have recently reported that PRL 3-globin can inhibit in situ gastric tumors expressed by PRL-3 formed by human gastric cancer cells (8). In addition, we found that PRL 3-globin also blocks recurrence of tumors expressing PRL-3 after resection.

In this study, to better summarize the clinically relevant therapeutic HCC treatment response 16, we established an in situ HCC model to test the ability of PRL 3-mab to inhibit liver tumors within its natural niche (fig. 21 a). Among the group screened for 6 human (1 mouse ⁺ MHCC-LM3 cells robustly formed liver tumors in a reasonable time frame (6 weeks) and were selected for subsequent treatment experiments. Similar to orthotopic gastric tumor 6, orthotopic liver tumor formation was significantly reduced in PRL 3-globin treated mice compared to untreated mice (fig. 21 c). Measurement of tumor volume showed a significant 7-fold reduction in mean tumor burden in treated mice compared to untreated mice (figure 21d 0.30 ± 0.36 vs 2.41 ± 1.20cm ³ P = 0.0001). To investigate whether treatment would have a long-term effect on mouse survival, we treated mice with PRL 3-globin for four weeks, stopped the treatment, and monitored the time required until the appearance of the pathological features ("death" events). According to this treatment regimen, with no treatmentCompared to treated mice, the overall survival was significantly prolonged with median survival of 12 and 8 weeks, respectively (figure 21e. Overall, our findings indicate that PRL 3-mab retains therapeutic efficacy, significantly reduces tumor burden and prolongs survival in this clinically relevant HCC model.

To understand the molecular mechanisms involved, we first performed in vitro assays to determine whether PRL 3-globin can directly inhibit PRL-3 ⁺ A cancer cell. Although PRL-3 ⁺ Tumors were significantly inhibited in vivo, but PRL 3-mab treatment did not inhibit PRL-3 in vitro ⁺ And PRL-3 cancer cell growth, even at high doses of 50mg/mL (fig. 24). In contrast, cisplatin treatment resulted in PRL-3 ⁺ And PRL-3 ^- Dose-dependent, non-specific cell killing of cells (fig. 24). This finding again confirms that PRL 3-globin, like other therapeutic antibodies, requires specific host factors to produce an anti-tumor effect 15. In conventional antibody therapy, fc receptors (fcrs) on immune cells bind to the constant (Fc) region of antigen-antibody complexes, causing them to recruit and activate effector pathways 17 of target antigen/cell clearance through antibody-dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP) 17. To investigate the mechanism of action of host FcR in the involvement of PRL 3-mab, we designed 2 complementation experiments (fig. 20 f), namely 1) co-treatment of mice vaccinated with PRL-3 orthotopic liver tumors with PRL 3-mab and anti-CD 16/32 antibody (2.4g2mab), effective IgG FcR-mediated immune clearance inhibitors by blocking the binding sites for Fc γ II and Fc γ III receptors 18,2) replacement of the intact PRL 3-mab with engineered (scFv-CH 3) 2PRL 3-minibodies lacking CH1 and CH2 domains, showing the necessity to bind Fc receptors of 19,20. Likewise, liver tumors treated with the (scFv-CH 3) 2PRL 3-minibody also showed no therapeutic response (FIG. 20 g). Notably, a similar lack of therapeutic effect was also evident for the in situ PRL-3SNU-484 gastric tumor (scFv-CH 3) 2PRL 3-minibody (fig. 25), suggesting that this is not a tissue-specific defect. Furthermore, the deletion of the CH1 and CH2 domains of PRL 3-globin does not affect the binding of the resulting minibody to PRL-3, e.g.Western blot, ELISA and immunofluorescence (data not shown), indicating that the loss of therapeutic efficacy is not due to potential antigen binding defects caused by antibody miniaturization. In summary, our results demonstrate that the interaction between the Fc domain of PRL 3-mab and the host Fc γ II/III receptor is essential for the anti-tumor effect of PRL 3-mab.

