CN105074469A - Anti-mucin antibodies for early detection and treament of pancreatic cancer - Google Patents

Abstract

The invention describes anti-mucin antibodies for early detection and treament of pancreatic cancer. Described herein are compositions and methods of use of anti-pancreatic cancer antibodies or fragments thereof, such as murine, chimeric, humanized or human PAM4 antibodies. The antibodies show novel and useful diagnostic characteristics, such as binding with high specificity to pancreatic and other cancers, but not to normal or benign pancreatic tissues and binding to a high percentage of early stage pancreatic cancers. Preferably, the antibodies bind to an epitope located within the second to fourth cysteine-rich domains of MUC5ac (amino acid residues 1575-2052) and are of use for the detection and diagnosis of early stage pancreatic cancer. In more preferred embodiments, the anti-pancreatic cancer antibodies can be used for immunoassay of serum samples, wherein the immunoassay detects a marker for early stage pancreatic cancer in serum. Most preferably, the serum is extracted with an organic phase, such as butanol, before immunoassay.

Description

For the anti-stick protein antibodies of cancer of pancreas early detection and treatment

Related application

Subject application requires according to 35U.S.C.119 (e) U.S. Patent Application Serial No. 61/807 that on April 1st, 2013 submits to, 176, the U.S. Patent Application Serial No. 61/818 submitted on May 2nd, 2013, the rights and interests of the U.S. Patent Application Serial No. 61/896,909 that on October 29th, 708 and 2013 submits to.Subject application is the part continuation application of the U.S. Patent Application Serial No. 14/036,765 that on September 25th, 2013 submits to.The text of each claimed priority application case is that the mode quoted in full is incorporated herein.

Sequence table

Subject application is containing ordered list, and it have been submitted to via EFS-Web with ASCII fromat and has been incorporated in the mode that this quotes in full.Described ASCII copy creating on March 28th, 2014, called after IMM343WO1_SL.txt, and size is 55,791 bytes.

Background of invention

Invention field

The present invention relates in conjunction with the mucinous anti-cancer of pancreas antibody of the MUC5ac in cancer of pancreas and its Fab.More preferably, described antibody or its fragment combine the epi-position being positioned at second to the 4th rich Cysteine domains (amino acid residue 1575-2052) of MUC5ac.Theme antibody or antibody fragment with high selectivity in conjunction with pancreatic cancer cell, thus allow cancer of pancreas very early time detect and/or diagnose this disease.Most preferably, for normal healthy controls, the mensuration based on antibody can detect the cancer of pancreas of about 85% or more, and false positive rate is about 5% or lower.In a particular embodiment, described method and composition can be used for being detected and/or diagnosis of pancreatic cancer from the blood serum sample of experimenter by screening, and can detect the I phase cancer of pancreas of 60% or more and the II phase cancer of pancreas of 80% or more preferably by blood serum sample analysis.In other embodiment, utilize anti-MUC5ac antibody immunoassay can with other pancreatic cancer marker of use, the immunodetection combination of such as CA19.9, thus improve cancer of pancreas recall rate not reducing in specific situation.In other embodiments, can be used for the reactivity of anti-cancer of pancreas antibody for the recessive cancer of pancreas of pancreatitis or optimum pancreatic regeneration background detection or tumour precursor lesion.

In preferred embodiments, described anti-cancer of pancreas antibody and PAM4 antibody are in conjunction with identical epi-position or competition binding MUC5ac, described PAM4 antibody comprises variable region of light chain complementarity-determining region (CDR) sequence C DR1 (SASSSVSSSYLY, SEQIDNO:1), CDR2 (STSNLAS, and CDR3 (HQWNRYPYT, SEQIDNO:3) SEQIDNO:2); And heavy CDR sequences CDR1 (SYVLH, SEQIDNO:4), CDR2 (YINPYNDGTQYNEKFKG, SEQIDNO:5) and CDR3 (GFGGSYGFAY, SEQIDNO:6).Most preferably, anti-cancer of pancreas antibody is humanization PAM4 (hPAM4) antibody, it comprises CDR sequence CDR1 (SASSSVSSSYLY, SEQIDNO:1), CDR2 (STSNLAS, and CDR3 (HQWNRYPYT, SEQIDNO:3) SEQIDNO:2); And heavy CDR sequences CDR1 (SYVLH, SEQIDNO:4), CDR2 (YINPYNDGTQYNEKFKG, and CDR3 (GFGGSYGFAY, SEQIDNO:6), and human antibody framework region (FR) and constant-region sequences SEQIDNO:5).

Association area

Cancer of pancreas is the malignant pancreatic growth mainly occurred in pancreatic ductal cell.This disease is the ninth-largest common cancer form, but is the fourth-largest and the fifth-largest main cause of cancer mortality respectively in masculinity and femininity.With monitoring and/or triage techniques other major cancers of having caused cancer related mortality rate to reduce unlike, the patient populations dying from cancer of pancreas every year continues rising (Jemal etc., 2009, CACancerJClin59:225-49).For cancer of pancreas, the overall survival rate after a year is only 20%, and is less than 4% after 5 years.The main cause of this poor prognosis comprises, and the succeed early detection of result of larger chance can not may be had to go out this disease in the measure of healing property, and lack effectively treatment to terminal illness.

In general, the patient suffering from early stage disease has higher survival rate than those patients suffering from terminal illness.Those patients of excision local disease have 5 years relative survival rates of 22%, and the patient suffering from unresectable advanced metastatic disease is 1% to 2% (Horner etc., 2009, SEERCancerStatisticsReview, 1975-2006, NCI, Bethesda, MD).For local disease, although early detection is successful treatment intervention provide more high likelihood, 5 years relative survival rates of 22% can be converted into the unacceptable high mortality (Bilimoria etc., 2007, AnnSurg246:173-80) of 78%.

The most common sympton of cancer of pancreas comprises jaundice, suffers from abdominal pain and lose weight, and they are nonspecific in itself together with other existing factor.Therefore, be often difficult to the early diagnosis cancer of pancreas at tumor growth, and need to carry out diagnostic test widely, generally include exploratory operation.Endoscopic ultrasonography and computer tomography are the non-invasive means of the best of available diagnostic cancer of pancreas now.But, be difficult to reliably detect little tumour, and differentiating pancreatic cancer and focal pancreatitis.At present, most pancreatic adenocarcinoma patients has extended to tunicle in tumour to be just diagnosed the outside late period invading peripheral organs and/or transfer on a large scale.(Gold etc., 2001, Crit.Rev.Oncology/Hematology, 39:147-54).

The treatment procedure being currently available for cancer of pancreas all can not be cured, or substantially improves the time-to-live.Excision is to provide the sole mode of chance of surviving.But, due to great tumor load, only there is the patient of 10% to 25% to become the candidate of " excision of healing property ".For those patients of experience operative treatment, five-year survival rate is still very low, and on average only about 10%.

The early detection of cancer of pancreas and diagnosis, and to this disease suitably by stages, will larger survival advantage be provided.Multiple laboratory has been attempted based on tumor associated marker to the release in blood flow, and develops diagnostic routine to the detection of biopsy samples internal labeling material.The best tumor associated marker previously characterized for cancer of pancreas is the immunoassays for CA19.9.Find this sialylated Le ^aepitopic structures level raises to some extent in the Pancreas cancer patients of 70%, but does not all find in checked any focal pancreatitis sample.But, find that CA19.9 level raises all to some extent in other pernicious and optimum symptom multiple, and this mensuration can not be used for diagnosis at present.This mensuration can be used for monitoring, and Post operation CA19.9 lasting serum levels increases instruction prognosis mala.Report multiple other monoclonal antibody (MAb) to be used from from immunoassays one and to diagnose in the different stages of development.These antibody include but not limited to DUPAN2, SPAN1, B72.3, Ia3 and various anti-CEA (carcinomebryonic antigen or CEACAM5) antibody.

The all multispecific antibody of extensive testing, especially MAb and through engineered antibody or antibody fragment, and be presented at various human disorders, comprising in the test-and-treat of cancer, autoimmune disease, communicable disease, inflammatory diseases and angiocardiopathy is valuable [Filpula and McGuire, Exp.Opin.Ther.Patents (1999) 9:231-245].The clinical practice of antibody or antibody derivating agent depends primarily on the ability that it combines the particular target antigen relevant to particular condition.Selectivity will be diagnosed or therapeutic agent for during the test-and-treat stage of human disorders, it is valuable that such as medicine, toxin, cell factor, hormone, hormone antagonist, enzyme, enzyme inhibitor, oligonucleotides, growth factor, radioactive nuclide, AI or metal are delivered to target position, particularly when diagnosis or therapeutic agent in body, normal structure is poisonous.Through radiolabeled antibody for Several Kinds of Malignancy, comprise oophoroma, colon cancer, medullary carcinoma of thyroid gland and lymph cancer, achieve certain success.Empirical tests, this technology also can be used for cancer of pancreas.But the antibody for pancreatic carcinoma antigen of previous report is not yet used successfully to effective treatment of cancer of pancreas or early detection and/or diagnosis.

In the art, the antibody that cancer of pancreas and other types of cancer represent high selectivity is still needed to compare with normal pancreatic tissue and other normal structure.Exactly, still need as useful diagnosis of pancreatic cancer and/or treatment tool (preferably in the very early time of this disease) and represent to strengthen to some extent the picked-up of target antigen and in healthy individuals blood, all multi-component combinations reduce and thus protect the antibody that normal structure and cell injure from these toxic therapeutic agent best when put together therewith by antibody-like for toxic therapeutic agent.Use this antibody-like to detect body fluid, especially the pancreas cancer-associated antigen in blood can improve the early diagnosis of this disease, as long as itself and benign disease are clearly distinguished, and can be used for monitoring the response to treatment, and prognosis can be strengthened by pointing out Disease Spectrum.

Summary of the invention

In various embodiments, the present invention relates in conjunction with MUC5ac pancreatic cancer cell mucin, preferably combine the antibody, antigen binding antibody fragment and the fusion that are positioned at the epi-position of second to the 4th rich Cysteine domains (amino acid residue 1575-2052) of MUC5ac.More preferably, theme antibody or its fragments specific in conjunction with pancreatic cancer cell, and seldom or not in conjunction with normal or non-superfluous natural disposition pancreatic cell.Described antibody can in conjunction with the cancer of pancreas of very early time, and the recall rate of PanIN-1A is the recall rate of about 50% to 60%, Pan1B is 70% to 80%, and the recall rate of PanIN-2 is 80% to 90%.More preferably, described antibody in conjunction with 80% to 90% or more mankind's aggressive cancer of pancreas, palilate myxoma formation in conduit, PanIN-1A, PanIN-1B and PanIN-2 pathology.Most preferably, described antibody can distinguish Early pancreatic carcinoma and non-malignant symptom, such as pancreatitis.

This antibody-like is particularly useful for the antidiastole between the early detection of cancer and Early pancreatic carcinoma and optimum pancreas symptom.In preferred embodiments, this antibody-like can be used for carrying out in body or ex vivo analyses to coming from the doubtful sample suffering from the individuality of Early pancreatic carcinoma or some other cancer.More preferably, described antibody can be used for carrying out diagnosis and detection Early pancreatic carcinoma by serum analysis sample.

In an alternate embodiment, described antibody, antibody fragment or fusion can binding synthetic peptide sequence, such as Phage Display Peptides, such as WTWNITKAYPLP (SEQIDNO:7) and ACPEWWGTTC (SEQIDNO:8).This type of synthetic peptide can be linear or ring-type, and can or cannot with antibody competition in conjunction with endogenous pancreatic carcinoma antigen.Amino acid on some position of described synthetic peptide sequence and other amino acid compare the importance that antibody is combined may be less.For example, in SEQIDNO:7, residue K, A and L on the position 7,8 and 11 of peptide sequence can change, and still keep antibody to combine simultaneously.Similarly, in SEQIDNO:8, the threonine residues on the position 8 and 9 of described sequence can change, but can be combined with peptide by appreciable impact antibody the replacement of the threonine on position 9.

Described antibody can by being suppressed with the such as agent treated target antigen such as dithiothreitol (DTT) (DTT) and/or periodates to the combination of target pancreatic carcinoma antigen.Thus, the combination of described antibody and pancreatic carcinoma antigen may depend on the existence of disulfide bond and/or the glycosylation state of target antigen.In a more preferred embodiment, the epi-position that theme antibody identifies not with other mucin-specific antibodies reported, such as MA5 antibody, CLH2-2 antibody and/or 45M1 antibody cross reaction (see such as Major etc., JHistochemCytochem.35:139-48,1987; Dion etc., Hybridoma10:595-610,1991).

Theme antibody or fragment can be naked antibody or fragment, or preferably put together at least one therapeutic agent and/or diagnosticum, so that by this drug delivery to target tissue.In an alternate embodiment, theme antibody or fragment can be parts for bispecific antibody, this bispecific antibody have for the epi-position of the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) being positioned at MUC5ac the first binding site and for can haptenic second binding site puted together of the construct of target.Describedly the construct of target can be connected at least one therapeutic agent and/or diagnosticum again, thus for pre-targeting technology.

In preferred embodiments, theme antibody, antibody fragment or fusion are humanization PAM4 antibody or fragment, it comprises variable region of light chain CDR sequence C DR1 (SASSSVSSSYLY, SEQIDNO:1), CDR2 (STSNLAS, and CDR3 (HQWNRYPYT SEQIDNO:2), and variable region of heavy chain CDR sequence C DR1 (SYVLH SEQIDNO:3), SEQIDNO:4), CDR2 (YINPYNDGTQYNEKFKG, and CDR3 (GFGGSYGFAY SEQIDNO:5), and human antibody framework region (FR) and constant-region sequences SEQIDNO:6).More preferably, the light chain of humanization PAM4 antibody or its fragment and the FR of variable region of heavy chain comprise amino acid that at least one amino acid residue 5,27,30,38,48,66,67 and 69 through muroid PAM4 variable region of heavy chain (SEQIDNO:12) replaces and/or at least one is selected from the amino acid of the amino acid residue 21,47,59,60,85,87 and 100 of muroid PAM4 variable region of light chain (SEQIDNO:10).Most preferably, described antibody or its fragment comprise the hPAM4V of SEQIDNO:19 _hthe hPAM4V of amino acid sequence and SEQIDNO:16 _kamino acid sequence.

In an alternate embodiment, described anti-cancer of pancreas antibody can be in conjunction with chimeric, the humanization of same antigen determinant (epi-position) or competition binding MUC5ac or human antibodies with chimeric PAM4 (cPAM4) antibody.Discuss as following, described cPAM4 antibody comprises variable region of light chain CDR sequence C DR1 (SASSSVSSSYLY, SEQIDNO:1), CDR2 (STSNLAS, and CDR3 (HQWNRYPYT SEQIDNO:2), and variable region of heavy chain CDR sequence C DR1 (SYVLH SEQIDNO:3), SEQIDNO:4), CDR2 (YINPYNDGTQYNEKFKG, and the antibody of CDR3 (GFGGSYGFAY, SEQIDNO:6) SEQIDNO:5).Antibody in conjunction with same antigen determinant can be differentiated by various technology as known in the art, such as by using cPAM4 antibody as competition antibody and using human pancreatic's mucin or MUC5ac to study as the competition binding of target antigen.The antibody that blocking-up (competition) cPAM4 antibody is combined with human pancreatic's mucin is called as cross-blocks antibody.Preferably, this type of cross-blocks antibody be combine be positioned at second to the 4th rich Cysteine domains of MUC5ac epi-position or with the antibody of PAM4 antibody competition in conjunction with amino acid residue 1575 to 2052.

Other embodiment relates to the treatment immunoconjugates of target cancer cell, and it comprises the antibody or its fragment or fusion that are combined with at least one therapeutic agent.Preferably, described therapeutic agent is selected from radioactive nuclide, immunomodulator, hormone, hormone antagonist, enzyme, the such as oligonucleotides such as antisense oligonucleotides or siRNA, enzyme inhibitor, photosensitivity therapeutic agent, the such as cytotoxic agent such as medicine or toxin, AI and short Apoptosis agent.Using in the embodiment more than a kind of therapeutic agent, described therapeutic agent can comprise multiple copy of same therapeutic agent or the combination of different therapeutic agent.

In one embodiment, as U.S. Patent number 5, the oligonucleotides that such as antisense molecule or siRNA etc. described in 734,033 (embodiment part is incorporated herein by reference) suppress bcl-2 to express can be connected to or be formed the therapeutic agent portion of immunoconjugates or antibody fusion protein.Alternatively, described oligonucleotides can with anti-cancer of pancreas antibody that is naked or that put together or antibody fragment (such as PAM4 antibody) simultaneously or sequential application.In a preferred embodiment, described oligonucleotides is the antisense oligonucleotides for oncogene or oncogene products (such as bcl-2, p53, ras or other oncogene known).

Preferably, described therapeutic agent is cytotoxic agent, such as medicine or toxin.Further preferably, described medicine is selected from nitrogen mustards, ethylenimine derivatives, alkyl sulfonic ester, nitroso ureas, gemcitabine, triazenes, folacin, anthracycline, taxane, cox 2 inhibitor, pyrimidine analogue, purine analogue, microbiotic, enzyme inhibitor, epipodophyllotoxin, platinum coordination complex, vinca alkaloids, the urea be substituted, methyl hydrazine derivatives, adrenal cortex inhibitor, hormone antagonist, Endostatin, taxol, camptothecine, SN-38, Doxorubicin and its analog, antimetabolite, alkylating agent, antimitotic agent, anti-angiogenic agent, tyrosine kinase inhibitor, bruton's tyrosine kinase inhibitor, mTOR inhibitors, heat shock protein (HSP90) inhibitor, proteasome inhibitor, hdac inhibitor, short Apoptosis agent, amethopterin, CPT-11, SN-38, 2-PDOX, pro-2-PDOX and its combination.

In another preferred embodiment, described therapeutic agent is the toxin being selected from ricin (WA), abrin, alpha toxin, saporin, ribonuclease (RNA enzyme), DNA enzymatic I, staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin and pseudomonad endotoxin and its combination.Or be selected from cell factor, stem cell factor, lymphocytotoxin, Hemopoietic factor, colony stimulating factor (CSF), interferon (IFN), stem cell factor, hematopoietin, thrombopoietin and its immunomodulator combined.

Alternatively, described therapeutic agent is the enzyme being selected from malic dehydrogenase, staphylococcal nuclease, δ-V-steroid isomeras, YAD, alpha-phosphate glycerol dehydrogenase, phosphotriose isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta galactosidase, ribonuclease, urease, hydrogen peroxidase, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase and acetylcholinesterase.This fermentoid can such as be used with the form of relative nontoxic and be become the prodrug composition of cytotoxic agent to use at target site by described enzymatic conversion.In other replacement scheme, medicine may be changed into the form of low toxicity by the endogenous enzyme in subject, but can change into cytotoxic form again by being treated property enzyme.