PRL 3-globin recruitment of B lymphocytes, natural killer cells, and M1 macrophages to PRL-3 expressing tumor niches for killing of cancer cells in vivo

The Fc-FcR interaction is important in the clearance of tumor cells by ADCC and ADCP. Although NK cells are the primary effector of ADCC, macrophages are the effector of ADCP, which is increasingly recognized as the primary mechanism of action behind most antibodies approved for the treatment of cancer 21. Tumor-associated macrophages (TAMs) are important elements of the tumor stoma and can exert dynamic, adverse effects during tumorigenesis, a difference 22 between immunostimulatory and tumoricidal activity (M1 macrophages) and immunosuppressive and prometastatic activity (M2 macrophages). To determine whether PRL 3-globin promotes infiltration and accumulation of macrophages and other immune cells in the tumor niche, immunofluorescence assays were performed on PRL-3+ mhcc liver tumor sections using various antibodies specific for different macrophage subtypes: m1 macrophages (CD 86), pan-macrophages (F4/80); m2 macrophages (CD 206), B cells (CD 45/B220) and NK cells (CD 335). Interestingly, significant increases in CD86+ M1 macrophages were evident (fig. 21c, F (5,12) =7.127, p < -0.0053), whereas no significant difference was observed between group means for accumulation of F4/80+ macrophages and CD206+ M2 macrophages (fig. 2a, b). Similarly, significant accumulation of B cells (fig. 21d (f (5,12) =40.14, p-straw 0.001) and NK cells (fig. 21e f (5,12) =7.386, p-straw 0.0046) was also observed in all treatment groups. Notably, the combination treatment of PRL 3-mab and 2.4g2mab resulted in a reversal of PRL 3-mab induced accumulation (fig. 2 c-e), confirming that PRL 3-mab promotes specific accumulation of these cells in an FcR-dependent manner. In summary, our results demonstrate that the interaction between the Fc domain of PRL 3-mab and Fc γ II/III receptors is essential for the recruitment of tumor killing M1 macrophages, B cells and NK cells, and that these are closely related to anti-tumor efficacy in vivo (fig. 21 f).

PRL-3 is a novel tumor target, frequently overexpressed in a variety of human cancers; urgent unmet medical need for PRL 3-globin to be used to treat these various PRL-3 positive human cancers

We have previously demonstrated the value of PRL-3 as a novel gastric cancer target, where PRL-3 expression was detected in 85% of freshly frozen gastric tumor tissue, but no PRL-3 expression was detected in patient-matched normal gastric tissue 6. Since elevated PRL-3 transcript expression has been described in many other tumor types 2, we sought to broadly characterize PRL-3 protein expression, particularly aggressive malignancies for which the medical needs are not met, in 110 freshly frozen patient tumor samples from 9 different cancer types that were difficult to obtain. In these randomly assigned fresh frozen samples from our clinical collaborators, we detected strong PRL-3 expression in 16/20 liver tumors (80%; fig. 23 a), 9/10 lung tumors (90%; fig. 23 b), 7/10 colon tumors (70%; fig. 23 c), 9/10 breast tumors (90%; fig. 23 d), 13/18 kidney tumors (72%; fig. 23e, f), 19/28 bladder tumors (68%; fig. 23 g), 6/12AML samples (50%; fig. 23 h), 5/6 stomach tumors (83%; fig. 23 i), and 4/4 prostate tumors (100%; fig. 23 j). For liver, lung, colon, breast and kidney tumors, we sought to obtain freshly frozen, patient-matched non-cancerous tissue from the same organ, which made the specificity of PRL-3 expression invaluable. Notably, despite high expression in the corresponding matched tumors, no PRL-3 was detected in any of the matched normal tissues (fig. 4 a-e). Taken together, these results indicate that PRL-3 is a broad, tumor-associated, broad target, with an average expression of >78% in 9 tumor types (Table 1), and highlight that PRL-3 is a superior target for a variety of cancer types, particularly those with urgent, unmet medical needs.