Other therapeutic agent comprises radioactive nuclide, such as ¹⁴c, ¹³n, ¹⁵o, ³²p, ³³p, ⁴⁷sc, ⁵¹cr, ⁵⁷co, ⁵⁸co, ⁵⁹fe, ⁶²cu, ⁶⁷cu, ⁶⁷ga, ⁶⁷ga, ⁷⁵br, ⁷⁵se, ⁷⁵se, ⁷⁶br, ⁷⁷as, ⁷⁷br, ^80mbr, ⁸⁹sr, ⁹⁰y, ⁹⁵ru, ⁹⁷ru, ⁹⁹mo, ^99mtc, ^103mrh, ¹⁰³ru, ¹⁰⁵rh, ¹⁰⁵ru, ¹⁰⁷hg, ¹⁰⁹pd, ¹⁰⁹pt, ¹¹¹ag, ¹¹¹in, ^113min, ¹¹⁹sb, ^121mte, ^122mte, ¹²⁵i, ^125mte, ¹²⁶i, ¹³¹i, ¹³³i, ¹⁴²pr, ¹⁴³pr, ¹⁴⁹pm, ¹⁵²dy, ¹⁵³sm, ¹⁶¹ho, ¹⁶¹tb, ¹⁶⁵tm, ¹⁶⁶dy, ¹⁶⁶ho, ¹⁶⁷tm, ¹⁶⁸tm, ¹⁶⁹er, ¹⁶⁹yb, ¹⁷⁷lu, ¹⁸⁶re, ¹⁸⁸re, ^189mos, ¹⁸⁹re, ¹⁹²ir, ¹⁹⁴ir, ¹⁹⁷pt, ¹⁹⁸au, ¹⁹⁹au, ¹⁹⁹au, ²⁰¹tl, ²⁰³hg, ²¹¹at, ²¹¹bi, ²¹¹pb, ²¹²bi, ²¹²pb, ²¹³bi, ²¹⁵po, ²¹⁷at, ²¹⁹rn, ²²¹fr, ²²³ra, ²²⁴ac, ²²⁵ac, ²⁵⁵fm or Th ²²⁷.

Known multiple tyrosine kinase inhibitor in this area, and this type of known treatment agent any can be utilized.Exemplary tyrosine kinase inhibitor includes but not limited to that how card is for Buddhist nun, Dasatinib, Erlotinib, Gefitinib, Imatinib, Lapatinib, leflunomide, nilotinib, pazopanib, SU5416, Sorafenib, Sutent, SU11248 and PTK787.A special category of tyrosine kinase inhibitor is bruton's tyrosine kinase inhibitor.Bruton's tyrosine kinase (Btk) has clear and definite effect in B cell is grown.Bu Ludun inhibitors of kinases includes but not limited to PCI-32765 (according to Shandong for Buddhist nun), PCI-45292, GDC-0834, LFM-A13 and RN486.

Theme antibody or fragment can diagnose (or detection) agent to put together with at least one.Preferably, described diagnosticum is selected from radioactive nuclide, contrast preparation, fluorescer, chemiluminescence agent, luminescent biological agent, paramagnetic ion, enzyme and photosensitivity diagnosticum.It is further preferred that described diagnosticum is the radioactive nuclide of energy between 20 and 4,000keV, or be selected from ¹¹⁰in, ¹¹¹in, ¹⁷⁷lu, ¹⁸f, ⁵²fe, ⁶²cu, ⁶⁴cu, ⁶⁷cu, ⁶⁷ga, ⁶⁸ga, ⁸⁶y, ⁹⁰y, ⁸⁹zr, ^94mtc, ⁹⁴tc, ^99mtc, ¹²⁰i, ¹²³i, ¹²⁴i, ¹²⁵i, ¹³¹i, ^154-158gd, ³²p, ¹¹c, ¹³n, ¹⁵o, ¹⁸⁶re, ¹⁸⁸re, ⁵¹mn, ^52mmn, ⁵⁵co, ⁷²as, ⁷⁵br, ⁷⁶br, ^82mrb, ⁸³the radioactive nuclide of Sr or other γ, β or positron emitter.In an especially preferred embodiment, described diagnostic radioactive nuclide ¹⁸f is for mark and PET imaging, as described in the following embodiments. ¹⁸f can by with the metal complex such as such as aluminium and ¹⁸f-metal complex is combined with the chelating moiety with targeting proteins matter, peptide or other molecular conjugate and is connected to antibody, antibody fragment or peptide.

Further preferably, described diagnosticum is paramagnetic ion, such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III), or radiopaque material, such as barium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid, iodamide, adipiodone, iodoxamic acid, iodine paddy amine, Iohexol, Iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamide meglumine, the U.S. acid in iodine west, Iotasul, iotetric acid, iotalamic acid, iotroxic acid, ioxaglic acid, Ioxotrizoic Acid, iopodic acid, meglumine, metrizamide, Sodium Metrizoate, propyl iodone and thallous chloride.

In other embodiments, described diagnosticum is the fluorescent labelling compound being selected from fluorescein isothiocynate, rhodamine, rhodophyll, phycocyanobilin, allophycocyanin, o-phthalaldehyde(OPA) and fluorescamine, be selected from the chemiluminescent labeling compound of luminol, different luminol, aromatics acridinium ester, imidazoles, acridinium salt and oxalate, or be selected from the bioluminescent compound of fluorescein, luciferase and aequorin.In another embodiment, described diagnosis immunoconjugates be in operation, endoscope or intravascular tumor diagnosis.

Also contemplate multivalence, multi-specificity antibody or its fragment, it comprises at least one can in conjunction with being positioned at the binding site of epi-position of the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) of MUC5ac and one or more haptens binding site hapten molecule to affinity.Preferably, described antibody or its fragment be fitted together to, humanization or completely human antibodies or its fragment.Described hapten molecule can put together in can target construct to send one or more therapeutic agents and/or diagnosticum.In certain preferred aspects, described multivalent antibody or its fragment can by DOCK-AND-LOCK as mentioned below ^tM(DNL ^tM) prepared by technology.Be incorporated to the exemplary DNL of hPAM4 antibody fragment ^tMconstruct called after TF10, as mentioned below.

Also contemplate bispecific antibody or its fragment, it comprises at least one has affinity binding site to the epi-position of the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) being positioned at MUC5ac, and at least one can have the binding site of affinity by target construct to what can transport at least one diagnosticum and/or therapeutic agent.What be applicable to use can be disclosed in such as U.S. Patent number 6,576,746,6,962,702,7,052,872,7 by target construct, 138,103,7,172,751,7,405,320,7,597,876,7,563,433,7,993,626,147,799,8,153,100,8,153,101,8,202,509,8,343,460,8,444,956,8,496,912,8,545,809,8,617,518 and 8,632, in 752, the embodiment part of each patent is incorporated herein by reference.

Other embodiment relates to fusion, and it comprises at least two kinds of anti-cancer of pancreas antibody as described in this article and its fragment.Alternatively, described fusion or its fragment can comprise at least one can in conjunction with the first antibody of epi-position of the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) or its fragment being positioned at MUC5ac, and at least one the 2nd MAb or its fragment.Preferably, described 2nd MAb in conjunction with tumor associated antigen, such as, is selected from CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, CEA (CEACAM5), CEACAM6, Le ^a, Lewis antigen Le (y), CSAp, IGF (IGF), Glycoproteins in Epithelial-1 (EGP-1), Glycoproteins in Epithelial-2 (EGP-2), CD-80, placenta growth factor (PlGF), carbonic anhydrase IX, tenascin, IL-6, HLA-DR, CD40, CD74 (such as, meter La Zhu monoclonal antibody), CD138 (syndecan-1), MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC16, MUC17, TAG-72, EGFR, platelet derived growth factor (PDGF), angiogenesis factor (such as, VEGF and PlGF), oncogene (such as, bcl-2, Kras, p53) product, cMET, HER2/neu and the antigen relevant to cancer of the stomach and colorectal cancer.Described 2nd MAb can also be different in conjunction with the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) from MUC5ac MUC5ac epi-position.Antibody fusion protein or its fragment can also comprise at least one diagnosticum and/or therapeutic agent.

Also describe the DNA sequence dna of the nucleic acid comprising coding anti-cancer of pancreas antibody as described in this article, fusion, multi-specificity antibody, bispecific antibody or its fragment herein.Other embodiment relates to the expression vector and/or host cell that comprise described antibody coding DNA sequence dna.In some preferred embodiment, described host cell can be adapted to carry out in serum free medium cell transformation and growth through sudden change Bcl-2 gene, such as, through Sp2/0 clone that treble genes mutation Bcl-2 gene (T69E, S70E, S87E) transforms.(see such as U.S. Patent number 7,531,327,7,537,930 and 7,608,425, the embodiment part of each patent is incorporated herein by reference.)

Another embodiment relates to the method for sending diagnosticum or therapeutic agent or its combination to target, comprising: (i) providing package contains the anti-cancer of pancreas antibody of epi-position or the composition of fragment that the combination of puting together with at least one diagnosticum and/or therapeutic agent is positioned at second to the 4th rich Cysteine domains (amino acid residue 1575-2052) of MUC5ac; (ii) to needing its experimenter to use as any one diagnosis in claimed antibody, antibody fragment or fusion or treatment conjugate herein.

Also contemplate and diagnosticum, therapeutic agent or its combination are delivered to target calibration method, comprising: (a) uses any one to experimenter to be had affinity to the epi-position of the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) being positioned at MUC5ac and comprises the multivalence of one or more haptens binding site, polyspecific or bispecific antibody or its fragment; B () waits for is enough to make the antibody in conjunction with MUC5ac clean the time of the blood flow of experimenter; (c) to described experimenter use comprise diagnosticum, therapeutic agent or its combination carrier molecule, described carrier molecule is in conjunction with the binding site of described antibody.Preferably, described carrier molecule is in conjunction with the more than one binding site of described antibody.

There is described herein a kind of for diagnosing or the method for Therapeutic cancer, comprising: (a) uses any one to experimenter claimedly herein to be had affinity to the epi-position of the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) being positioned at MUC5ac and comprises the multivalence of one or more haptens binding site, multi-specificity antibody or its fragment; B () waits for is enough to make non-binding antibody clean the time of the blood flow of experimenter; (c) to described experimenter use comprise diagnosticum, therapeutic agent or its combination carrier molecule, described carrier molecule is in conjunction with the binding site of described antibody.In a preferred embodiment, described cancer is cancer of pancreas.Further preferably, described method can be used for the endoscope discriminating of discriminating, pathological tissues in the operation of pathological tissues or the Ink vessel transfusing discriminating of pathological tissues.

Another embodiment is the method for the malignant tumour in treatment experimenter, comprise the epi-position combining the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) being positioned at MUC5ac to described experimenter's administering therapeutic effective dose, the antibody optionally puted together with at least one therapeutic agent or its fragment.Alternatively, described antibody or its fragment can be naked antibody or its fragment.In a more preferred embodiment, described antibody or fragment before using another kind of therapeutic agent as above, simultaneously or use afterwards.

Contemplate the malignant tumour in diagnosis experimenter herein, especially the method for cancer of pancreas, comprise: (a) uses to described experimenter that comprise can in conjunction with the antibody of epi-position of the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) or the diagnosis conjugate of its fragment being positioned at MUC5ac, wherein said MAb or its fragment and at least one diagnosticum are puted together; (b) existence through labelled antibody that is combined with pancreatic cancer cell or other malignant cell is detected, the existence in conjunction with diagnosable cancer of pancreas or another kind of malignant tumour of wherein said antibody.In preferred embodiments, described antibody or fragment in conjunction with cancer of pancreas not in conjunction with normal pancreatic tissue, pancreatitis or other non-malignant symptom.In less preferred embodiment, compared with non-malignant cell, described antibody or fragment with significantly higher horizontal integration cancer cell, thus allow antidiastole cancer and non-malignant symptom.In the most preferred embodiment, described diagnosticum can be the molecule through F-18 mark by PET image checking.

In a more preferred embodiment, very early time at malignant disease can be allowed with high specific and sensitivity Detection and/or diagnosis of pancreatic cancer in conjunction with the use of anti-cancer of pancreas antibody of epi-position of the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) being positioned at MUC5ac.Preferably, described diagnosis antibody or fragment can mark at least 70%, more preferably at least 80%, more preferably at least 90%, the good differentiation of more preferably at least 95%, most preferably about 100%, the cancer of pancreas of moderate differentiation and bad differentiation and the aggressive cancer of pancreas of 90% or more.The anti-cancer of pancreas antibody used preferably can detect PanIN-1A, PanIN-1B, PanIN-2, IPMN and MCN precursor lesion of 85% or more.Most preferably, the immunoassays of anti-cancer of pancreas antibody are used can to detect the MCN of total PanIN of 89% or more, the IPMN and 92% of 86% or more or more.

An alternate embodiment in individuality, detects the existence of PAM4 in conjunction with MUC5ac and/or the method for diagnosis of pancreatic cancer by analyzed in vitro blood, blood plasma or blood serum sample.Preferably, described sample, through using the organic solvents such as such as butanols to carry out organic extractant phase, then carries out immune detection through process for anti-cancer of pancreas antibody such as using such as PAM4 antibody.After organic extractant phase, use any one in panimmunity determination techniques as known in the art, such as ELISA, sandwich immunoassay, solid-phase RIA and similar techniques, analyze the existence of the MUC5ac epi-position be combined with PAM4 in described sample to the aqueous phase through extraction.Surprisingly, organic extractant phase eliminates the PAM4 inhibitor be combined with MUC5ac, thus allows to detect the MUC5ac in fresh serum sample.More surprisingly, use analyzed in vitro technology described herein, can serum analysis sample in case cancer of pancreas very early time detect and/or diagnosis experimenter in cancer of pancreas.These unpredictable consequences provide first detection technique based on serum of the existence of diagnosable Early pancreatic carcinoma.

Another embodiment is the method for cancer cell in treatment experimenter, comprises using to described experimenter that comprise can in conjunction with the naked antibody of epi-position of the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) or the composition of its fragment or naked antibody fusion protein or its fragment being positioned at MUC5ac.Preferably, described method also comprises using and is selected from the second following naked antibody or its fragment: CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, anti-CEA, anti-CEACAM6, anti-EGP-1, anti-EGP-2, anti-Le ^a, the antibody to be limited by Lewis antigen Le (y) and for CSAp, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC16, MUC17, TAG-72, EGFR, CD40, HLA-DR, CD74, CD138, angiogenesis factor (such as, VEGF and placenta like growth factor (PlGF)), IGF (IGF), tenascin, platelet derived growth factor, IL-6, the product of oncogene, the antibody of cMET and HER2/neu.

Other embodiment relates to the method for malignant tumour in diagnosis experimenter, comprising: (i) can carry out in-vitro diagnosis mensuration in conjunction with the antibody of epi-position of the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) or the composition of its fragment being positioned at MUC5ac to the sample deriving from described experimenter with comprising; (ii) existence of antibody that malignant cell in described sample is combined or fragment is detected.Preferably, described malignant tumour is cancer.More preferably, described cancer is cancer of pancreas.

Accompanying drawing is sketched

Figure 1A. the variable region cDNA sequence (SEQIDNO:9) of muroid PAM4Vk and derivation amino acid sequence (SEQIDNO:10).Provided with using single letter code form below nucleotide sequence by the amino acid sequence of corresponding DNA sequence encoding.The numbering of nucleotide sequence is on right side.Amino acid residue in CDR district illustrates with runic and underscore.The Ig molecule numbering of Kabat is used for the amino acid residue shown by the numbering above amino acid residue.It is the insertion residue defined by Kabat numbering plan with the amino acid residue of an alpha code.The numeral inserted before residue is identical with residue above.

Figure 1B. the variable region cDNA sequence (SEQIDNO:11) of muroid PAM4VH and derivation amino acid sequence (SEQIDNO:12).Provided with using single letter code form below nucleotide sequence by the amino acid sequence of corresponding DNA sequence encoding.The numbering of nucleotide sequence is on right side.Amino acid residue in CDR district illustrates with runic and underscore.The Ig molecule numbering of Kabat is used for the amino acid residue shown by the numbering above amino acid residue.It is the insertion residue defined by Kabat numbering plan with the amino acid residue of an alpha code.The numeral inserted before residue is identical with residue above.

Fig. 2 A. is fitted together to the amino acid sequence (SEQIDNO:13) of PAM4 (cPAM4) Vk.Described sequence provides with using single letter code form.Amino acid residue in CDR district illustrates with runic and underscore.The Ig molecule numbering plan of Kabat is used to be numbered residue.

The amino acid sequence (SEQIDNO:14) of Fig. 2 B.cPAM4VH.Described sequence provides with using single letter code form.Amino acid residue in CDR district illustrates with runic and underscore.The Ig molecule numbering plan of Kabat is used to be numbered residue.

The V of Fig. 3 A. human antibodies Walker (SEQIDNO:15) and PAM4 (SEQIDNO:10) and hPAM4 (SEQIDNO:16) _kthe comparison of amino acid sequence.The PAM4 residue that some instruction is same with the corresponding residue of the mankind or humanized antibody.Square areas represents CDR district.The N end of hPAM4 and C terminal residue (underscore) all pass through used staging vector to be fixed.The Ig molecule numbering plan of Kabat is used to be numbered residue.

The comparison of the VH amino acid sequence of Fig. 3 B. human antibodies Wil2 (FR1-3) (SEQIDNO:17) and NEWM (FR4) (SEQIDNO:18) and PAM4 (SEQIDNO:12) and hPAM4 (SEQIDNO:19).The PAM4 residue that some instruction is same with the corresponding residue of the mankind or humanized antibody.Square areas represents CDR district.The N end of hPAM4 and C terminal residue (underscore) all pass through used staging vector to be fixed.The Ig molecule numbering plan of Kabat is used to be numbered residue.

The DNA sequence dna (SEQIDNO:20) of Fig. 4 A. humanization PAM4 (hPAM4) Vk and amino acid sequence (SEQIDNO:16).The numbering of nucleotide sequence is on right side.Provided with using single letter code form by the amino acid sequence of corresponding DNA sequence encoding.Amino acid residue in CDR district illustrates with runic and underscore.The Ig molecule numbering plan of Kabat is used for amino acid residue.

The DNA sequence dna (SEQIDNO:21) of Fig. 4 B.hPAM4VH and amino acid sequence (SEQIDNO:19).The numbering of nucleotide sequence is on right side.Provided with using single letter code form by the amino acid sequence of corresponding DNA sequence encoding.Amino acid residue in CDR district illustrates with runic and underscore.The Ig molecule numbering plan of Kabat is used for amino acid residue.

Fig. 5. humanization PAM4 antibody hPAM4 compares with the binding activities of chimeric PAM4cPAM4.HPAM4 illustrates with rhombus, and cPAM4 illustrates with solid annular.Result instruction when with ¹²⁵during I-cPAM4 competition binding CaPan1 antigen, the binding activities of hPAM4 antibody and cPAM4 is suitable.

Fig. 6. use gradation ⁹⁰the PET/CT merging image of the patient suffering from unsuitable operation type metastatic cancer of pancreas that Y-hPAM4 adds gemcitabine treatment (left side) and treatment afterwards (right side) before the treatment.The position of annular instruction novo lesions, it illustrates PET/CT intensity and significantly reduces after the treatment.

Fig. 7. use gradation ⁹⁰the patient suffering from unsuitable operation type metastatic cancer of pancreas that Y-hPAM4 adds gemcitabine treatment before the treatment (left side) and treat the 3DPET image on afterwards (right side).Arrow points novo lesions (right side) and transfer (left side) position, each illustrate with add through radiolabeled hPAM4 gemcitabine treatment after PET image intensity significantly reduce.

Fig. 8 A. when have or cancer of pancreas MUC5ac antibody anti-without pre-targeting TF10 bispecific use warp ¹¹¹the diHSG peptide (IMP288) of In mark carries out tumour in-vivo imaging.Fig. 8 A illustrates the mouse of display knub position (arrow).

Fig. 8 B. when have or cancer of pancreas MUC5ac antibody anti-without pre-targeting TF10 bispecific use warp ¹¹¹the diHSG peptide (IMP288) of In mark carries out tumour in-vivo imaging.Warp when Fig. 8 B shows existence (upper figure) or there is not (figure below) TF10 bispecific antibody ¹¹¹the tumour that the MP288 that In marks detects.

Fig. 9 .TF10, PAM4-IgG, PAM4-F (ab') ₂with the exemplary combination curve of the chemically conjugated thing of unit price bsPAM4 (PAM4-Fab' × anti-DTPA-Fab').The combination of measuring target mucin antigen is measured by ELISA.

The immunoscintigraphy of Figure 10 A.CaPan1 human pancreatic adenocarcinoma xenograft (~ 0.25g).Injection bispecific TF10 (80 μ g, 5.07 × 10 ^-10mol) use after 16h afterwards ¹¹¹in-IMP-288 (30 μ Ci, 5.07 × 10 ^-11the image of mouse mol).Image is gathered after 3h.Increase the intensity of image background, so that coupling is used separately ¹¹¹in-IMP-288 (30 μ Ci, 5.07 × 10 ^-11nmol) image intensity obtained time.

The immunoscintigraphy of Figure 10 B.CaPan1 human pancreatic adenocarcinoma xenograft (~ 0.25g).Give separately ¹¹¹target is not observed in the mouse of In-MP-288.

The immunoscintigraphy of Figure 10 C.CaPan1 human pancreatic adenocarcinoma xenograft (~ 0.25g).Give ¹¹¹in-DOTA-PAM4-IgG (20 μ Ci, 50 μ g), carries out the mouse images that imaging obtains after 24h.Although visual tumors, still there is considerable background activity in this time point.

In the nude mice of Figure 11 A. with CaPan1 human pancreatic adenocarcinoma xenograft (the average tumor weight +/-standard deviation of pre-targeting animal and IgG treated animal is respectively 0.28+/-0.21 and 0.10+/-0.06g) ¹¹¹in-DOTA-PAM4-IgG (20 μ Ci, 50 μ g) and TF10 pre-targeting ¹¹¹in-IMP-288 (80 μ g, 5.07 × 10 ^-10after molTF10,16h, 30 μ Ci, 5.07 × 10 ^-11mol ¹¹¹in-IMP-288) extension bio distribution.Figure 11 A shows the predose number percent of every gram of tissue in the tumour (hollow circular) with PAM4IgG, the blood (open squares) with PAM4IgG, the tumour (solid annular) with pre-targeting peptide and the blood (filled squares) with pre-targeting peptide.

In the nude mice of Figure 11 B. with CaPan1 human pancreatic adenocarcinoma xenograft (the average tumor weight +/-standard deviation of pre-targeting animal and IgG treated animal is respectively 0.28+/-0.21 and 0.10+/-0.06g) ¹¹¹in-DOTA-PAM4-IgG (20 μ Ci, 50 μ g) and TF10 pre-targeting ¹¹¹in-IMP-288 (80 μ g, 5.07 × 10 ^-10after molTF10,16h, 30 μ Ci, 5.07 × 10 ^-11mol ¹¹¹in-IMP-288) extension bio distribution.Figure 11 B shows the predose number percent of every gram of tissue in the liver (hollow triangle) with PAM4IgG, the kidney (open diamonds) with PAM4IgG, the liver (black triangle) with pre-targeting peptide and the kidney (solid diamond) with pre-targeting peptide.

In the nude mice of Figure 11 C. with CaPan1 human pancreatic adenocarcinoma xenograft (the average tumor weight +/-standard deviation of pre-targeting animal and IgG treated animal is respectively 0.28+/-0.21 and 0.10+/-0.06g) ¹¹¹in-DOTA-PAM4-IgG (20 μ Ci, 50 μ g) and TF10 pre-targeting ¹¹¹in-IMP-288 (80 μ g, 5.07 × 10 ^-10after molTF10,16h, 30 μ Ci, 5.07 × 10 ^-11mol ¹¹¹in-IMP-288) extension bio distribution.Figure 11 C shows the microcurie mileage of every gram of tissue in the tumour (hollow circular) with PAM4IgG, the blood (open squares) with PAM4IgG, the tumour (solid annular) with pre-targeting peptide and the blood (filled squares) with pre-targeting peptide.

In the nude mice of Figure 11 D. with CaPan1 human pancreatic adenocarcinoma xenograft (the average tumor weight +/-standard deviation of pre-targeting animal and IgG treated animal is respectively 0.28+/-0.21 and 0.10+/-0.06g) ¹¹¹in-DOTA-PAM4-IgG (20 μ Ci, 50 μ g) and TF10 pre-targeting ¹¹¹in-IMP-288 (80 μ g, 5.07 × 10 ^-10after molTF10,16h, 30 μ Ci, 5.07 × 10 ^-11mol ¹¹¹in-IMP-288) extension bio distribution.Figure 11 D shows the microcurie mileage of every gram of tissue in the liver (hollow triangle) with PAM4IgG, the kidney (open diamonds) with PAM4IgG, the liver (black triangle) with pre-targeting peptide and the kidney (solid diamond) with pre-targeting peptide.

Figure 12. use 0.15mCi ⁹⁰y-hPAM4IgG or 0.25 or 0.50mCiTF10 pre-targeting ⁹⁰y-IMP-288 is to clear and definite (~ 0.4cm ³) CaPan1 tumour carries out the therapeutic activity of single therapy.

The gemcitabine enhancement effect of Figure 13 .PT-RAIT therapy.

Figure 14. the effect that Cetuximab and gemcitabine and PT-RAIT combine.

Figure 15. use the immunoassays based on PAM4 to carry out antidiastole to cancer of pancreas.Horizontal line shows analyzes based on ROC the cutoff level aligning result and select.

Figure 16. derive from healthy volunteer and not the same period cancer of pancreas individuality patients serum in the frequency distribution of PAM4 antigen.

The ROC curve that Figure 17 .PAM4 sero-immunity measures, it illustrates detection sensitivity is 81.6%, and specificity is 84.6%.

The accuracy of Figure 18 .PAM4 immunoassays is defined as in be equal to or greater than the nominal concentration checked under the cutoff of 2.40 units/mL 10%.With equation y=0.965x+0.174 and degree of fitting r ²=0.999 calculates linear trend.

Figure 19. the frequency distribution of PAM4 active antigens in the patients serum of each staging.Cutoff=2.4 unit/mL (horizontal line).Show the intermediate value (unit/mL) of each seminar.

Figure 20. based on recipient's operating characteristics (ROC) curve of the performance of the immunoassays of PAM4; Cancer of pancreas contrasts healthy adult.Provide area under curve (AUC) value and 95% fiducial limit.

Figure 21 A. uses ⁹⁰y-PAM4-IgG adds gemcitabine treatment Posterior circle PAM4 antigen levels and gross tumor volume (CT) relation that is in progress/disappears.Patient 076-001 responds to therapy and blood-serum P AM4 antigen reduces.Blood-serum P AM4 level is relevant to gross tumor volume.

Figure 21 B. uses ⁹⁰y-PAM4-IgG adds gemcitabine treatment Posterior circle PAM4 antigen levels and gross tumor volume (CT) relation that is in progress/disappears.Patient 1810002 shows the initial reaction to therapy, subsequently tumor recurrence.Blood-serum P AM4 level is relevant to gross tumor volume.

Figure 22 .PAM4 and the reactivity of mucin reference material when presence or absence palmitic acid.

Figure 23 A.PDAC contrasts PAM4 detection sensitivity and the specificity of chronic pancreatitis (CP).

Figure 23 B.PDAC contrasts PAM4 detection sensitivity and the specificity of all benign tissue samples.

Figure 24. the antibody utilizing PAM4 antibody to contrast for MUC1, MUC4, CEACAM6 and CA19-9 contrasts the comparative mark that carries out of non-superfluous natural disposition prostata tissue to PDAC.

Figure 25. the reactivity of the mucinous fraction of pbz polymer amount (CPM1) that several anti-stick albumen MAb is separated with from Capan-1 human pancreatic adenocarcinoma.MAb is differentiated, with horizontal stripe Indicator Reaction mucin kind below MAb clone name by clone name.Except PAM4, antagonism MUC1, MUC5ac and CEACAM6 antibody has observed substantive reaction.All MAb use with constant 10 μ g/mL.

Figure 26. several anti-stick albumen MAb and PAM4 captures the reaction of antigen.Mucin antigen is trapped on the plate of hPAM4 coating, and subsequently with several muroid anti-stick albumen MAb detection reaction signal.Anti-MUC5acMAb (2-11M1 and 45M1) captures mucin with hPAM4 and is combined, and anti-MUC1MAb (MA5 and KC4) does not combine.Homology hPAM4/mPAM4 captures/detects immunoassays and do not send signal, shows that the density of the PAM4 epi-position in described mucin may be lower, only may have Single locus.Anti-for rabbit polyclonal CPM1IgG is used as the positive control of the reaction of capturing antigen with hPAM4.

The anti-stick albumen MAb of Figure 27 A. muroid is to the suppression of hPAM4/ antigen consolidation reaction.Anti-stick albumen mMAb (purified IgG) is joined through CPM1 coating plate on as potential inhibitor, add hPAM4 subsequently.MPAM4 almost suppresses hPAM4 and the reaction between antigen and the anti-MUC5ac of 45M1 completely, thus provides limited depression effect (IC _max=25.5%).The anti-MUC1MAb of 2-11M1 anti-MUC5ac or MA5 and KC4 can not suppress specificity hPAM4/ antigen-reactive.

The anti-stick albumen MAb of Figure 27 B. muroid is to the suppression of hPAM4/ antigen consolidation reaction.The several anti-MUC5acMAb being used as the acquisition of ascites fluid form carries out similar suppression research.The anti-MUC5ac of MAb21M1,62M1 and 463M1 provides and the similar substantive inhibitory effect observed from suppressing at mPAM4IgG.The ascites of 45M1 creates the inhibitory effect similar with purified IgG.Use the ascites containing anti-alpha-fetoprotein as negative contrast.

The expression of Figure 28 .MUC5ac glycoprotein structural domain, wherein indicates the reactive epi-position of several anti-MUC5acMAb.The position of the defined epitope for the anti-MUC5acMAb in current research has been identified by the plasmid vector of cDNA held with the 3' containing MUC5ac and the data of carrying out transfection by the derivative cDNA carrier that restriction enzyme digestion obtains and drawing.Specific inhibition research shows that PAM4 epi-position is in the C terminal domains of rich halfcystine.

Describe in detail

Definition

Unless otherwise indicated, otherwise " one (kind) " mean one (kind) or multiple (kind).

As used herein, " about " means to add deduct 10%.For example, " about 100 " will comprise any numeral between 90 and 110.

As described in this article, term " PAM4 antibody " comprises muroid, chimeric, humanization and mankind PAM4 antibody.In preferred embodiments, described PAM4 antibody or its Fab comprise the CDR sequence of SEQIDNO:1 to SEQIDNO:6.

As used herein, " anti-cancer of pancreas antibody " represents the diagnosis identical with PAM4 antibody, to treat and in conjunction with the antibody of feature.In preferred embodiments, described " anti-cancer of pancreas antibody " combines the epi-position being positioned at second to the 4th rich Cysteine domains (amino acid residue 1575-2052) of MUC5ac.

" non-incretion cancer of pancreas " generally refers to the cancer that exocrine pancreas produces.This term does not comprise pancreatic insulin knurl and comprises cancer of pancreas knurl, cancer of pancreas, adenosquamous carcinoma, squamous cell carcinoma and carcinoma gigantocellulare and precursor lesion, mucinous tumors (IPMN) in such as, in pancreas epithelium neoplasia (PanIN), mucus cystoma (MCN) and pancreas, they are superfluous natural disposition but are not pernicious.Term " cancer of pancreas " and " non-incretion cancer of pancreas " are used interchangeably in this article.

As described in this article, antibody refers to that total length (namely, naturally occurring or to be formed by normal immunoglobulin gene fragment recombinatorial processes) immunoglobulin molecules is (such as, IgG antibody) or immunoglobulin molecules immunocompetence (namely, specific binding) part, as antibody fragment.

Antibody fragment is the part of antibody, such as F (ab') ₂, Fab', Fab, Fv, sFv etc.Regardless of structure, antibody fragment is in conjunction with the same antigen of full length antibody identification.Term " antibody fragment " also comprises the isolated fragment be made up of the variable region of antibody, the recombinant single chain peptide molecule (" scFv albumen ") that " Fv " fragment be such as made up of heavy chain and variable region of light chain is connected by peptide connexon with variable region of heavy chain with light chain.The another kind of form of antibody fragment is single domain antibody (nano antibody).

Naked antibody is the antibody or its fragment do not puted together with therapeutic agent or diagnosticum.In general, the Fc part of antibody molecule provides effector function, and the cytotoxicity (CDC) of such as complement-mediated and ADCC (antibody dependent cellular cytotoxicity), it can cause cytolytic mechanism to put into practice.But, when other mechanism such as the Apoptosis that such as intracellular signaling is induced work, Fc part may not be needed to realize treatment function.Naked antibody comprises both polyclonal antibody and monoclonal antibody, and fusion and some recombinant antibodies, such as chimeric, humanization or human antibodies.

Chimeric antibody is containing comprising the antibody deriving from species, the preferably recombinant protein of the variable domains of the complementarity-determining region (CDR) of rodent animal antibody, and the constant domain of described antibody molecule derives from the constant domain of human antibodies.For animal doctor's application, the constant domain of described chimeric antibody can derive from the constant domain of such as other species such as cat or dog.

Humanized antibody be from species antibody (such as, rodent animal antibody) CDR be transferred to recombinant protein in human heavy chain and light variable domains (such as, framework sequence) from the heavy chain of described rodent animal antibody and variable region of light chain.The constant domain of described antibody molecule derives from the constant domain of human antibodies.In certain embodiments, a limited number of Framework Region amino acid residue deriving from parent (rodent) antibody can be substituted by human antibody framework region sequence.

Human antibodies is such as from the antibody obtained with the transgenic mice producing particular person antibody-like in response to antigen stimulation through " engineered ".In this technology, the element of human heavy chain and light chain gene seat is introduced in the mouse species of the embryonic stem cell line of the targeted disruption derived from containing endogenous muroid heavy chain and light chain gene seat.Transgenic mice can synthesize and has specific human antibodies to specific antigen, and described mouse can be used for the hybridoma producing secretion human antibodies.Green etc., NatureGenet.7:13 (1994); Lonberg etc., Nature368:856 (1994); The method obtaining human antibodies from transgenic mice is described with Taylor etc., Int.Immun.6:579 (1994).Can also pass through gene or chromosomal transfection methods, and display technique of bacteriophage builds complete human antibodies, all methods are as known in the art.About by the external generation human antibodies of immunoglobulin variable domain domain gene pedigree and its fragment of deriving from non-immune donors, see such as McCafferty etc., Nature348:552-553 (1990).In this technology, antibody variable domain gene is cloned in the main of filobactivirus or secondary coat protein gene with frame, and is rendered as function antibody fragment on the surface at phage particle.Because filamentous particle comprises the single-stranded DNA copy of phage genome, so carry out selecting also to cause selecting the gene representing the antibody of those character of encoding based on the functional character of antibody.Like this, bacteriophage simulates the properties of B cell.Phage display can be undertaken by various ways, about summary, see such as Johnson and Chiswell, CurrentOpinioninStructuralBiology3:5564-571 (1993).Human antibodies can also be produced by Activated in Vitro B cell.See U.S. Patent number 5,567,610 and 5,229,275, embodiment part is incorporated herein by reference.

Therapeutic agent to separate with antibody moiety, simultaneously or sequential application, or put together with antibody moiety (that is, antibody or antibody fragment or subfragrnent), and can be used for the compound of disease therapy, molecule or atom.The example of therapeutic agent comprises antibody, antibody fragment, cytotoxic agent, medicine, toxin, nuclease, hormone, immunomodulator, short Apoptosis agent, anti-angiogenic agent, boron compound, photosensitizer or dyestuff and radioactive isotope.The therapeutic agent used below is described in more detail.

Diagnosticum be can with antibody moiety or can target construct put together use and the molecule that can be used for being detected by the cell of locating containing target antigen or diagnose the illness, atom or other can detecting portion.The reinforcing agent (such as, paramagnetic ion) that available diagnosticum includes but not limited to radioactive isotope, dyestuff, contrast preparation, fluorescent chemicals or molecule and scans for magnetic resonance imaging (MRI) or positron emission tomography (PET).Preferably, diagnosticum is selected from radioactive isotope, for the reinforcing agent of magnetic resonance or PET imaging and fluorescent chemicals.In order to make radioactive metal, paramagnetic ion or other diagnosis kation load antibodies component, be necessary the reagent reacting making it Yu there is the long-tail portion being connected with multiple chelation group for coupled ion.This type of afterbody can be polymkeric substance, such as polylysine, glycan or other have and can be combined with chelation group such as ethylenediamine tetraacetic acid (EDTA), diethylene-triamine pentaacetic acid (DTPA), DOTA, NOTA, NETA, porphyrin, polyamine, crown ether, two thiosemicarbazones, poly-oxime and knownly can be used for the derivative of the side base of the similar group of this object or can derivative chain.Standard chemical reaction is used to make chelate and antibody coupling.Chelate has minimum immunoreactivity and loses and seldom to assemble and/or the group of internal crosslinking is connected with antibody normal through making it possible to form key with molecule.U.S. Patent number 4,824, disclose other method be more of little use and reagent of making chelate and antibody conjugate in 659, the embodiment part of this patent is incorporated herein by reference.The combination of useful especially metal-chelate comprises 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, its with such as ¹²⁵i, ¹³¹i, ¹²³i, ¹²⁴i, ⁶²cu, ⁶⁴cu, ¹⁸f, ¹¹¹in, ⁶⁷ga, ⁶⁸ga, ^99mtc, ^94mtc, ¹¹c, ¹³n, ¹⁵o, ⁷⁶the diagnosis isotope one within the scope of the Universal Energy of 60 to 4,000keV such as Br is used from radiological imaging.When using together with antibody of the present invention, identical chelate is can be used for MRI with during the non-radioactive metal complexings such as such as manganese, iron and gadolinium.Such as NOTA (1,4,7-7-triazacyclononane-N, N', N "-triacetic acid), the macrocyclic chelate thing such as DOTA (Isosorbide-5-Nitrae, 7; 10-tetraazacyclododecanand tetraacethyl) and TETA (to bromoacetamido-benzyl-triethylammonium tetrakis tetraacethyl) can with various metals and radioactive metal, use together with copper with radioactive nuclide gallium, yttrium the most respectively.Can it be made to be suitable for associated metal by customization ring size and make this type of metal-chelate complex compound highly stable.Other ring-like chelate, such as macrocyclic polyether is contained in the present invention, and interested is that it can stably in conjunction with nucleic, such as ²²³ra, for Radioimmunotherapy (RAIT).Recently, U.S. Patent number 7,563,433,7,597,876 and 7,993, describe use in 626 ¹⁸the in fact any molecule of F atom mark is for the current techique of PET imaging, and the embodiment part of each patent is incorporated herein by reference.

Immunoconjugates is antibody, antibody fragment or antibody fusion protein and at least one therapeutic agent and/or diagnosticum conjugate.Described diagnosticum and/or therapeutic agent are as defined above.

Expression vector is the DNA molecular comprising gene expressed in host cell.Typically, gene expression is at some controlling element, carries out under comprising the control of composition or inducible promoter, Tissue-specific regulatory element and enhancer.This genoid is called as " being operably connected to " controlling element.