Table 1: summary of PRL3 expression in different tumor types

Discussion of the related Art

This study was based on our previous work and provided conclusive evidence on further profiling how possible antibodies target the acting molecules of "intracellular oncoproteins" and the future therapeutic value of PRL 3-globin on a variety of PRL-3 positive human cancer types. Our findings for PRL-3 are tumor-associated targets, present at >78% frequency in 110 randomly analyzed samples of fresh frozen human cancers, and demonstrated significant therapeutic benefit of PRL 3-globin in orthotopic liver and lung tumors using the orthotopic gastric tumor model in this and previous studies (reference), we again demonstrated that PRL 3-globin is a breakthrough immunotherapeutic candidate for these acute malignancies, with other urgent medical needs, in addition to other cancers.

The pathophysiological complexity of HCC, including potential functional hepatic insufficiency, makes the treatment of HCC challenging. Recurrence of HCC after transplantation is also a clinically relevant problem. Previous efforts to identify specific molecular changes involved in the progression of HCC have resulted in few practical hits, particularly due to the different etiologies of HCC: more than 90% of HCC is caused by cirrhosis, which in turn is caused by a variety of factors, including alcoholism, hepatitis b or c infection, or the accumulation of fat in the liver. Evidence for HCC heterogeneity, at least five major phase III trials of novel molecular targeted drugs against advanced liver cancer have failed over the past six years 32. Sorafenib was the initial therapy, indicating an improvement in mortality of advanced HCC, with a median survival extension of 2.8 months by 12. However, treatment of sorafenib in patients with advanced HCC and liver dysfunction (Child-Pugh B patients) resulted in poorer survival outcomes 33. Therefore, there is an urgent need to find novel molecular targeted drugs with high therapeutic efficacy and low toxicity to HCC patients. Here, the detection of PRL-3 overexpression in 80% of randomly analyzed liver cancer patient samples provides the first clinical evidence that PRL-3 protein may be a common marker of this pathological condition. Notably, due to the lack of PRL-3 protein expression 6 in most major human organs, PRL 3-globin has been demonstrated to be well tolerated in non-human primate toxicology studies, where no adverse event level (NOAEL) doses up to 36mg/kg were observed (unpublished observations). The anti-tumor efficacy of PRL 3-globin in the orthotopic mouse model reported here provides a strong support for PRL3-HCC patients to start early trials of PRL 3-globin as a safe and effective treatment modality.

To address how antibodies recognize intracellular oncoproteins, we previously provided three possible models of mechanism of action (MOA), including that antibodies can be taken up by cancer cells (CBT, 2008). In this study, we consolidated MOA by providing evidence on how to externalize ' intracellular oncoproteins into ' extracellular oncoproteins ', and therefore, following the classical pathway of cancer cell killing effects, the mechanistic explanation for the safe and effective anti-tumor effects of PRL 3-globin was based on the specific and consistent upregulation of PRL-3 in tumor but not normal tissues. Indeed, cell surface relocation of tumor-associated intracellular antigens provides new opportunities for therapeutic intervention. Darkening, tumor necrosis, tumor cell killing lysate, apoptosis, and may also promote leakage of intracellular proteins into the tumor microenvironment and initiate immune responses in vivo. In addition to PRL-3, in fact, cell surface relocation of other intracellular proteins associated with tumors is described, heat shock protein 70 (HSP 70), heat shock protein 90 (HSP 90), glucose regulatory protein 78 (GRP 78), actin, cytokeratin, vimentin, nucleolin, nucleosome, estrogen receptor-alpha variant 36 (ER-alpha 36), and fetal-acinar trypsin (FAPP) (5). As much effort has been devoted to identifying new antigenic targets suitable for antibody-based therapy in cancer, our results reiterate here that cell-surface translocation of classical "intracellular" cytoplasmic and nuclear proteins during malignant progression may be a common tumor-specific phenomenon of greater concern and lay the foundation for increasing innovation and rational drug design to reduce cancer morbidity and mortality.