Recombinant host can be any protokaryon containing cloning vector or expression vector or eukaryotic.This term also comprises those protokaryons or eukaryotic, and through genetic engineering modified and containing the transgenic animals of clone gene in the chromosome or genome of host cell.Suitable mammalian host cell comprises myeloma cell, such as SP2/0 cell and NS0 cell, and Chinese hamster ovary (CHO) cell, hybridoma cell line and can be used for expressing other mammalian host cell of antibody.What be also specially adapted to express mAb and other fusion is through inhibitors of apoptosis transfections such as such as Bcl-EEE genes and is suitable for growth and through the Sp2/0 cell of further transfection under serum-free condition, as U.S. Patent number 7,531,327,7,537,930 and 7,608, described in 425, the embodiment part of each patent is incorporated herein by reference.

PAM4 antibody

Various embodiments of the present invention relate to normal or benign pancreatic tissue is contrary, the antibody reacted with high selectivity and cancer of pancreas.Anti-cancer of pancreas antibody and its fragment preferably produce for the rough mucin preparation from human pancreatic tumors, but can utilize through partial purification or even purified MUC5ac.The limiting examples of this antibody-like is PAM4 antibody.

Originally muroid PAM4 (mPAM4) antibody was developed as immunity by adopting the cancer of pancreas mucin deriving from heteroplastic RIP-1 human pancreatic adenocarcinoma.(Gold,Int.J.Cancer,57:204-210,1994。) as discussed below, antibody cross reaction and immunohistochemical staining research show the epi-position of uniqueness on PAM4 antibody recognition MUC5ac and novelty.Immunohistochemical staining is studied, and all as described in Example 2 those, have shown PAM4MAb in conjunction with the antigen expressed by mammary gland, pancreas and other cancer cell, and with Normal Human Tissue in conjunction with limited.But most high expressed is realized by pancreatic cancer cell usually.Therefore, PAM4 antibody has relative specificity to cancer of pancreas, and preferentially in conjunction with pancreatic cancer cell.PAM4 antibody with the target epi-position of internalization can have reactivity.This epi-position is primarily of relevant to cancer of pancreas and irrelevant with focal pancreatitis or normal pancreatic tissue antigen presentation.The combination of PAM4 antibody and PAM4 epi-position is subject to the suppression with DTT or periodates process antigen.Use to position through radiolabeled PAM4MAb in animal model and shown cancer target and therapeutic efficiency with Therapy study.

PAM4 antibody is in conjunction with the epi-position (being positioned at the second to the 4th rich Cysteine domains) of MUC5ac, and this antigen is expressed by multiple organ and tumor type, but preferentially expresses in pancreatic cancer cell.Utilize the research of PAM4MAb, such as in embodiment 3, show that described antibody presents multiple critical nature, this becomes the good candidate of clinical diagnosis and treatment use.Described epi-position is available Diagnosis and Treat target for cancer of pancreas and other cancer.Provide the useful target of pancreas and other cancer diagnosis and treatment.The MUC5ac epi-position that PAM4 antibody identifies obviously is different from non-PAM4 anti-cancer of pancreas antibody (e.g., CA19.9, DUPAN2, SPAN1, Nd2, CEACAM5, B72.3, anti-Le ^awith other anti-Lewis antigen) epi-position that identifies.

Surprisingly, following examples show that the MUC5ac epi-position that PAM4 combines is present in the serum of pole Early pancreatic carcinoma patient with detectable concentration.Also surprisingly, be obviously present in fresh serum human in conjunction with the endogenous inhibitor of the PAM4 antibody of MUC5ac.Described inhibitor is by carrying out extended refrigerated storage to blood serum sample, or removes by carrying out organic extractant phase to fresh serum.These unpredictable consequences provide the basis of the relative Non-Invasive cancer of pancreas early detection test using blood, serum or plasma sample.In an alternate embodiment, PAM4 antibody can separately or with one or more other antibody, the antibody combined use of such as CA19.9, to detect the pancreatic cancer marker in serum.

Treatment is used, is applicable to comprising such as antibody CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, anti-CEA, anti-CEACAM6, anti-Le with the antibody of PAM4 Antibody Combination or conbined usage ^a, anti-HLA-DR, anti-CD40, anti-CD74, anti-CD138 and the antibody defined by Lewis antigen Le (y), or for colon-specific antigen-p (CSAp), MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC16, MUC17, EGP-1, EGP-2, HER2/neu, EGFR, angiogenesis factor (such as, VEGF and PlGF), IGF (IGF), tenascin, platelet derived growth factor and IL-6, and oncogene (bcl-2, Kras, p53) product, the antibody of cMET and the antibody for neoplasm necrosis material, patent (the U.S. Patent number 6 of such as Epstein etc., 071, 491, 6, 017, 514, 5, 019, 368 and 5, 882, 626) described in.This antibody-like will can be used for supplementary PAM4 antibody mediated immunity and detect and immunotherapy method.When before using PAM4 antibody, simultaneously or when using afterwards, these and other therapeutic agent can act synergistically with the anti-cancer of pancreas antibody such as such as PAM4 antibody.

In treatment use, to the immunomodulator related to for the effector cell function of tumour cell in competition or the antibody of Antagonism also may with use separately or with the PAM4 Antibody Combination that other TAA combines, example is the antibody for CD40.Todryk etc., J.ImmunolMethods, 248:139-147 (2001); Turner etc., J.Immunol, 166:89-94 (2001).Also use the antibody for oncogene (such as, bcl-2, Kras, p53, cMET) label or product, or for the antibody of the such as angiogenesis factor such as VEGFR and placenta like growth factor (PlGF).

Important for the two determinant enzyme linked immunosorbent assay (ELISA) (ELISA) of exploitation for the MUC5ac in clinical sample in conjunction with the availability of the another kind of PAM4 sample antibody of the different epi-positions of MUC5a.ELISA experiment is described in embodiment 1 and 5.

Muroid described herein, chimeric, humanization and mankind PAM4 antibody and fragment thereof are the examples of the anti-cancer of pancreas antibody used in diagnosis and/or methods for the treatment of completely.Following examples disclose the preferred embodiment building and use humanization PMA4 antibody.Because non-human monoclonal antibodies may be identified as extraneous protein by human host, and duplicate injection may cause harmful allergic reaction, so carry out humanization to rodent antibody sequence can reduce the unfavorable immune response that patient may experience.For the monoclonal antibody based on muroid, this is commonly referred to mankind's anti-mouse antibody (HAMA) reaction.Preferably, in the framework region of the anti-cancer of pancreas antibody of humanization or its fragment, some human residue are replaced by the homologue of its mouse.Combination from the Frame sequence of two kinds of different people antibody-likes is used for V _halso be preferred.The constant domain of antibody molecule derives from the constant domain of human antibodies.

Prepared by antibody

The monoclonal antibody of specific antigen obtains by method known to those skilled in the art.See such as Kohler and Milstein, Nature256:495 (1975); With (volumes) such as Coligan, CURRENTPROTOCOLSINIMMUNOLOGY, the 1st volume, 2.5.1-2.6.7 page (JohnWiley & Sons1991) (" Coligan " hereinafter).In brief, anti-cancer of pancreas MAb is obtained: with including the cancer of pancreas mucin potpourri that comprises MUC5ac or the MUC5ac through purifying or injecting mouse corresponding to the peptide of epi-position of the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) or the composition of protein that are positioned at MUC5ac by such as under type, the existence that antibody produces is verified by removing blood serum sample, take out spleen to obtain B-lymphocyte, make B-lymphocyte and myeloma cell fusion to produce hybridoma, clone hybridization knurl, select the positive colony of the antibody created for MUC5ac, cultivate the clone of the antibody of the epi-position produced for the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) being positioned at MUC5ac, anti-cancer of pancreas antibody is separated with from Hybridoma culture.

Tentatively producing after for immunogenic antibody, can check order to described antibody, and being prepared by recombinant technique subsequently, to produce chimeric or humanized antibody.Rodent antibody and the chimeric of antibody fragment are well known to those skilled in the art.The use deriving from the antibody component of chimeric mAb decreases the potential problems relevant to the immunogenicity of muroid constant region.

Current techique for cloning muroid immunoglobulin variable domain territory is described in such as publication Orlandi etc., and in ProcNat'lAcad.Sci.USA86:3833 (1989), the document is incorporated herein by reference.In general, the V of mouse-anti body _k(variable light) and V _h(variable heavy chain) sequence obtains by different kinds of molecules Cloning processes such as such as RT-PCR, 5'-RACE and cDNA library screenings.Specifically, by RT-PCR by carrying out the V of pcr amplification to muroid PAM4MAb from hybridoma _hand V _ksequence is cloned, and determines its sequence by DNA sequencing.In order to confirm its authenticity, can by the V of clone in cell culture _kand V _hgene expression is chimeric Ab, described by (ProcNatl.Acad.Sci., USA, 86:3833,1989) such as Orlandi.

In a preferred embodiment, chimeric PAM4 antibody or antibody fragment comprise the complementarity-determining region (CDR) of muroid PAM4MAb and the light chain of framework region (FR) and human antibodies and CH, wherein the variable region of light chain CDR of chimeric PAM4 comprises CDR1 (SASSSVSSSYLY, SEQIDNO:1), CDR2 (STSNLAS, and CDR3 (HQWNRYPYT, SEQIDNO:3) SEQIDNO:2); And the variable region of heavy chain CDR of chimeric PAM4MAb comprises CDR1 (SYVLH, SEQIDNO:4), CDR2 (YINPYNDGTQYNEKFKG, SEQIDNO:5) and CDR3 (GFGGSYGFAY, SEQIDNO:6).The use deriving from the antibody component of chimeric mAb decreases the potential problems relevant to muroid constant region immunogenicity.

The humanization of rodent antibody and antibody fragment is also well known to those skilled in the art.Technology for generation of humanization MAb is disclosed in such as with in Publication about Document: Carter etc., ProcNat'lAcad.Sci.USA89:4285 (1992); Singer etc., J.Immun.150:2844 (1992); Mountain etc., BiotechnolGenetEngRev.10:1 (1992); And Coligan, 10.19.1-10.19.11 page, each document is incorporated herein by reference.For example, can by the muroid complementary determining region from mouse immunoglobulin heavy and variable region of light chain be transferred in human variable-domain, and subsequently human residue selected by framework region is substituted by its muroid FR homologue and carrys out generating humanized monoclonal antibody.Except human constant region sequences, the use of human framework's region sequence also reduces the chance of induction HAMA reaction.

Can design and build humanized antibody described by (MolImmunol.32:1413 (1995)) such as Leung, the document is incorporated to herein by reference.Example 1 describes the humanization approach for building hPAM4MAb.

Nucleotide sequence for the preparation of the primer of hPAM4 antibody is discussed in following examples 1.In a preferred embodiment, humanization PAM4 antibody or antibody fragment comprise above disclosed light chain and heavy CDR sequences (SEQIDNO:1 to SEQIDNO:6).Further preferably, the light chain of humanized antibody and the FR of variable region of heavy chain comprise the amino acid that at least one is replaced by the described corresponding FR of muroid PAM4MAb.

Complete human antibodies, such as mankind PAM4 can available from nonhuman transgenic animal.See such as Mendez etc., NatureGenetics, 15:146-156 (1997); U.S. Patent number 5,633,425.For example, human antibodies can be reclaimed from the transgenic mice with human immunoglobulin gene seat.Mouse humoral immune system humanization is made by making endogenous immunoglobulin genes inactivation and introducing human immunoglobulin gene seat.Human immunoglobulin gene seat is too complicated, and comprises a large amount of discrete section, and they together occupy the human genome of almost 0.2%.Enough antibody pedigrees can be produced in order to ensure transgenic mice, most of human heavy chain and light chain gene seat must be incorporated in mouse genome.This realizes with Step wise procedure, and described method starts from the yeast artificial chromosome (YAC) being formed in embryonal system configuration and contain human heavy chain or light chain immunoglobulins locus.Because each insetion sequence is approximately lMb size, therefore YAC structure needs to carry out homologous recombination to the overlapping fragments of immunoglobulin loci.These two kinds of YAC, one is containing heavy chain gene seat and one contains light chain gene seat, is to introduce respectively in mouse via containing the yeast spheroblasts of YAC and the fusion of mouse embryo stem cell.Subsequently by micro-for Embryonic stem cell clones injection mouse blastocyst.The ability being propagated YAC by its embryonal system of screening gained chimeric males, and make itself and mouse-anti body produce the mouse hybrid of defect.Two kinds of transgenic strains, a kind of containing human heavy chain loci and the another kind of hybridization containing Human light chain genes's seat creates the filial generation that can produce human antibodies in response to immunity.But these technology are nonrestrictive, and other method for generation of human antibodies as known in the art, such as use phage display, also can be used for producing the anti-cancer of pancreas antibody of the mankind.

Method as known in the art can be used to produce antibody by cell culture technology.In one embodiment, transfectoma cultures is made to adapt to serum free medium.For the generation of humanized antibody, cell can be made in the roller bottle using HSFM to be grown to 500ml culture.Carry out centrifugal to culture, and make supernatant by 0.2 μm of membrane filtration.Make through filtration nutrient culture media with the flow velocity of 1ml/min by albumin A post (1 × 3cm).Use the PBS washing resin of about 10 times of column volumes subsequently, and with containing the antibody of 0.1M glycine buffer (pH3.5) from eluted protein A combination post of 10mMEDTA.By 1.0ml fraction collector in the pipe comprising 10 μ l3MTris (pH8.6), and by the absorptance determination protein concentration under 280/260nm.Collect peak fraction, relative to PBS dialysis, and with such as Centricon30 filtrator (Amicon, Beverly, MA) concentrated antibody.Measure antibody concentration by ELISA, and use PBS that its concentration is adjusted to about 1mg/ml.0.01% (w/v) sodium azide is added easily as antiseptic in sample.

Can by the technology of multiple establishment abstraction and purification antibody from Hybridoma culture.This type of isolation technics comprises the affinity chromatography, size exclusion chromatography (SEC) and the ion-exchange chromatography that utilize Protein-A Sepharose.See such as Coligan, 2.7.1-2.7.12 page and 2.9.1-2.9.3 page.Also see Baines etc., " PurificationofImmunoglobulinG (IgG) ", METHODSINMOLECULARBIOLOGY, the 10th volume, 79-104 page (TheHumanaPress, Inc.1992).

Anti-cancer of pancreas MAb can be characterized by multiple technologies well known to those skilled in the art.For example, antibody combines the ability being positioned at the epi-position of second to the 4th rich Cysteine domains (amino acid residue 1575-2052) of MUC5ac and can use indirect enzyme immunoassays, flow cytometry, ELISA or western blot analysis to verify.

Antibody fragment

Antibody fragment is the antigen-binding portion thereof of antibody, such as F (ab') ₂, Fab', F (ab) ₂, Fab, Fv, sFv, scFv etc.Identify that the antibody fragment of defined epitope can be produced by known technology.For example, F (ab') ₂fragment can be produced by the pepsin digestion of antibody molecule.For example, Goldenberg, U.S. Patent number 4,036,945 and 4,331,647 and wherein contained list of references in describe these and other method.Also see Nisonoff etc., ArchBiochem.Biophys.89:230 (1960); Porter, Biochem.J.73:119 (1959); Edelman etc., METHODSINENZYMOLOGY the 1st volume, the 422nd page of (AcademicPress1967) and Coligan, 2.8.1-2.8.10 page and 2.10.-2.10.4 page.Alternatively, Fab' expression library (Huse etc., 1989, Science, 246:1274-1281) can be built, to allow fast and easily to differentiate monoclonal Fab' fragment by required specificity.

Single Chain Fv Molecule A (scFv) comprises V _ldomain and V _hdomain.V _ldomain and V _hdomain associates and forms target binding site.These two domains are also covalently bound by peptide connexon (L).If V _ldomain is the N-end section of scFv molecule, then scFv molecule is expressed as V _l-L-V _hif, or V _hdomain is the N-end section of scFv molecule, be then expressed as V _h-L-V _l.Be described in in Publication about Document for the manufacture of scFv molecule and the method designing suitable peptide connexon: U.S. Patent number 4,704,692; U.S. Patent number 4,946,778; R.Raag and M.Whitlow, " SingleChainFvs " .FASEB the 9th volume: 73-80 (1995); With R.E.Bird and B.W.Walker, SingleChainAntibodyVariableRegions, TIBTECH, the 9th volume: 132-137 (1991).

Other antibody fragment, such as single domain antibody fragment are known in ability domain and can be used for claimed construct.Single domain antibody (VHH) can such as by standard immunoassay techniques available from camel, alpaca or llama.(see such as Muyldermans etc., TIBS26:230-235,2001; Yau etc., JImmunolMethods281:161-75,2003; Maass etc., JImmunolMethods324:13-25,2007).VHH can have powerful antigen binding capacity, and can with can not enter conventional V _h-V _lthe novel epi-position of pairing interacts (Muyldermans etc., 2001).Sheep rides serum IgG containing 50% only camel heavy chain IgG antibody (HCAb) (Maass etc., 2007) of having an appointment.Alpaca can carry out immunity with known antigens such as such as TNF-α, and can be separated the VHH (Maass etc., 2007) combined with target antigen also.Identify the PCR primer of all alpaca VHH coded sequences that in fact can increase, and can be used for building alpaca VHH phage display library, this storehouse can be used for carrying out antibody fragment separation (Maass etc., 2007) by standard biological panning technique well known in the art.

Antibody fragment can also be prepared by the proteolysis of full length antibody or by the DNA expressing the described fragment of coding in Escherichia coli or another kind of host.Antibody fragment can be obtained by the pepsin of full length antibody or papain digestion by conventional method.For example, antibody fragment by carrying out enzymatic lysis with pepsin antagonist, thus can provide with F (ab') ₂the about 100Kd fragment represented produces.This fragment can use thiol reductant and optionally for the further cracking of end-capping group of the sulfydryl produced by disulfide bond cracking, produce about 50KdFab' monovalent fragment.Alternatively, use papain to carry out enzymatic lysis and directly produce two monovalent Fab fragment and a Fc fragment.

Other antibody cleavage method can also be used, be such as separated heavy chain to form monovalent light-heavy chain fragment, further crack fragment or other enzymatic, chemistry or gene technology, as long as the antigen that described fragment identifies in conjunction with complete antibody.

Antibody fusion protein and multivalent antibody

The fusion comprising relevant anti-cancer of pancreas antibody can be prepared by multiple conventional program, is bonded between functional group has more specific binding from glutaraldehyde.Comprise the antibody of fusion described herein and/or antibody fragment preferably directly or by connexon part, by the one or more functional groups on antibody or fragment, such as amine, carboxyl, phenyl, mercaptan or hydroxyl covalent bond each other.The various conventional linkers except glutaraldehyde can be used, such as diisocyanate, diisothio-cyanate, two (N-Hydroxysuccinimide) ester, carbodiimide, maleimide hydroxysuccinimide eater etc.

Straightforward procedure for generation of fusion is mixed antibody or fragment under the existence of glutaraldehyde.Initial schiff bases binding can such as become secondary amine by borohydride reduction and be stablized.Diisothio-cyanate or carbodiimide can be used to replace glutaraldehyde as non-site specific linker.In one embodiment, antibody fusion protein comprises anti-cancer of pancreas MAb or its fragment, and wherein said MAb combines the epi-position being positioned at second to the 4th rich Cysteine domains (amino acid residue 1575-2052) of MUC5ac.This fusion and its fragment are preferentially in conjunction with pancreatic cancer cell.This unit price monospecific MAb can be used for direct targeting antigen, and wherein said MAb is connected to therapeutic agent, diagnosticum or its combination, and described protein is directly applied to patient.

Described fusion alternatively can comprise the anti-cancer of pancreas MAb of the different epi-positions of at least two basic change MUC5ac.For example, MAb can produce antigentic specificity bivalent antibody, trivalent antibodies and tetravalent antibody, and they are multivalence, but have monospecific to MUC5ac.The noncovalent associations of two or more scFv molecules can form function bivalent antibody, trivalent antibodies and tetravalent antibody.Monospecific diabody is the homodimer of identical scFv, and wherein each scFv comprises the V of selected antibody _hdomain, this V _hdomain is connected to the V of same antibody by short connexon _ldomain.Bivalent antibody is by two scFv noncovalent associations, thus the divalence dimer producing two Fv binding sites and formed.Trivalent antibodies by the trivalent tripolymer forming three scFv, thus produces three binding sites and obtains, and tetravalent antibody is the tetravalence tetramer of four scFv, thus produces four combinations site.Use containing comprising V _h1-connexon-V _lthe expression vector of the recombination construct of 1 has prepared several monospecific diabody, see Holliger etc., ProcNatl.Acad.SciUSA90:6444-6448 (1993); Atwell etc., MolecularImmunology33:1301-1302 (1996); HolIiger etc., NatureBiotechnology15:632-631 (1997); Helfrich etc., IntJCancer76:232-239 (1998); Kipriyanov etc., IntJCancer77:763-772 (1998); Holiger etc., CancerResearch59:2909-2916 (1999).U.S. Patent number 4,946,778 (1990) and U.S. Patent number 5,132, disclose the method building scFv in 405 (1992), the embodiment part of each patent is incorporated herein by reference.U.S. Patent number 5,837,242 (1998), U.S. Patent number 5,844,094 (1998) and WO-98/44001 (1998) in disclose and produce based on the method for the multivalence Mono-specific antibodies of scFv, the embodiment part of each patent is incorporated herein by reference.Multivalence Mono-specific antibodies fusion is in conjunction with the epi-position of two or more identical types, and described epi-position can be positioned on antigen in same antigen or independent.Valent increase allows extra interaction, affinity increases and extended residence time.These antibody fusion proteins can be used for direct targeted system, and wherein antibody fusion protein is puted together in therapeutic agent, diagnosticum or its combination, and is directly applied to the patient needing it.

Preferred embodiment is multivalence multi-specificity antibody or its fragment, and it comprises and one or morely PAM4 target epi-position is had to the antigen binding site of affinity and one or more extra binding site for other epi-position relevant to cancer of pancreas.This fusion is polyspecific, because the epi-position that its combination at least two is different, described epi-position can be on identical or different antigen.For example, fusion can comprise more than one antigen binding site, first has affinity to the epi-position of the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) being positioned at MUC5ac, and second has affinity to another target antigens such as such as TAG-72 or CEA.Another example is bispecific fusion protein, and it can comprise CA19.9MAb (or its fragment) and PAM4MAb (or its fragment).This type of fusion will have affinity to CA19.9 and MUC5ac.Antibody fusion protein and its fragment can be used for direct targeted system, and wherein said antibody fusion protein is puted together in therapeutic agent, diagnosticum or its combination, and is directly applied to the patient needing it.

Another preferred embodiment is multivalence multi-specificity antibody, and it comprises at least one and has the binding site of affinity to PAM4 target epi-position and at least one has the haptens binding site of affinity to hapten molecule.For example, bispecific fusion protein can comprise 679MAb (or its fragment) and PAM4MAb (or its fragment).Monoclonal 679 antibody combines the molecule containing three peptide moiety histamine succinyls glycyl (HSG) with high-affinity.This type of bispecific PAM4 antibody fusion protein can such as by obtaining F (ab') from 679 ₂prepared by fragment, as described above.679F (ab') is leniently reduced with DTT ₂the interchain disulfide bridge of fragment, carefully avoids light-heavy chain binding to form Fab'-SH fragment.SH group is activated with excessive bismaleimides connexon (1,1'-(methylene two-4,1-phenylene) bismaleimides).PAM4MAb is converted into Fab'-SH, and reacts with the 679Fab'-SH fragment of activation subsequently, obtain bispecific fusion protein.Bispecific fusion protein, such as this is a kind of, can be used for affinity and strengthen system, wherein with described fusion pre-targeting target antigen, and subsequently with the diagnosticum be connected with containing the haptenic carrier part of one or more HSG (can target construct) or therapeutic agent target.In alternative preferred embodiment, such as TF10 etc. are based on DNL ^tMhPAM4-679 construct can prepare as described in the following embodiments and use.

Bispecific antibody manufactures by multiple conventional method, such as complete IgG or preferably F (ab') ₂the disulfide cleavage of the potpourri of fragment and again formed, can produce to be formed the polyoma had more than a species specific antibody more than a kind of hybridoma fusion, and genetic engineering.Prepare bispecific fusion protein by the oxicracking of the Fab' fragment obtained by the reductive cleavage of different antibodies.This carries out advantageous by with under type: two kinds that are produced by the pepsin digestion by two kinds of different antibody different F (ab') ₂fragment mixes, and reductive cleavage, to form the potpourri of Fab' fragment, is oxidized the F (ab') again being formed to produce and comprise bispecific fusion protein subsequently to disulfide bond ₂the potpourri of fragment, described fusion comprises and has specific Fab' part to each former epi-position.Current techique for the preparation of antibody fusion protein is found in such as with in Publication about Document: Nisonoff etc., ArchBiochemBiophys.93:470 (1961); Hammerling etc., JExpMed.128:1461 (1968); With U.S. Patent number 4,331,647.

Have more optionally binding to realize by using the Heterobifunctional connexons such as such as maleimide hydroxysuccinimide eater.The reaction of ester and antibody or fragment is by the amido on derived antibody or fragment, and derivant can react with the Fab fragments (or sulfydryl is attached to more large fragment above or complete antibody by such as Traut reagent) such as with free sulfhydryl groups subsequently.This type of connexon unlikely makes the group in same antibody be cross-linked, and improves the selectivity of binding.

Advantageously connecting antibody or fragment away from the site of antigen binding site.This can by such as realizing with the interchain sulfydryl binding of cracking as above.Another kind method relates to the antibody making to have already oxidised carbohydrate portions and another antibody response with at least one free amine function.This generates initial schiff bases binding, it is preferably by being reduced into secondary amine, such as, by borohydride reduction, is stablized to form resulting composite.For Small molecular, this type of locus specificity binding is disclosed in U.S. Patent number 4,671, in 958, for larger condiment, is then disclosed in U.S. Patent number 4,699, and in 784, the embodiment part of each patent is incorporated herein by reference.

There is the V that ScFv that length is greater than the connexon (such as, the connexon of 15 or 18 residues) of 12 amino acid residues allows on same chain _hand V _linteraction between domain, and generally form monomer, dimer (being called bivalent antibody) and a small amount of more polymeric potpourri of high-quality (Kortt etc., EurJBiochem. (1994) 221:151-157).But the ScFv with the connexon of less than 5 amino acid residues prevents the V on same chain _hand V _lpairing in the molecule of domain, thus force and the V on different chain _hand V _lpairing.Connexon between 3 and 12 residues mainly forms dimer (Atwell etc., ProteinEngineering (1999) 12:597-604).When the connexon between 0 and 2 residue, define the trimerization (being called trivalent antibodies) of scFv, four poly-(being called tetravalent antibody) or higher oligomer structures; But except connexon length, the definite pattern of oligomerization seems also to depend on composition and the orientation of V-domain.

Recently, U.S. Patent number 7,550,143,7,521,056,7,534,866,7,527,787 and 7,666, described the new technology of the potpourri for building antibody, antibody fragment and/or other effect part in fact in any array configuration in 400, the embodiment part of each patent is incorporated herein by reference.Be commonly referred to as DOCK-AND-LOCK ^tM(DNL ^tM) technology relate to the fusion producing and to comprise one of two complementary peptide sequences being called as dimerization and docking structure territory (DDD) and anchoring domain (AD) sequence at N or C end.In preferred embodiments, DDD sequence derives from the adjustment subunit of cAMP deopendent protein kinase, and AD sequence derives from the sequence of A kinase anchoring protein (AKAP).DDD sequence forms the dimer be combined with AD sequence, and this just allows to form any one in tripolymer, the tetramer, six aggressiveness or other compound multiple.By the such as effect such as antibody or antibody fragment part is connected to DDD and AD sequence, any selected compound combined by antibody or antibody fragment can be formed.DNL ^tMcompound can be stablized with covalent manner by formation disulfide bond or other binding.

Pre-targeting

Bispecific or multi-specificity antibody can be used for pre-targeting technology.Pre-targeting is multistage method, and originally exploitation is removed for the slow blood solving direct targeting antibodies, and this causes unwanted toxicity to normal structures such as such as marrow.When pre-targeting, radioactive nuclide or other therapeutic agent are connected to littlely sends molecule (can target construct or can target conjugate) by what removed from blood in several minutes.First use and have for can the pre-targeting bispecific of binding site of target construct and target antigen or multi-specificity antibody, allow free antibodies to remove from circulation, and use subsequently can target construct.

Pre-targeting method is known in the art, such as, as with disclosed in Publication about Document: Goodwin etc., U.S. Patent number 4,863,713; Goodwin etc., J.Nucl.Med.29:226,1988; Hnatowich etc., J.Nucl.Med.28:1294,1987; Oehr etc., J.Nucl.Med.29:728,1988; Klibanov etc., J.Nucl.Med.29:1951,1988; Sinitsyn etc., J.Nucl.Med.30:66,1989; Kalofonos etc., J.Nucl.Med.31:1791,1990; Schechter etc., Int.J.Cancer48:167,1991; Paganelli etc., CancerRes.51:5960,1991; Paganelli etc., Nucl.Med.Commun.12:211,1991; U.S. Patent number 5,256,395; Stickney etc., CancerRes.51:6650,1991; Yuan etc., CancerRes.51:3119,1991; U.S. Patent number 6,077,499; U.S. Patent number 6,090,381; U.S. Patent number 6,472,511; With U.S. Patent number 6,962,702.

Treat or diagnose the disease of experimenter or the pre-targeting method of illness to provide in the following manner: (I) uses bispecific antibody or antigen binding antibody fragment to described experimenter; (2) optionally use removing composition to described experimenter, and allow said composition to remove antibody from circulation; (3) to experimenter use containing one or more chelatings or chemical bond treatment or diagnosticum can target construct.This technology also can by use with can the enzyme puted together of target construct, using subsequently can by the prodrug of the Viability form of described enzymatic conversion for antibody dependent enzyme prodrug therapy (ADEPT).

Known antibody

In various embodiments, claimed method and composition can utilize any one in Multiple Antibodies as known in the art, such as, for combinatorial antibody therapy.The antibody used can be commercially available from known source.For example, Multiple Antibodies secretion hybridoma system can from American type culture collection (AmericanTypeCultureCollection, ATCC; Manassas, VA) obtain.For various disease targets, include but not limited to that the lot of antibodies of tumor associated antigen is deposited with ATCC and/or has disclosed variable region sequences, and can be used for claimed method and composition.See such as U.S. Patent number 7,312,318, 7,282,567, 7,151,164, 7,074,403, 7,060,802, 7,056,509, 7,049,060, 7,045,132, 7,041,803, 7,041,802, 7,041,293, 7,038,018, 7,037,498, 7,012,133, 7,001,598, 6,998,468, 6,994,976, 6,994,852, 6,989,241, 6,974,863, 6,965,018, 6,964,854, 6,962,981, 6,962,813, 6,956,107, 6,951,924, 6,949,244, 6,946,129, 6,943,020, 6,939,547, 6,921,645, 6,921,645, 6,921,533, 6,919,433, 6,919,078, 6,916,475, 6,905,681, 6,899,879, 6,893,625, 6,887,468, 6,887,466, 6,884,594, 6,881,405, 6,878,812, 6,875,580, 6,872,568, 6,867,006, 6,864,062, 6,861,511, 6,861,227, 6,861,226, 6,838,282, 6,835,549, 6,835,370, 6,824,780, 6,824,778, 6,812,206, 6,793,924, 6,783,758, 6,770,450, 6,767,711, 6,764,688, 6,764,681, 6,764,679, 6,743,898, 6,733,981, 6,730,307, 6,720,155, 6,716,966, 6,709,653, 6,693,176, 6,692,908, 6,689,607, 6,689,362, 6,689,355, 6,682,737, 6,682,736, 6,682,734, 6,673,344, 6,653,104, 6,652,852, 6,635,482, 6,630,144, 6,610,833, 6,610,294, 6,605,441, 6,605,279, 6,596,852, 6,592,868, 6,576,745, 6,572, 856, 6,566,076, 6,562,618, 6,545,130, 6,544,749, 6,534,058, 6,528,625, 6,528,269, 6,521,227, 6,518,404, 6,511,665, 6,491,915, 6,488,930, 6,482,598, 6,482,408, 6,479,247, 6,468,531, 6,468,529, 6,465,173, 6,461,823, 6,458,356, 6,455,044, 6,455,040, 6,451,310, 6,444,206, 6,441,143, 6,432,404, 6,432,402, 6,419,928, 6,413,726, 6,406,694, 6,403,770, 6,403,091, 6,395,276, 6,395,274, 6,387,350, 6,383,759, 6,383,484, 6,376,654, 6,372,215, 6,359,126, 6,355,481, 6,355,444, 6,355,245, 6,355,244, 6,346,246, 6,344,198, 6,340,571, 6,340,459, 6,331,175, 6,306,393, 6,254,868, 6,187,287, 6,183,744, 6,129,914, 6,120,767, 6,096,289, 6,077,499, 5,922,302, 5,874,540, 5,814,440, 5,798,229, 5,789,554, 5,776,456, 5,736,119, 5,716,595, 5,677,136, 5,587,459, 5,443,953, 5,525,338, the embodiment part of each patent is incorporated herein by reference.These are only exemplary, and other antibody multiple and their hybridoma are well known in the art.Technician will recognize, almost can by carrying out simple search to obtain to the associated antibodies for selected disease association target at ATCC, NCBI and/or USPTO database for the antibody sequence of any Disease associated antigens or antibody-secreting hybridoma.The antigen-binding domains of clonal antibody can use standard technique well known in the art to carry out increasing, excises, is connected in expression vector, transfection is in the host cell through transformation, and produce (see such as U.S. Patent number 7 for protein, 531,327,7,537,930,7,608,425 and 7,785,880, the embodiment part of each patent is incorporated herein by reference).

The specific antibodies that can be used for cancer therapy in the scope of claimed method and composition includes but not limited to LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), dimension trastuzumab (hA20, anti-CD20), Rituximab (anti-CD20), trastuzumab (GA101 difficult to understand, anti-CD20), langley pearl monoclonal antibody (anti-PD-1 acceptor), Buddhist nun Shandong monoclonal antibody (anti-PD-1 acceptor), her monoclonal antibody (anti-CTLA-4), RS7 (anti-Glycoproteins in Epithelial-1 (EGP-1, also referred to as TROP-2)), KC4 (anti-stick albumen), MN-14 (anti-carcinoembryonic antigen, anti-CEA, also referred to as CD66e or CEACAM5), MN-15 or MN-3 (anti-CEACAM6), Mu-9 (anti-colon-specific antigen p), Immu31 (anti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19), TAG-72 (such as, CC49), Tn, J591 or HuJ591 (anti-PSMA (prostate specific membrane antigen)), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IXMAb), L243 (anti-HLA-DR), alemtuzumab (anti-CD52), Avastin (anti-vegf), Cetuximab (anti-EGFR), WAY-CMA 676 (anti-CD 33), ibritumomab tiuxetan (anti-CD20), Victibix (anti-EGFR), tositumomab (anti-CD20), PAM4 (has another name called Ke Liwo monoclonal antibody, anti-MUC5ac) and Herceptin (anti-ErbB).This antibody-like is well known in the art (such as, U.S. Patent number 5,686,072,5,874,540,6,107,090,6,183,744,6,306,393,6,653,104,6,730.300,6,899,864,6,926,893,6,962,702,7,074,403,7,230,084,7,238,785,7,238,786,7,256,004,7,282,567,7,300,655,7,312,318,7,585,491,7,612,180,7,642,239; With U.S. Patent Application Publication No. 20050271671,20060193865,20060210475,20070087001; Respective embodiment part is incorporated herein by reference).Concrete known useful antibody comprises hPAM4 (U.S. Patent number 7,282,567), hA20 (U.S. Patent number 7,251,164), hA19 (U.S. Patent number 7,109,304), hIMMU-31 (U.S. Patent number 7,300,655), hLL1 (U.S. Patent number 7,312,318), hLL2 (U.S. Patent number 7,074,403), hMu-9 (U.S. Patent number 7,387,773), hL243 (U.S. Patent number 7,612,180), hMN-14 (U.S. Patent number 6,676,924), hMN-15 (U.S. Patent number 7,541,440), hR1 (U.S. Patent application 12/772,645), hRS7 (U.S. Patent number 7,238,785), hMN-3 (U.S. Patent number 7,541,440), AB-PG1-XG1-026 (U.S. Patent application 11/983,372, deposits as ATCCPTA-4405 and PTA-4406), D2/B (WO2009/130575), BWA-3 (anti-histone H4), LG2-1 (anti-histone H3) and LG2-2 (anti-histone H2B) (U.S. Patent Application Serial Number 14/180,646, on February 14th, 2014 submits to), be incorporated herein by reference about figure and the text of embodiment part in each described patent or application.

Other available antigen of described conjugate target can be used to comprise carbonic anhydrase IX, B7, CCL19, CCL21, CSAp, HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5MAbs), CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD47, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM6, CTLA-4, CXCR4, alpha-fetoprotein (AFP), VEGF (such as, fibronectin splicing variants), ED-B Fibronectin (such as, L19), EGP-1 (TROP-2), EGP-2 (such as, 17-1A), EGF acceptor (ErbB1) (such as, ), ErbB2, ErbB3, factor H, FHL-1, Flt-3, folacin receptor, Ga733, GRO-β, HMGB-1, Hypoxia-Inducible Factor (HIF), HM1.24, HER-2/neu, histone H2B, histone H 3, histone H 4, IGF (ILGF), IFN-γ, IFN-α, IFN-β, IFN-λ, IL-2R, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, IGF-1R, Ia, HM1.24, gangliosides, HCG, the HLA-DR antigen that L243 combines, CD66 antigen (i.e. CD66a-d) or its combination, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, macrophage migration inhibition factor (MIF), MUC1, MUC2, MUC3, MUC4, MUC5ac, placenta growth factor (PlGF), PSA (prostate specific antigen), PSMA, PD-1, PD-L1, NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1, Le (y), mesothelin, S100, tenascin, TAC, Tn antigen, Thomas-Friedenreich antigen, tumor necrosis antigens, Tumor Angiongesis antigen, TNF-α, TRAIL acceptor (R1 and R2), TROP-2, VEGFR, RANTES, T101 and cancer stem cell antigen, complement factor C_3, C3a, C3b, C5a, C5 and oncogene products.

As shown in by flow cytometry, and what can instruct the immunotherapy selecting suitable antibody for drug conjugate is Craig and Foon to the comprehensive analysis of suitable antigen (title of trooping or the CD) target on hematopoiesis malignant cell, Blood, online pre-publication on January 15th, 2008; DOL10.1182/blood-2007-11-120535.