We understand the challenge of reflecting in vivo tumor cell killing events in a cell culture system that artificially conditions limit cell types in 10-percent fbs medium, and cannot mimic in vivo complexity. We have recognized that the drug that kills cancer cells in the culture dish may be due to the toxicity of the drug itself. However, we show that although treatment of PRL 3-mab to PRL-3+ cancer cells in vitro does not result in any inhibition of cell growth, tumors from these cells can be effectively inhibited in vivo by PRL 3-mab.

Our findings provide two explanations for this phenomenon. First, the amount of 'extracellular PRL-3' is not significant in culture systems and thus insufficient for ADCC in vitro, but is highly upregulated in tumor cells to a level sufficient to trigger PRL 3-mab mediated killing of cancer cells. Second, unlike in vivo systems, in vitro systems fail to encompass the complex host factors necessary for PRL 3-globin to induce immune-mediated killing of tumor cells.

These results provide reliable evidence that the in vivo environment plays an important role in influencing the potency of the target protein and its therapeutic response, a phenomenon that may be overlooked in experiments based on simplified culture conditions. In this context, we found that Fc-host Fc γ R interaction is essential for the anti-tumor effect of PRL 3-mab, and that blocking Fc γ R in host cells leads to complete loss of the anti-tumor effect of PRL 3-mab with concomitant reduction of infiltration of B-cells, NK-cells and M1 macrophages, which is important for participation in ADCC and ADCP. Macrophages are one of the major populations of tumor-infiltrating immune cells, and generally favor tumor growth and metastasis. This is primarily because macrophage polarization events during tumor progression are observed in advanced cancers that promote tumor escape by inducing differentiation from the M1 to M2 phenotype. The M1 cell has high microbicidal activity, immunostimulation function and tumor cytotoxicity. A recent meta-analysis study has established a significant correlation between increased tumoricidal M1 infiltration and favorable survival in patients with lung 23 and stomach 24 cancers. Importantly, PRL 3-globin treatment resulted in an increase in specificity for M1 but not M2, and accumulation of macrophages. Whether this reflects a reversal of M1/M2 polarization towards an anti-tumor phenotype, or a specific promotion of M1 macrophage recruitment, requires further investigation. Interestingly, this enhancement of M1 tumoricidal activity by PRL 3-globin juxtaposies it with other sophisticated TAM targeting anti-tumor strategies (e.g., targeting NF-kB and STAT1 pathways), as well as treatment with cytokines (e.g., GM-CSF, IFN-g, IL-12) to promote M1TAM polarization 25. In addition to M1 macrophages, PRL 3-globin promotes infiltration of NK and B cells in an Fc γ R dependent manner as well as anti-tumor effects. Although NK cells are well known as the major effector of ADCC, little is known about the function of B cells in anti-tumor responses. Previously, we suggested that B cells have an anti-tumor effect when we found that anti-PRL-3 monoclonal antibodies were unable to inhibit PRL-3+ tumors in genetically engineered mouse strains (muMT mice) deficient in B cell maturation and activation. Studies have shown that tumor-associated B cells can promote cancer immune surveillance and inhibit metastasis. Higher infiltration of B cells into primary human breast and ovarian tumors has been found to correlate with better prognosis. Chemotherapy has been shown to promote anti-tumor B cell activation and intratumoral accumulation in a manner correlated with a better anti-tumor response. In contrast, B cell depletion compromises T cell-dependent anti-tumor cytotoxic responses and promotes tumor growth. Interestingly, in a retrospective analysis of lymphoma patients receiving high dose chemotherapy followed by autologous transplantation, it was noted that depletion of B cells during the high dose chemotherapy regimen resulted in a significant increase in the incidence of solid tumors. In summary, we hypothesized that B cells play an important but not well-understood role in the mechanical efficacy of general anti-tumor therapies.

Important evidence of antibody and Fc receptor Fc function in this study, as well as key immune cell recruitment, we propose that the mechanism of action of PRL 3-globin primarily involves binding to cell surface PRL-3 followed by anti-tumor clearance by classical ADCC or ADCP, similar to other receptor targeting antibodies such as trastuzumab and rituximab. Our unconventional antibody target 'intracellular oncoprotein' ensures further study of the intracellular great reservoir of potential cancer-specific therapeutic targets for antibody therapy, as both "intracellular and extracellular oncoproteins" track immune-mediated tumor cell killing through ADCC and/or ADCP. Our pioneering new cancer treatments will await a new era of cancer immunotherapy in order to benefit cancer patients as soon as possible.

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20 25 30

Glu Asn Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Ser Pro Lys Leu

35 40 45

Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro

85 90 95

Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 8

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized light chain protein HZD _ K2

<400> 8

Asp Thr Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Cys Lys Ala Ser Gln Ser Val Glu Asp Asp Gly

20 25 30

Glu Asn Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Ser Pro Lys Leu

35 40 45

Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro

85 90 95

Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 9

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized light chain protein HZD _ K3

<400> 9

Asp Thr Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Cys Lys Ala Ser Gln Ser Val Glu Asp Asp Gly

20 25 30

Glu Asn Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Ser Pro Lys Leu

35 40 45

Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro

85 90 95

Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 10

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized light chain protein HZD _ K4

<400> 10

Asp Thr Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Cys Lys Ala Ser Gln Ser Val Glu Asp Asp Gly

20 25 30

Glu Asn Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Ser Pro Lys Leu

35 40 45

Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro

85 90 95

Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 11

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized light chain protein HZD _ K5

<400> 11

Asp Thr Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Cys Lys Ala Ser Gln Ser Val Glu Asp Asp Gly

20 25 30

Glu Asn Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Ser Pro Lys Leu

35 40 45

Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro

85 90 95

Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 12

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized light chain protein HZD _ K6

<400> 12

Asp Thr Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Cys Lys Ala Ser Gln Ser Val Glu Asp Asp Gly

20 25 30

Glu Asn Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Ser Pro Lys Leu

35 40 45

Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro

85 90 95

Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 13

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized light chain protein K

<400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Glu Asp Asp

20 25 30

Gly Glu Asn Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr

<210> 14

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized light chain protein L1

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Glu Asp Asp

20 25 30

Gly Glu Asn Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg

100 105 110

Thr

<210> 15

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized light chain protein L2

<400> 15

Asp Thr Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Glu Asp Asp

20 25 30

Gly Glu Asn Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg

100 105 110

Thr

<210> 16

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized heavy chain protein HZD _ H1

<400> 16

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Leu Glu Trp Met Gly

35 40 45

Trp Ile Tyr Pro Gly Asn Val Asn Thr Tyr Tyr Asn Glu Lys Phe Arg

50 55 60

Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr Met

65 70 75 80

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser

85 90 95

Glu Glu Lys Asn Tyr Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Ser

115

<210> 17

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized heavy chain protein HZD _ H2

<400> 17

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Leu Glu Trp Ile Gly

35 40 45

Trp Ile Tyr Pro Gly Asn Val Asn Thr Tyr Tyr Asn Glu Lys Phe Arg

50 55 60

Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala Tyr Met

65 70 75 80

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser

85 90 95

Glu Glu Lys Asn Tyr Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Ser

115

<210> 18

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized heavy chain protein HZD _ H3

<400> 18

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Leu Glu Trp Ile Gly

35 40 45

Trp Ile Tyr Pro Gly Asn Val Asn Thr Tyr Tyr Asn Glu Lys Phe Arg

50 55 60

Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr Met

65 70 75 80

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser

85 90 95

Glu Glu Lys Asn Tyr Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Ser

115

<210> 19

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized heavy chain protein HZD _ H4