CD66 antigen is made up of the five kinds of different sugar PROTEIN C D66a-e with similar structures encoded by carcinomebryonic antigen (CEA) gene family member BCG, CGM6, NCA, CGM1 and CEA respectively.These CD66 antigens (such as CEACAM6) are mainly expressed in the tumour cell of granulocyte, gastral normal epithelium cell and various tissue.Suitable cancer target also comprises Cancer Testis Antigens, such as NY-ESO-1 (Theurillat etc., Int.J.Cancer2007; 120 (11): 2411-7) and myelomatosis (Kozlov etc., CancerGenet.Cytogenet.2005; 163 (1): 62-7) CD79a in B cell disease is also had, and the CD79b of non-Hodgkin's lymphoma (Poison etc., Blood110 (2): 616-623).Much above-mentioned antigen is disclosed in the U.S.Provisional Serial 60/426 of " UseofMulti-specific, the Non-covalentComplexesforTargetedDeliveryofTherapeutics " by name submitted on November 15th, 2002, in 379.Be attributed to forerunner's malignant cell colony (Hill and Perris, J.Natl.CancerInst.2007 that treatment resistance is larger; Cancer stem cell 99:1435-40) has in some cancer types can the antigen of target, such as prostate cancer (Maitland etc., ErnstScheringFound.Sympos.Proc.2006; 5:155-79), non-small cell lung cancer (Donnenberg etc., J.ControlRelease2007; 122 (3): 385-91) and glioblastoma (Beier etc., CancerRes.2007; 67 (9): 4010-5) CD133 in, and colorectal cancer (Dalerba etc., Proc.Natl.Acad.Sci.USA2007; 104 (24) 10158-63), cancer of pancreas (Li etc., CancerRes.2007; 67 (3): 1030-7) and SCCHN (Prince etc., Proc.Natl.Acad.Sci.USA2007; 104 (3) 973-8) in CD44.Another available target of breast cancer therapeutics is (the Biochem.J.2003 such as Taylor; LIV-1 antigen 375:51-9).

For Huppert's disease therapy, describe for such as CD38 and CD138 (Stevenson, MolMed2006; 12 (11-12): 345-346; Tassone etc., Blood2004; 104 (12): 3688-96), CD74 (Stein etc., ibid), CS1 (Tai etc., Blood2008; 112 (4): 1329-37) and CD40 (Tai etc., 2005; Suitable targeting antibodies CancerRes.65 (13): 5898-5906).

Macrophage migration inhibition factor (MIF) is congenital and adaptive immunity and apoptotic important regulatory factor.Report the endogenous receptor (Leng etc., 2003, JExpMed197:1467-76) that CD74 is MIF.The result for the treatment of of the anti-CD74 antibody of Antagonism to the intracellular pathway that MIF mediates can be used for treating various disease states, such as carcinoma of urinary bladder, prostate cancer, breast cancer, lung cancer, colon cancer and chronic lymphocytic leukemia are (such as, Meyer-Siegler etc., 2004, BMCCancer12:34; Shachar and Haran, 2011, LeukLymphoma52:1446-54); Autoimmune disease, such as arthritis deformans and systemic lupus erythematosus (Morand and Leech, 2005, FrontBiosci10:12-22; Shachar and Haran, 2011, LeukLymphoma52:1446-54); Kidney trouble, such as kidney allograft repels (Lan, 2008, NephronExpNephrol.109:e79-83); With many inflammatory diseases (Meyer-Siegler etc., 2009, MediatorsInflamm, on March 22nd, 2009 Electronically announces; Takahashi etc., 2009, RespirRes10:33); meter La Zhu monoclonal antibody (hLL1) is the exemplary anti-CD74 antibody disease for the treatment of MIF mediation to therapeutic use.

Anti-TNF-α antibody is well known in the art, and may be used for treating immunological diseases, such as autoimmune disease, immune dysfunction (such as, graft is to versus-host disease, organ-graft refection) or diabetes.Known antibodies for TNF-α comprises human antibodies CDP571 (Ofei etc., 2011, Diabetes45:881-85); Rodent antibody MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B and M303 (ThermoScientific, Rockford, IL); Infliximab (Centocor, Malvern, PA); Pegylation match trastuzumab (UCB, Brussels, Belgium); With adalimumab (Abbott, AbbottPark, IL).These and other known anti-TNF-α antibodies many may be used in claimed method and composition.Other antibody being used for the treatment of immune disorder or autoimmune disease includes but not limited to anti-B cell antibody, such as ties up trastuzumab, epratuzumab, meter La Zhu monoclonal antibody or hL243; Holder pearl monoclonal antibody (anti-IL-6 acceptor); Basiliximab (anti-CD25); Daclizumab (anti-CD25); Efalizumab (anti-CD11a); Muromonab-CD3 (AntiCD3 McAb acceptor); Anti-CD 40 L (UCB, Brussels, Belgium); Natalizumab (anti alpha 4 integrin) and agate pearl monoclonal antibody difficult to understand (anti-IgE).

Checkpoint inhibitor antibody is mainly used in cancer therapy.Immunologic test point refers to that in immune system, responsible maintenance self-tolerance and immunity moderation system response degree react thus damaging surrounding tissue reduced to minimum suppression approach.But all right activated immune system checkpoint of tumour cell is to reduce the immunoreactive validity for tumor tissues.For cytotoxic T lymphocyte epitope (CTLA4, also referred to as CD152), apoptosis albumen 1 (PD1, also referred to as CD279) and the exemplary checkpoint inhibitor antibody of apoptosis 1 ligand 1 (PD-L1, also referred to as CD274) can with one or more other pharmaceutical agent combinations for improving the immunoreactive validity to disease cells, tissue or pathogen.Exemplary anti-PD1 antibody comprises langley pearl monoclonal antibody (MK-3475, MERCK), Buddhist nun Shandong monoclonal antibody (BMS-936558, BRISTOL-MYERS, SQUIBB), AMP-224 (MERCK) and skin profit pearl monoclonal antibody (CT-011, CURETECHLTD.).Anti-PD1 antibody can purchased from such as (AB137132), (EH12.2H7, RMP1-14) and AFFYMETRIXEBIOSCIENCE (J105, J116, MIH4).Exemplary anti-PD-L1 antibody comprises MDX-1105 (MEDAREX), MEDI4736 (MEDIMMUNE), MPDL3280A (GENENTECH) and BMS-936559 (BRISTOL-MYERSSQUIBB).Anti-PD-L1 antibody also can purchased from such as AFFYMETRIXEBIOSCIENCE (MIH1).Exemplary anti-CTLA 4 antibody comprises her monoclonal antibody (Bristol-MyersSquibb) and Sibutramine Hydrochloride not monoclonal antibody (PFIZER).Anti-PD1 antibody can purchased from such as (AB134090), SINOBIOLOGICALINC. (11159-H03H, 11159-H08H) and THERMOSCIENTIFICPIERCE (PA5-29572, PA5-23967, PA5-26465, MA1-12205, MA1-35914).Her monoclonal antibody has received FDA approval (Wada etc., 2013, JTranslMed11:89) being used for the treatment of metastatic melanoma recently.

Other useful antibody can comprise histonic antibody and/or its Fab, such as BWA-3 (anti-H4), LG2-1 (anti-H3) and LG2-2 (anti-H2B) antibody.Exemplary histonic antibody is disclosed in the U.S. Patent Application Serial Number 14/180,646 (embodiment part is incorporated herein by reference) that such as on February 14th, 2014 submits to.

In another preferred embodiment, the antibody used is by quick internalization and expressed subsequently again, process and be presented on cell surface, thus makes described cell can constant absorption and build-up cycle conjugate.An example of most preferred antibody/antigen pairing is LL1, and anti-CD74MAb (invariant chain, II class Specific Chaperones, Ii) is (see such as U.S. Patent number 6,653,104,7,312,318; Respective embodiment part is incorporated herein by reference).CD74 antigen height is expressed in B cell lymphoma (comprising Huppert's disease) and leukaemia, some t cell lymphoma, melanoma, colon cancer, lung cancer and kidney, glioblastoma and some other cancer (Ong etc., Immunology98:296-302 (1999)).In cancer, the summary of CD74 antibody is used to list in the Stein etc. be incorporated herein by reference, ClinCancerRes.2007 September 15; 13 (18Pt2): in 5556s-5563s.

Preferably include but not limited to non_hodgkin lymphoma, Hodgkin's disease, melanoma, lung cancer, kidney, colon cancer, glioblastoma multiforme, histocytoma, myelomatosis and Huppert's disease by the disease of anti-CD74 Antybody therapy.CD74 antigen on target cells within short-term continuous expression, carry out antigen internalization and antigen subsequently and express again and make target LL1 antibody can with any chemotherapy part internalization of its delivery.This just allows the LL1-chemotherapeutics conjugate of high treatment concentration at this type of intracellular accumulation.The LL1-chemotherapeutics conjugate of internalization is by lysosome and endosome circulation, and chemotherapy part discharges with active form in target cell.

The treatment of antibody and diagnostic uses

Some embodiment relates to the method for the malignant tumour of diagnosis or treatment experimenter, and comprise and use anti-cancer of pancreas MAb, fusion or its fragment to experimenter, wherein said MAb, fusion or fragment diagnose with at least one and/or therapeutic agent is combined.The further preferably method of diagnosis or Therapeutic cancer, comprise and use multivalence, multi-specificity antibody or its fragment to experimenter, described antibody or its fragment comprise one or more for being positioned at the antigen binding site of epi-position of the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) of MUC5ac and one or more haptens binding site; Wait for the enough time so that unconjugated antibody cleans the blood flow of experimenter; Use subsequently to described experimenter and comprise diagnosticum, therapeutic agent or its carrier molecule combined, described carrier molecule is combined with the haptens binding site of localization antibody.In a preferred embodiment, cancer is non-incretion cancer of pancreas.

MAb is used for in-vitro diagnosis and knows.See such as Carlsson etc., Bio/Technology7 (6): 567 (1989).For example, MAb can be used for detecting the existence from tumor associated antigen in the tissue of biopsy samples.MAb also can be used for using the technology such as such as radioimmunoassay, enzyme linked immunosorbent assay (ELISA) and fluorescence immunoassay to measure the amount of tumor associated antigen in clinical fluid sample such as such as blood or serum etc.Hereinafter discuss in vitro and in vivo diagnostic method in more detail.

The conjugate of cancer target MAb and toxin can be used for selectivity in vivo and kills cancer cell (Spalding, Bio/Technology9 (8): 701 (1991); Goldenberg, ScientificAmericanScience & Medicine1 (1): 64 (1994)).For example, the Therapy study in experimental animal model has demonstrated the antitumor activity of the antibody being loaded with cytotoxic radionuclides.(Goldenberg etc., CancerRes.41:4354 (1981); Cheung etc., J.Nat'lCancerInst.77:739 (1986); With Senekowitsch etc., J.Nucl.Med.30:531 (1989)).In a preferred embodiment, conjugate comprise through ⁹⁰the hPAM4 antibody of Y mark.Conjugate is optionally used with one or more other therapeutic agent.In a preferred embodiment, warp ⁹⁰the hPAM4 of Y mark is applied to Pancreas cancer patients together with gemcitabine or 5 FU 5 fluorouracil.In a preferred embodiment, ⁹⁰y puts together in DOTA chelate, to be connected to hPAM4.In a preferred embodiment, ⁹⁰y-DOTA-hPAM4 combine with the fractionated dose and the gemcitabine that form treatment circulation, such as repetition, lower, compared with the gemcitabine of hypotoxicity dosage and lower, fractionated dose ⁹⁰y-DOTA-hPAM4 combines.Alternatively, through radiolabeled or other PAM4 antibody puted together can with another kind of immunoconjugates, such as SN-38 conjugation of antibodies combined administration.Particularly preferred combination is ⁹⁰y-hPAM4 and SN-38-hRS7 (anti-TROP2 antibody) (see such as U.S. Patent number 8,586,050, embodiment part is incorporated herein by reference).

When tolerating, the repetitive cycling of instruction fractionated dose scheme.For example, the 200mg/m of 4 parts of weekly doses ²the 8mg/m of gemcitabine and three parts of weekly doses ²⁹⁰y-DOTA-hPAM4 combines, and the latter, from the second week that gemcitabine is used, forms single treatment circulation.Other dosage (each component is higher or lower) also can form fractionated dose, it determines (see such as U.S. Patent number 6 by the conventional means of assessment hematopoietic toxicity, 649,352,7,112,409,7,279,289,7,465,551), because the myelosuppressive effects of two kinds of reagent can be accumulated.The skilled doctor of this type of Results based on the bone marrow status of patient and general health state, based on many factors, can comprise the previous exposure to myelosuppresive therapy agent to adjust these dosage.These principles can also be applied to through radioactive label hPAM4 and other therapeutic agent, comprise the combination of radiosensitizing medicine such as such as 5 FU 5 fluorouracil and cis-platinum etc.

Chimeric, humanization and human antibodies and its fragment are for interior therapeutic and diagnostic method.Therefore contemplate and diagnosis or therapeutic agent or its combination are delivered to target calibration method, comprise (i) and composition is provided, said composition comprises anti-cancer of pancreas antibody or its fragments such as such as chimeric, humanization or mankind PAM4 antibody with at least one diagnose and/or the conjugate of therapeutic agent, and (ii) uses to experimenter and diagnose or treat antibody conjugates.In a preferred embodiment, anti-cancer of pancreas antibody and its fragment are humanized or the whole mankind.

Another embodiment relates to the method for the treatment of malignant tumour, comprise separately or use with one or more other therapeutic agent can in conjunction with naked or that put together anti-cancer of pancreas antibody, antibody fragment or the fusion of epi-position of the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) being positioned at MUC5ac, such as PAM4 antibody.Other therapeutic agent can before described antibody, simultaneously or add afterwards.In a preferred embodiment, described therapeutic agent is gemcitabine, and in a preferred embodiment, gemcitabine and hPAM4 radiate conjugate and give with fractionated dose scheme, and its dosage gives 800-1 weekly, 000mg/m lower than routine ²gemcitabine dosage continues 6 weeks.For example, when with interval procedure dosage ⁹⁰during Y-PAM4 combination, inject the fractionated dose repeated, be intended to be used as 200-380mg/m ²the Radiosensitizers of gemcitabine.Technician it should be understood that described herein and claimed antibody, fusion and/or its fragment can with any known or therapeutic agent of describing, include but not limited to that heat shock protein 90 (Hsp90) is used together.

In the another kind of form of multi-mode treatment, experimenter accepts immunoconjugates and the chemotherapeutic combination of standard cancer.For example, " CVB " (1.5g/m ²endoxan, 200-400mg/m ²etoposide and 150-200mg/m ²bCNU) be the scheme being used for the treatment of non-Hodgkin's lymphoma.Patti etc., Eur.J.Haematol.51:18 (1993).Other suitable combination chemotherapy scheme is well known to those skilled in the art.See such as Freedman etc., " Non-Hodgkin'sLymphomas ", CANCERMEDICINE, the 2nd volume, the 3rd edition, Holland etc. (volume), 2028-2068 page (Lea & Febiger1993).Illustratively, the first generation chemotherapy regimen being used for the treatment of intermediate non-Hodgkin's lymphoma (NHL) comprises C-MOPP (endoxan, vincristine, procarbazine and metacortandracin) and CHOP (endoxan, Doxorubicin, vincristine and metacortandracin).Available second generation chemotherapy regimen is m-BACOD (methotrexate (MTX), bleomycin, Doxorubicin, endoxan, vincristine, dexamethasone and formyl tetrahydrofolic acid), and suitable third generation scheme is MACOP-B (methotrexate (MTX), Doxorubicin, endoxan, vincristine, metacortandracin, bleomycin and formyl tetrahydrofolic acid).Other available medicine comprises phenyl butyrate, bendamustine and Bryostatin-1.

Present invention contemplates anti-cancer of pancreas antibody separately, to use as naked antibody or antibody fragment with its fragment (comprising fusion and its fragment), or use as multi-mode therapy.Preferably, antibody is humanization or whole mankind PAM4 antibody or its fragment.Multi-mode therapy also comprises and utilizes naked anti-cancer of pancreas antibody and supplementation administration is naked antibody, fusion or the immunotherapy of other antibody in immunoconjugates form.For example, humanization or completely mankind PAM4 antibody can with the naked antibody of another kind or humanization PAM4 or other antibody puted together with isotope, one or more chemotherapeutics, cell factor, toxin or its combine.For example, present invention contemplates at other pancreatic neoplasm associated antibodies, such as CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, anti-Le ^aantibody and for other Lewis antigen (such as, Le (y)) antibody and for carcinomebryonic antigen (CEA or CEACAM5), CEACAM6, colon-specific antigen-p (CSAp), MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC16, MUC17, HLA-DR, CD40, CD74, CD138, HER2/neu, EGFR, EGP-1, EGP-2, angiogenesis factor (such as, VEGF, PlGF), IGF (IGF), tenascin, platelet derived growth factor and IL-6 and oncogene are (such as, bcl-2, Kras, p53) product, before the antibody of cMET and the antibody for neoplasm necrosis material, combination or treat with PAM4 antibody that is naked or that put together or its fragment afterwards.

These solid tumor antibody can be naked or put together in especially medicine, toxin, isotope, radioactive nuclide or immunomodulator.Multiple different Antibody Combination can be built, and use with form that is naked or that put together.Alternatively, different naked Antibody Combination may be used for and other therapeutic agent, such as cytotoxic drug or with continuously, simultaneously or the radiating composite in succession given use.

Antibody and its fragment use can by intravenous, artery, in peritonaeum, in muscle, in subcutaneous, pleura, in sheath, through region catheter infusion or directly intralesional injection realize.When by injection administration of antibodies, use and can be undertaken by continuous infusion or injected by single or multiple.

Immunoconjugates of the present invention can through preparation so that via such as injecting or continuous infusion and using through intravenous.Preferably, antibody infusion of the present invention was less than in the time period of about 4 hours, and more preferably, was less than the time period of about 3 hours.For example, first can at 30 minutes, preferably even infusion 25-50mg in 15 minutes, all the other are infusion in ensuing 2-3 hour.Ejection preparation can be unit dosage forms, as in the ampoule bottle or the multi-dose container form that add antiseptic.Described composition in suspending liquid, solution or the emulsion form such as in oiliness or aqueous vehicles, and can contain preparaton, such as suspending agent, stabilizing agent and/or spreading agent.Alternatively, active component can in powder type so that before use with suitable mediator, and such as aseptic apirogen water is restored.

Other pharmaceutical methods can be adopted control the acting duration for the treatment of conjugate.Can complexing be carried out by using polymkeric substance or adsorb immunoconjugates and prepare Co ntrolled release preparation.For example, biocompatible polymer comprises the polyanhydride copolymer matrix of poly-(ethane-acetic acid ethyenyl ester) copolymer matrix and stearic acid dimer and decanedioic acid.Sherwood etc., Bio/Technology10:1446 (1992).The speed that immunoconjugates or antibody discharge from this type of matrix depends on the molecular weight of immunoconjugates or antibody, the amount of Medium Culture immunoconjugates or antibody and the size of discrete particles.Saltzman etc., Biophys.J.55:163 (1989); Sherwood etc., ibid.Other solid dosage forms is described in Ansel etc., PHARMACEUTICALDOSAGEFORMSANDDRUGDELIVERYSYSTEMS, the 5th edition (Lea & Febiger1990); With Gennaro (volume), REMINGTON ' SPHARMACEUTICALSCIENCES, in the 18th edition (MackPublishingCompany1990) and its revised edition.

More generally, the factors such as such as patient age, body weight, height, sex, general medicine symptom and medical history will be depended on to the dosage of the immunoconjugates of human administration and change.May need to provide the immunoconjugates in about 1mg/kg to 25mg/kg scope, antibody fusion protein dosage in infusion format in single dose intravenous to experimenter, but also can depend on the circumstances and use lower or higher dosage.The dosage of 1-20mg/kg is such as 70-1,400mg for 70kg patient or is 41-824mg/m for 1.7m patient ².Dosage can optionally repeat, and such as, the weekly 4-10 of continuing week, weekly lasting 8 weeks or weekly continue 4 weeks.Also can give by lower frequency, such as continue some months week about or monthly or per once continue many individual month season, depending on the needs in maintenance therapy.