<400> 19

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Leu Glu Trp Ile Gly

35 40 45

Trp Ile Tyr Pro Gly Asn Val Asn Thr Tyr Tyr Asn Glu Lys Phe Arg

50 55 60

Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala Tyr Met

65 70 75 80

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser

85 90 95

Glu Glu Lys Asn Tyr Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Ser

115

<210> 20

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized heavy chain protein HZD _ H5

<400> 20

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln Leu Glu Trp Ile Gly

35 40 45

Trp Ile Tyr Pro Gly Asn Val Asn Thr Tyr Tyr Asn Glu Lys Phe Arg

50 55 60

Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala Tyr Met

65 70 75 80

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser

85 90 95

Glu Glu Lys Asn Tyr Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Ser

115

<210> 21

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized heavy chain protein HZD _ H6

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Arg Pro Gly Gln Leu Glu Trp Ile Gly

35 40 45

Trp Ile Tyr Pro Gly Asn Val Asn Thr Tyr Tyr Asn Glu Lys Phe Arg

50 55 60

Gly Lys Ala Thr Ile Thr Ala Asp Lys Ser Ala Ser Thr Ala Tyr Met

65 70 75 80

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser

85 90 95

Glu Glu Lys Asn Tyr Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Ser

115

<210> 22

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized heavy chain protein HZD _ H7

<400> 22

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Glu Glu Lys Asn Tyr Pro Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Ser

115

<210> 23

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized heavy chain protein H1

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Ile Thr Ala Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Glu Glu Lys Asn Tyr Pro Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser

115 120

<210> 24

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized heavy chain protein H2

<400> 24

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Glu Glu Lys Asn Tyr Pro Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser

115 120

<210> 25

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized heavy chain protein H3

<400> 25

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Glu Glu Lys Asn Tyr Pro Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser

115 120

<210> 26

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> important light chain Domain sequence

<400> 26

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

1 5 10 15

<210> 27

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> important light chain Domain sequence

<400> 27

Lys Ala Ser Gln Ser Val Glu Asp Asp Gly Glu Asn Tyr Met Asn Trp

1 5 10 15

Tyr Gln Gln Lys

20

<210> 28

<211> 35

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> important light chain Domain sequence

<400> 28

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

1 5 10 15

Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Glu Asp

20 25 30

Pro Phe Thr

35

<210> 29

<211> 33

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> important heavy chain Domain sequence

<400> 29

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val

1 5 10 15

Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Met His Trp

20 25 30

Val

<210> 30

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> important heavy chain Domain sequence

<400> 30

Trp Ile Tyr Pro Gly Asn Val Asn Thr Tyr Tyr Asn Glu Lys Phe Arg

1 5 10 15

<210> 31

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> important heavy chain Domain sequence

<400> 31

Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

1 5 10

<210> 32

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> important heavy chain Domain sequence

<400> 32

Ala Ser Glu Glu Lys Asn Tyr Pro Trp Phe Ala Tyr Trp Gly Gln Gly

1 5 10 15

Thr Leu Val Thr

20

<210> 33

<211> 157

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> monoclonal antibody 223 heavy chain variable region

<400> 33

Glu Phe Met Glu Trp Ser Trp Val Ile Leu Phe Leu Leu Ser Ile Ile

1 5 10 15

Ala Gly Val His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu

20 25 30

Val Lys Pro Gly Ala Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Thr Phe Thr Ser Tyr Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln

50 55 60

Gly Leu Glu Trp Ile Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Glu

65 70 75 80

Tyr Asn Glu Lys Phe Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser

85 90 95

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser

100 105 110

Ala Val Tyr Phe Cys Ala Ser Glu Glu Arg Asn Tyr Pro Trp Phe Ala

115 120 125

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr

130 135 140

Pro Pro Pro Val Tyr Pro Leu Val Pro Gly Ser Leu Gly

145 150 155

<210> 34

<211> 152

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> monoclonal antibody 223 light chain variable region