Alternatively, antibody can use a dosage in every 2 or 3 weeks, repeats at least 3 dosage altogether.Or antibody can use twice weekly, continue 4-6 week.If dosage is down to about 200-300mg/m ²(for 1.7m patient per dose 340mg, or for 70kg patient 4.9mg/kg), then can use weekly once or even also continue for 4 to 10 weeks twice.Alternatively, dosage can reduce, and namely within every 2 or 3 weeks, once also continues the 2-3 month.But determined even higher dosage, such as 20mg/kg is weekly or every 2-3 week once can use repeat administration circulation by slow intravenous infusion.Dosage regimen can optionally repeat with other interval, and dosage can be given by various parenteral route when suitably adjusting dosage and scheme

Immunoconjugates

Anti-cancer of pancreas antibody and its fragment can be puted together at least one treatment and/or diagnosticum for treatment or diagnosis.For immunotherapy, target is by the radioactivity of cytotoxicity dosage, toxin, antibody and/or drug delivery to target cell, makes to minimize the exposure of non-target tissue simultaneously.Preferably, anti-cancer of pancreas antibody is used for diagnosis and/or treatment pancreatic neoplasm.

Any antibody, antibody fragment and fusion can use multiple technologies as known in the art and one or more are treated or diagnosticum is puted together.One or more treatments or diagnosticum can be connected to each antibody, antibody fragment or fusion, such as, by reagent being puted together the carbohydrate portions in antibody Fc district.If Fc district does not exist (such as when some antibody fragment), likely carbohydrate portions is introduced and may be connected with in treatment or the antibody of diagnosticum or the variable region of light chain of antibody fragment.See such as Leung etc., JImmunol.154:5919 (1995); Hansen etc., U.S. Patent number 5,443,953 (1995); Leung etc., U.S. Patent number 6,254,868, the embodiment part of each patent is incorporated herein by reference.

For via antibody carbohydrate part, by peptide, the method for puting together in antibody component is well known to those skilled in the art.See such as Shih etc., IntJCancer41:832 (1988); Shih etc., IntJCancer46:1101 (1990); With Shih etc., U.S. Patent number 5,057,313, the embodiment part of this patent is incorporated to herein by reference.Conventional method relates to the antibody component that makes to have already oxidised carbohydrate portions and has at least one free amine function and load has the carrier polymer of multiple therapeutic agent (such as peptide or medicine) to react.This reaction produces initial schiff bases (imines) binding, and this binding can be stablized by being reduced into secondary amine, to form final conjugate.

Antibody fusion protein or multi-specificity antibody comprise two or more antibody or its fragment, can be connected at least one therapeutic agent and/or diagnosticum separately.Therefore, one or more in the antibody of antibody fusion protein or its fragment can be connected with more than one treatment and/or diagnosticum.In addition, therapeutic agent without the need to identical, but can be different therapeutic agents, such as, medicine and radioactive isotope can be connected to same fusion.For example, IgG can use ¹³¹i carries out radioactive label and is connected to medicine. ¹³¹i can be incorporated to the tyrosine of IgG and be connected in the medicine of ε amino of IgG lysine.Both therapeutic agent and diagnosticum can also be connected to the SH group through reduction and the carbohydrate side chain of antibody.Alternatively, bispecific antibody can comprise one for the antibody of disease antigen or its fragment, and another can the haptenic antibody of target construct or its fragment for being connected to, for pre-targeting technology discussed above.

Treatment or diagnosticum can be connected to the hinge area of the antibody component through reduction via disulfide formation.As an alternative, this type of reagent can use heterobifunctional cross-linker, and such as N-succinyl 3-(2-pyridine two sulfo-) propionic ester (SPDP) is connected to antibody component.Yu etc., Int.J.Cancer56:244 (1994).The general technology puted together for this type of is known in the art.See such as Wong, CHEMISTRYOFPROTEINCONJUGATIONANDCROSS-LINKING (CRCPress1991); Upeslacis etc., " ModificationofAntibodiesbyChemicalMethods ", MONOCLONALANTIBODIES:PRINCIPLESANDAPPLICATIONS, Birch etc. (volume), 187-230 page (Wiley-Liss, Inc.1995); Price, " ProductionandCharacterizationofSyntheticPeptide-DerivedA ntibodies ", MONOCLONALANTIBODIES:PRODUCTION, ENGINEERINGANDCLINICALAPPLICATION, Ritter etc. (volume), 60-84 page (CambridgeUniversityPress1995).

Click chemistry

Alternative method chelating moiety, medicine or other functional group being connected to antibody, fragment or fusion relates to the reaction of use click chemistry.Click chemistry method is envisioned at first by little subunit being bonded together in modular fashion and produces the method for compound substance fast.(see such as Kolb etc., 2004, AngewChemIntEd40:3004-31; Evans, 2007, AustJChem60:384-95.) click chemistry reaction various forms be well known in the art, such as Huisgen1,3-dipole-diople interaction catalysed reaction of copper (Tornoe etc., 2002, JOrganicChem67:3057-64), this is commonly called " click-reaction ".Other replacement scheme comprises cycloaddition reaction, such as Diels-Alder, nucleophilic substitution (especially small tension receiving coil ring, as epoxy and aziridine cpd), the carbonylation of carbamide compound forms and relates to carbon-to-carbon double bond, the reaction of the alkynes in such as mercaptan-alkyne reaction.

Nitrine alkynes Huisgen cycloaddition reaction use copper catalyst in the presence of a reducing agent catalysis terminal alkyne group is connected to the reaction of the first molecule.Under the dimolecular existence comprising azide moiety, the alkynes of nitrine and activation reacts and is formed through Isosorbide-5-Nitrae-dibasic 1,2,3-triazoles.Catalysed reaction of copper at room temperature carries out, and specificity is enough to make usually without the need to carrying out purifying to reaction product.(Rostovstev etc., 2002, AngewChemIntEd41:2596; Tornoe etc., 2002, JOrgChem67:3057.) nitrine and alkynes functional group be inertia to a great extent to the biomolecule in aqueous medium, thus allow reaction to carry out in complex solution.The triazole formed is stable in the chemically, and can not experience enzymatic lysis, makes click chemistry product highly stable in living things system.Although copper catalyst is poisonous to living cells, the click chemistry reaction based on copper can be formed for immunoconjugates in vitro.

Propose without the covalent modification of copper click-reaction for biomolecule.(see such as Agard etc., 2004, JAmChemSoc126:15046-47).Ring strain is used to replace copper catalyst to promote [3+2] nitrine-alkynes cycloaddition reaction (ditto) without copper reaction.For example, cyclooctyne is 8 carbocyclic ring structure comprising inner acetylene bond.Closed loop configuration induction has highly reactive acetylene to azido group and substantive bond-angle deformation occurs, to form triazole.Thus, cyclooctyne derivant can be used for without copper click-reaction (ditto).

Reporting to some extent in (2010, AngewChemIntEd49:3065-68) such as Ning without copper click-reaction of another kind of type, it relates to alkynes-nitrone cycloaddition reaction that tension force promotes.In order to solve the sluggish speed of initial cyclooctyne, electron withdraw group is made to be connected to triple bond adjacent (ditto).The example of this type of cyclooctyne be substituted comprises bifluoride cyclooctyne, 4-dibenzo cyclooctyne alcohol and azacyclo-octyne (ditto).Substitute the alkynes-nitrone cycloaddition reaction relating to tension force promotion without copper reaction, obtain N-alkylation isoxazoline (ditto).It is reported, this reaction has abnormal reaction kinetics fast, and one pot of three step scheme (ditto) of site-specific sex modification for peptides and proteins.Prepare nitrone by the condensation of suitable aldehyde and N-methyl hydroxylamine, and cycloaddition reaction is carried out (ditto) in the potpourri of acetonitrile and water.The reaction of these and other known click chemistry may be used for chelating moiety being connected to antibody or other targeted molecular in vitro.

Therapeutic agent

Multiple treatment reagent can simultaneously or sequential application, maybe should put together in antibody of the present invention, such as medicine, toxin, oligonucleotides (such as, siRNA), immunomodulator, hormone, hormone antagonist, enzyme, enzyme inhibitor, radioactive nuclide, angiogenesis inhibitors, short Apoptosis agent etc.Therapeutic agent cited herein can be used for puting together in antibody, fragment or fusion, or for those reagent with naked antibody separate administration as above.

Therapeutic agent comprises such as chemotherapeutics, such as vinca alkaloids, anthracene nucleus, gemcitabine, table podophyllotoxin, taxane, antimetabolite, alkylating agent, microbiotic, SN-38, cox 2 inhibitor, antimitotic agent, Anti-angiogenesis and Apoptosis agent (particularly Doxorubicin, methotrexate (MTX), taxol, CPT-11, camptothecine), proteasome inhibitor, mTOR inhibitors, hdac inhibitor, tyrosine kinase inhibitor and the other medicines from these classifications and other classification anticancer.

Other available cancer chemotherapeutic drug comprises mustargen, alkyl sulfonic ester, nitroso ureas, triazenes, folacin, cox 2 inhibitor, antimetabolite, pyrimidine analogue, purine analogue, platinum coordination complex, mTOR inhibitors, tyrosine kinase inhibitor, proteasome inhibitor, hdac inhibitor, camptothecine and hormone.Suitable chemotherapeutics is described in the revised edition of REMINGTON'SPHARMACEUTICALSCIENCES the 19th edition (MackPublishingCo.1995) and GOODMANANDGILMAN'STHEPHARMACOLOGICALBASISOFTHERAPEUTICS the 7th edition (MacMillanPublishingCo.1985) and these publications.Other suitable chemotherapeutics, such as Experimental agents are well known by persons skilled in the art.

Useful certain drug can comprise 5 FU 5 fluorouracil, Ah method is for Buddhist nun, aplidin, azaribine, Anastrozole, anthracycline, Axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, Bryostatin-1, busulfan, calicheamycin, camptothecine, carboplatin, 10-hydroxycamptothecine, carmustine, Celebrex, Chlorambucil, neoplatin (CDDP), Cox-2 inhibitor, Irinotecan (CPT-11), SN-38, carboplatin, Cladribine, camptothecine, gram azoles is for Buddhist nun, endoxan, cytarabine, Dacarbazine, Dasatinib, dinaciclib, docetaxel, dactinomycin D, daunorubicin, Doxorubicin, 2-pyrrolin Doxorubicin (2-PDOX), pro-2PDOX, cyano group morpholinyl Doxorubicin, Doxorubicin glucosiduronic acid, epirubicin glucuronide, Erlotinib, estramustine, epipodophyllotoxin, Erlotinib, grace is for Nuo Te, estrogen receptor binding agents, Etoposide (VP16), Etoposide glucosiduronic acid, etoposide phosphate, Exemestane, FTY720, floxuridine (FUdR), 3', 5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, Flutamide, farnesyl protein transferase inhibitor, Flavopiridol, good fortune he for Buddhist nun, ganetespib, GDC-0834, GS-1101, Gefitinib, gemcitabine, hydroxycarbamide, according to Shandong for Buddhist nun, idarubicin, Ai Dailalisi, ifosfamide, Imatinib, leunase, Lapatinib, lenalidomide, formyl tetrahydrofolic acid, LFM-A13, lomustine, mustargen, melphalan, purinethol, Ismipur, amethopterin, mitoxantrone, mithramycin, mitomycin, mitotane, NVB, HKI-272, nilotinib, nitroso ureas, Aura handkerchief Buddhist nun, plicamycin, procarbazine, taxol, PCI-32765, Pentostatin, PSI-341, Raloxifene, Semustine, Sorafenib, streptozotocin, SU11248, Sutent, Tamoxifen, Temozolomide (aqueous form of DTIC), anti-platinum, Thalidomide, thioguanine, thiotepa, Teniposide, topotecan, uracil mastard, PTK787, vinorelbine, vinblastine, vincristine, vinca alkaloids and ZD1839.

In preferred embodiments, the conjugate of the related compounds such as camptothecine and such as SN-38 can be puted together in hPAM4 or other anti-cancer of pancreas antibody, such as, as U.S. Patent number 7,591, the U.S. Patent Application Serial Number 11/388 that on March 23rd, 994 and 2006 submits to, disclosed in 032, the embodiment part of each patent is incorporated herein by reference.

In another preferred embodiment, as U.S. Patent Application Serial Number 14/175, the prodrug forms of 2-PDOX disclosed in 089 (embodiment part is incorporated herein by reference) can in can use in conjunction with the form of immunoconjugates of anti-cancer of pancreas antibody of epi-position of the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) being positioned at MUC5ac.

In another preferred embodiment, hPAM4 antibody gives together with gemcitabine, and gemcitabine can before chimeric, humanization that is naked or that put together or mankind PAM4 antibody, give afterwards or simultaneously.Preferably, the hPAM4 antibody puted together or antibody fragment put together in radioactive nuclide.

Toxin can derive from animal, plant or microorganism.The toxin such as such as Pseudomonas exotoxin also can be complexed to or be formed the therapeutic agent portion of the immunoconjugates of anti-cancer of pancreas and hPAM4 antibody.Other toxin being applicable to prepare this type of conjugate or other fusion comprises ricin (WA), abrin, ribonuclease (RNA enzyme), DNA enzymatic I, staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, ranpirnase, Pseudomonas exotoxin and pseudomonad endotoxin.See such as Pastan etc., Cell47:641 (1986); Goldenberg, CA--ACancerJournalforClinicians44:43 (1994); Sharkey and Goldenberg, CA--ACancerJournalforClinicians56:226 (2006).Other toxin be suitable for is well known by persons skilled in the art, and is disclosed in U.S. Patent number 6,077, and in 499, embodiment part is incorporated herein by reference.

The immunomodulators such as such as cell factor also can be puted together in or form the therapeutic agent portion of immunoconjugates, or can use together with antibody, antibody fragment or fusion, but do not put together.Fusion can comprise one or more antibody or its fragment of being combined with in not synantigen.For example, fusion can in conjunction with the epi-position of the second to the 4th rich Cysteine domains (amino acid residue 1575-2052) being positioned at MUC5ac, and immunity regulatory cell or the factor.Alternatively, experimenter can accept the cell factor of naked antibody, antibody fragment or fusion and separate administration, described cell factor can before naked antibody is used, simultaneously or use afterwards.As used herein, term " immunomodulator " comprises cell factor, lymphokine, monokine, stem cell factor, lymphotoxin, Hemopoietic factor, colony stimulating factor (CSF), interferon (IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxain, relaxation precipitinogen, follicle-stimulating hormone (FSH) (FSH), thyrotropic hormone (TSH), luteinizing principle (LH), hepatocyte growth factor, prostaglandin, fibroblast growth factor, prolactin, galactagogin, OB albumen, TGF (TGF), TGF-α, TGF-β, IGF (IGF), hematopoietin, TPO, TNF (TNF), TNF-α, TNF-β, Miao Le Shi pipe inhibiting substances, mouse promoting sexual gland hormone related peptide, inhibin, activin, vascular endothelial growth factor, integrin, interleukin (IL), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), interferon-' alpha ', interferon-beta, interferon-γ, the S1 factor, IL-1, IL-1cc, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21 and IL-25, LIF, kit-ligand, FLT-3, angiostatin, thrombospondin, Endostatin and lymphotoxin.

Therapeutic agent can comprise one or more radioactive isotopes that can be used for treating pathological tissues.Especially can radiotherapy nucleic include but not limited to ¹¹¹in, ¹⁷⁷lu, ²¹²bi, ²¹³bi, ²¹¹at, ⁶²cu, ⁶⁴cu, ⁶⁷cu, ⁹⁰y, ¹²⁵i, ¹³¹i, ³²p, ³³p, ⁴⁷sc, ¹¹¹ag, ⁶⁷ga, ¹⁴²pr, ¹⁵³sm, ¹⁶¹tb, ¹⁶⁶dy, ¹⁶⁶ho, ¹⁸⁶re, ¹⁸⁸re, ¹⁸⁹re, ²¹²pb, ²²³ra, ²²⁵ac, ⁵⁹fe, ⁷⁵se, ⁷⁷as, ⁸⁹sr, ⁹⁹mo, ¹⁰⁵rh, ¹⁰⁹pd, ¹⁴³pr, ¹⁴⁹pm, ¹⁶⁹er, ¹⁹⁴ir, ¹⁹⁸au, ¹⁹⁹au, ²¹¹pb and ²²⁷th.The decay energy that radiotherapy nucleic has, preferably 20 to 6, within the scope of 000keV, for Auger emitter preferably in the scope of 60 to 200keV, is 100-2,500keV for beta emitter, and is 4,000-6,000keV for alpha emitter.The maximum decay energy that available beta particle launches nucleic is preferably 20-5,000keV, is more preferably 100-4,000keV, and is most preferably 500-2,500keV.Radioactive nuclide in fact with the decay of Auger emitted particle is also preferred.For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161, Os-189m and Ir-192.Available beta particle launches decay energy preferably <1, the 000keV of nucleic, more preferably <100keV, and most preferably <70keV.Produce with alpha particle in fact and the radioactive nuclide of decay is also preferred.This type of radioactive nuclide includes but not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, Th-227 and Fm-255.The decay energy that available alpha particle launches radioactive nuclide is preferably 2,000-10,000keV, be more preferably 3,000-8,000keV, and be most preferably 4,000-7,000keV.

For example, ⁶⁷cu is regarded as one of more promising Radioimmunotherapy radioactive isotope due to its 61.5 hr half-life and abundant β particle and gamma-rays supply, and it can use sequestrant to put together in antibody bromoacetamido-benzyl-triethylammonium tetrakis tetraacethyl (TETA).Alternatively, ⁹⁰y launches energetic beta particle, and it can use diethylene-triamine pentaacetic acid (DTPA), or more preferably uses DOTA to be coupled to antibody, antibody fragment or fusion.Make ⁹⁰y is coupled to antibody or can the method for target construct be well known in the art, and can use this type of known method any.(see such as U.S. Patent number 7,259,249, embodiment part is incorporated to herein by reference.Also see Lind é n etc., ClinCancerRes.11:5215-22,2005; Sharkey etc., JNuclMed.46:620-33,2005; Sharkey etc., JNuclMed.44:2000-18,2003.)

Other potential radiotherapy isotope comprises ¹¹c, ¹³n, ¹⁵o, ⁷⁵br, ¹⁹⁸au, ²²⁴ac, ¹²⁶i, ¹³³i, ⁷⁷br, ^113min, ⁹⁵ru, ⁹⁷ru, ¹⁰³ru, ¹⁰⁵ru, ¹⁰⁷hg, ²⁰³hg, ^121mte, ^122mte, ^125mte, ¹⁶⁵tm, ¹⁶⁷tm, ¹⁶⁸tm, ¹⁹⁷pt, ¹⁰⁹pd, ¹⁰⁵rh, ¹⁴²pr, ¹⁴³pr, ¹⁶¹tb, ¹⁶⁶ho, ¹⁹⁹au, ⁵⁷co, ⁵⁸co, ⁵¹cr, ⁵⁹fe, ⁷⁵se, ²⁰¹tl, ²²⁵ac, ⁷⁶br, ¹⁶⁹yb etc.