<400> 34

Trp Glu Phe Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu

1 5 10 15

Trp Val Pro Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala

20 25 30

Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala

35 40 45

Ser Gln Ser Val Glu Asp Asp Gly Glu Asn Tyr Met Asn Trp Tyr Gln

50 55 60

Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn

65 70 75 80

Leu Glu Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr

85 90 95

Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr

100 105 110

Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro Phe Thr Phe Gly Ser Gly

115 120 125

Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile

130 135 140

Phe Pro Pro Ser Ser Lys Leu Gly

145 150

<210> 35

<211> 154

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> monoclonal antibody 318 heavy chain variable region

<400> 35

Glu Phe Met Glu Trp Ser Trp Val Phe Leu Phe Leu Leu Ser Ile Ile

1 5 10 15

Ala Gly Val His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu

20 25 30

Val Lys Pro Gly Ala Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Thr Phe Thr Asn Tyr Tyr Met His Trp Val Lys Gln Arg Pro Gly Gln

50 55 60

Gly Leu Glu Trp Ile Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Tyr

65 70 75 80

Tyr Asn Glu Lys Phe Arg Ala Arg Pro His Leu Gln Thr Asn Pro Pro

85 90 95

Ala Gln Pro Thr Cys Ser Ser Ala Ala Pro Leu Arg Thr Leu Arg Ser

100 105 110

Ile Ser Val Gln Val Arg Arg Glu Leu Pro Leu Val Cys Leu Leu Gly

115 120 125

Pro Arg Asp Ser Gly His Cys Leu Cys Ser Gln Asn Asp Thr Pro Ile

130 135 140

Arg Leu Ser Pro Gly Pro Trp Lys Leu Gly

145 150

<210> 36

<211> 131

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> monoclonal antibody 318 light chain variable region

<400> 36

Leu Val Asp Met Glu Ser Asp Thr Leu Leu Leu Trp Val Leu Leu Leu

1 5 10 15

Trp Val Pro Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala

20 25 30

Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Tyr Arg Ala

35 40 45

Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His Trp Asn Gln

50 55 60

Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr Leu Val Ser Asn

65 70 75 80

Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr

85 90 95

Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr

100 105 110

Tyr Tyr Cys Gln His Ile Arg Glu Leu Thr Arg Ser Glu Gly Gly Pro

115 120 125

Ser Trp Lys

130

<210> 37

<211> 173

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> human PRL3 sequence

<400> 37

Met Ala Arg Met Asn Arg Pro Ala Pro Val Glu Val Ser Tyr Lys His

1 5 10 15

Met Arg Phe Leu Ile Thr His Asn Pro Thr Asn Ala Thr Leu Ser Thr

20 25 30

Phe Ile Glu Asp Leu Lys Lys Tyr Gly Ala Thr Thr Val Val Arg Val

35 40 45

Cys Glu Val Thr Tyr Asp Lys Thr Pro Leu Glu Lys Asp Gly Ile Thr

50 55 60

Val Val Asp Trp Pro Phe Asp Asp Gly Ala Pro Pro Pro Gly Lys Val

65 70 75 80

Val Glu Asp Trp Leu Ser Leu Val Lys Ala Lys Phe Cys Glu Ala Pro

85 90 95

Gly Ser Cys Val Ala Val His Cys Val Ala Gly Leu Gly Arg Ala Pro

100 105 110

Val Leu Val Ala Leu Ala Leu Ile Glu Ser Gly Met Lys Tyr Glu Asp

115 120 125

Ala Ile Gln Phe Ile Arg Gln Lys Arg Arg Gly Ala Ile Asn Ser Lys

130 135 140

Gln Leu Thr Tyr Leu Glu Lys Tyr Arg Pro Lys Gln Arg Leu Arg Phe

145 150 155 160

Lys Asp Pro His Thr His Lys Thr Arg Cys Cys Val Met

165 170

<210> 38

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> binding sequence

<400> 38

Lys Ala Lys Phe Tyr Asn

1 5

<210> 39

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> binding sequence

<400> 39

His Thr His Lys Thr Arg

1 5

Claims (16)

1.A humanized antibody or antigen-binding fragment that binds PRL3, the antibody or antigen-binding fragment having at least one light chain variable region comprising the following CDRs:

LC-CDR1：KASQSVEDDGENYMN（SEQ ID NO：4）

LC-CDR2：AASNLES（SEQ ID NO：5）

LC-CDR3: QQSNEDPFT (SEQ ID NO: 6); and

at least one heavy chain variable region comprising the following CDRs:

HC-CDR1：GYTFTNYYMH（SEQ ID NO：1）

HC-CDR2：WIYPGNVNTYYNEKFRG（SEQ ID NO：2）

HC-CDR3：EEKNYPWFAY（SEQ ID NO：3），

wherein the humanized antibody is capable of binding PRL3 in vivo.

2. The humanized antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises CH1 and CH2 domains.

3. The humanized antibody or antigen-binding fragment of claim 1, which binds to an epitope comprising the amino acid sequence KAKFYN and/or HTHKTR.

4. An in vitro complex comprising the antibody or antigen-binding fragment of any one of the preceding claims bound to PRL3.

5. An antibody or antigen-binding fragment comprising the amino acid sequence set forth in SEQ ID NO: 16. SEQ ID NO: 17. the amino acid sequence of SEQ ID NO: 18. the amino acid sequence of SEQ ID NO: 19. the amino acid sequence of SEQ ID NO: 20. the amino acid sequence of SEQ ID NO: 21. SEQ ID NO: 22. the amino acid sequence of SEQ ID NO: 23. SEQ ID NO:24 or SEQ ID NO:25, and the heavy chain variable region sequence set forth in SEQ ID NO: 7. SEQ ID NO: 8. the amino acid sequence of SEQ ID NO: 9. the amino acid sequence of SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO:14 or SEQ ID NO:15, or a light chain variable region sequence set forth in seq id no.

6. Use of the humanized antibody or antigen-binding fragment of claim 1 in the manufacture of a medicament for the treatment of a PRL 3-positive cancer.

7. The use of claim 6, wherein the cancer is gastric cancer.

8. The use of claim 6, wherein the cancer is gastric cancer metastasis.

9. The use of claim 6, wherein the humanized antibody or antigen-binding fragment is administered intravenously.

10. The use of claim 6, wherein the humanized antibody or antigen binding fragment is administered to a site remote from the cancer to be treated.

11. The use of claim 6, wherein the humanized antibody or antigen-binding fragment is administered to a patient with gastric cancer, wherein the patient has not previously received an anti-metabolite therapy agent, the humanized antibody or antigen-binding fragment being a humanized anti-PRL 3 antibody or antigen-binding fragment.

12. The use of claim 6, wherein the humanized antibody or antigen-binding fragment is administered to a patient having gastric cancer, wherein the patient has not previously received an anti-metabolite therapy agent against gastric cancer, the humanized antibody or antigen-binding fragment being a humanized anti-PRL 3 antibody or antigen-binding fragment.

13. The use of claim 11 or 12, wherein the antimetabolite therapeutic is 5-FU.

14. The use of claim 6, wherein the humanized antibody or antigen-binding fragment is administered to a patient who has been determined to have no impairment of the immune system, the humanized antibody or antigen-binding fragment being a humanized anti-PRL 3 antibody or antigen-binding fragment.

15. Use of the humanized antibody or antigen binding fragment of any one of claims 1-3 in the manufacture of a medicament for use in a method of diagnosing cancer, wherein the method comprises determining in vitro the cellular localization of PRL3 in a cell, wherein expression of PRL3 at the surface of the cell indicates that the cell is cancerous.

16. The use of claim 15, wherein PRL3 expression on the cell surface is increased 2-fold compared to a control cell.