In another embodiment, radiosensitizer can combinationally use with antibody that is naked or that put together or antibody fragment.For example, radiosensitizer can combinationally use with through radiolabeled antibody or antibody fragment.When with independent compared with radiolabeled antibody or antibody fragment are treated time, adding radiosensitizer can strengthen effect.Radiosensitizer is described in D.M.Goldenberg (volume), in CANCERTHERAPYWITHRADIOLABELEDANTIBODIES, CRCPress (1995).Comprise gemcitabine, 5 FU 5 fluorouracil and cis-platinum to other relevant typical radiosensitizer that this technology uses together, and combine with external irradiation and be used for the treatment of various cancer, comprise cancer of pancreas.Therefore, we have studied gemcitabine and (it is believed that it is radiosensitizing dosage (weekly 200mg/m ², 4 weeks) gemcitabine) with fractionated dose ⁹⁰the combination of Y-hPAM4, and the objective evidence (by NCI-CTCV.3 standard, without 3-4 level toxicity) observing that after the single circulation being proved to be the good this combination of tolerance cancer of pancreas alleviates.

For thermal neutron activation therapy, having load has the antibody of the accrete carrier of boron or its fragment will be influenced in a similar fashion usually.But, should wait for until non-targeted immunoconjugates is eliminated before underway son irradiation.Use can be able to be accelerated to remove in conjunction with the anti-id AB of anti-cancer of pancreas antibody.About the description of this General Principle, see U.S. Patent number 4,624,846.For example, the boron condiment such as such as carborane can be connected to antibody.As is well known in the art, carborane can be prepared as and have carboxyl functional group on pendant side chain.The connection of the carriers such as carborane and such as glycosaminoglycan can realize by the carboxyl of activated carbon borine with the amine condensation on carrier.Middle conjugate is made to put together in antibody subsequently.After administration of antibodies conjugate, irradiate activation boron condiment by thermal neutron, and be converted into radioactive atom, it is by α-emission decay, thus produces high toxicity short-range effect.

RNA interfering

The therapeutic agent of another kind of type is RNAi or siRNA.RNA interference (RNAi) is silencing complex (RISC) mediation of being induced by RNA, and by causing (Rand etc., 2005, Cell123:621-29) with the short dsrna molecule of catalysis RISC component Ah lattice protein-interacting.The type of RNAi molecule comprises microRNA (miRNA) and siRNA (SiRNA).RNAi material by complementary base pairing in conjunction with mRNA (mRNA), and can carry out inhibition of gene expression by PTGS.After being combined with complementary mRNA material, RNAi is by the cracking of Ah lattice's protein component induction mRNA molecule of RISC.In further feature, for specific gene target, the degrees of specificity of miRNA with siRNA to specific gene target is different, and wherein siRNA has relative specificity to particular target gene, and miRNA suppresses the translation of multiple mRNA material.

RNAi has attempted for various disease states by suppressing the therapeutical uses of selected gene expression realization, such as macular degeneration and respiratory syncytial virus infection (Sah, 2006, LifeSci79:1773-80).Show, the function of siRNA in host cell is that defend against computer virus infects, and has extensively verified that siRNA can be used as an approach of antiviral therapy (see such as Zhang etc. 2004, NatureMed11:56-62; Novina etc., 2002, NatureMed8:681-86; Palliser etc., 2006, Nature439:89-94).Also attempted siRNA for cancer therapy.The siRNA transfection HPV positive cervical cancer cell of Fujii etc. (2006, IntJOncol29:541-48) for HPVE6 and E7, and inhibit tumor growth.It is reported, in the breast cancer cell of siRNA mediation, different mucin is expressed to strike and is lowly inhibit experimental lung metastasis of cancer (Brown and Ruoslahti, 2004, CancerCell5:365-74).

Attempt providing siRNA targeted delivery, to reduce the possibility of toxicity of missing the target.SiRNA is delivered to circulating cells by the Fab fragment for HIV envelope protein that Song etc. (2005, NatBiotechnol23:709-17) use nucleoprotamine to put together.RGD peptide is puted together in nano particle by Schiffelers etc. (2004, NuclAcidsRes32:e149), anti-vegf R2siRNA is delivered to tumour, and suppresses the neonate tumour blood vessel in nude mice and growth rate.Dickerson etc. (2010, Cancer10:10) use through the functionalized nanogel of anti-eph A2 acceptor peptide, to carry out chemical sensitization with the siRNA for EGFR to ovarian cancer cell.The magnetic nano-particle that dendritic macromole is puted together has been applied to the targeted delivery (Pan etc., 2007, CancerRes67:8156-63) of antisense survivin oligodeoxynucleotide.

Technician it should be understood that any siRNA or RNA interfering material all can be connected to theme antibody.SiRNA and RNAi material for multiple target is well known in the art, and this type of known oligonucleotides material any all can be used in claimed method and composition.

The known siRNA material with potential use comprise to following thing have specific those: IKK-γ (United States Patent (USP) 7,022,828); VEGF, Flt-1 and Flk-1/KDR (United States Patent (USP) 7,148,342); Bcl2 and EGFR (United States Patent (USP) 7,541,453); CDC20 (United States Patent (USP) 7,550,572); Transducin (β) sample 3 (United States Patent (USP) 7,576,196); KRAS (United States Patent (USP) 7,576,197); Carbonic anhydrase II (United States Patent (USP) 7,579,457); Complement component 3 (United States Patent (USP) 7,582,746); Interleukin 1 receptor associated kinase 4 (IRAK4) (United States Patent (USP) 7,592,443); Survivin (United States Patent (USP) 7,608,7070); Superoxide dismutase 1 (United States Patent (USP) 7,632,938); MET proto-oncogene (United States Patent (USP) 7,632,939); Kind of starch β forerunner albumen (APP) (United States Patent (USP) 7,635,771); IGF-1R (United States Patent (USP) 7,638,621); ICAM1 (United States Patent (USP) 7,642,349); Complement factor B (United States Patent (USP) 7,696,344); P53 (7,781,575) and apolipoprotein B (7,795,421), the embodiment part of each patent is incorporated herein by reference.

Other siRNA material can purchased from known commercial source, such as Sigma-Aldrich (StLouis, MO), Invitrogen (Carlsbad, CA), SantaCruzBiotechnology (SantaCruz, CA), Ambion (Austin, TX), Dharmacon (ThermoScientific, Lafayette, CO), Promega (Madison, WI), MirusBio (Madison, and Qiagen (Valencia, CA) etc. WI).The siRNA Material Source that other can openly obtain comprises the siRNAdb database at bioinformatics center, Stockholm, MIT/ICBPsiRNA database, the RNAi consortium shRNA storehouse of Boulder research institute and the Probe database of NCBI.For example, in NCBIProbe database, 30 are had, 852 kinds of siRNA materials.Technician it should be understood that for any related gene, has devised any one siRNA material, and the Software tool that can openly obtain maybe can be used easily to design.This type of siRNA material any can use experimenter DNL ^tMcompound is sent.

The exemplary siRNA material reported is listed in Table 1.Although siRNA sends with two-chain molecular form, for simplicity, illustrate only sense strand sequence in table 1.

The exemplary siRNA sequence of table 1.

Diagnosticum

In the linguistic context of the application, term " diagnosis " or " detection " are used interchangeably.In view of diagnosis typically refers to the particular pathologies state of definition tissue, therefore detect and identify and locate tissue containing specific antigen, pathology or biosome.

Theme antibody and fragment can carry out detectable label by antibody is connected to enzyme.When hatching antibody-enzyme conjugate when there is suitable substrate, enzyme part and substrate reactions, produce the chemical part that such as can pass through spectrophotometric, fluorescence or range estimation means and detect.The example that can be used for the enzyme that can detect ground labelled antibody comprises the enzyme of malic dehydrogenase, Golden yellow staphylococcal nuclease, δ-V-steroid isomeras, Alcohol Dehydrogenase from Yeast, α-glycerophosphate dehydrogenase, triose-phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, alpha-galactosidase, ribonuclease, urease, hydrogen peroxidase, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase and acetylcholinesterase.

Immunoconjugates can comprise one or more radioactive isotopes that can be used for detecting pathological tissues.Useful especially diagnostic radioactive nucleic includes but not limited to ¹¹⁰in, ¹¹¹in, ¹⁷⁷lu, ¹⁸f, ⁵²fe, ⁶²cu, ⁶⁴cu, ⁶⁷cu, ⁶⁷ga, ⁶⁸ga, ⁸⁶y, ⁹⁰y, ⁸⁹zr, ^94mtc, ⁹⁴tc, ^99mtc, ¹²⁰i, ¹²³i, ¹²⁴i, ¹²⁵i, ¹³¹i, ^154-158gd, ³²p, ¹¹c, ¹³n, ¹⁵o, ¹⁸⁶re, ¹⁸⁸re, ⁵¹mn, ^52mmn, ⁵⁵co, ⁷²as, ⁷⁵br, ⁷⁶br, ^82mrb, ⁸³sr or other γ-, β-or positron emitter, its decay energy preferably 20 to 4, in the scope of 000keV, more preferably 25 to 4, in the scope of 000keV, even more preferably 25 to 1, in the scope of 000keV, and more preferably in the scope of 70 to 700keV.Total decay energy preferably <2,000keV of available positron-emitting radioactive nucleic, is more preferably less than 1,000keV, and most preferably <700keV.The radioactive nuclide that can be used as diagnosticum when utilizing gamma-radiation to detect includes but not limited to: ⁵¹cr, ⁵⁷co, ⁵⁸co, ⁵⁹fe, ⁶⁷cu, ⁶⁷ga, ⁷⁵se, ⁹⁷ru, ^99mtc, ¹¹¹in, ^114min, ¹²³i, ¹²⁵i, ¹³¹i, ¹⁶⁹yb, ¹⁹⁷hg and ²⁰¹tl.The decay energy that available gamma-radiation launches radioactive nuclide is preferably 20-2000keV, is more preferably 60-600keV, and is most preferably 100-300keV.

Diagnosis experimenter in cancer method can by use diagnosis immunoconjugates and detection and location realize in the diagnostic flag be connected with immunoconjugates of cancer or tumour.Antibody, antibody fragment and fusion can be puted together in diagnosticum, maybe can use can using in pre-targeting technology by target construct of being connected with diagnosticum.Discuss the radioreagent that can be used as diagnosticum above.Suitable on-radiation diagnosticum is applicable to magnetic resonance imaging, X-ray, computer tomography or ultrasonic contrast preparation.For example, magnetic imaging agent comprises the complex compound that the non-radioactive metal such as such as manganese, iron and gadolinium combine with the metal-chelate comprising 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs.See the U.S. Patent Application Serial Number 09/921,290 (now abandoning) that October 10 calendar year 2001 submits to, embodiment part is incorporated herein by reference.Can also use other preparation, such as PET scans nucleotide, preferably ¹⁸f.

The contrast preparation such as such as MRI contrast agent comprise such as gadolinium ion, lanthanum ion, dysprosium ion, ferric ion, manganese ion or other suitable mark, and CT contrast preparation and acoustic contrast agent can be used as diagnosticum.The paramagnetic ion be suitable for comprises chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III), and wherein gadolinium is particularly preferred.

Can be used for other linguistic context, such as the ion of X-radial imaging, include but not limited to lanthanum (III), gold (III), plumbous (II) and bismuth (III).Fluorescence labeling comprises rhodamine, fluorescein and renographin.Rhodamine and fluorescein are connected via isothiocyanates intermediate usually.

Metal also can be used for diagnosticum, comprises those metals for mr imaging technique.These metals include but not limited to: gadolinium, manganese, iron, chromium, copper, cobalt, nickel, dysprosium, rhenium, europium, terbium, holmium and neodymium.In order to make antibody bearing radioactive metal or paramagnetic ion be loaded into, be necessary to make it and have to be connected with multiple chelation group so that the reagent reacting in the long-tail portion in conjunction with described ion.This type of afterbody can be polymkeric substance, such as polylysine, polysaccharide or have can with such as ethylenediamine tetraacetic acid (EDTA), diethylene-triamine pentaacetic acid (DTPA), porphyrin, polyamine, crown ether, two thiosemicarbazones, poly-oxime and known can be used for the side base that the chelation groups such as the similar group of this object are combined other to derive or can derivative chain.

Standard chemical reaction is used to make chelate and antibody, fusion or its fragment coupling.Chelate normal through make it possible to when exist the loss of few immunoreactivity and few assemble and/or internal crosslinking form key with described molecule group be connected to antibody.For chelate being puted together the U.S. Patent number 4 being disclosed in " AntibodyConjugates " by name authorizing Hawthorne on April 25th, 1989 in other rarer method of antibody and reagent, 824, in 659, the embodiment part of this patent is incorporated herein by reference.The combination of useful especially metal-chelate comprises 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs and the diagnosis isotope within the scope of 20 to 2,000keV Universal Energy and uses together.This chelate is when can be used for MRI with during the non-radioactive metal complexings such as such as manganese, iron and gadolinium.The macrocyclic chelate things such as such as NOTA, DOTA and TETA and various metals and radioactive metal, use together with copper with radioactive nuclide gallium, yttrium the most respectively.Its applicable associated metal can be made to make this type of metal-chelate complex compound highly stable by customization ring size.Other ring-like chelate, such as macrocyclic polyether is also contained in the present invention, and it is concerned for RAIT because stably combining the nucleic such as such as 223Ra.

Radiopaque material and Marker material are for strengthening X-ray and computer tomography, and comprise iodine compound, barium compound, gallium compound, thallium compound etc.Specific compound comprises barium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid, iodamide, adipiodone, iodoxamic acid, iodine paddy amine, Iohexol, Iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamide meglumine, the U.S. acid in iodine west, Iotasul, iotetric acid, iotalamic acid, iotroxic acid, ioxaglic acid, Ioxotrizoic Acid, iopodic acid, meglumine, metrizamide, Sodium Metrizoate, propyl iodone and thallous chloride.

Antibody, antibody fragment and fusion can also be marked with fluorescent chemicals.Existence through fluorescently-labeled MAb is exposed to the light of suitable wavelength by making antibody and detects gained fluorescence and determine.Fluorescent labelling compound comprises Alexa350, Alexa430, AMCA, aminacrine, BODIPY630/650, BODIPY650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, chloro-2', the 7'-dimethoxyfluorescein of 5-carboxyl-4', 5'-bis-, 5-carboxyl-2', 4', 5', 7'-tetrachlorofluorescein, CF, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxyl tetramethyl is amino, waterfall is blue, Cy2, Cy3, Cy5, 6-FAM, dansyl Cl, fluorescein, fluorescein isothiocynate, fluorescamine, HEX, 6-JOE, NBD (7-nitro benzo-2-oxa--1,3-diazole), Russia strangles green 488, Russia strangles green 500, Russia strangles green 514, the Pacific Ocean is blue, phthalic acid, terephthalic acid (TPA), m-phthalic acid, cresols is solid purple, cresols royal purple, brilliant cresyl blue, p-aminobenzoic acid, erythrosine, phthalocyanine, phthaladehyde, azomethine, Hua Jing, xanthine, succinylfluoresceins, rare earth metal cryptate, terpyridyl diamines europium, europium cryptate or chelate, diamines, dicyanin, the blue dyestuff in La Jolla, allophycocyanin, different azurin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrocyanin (pec), phycoerythrin R, REG, rhodamine is green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (the different mercaptan of tetramethylrhodamin), tetramethylrhodamin and Texas red.Be particularly useful for flow cytometry through fluorescently-labeled antibody, but also can be used for endoscope and Ink vessel transfusing detection method.

Alternatively, antibody, antibody fragment and fusion can by making antibody and chemiluminescence compound coupling and carrying out marking with detecting.Existence through the MAb of chemiluminescent labeling is that the existence by detecting the luminescence occurred in chemical reaction process is determined.The example of chemiluminescent labeling compound comprises luminol, different luminol, aromatics acridinium ester, imidazoles, acridinium salt and oxalate.

Similarly, bioluminescent compound can be used for labelled antibody and its fragment.Bioluminescence can improve at catalytic protein the chemiluminescence type found in the biosystem of the efficiency of chemiluminescence reaction.The existence of bioluminescent protein determines by detecting luminous existence.The bioluminescent compound that can be used for marking comprises fluorescein, luciferase and aequorin.

Because described herein the method for malignant tumour in diagnosis experimenter, the composition comprised with comprising anti-cancer of pancreas MAb, fusion or its fragment carries out in-vitro diagnosis mensuration to the sample (body fluid, tissue or cell) from experimenter.The existence of the antibody that immunohistochemistry can be used for by having combined detects the existence of PAM4 antigen in cell or tissue.Preferably, the malignant tumour diagnosed is cancer.Most preferably, cancer is cancer of pancreas.

In addition, the sequestrant such as such as DTPA, DOTA, TETA or NOTA or the suitable peptide that is connected with the detectable labels such as such as fluorescence molecule or the such as cytotoxic agent such as heavy metal or radioactive nuclide can be puted together in theme antibody.For example, treat available immunoconjugates to obtain in antibody fusion protein by making photosensitizer or dyestuff put together.Fluorescent composition or the such as porphyrins etc. such as such as fluorophor and other chromogen to the dyestuff of visible ray sensitivity by making suitable light point to pathology for test-and-treat pathology.In treatment, this is called as optical radiation, light therapy or photokinesis therapy (Jori etc. (volume), PHOTODYNAMICTHERAPYOFTUMORSANDOTHERDISEASES (LibreriaProgetto1985); VandenBergh, Chem.Britain22:430 (1986)).In addition, the dye-coupling of monoclonal antibody and photoactivation has been made, to realize light therapy.Mew etc., J.Immunol.130:1473 (1983); The same, CancerRes.45:4380 (1985); Oseroff etc., ProcNatl.Acad.Sci.USA83:8744 (1986); The same, Photochem.Photobiol.46:83 (1987); Hasan etc., Prog.Clin.Biol.Res.288:471 (1989); Tatsuta etc., LasersSurg.Med.9:422 (1989); Pelegrin etc., Cancer67:2529 (1991).

Be particularly useful for that endoscope is carried out to the pathological tissues such as such as malignant tumour or the relevant target antigen of cell cluster to antibody conjugate or for the fluorescence of bispecific pre-targeting method and radioreagent, perform the operation in or Ink vessel transfusing detect, as GoIdenberg U.S. Patent number 5,716,595,6,096,289 and 6,387, disclosed in 350, the embodiment part of each patent is incorporated herein by reference, particularly γ-, β-and positron emitter.When being diffused into such as colon etc. and allowing the structure of endoscope, endoscopic applications can be used.Radioactive nuclide for positron emission tomography includes but not limited to: F-18, Mn-51, Mn-52m, Fe-52, Co-55, Cu-62, Cu-64, Ga-68, As-72, Br-75, Br-76, Rb-82m, Sr-83, Y-86, Zr-89, Tc-94m, In-110, I-120 and I-124.Total decay energy preferably <2,000keV of available positron-emitting radioactive nucleic, is more preferably less than 1,000keV, and most preferably <700keV.The radioactive nuclide that can be used as diagnosticum when utilizing gamma-radiation to detect includes but not limited to: Cr-51, Co-57, Co-58, Fe-59, Cu-67, Ga-67, Se-75, Ru-97, Tc-99m, In-111, In-114m, I-123, I-125, I-131, Yb-169, Hg-197 and Tl-201.The decay energy that available gamma-radiation launches radioactive nuclide is preferably 20-2000keV, is more preferably 60-600keV, and is most preferably 100-300keV.

In-vitro diagnosis

Present invention contemplates the existence using anti-cancer of pancreas antibody to screen PAM4 antigen in biological sample in vitro.In these type of immunoassays, antibody, antibody fragment or fusion can use in liquid phase or be combined with solid phase carrier, as described below.For the object of in-vitro diagnosis, the antibody of any type can be used, such as muroid, chimeric, humanization or the mankind, because do not need to consider host immune response.

For determining that the example whether biological sample contains the screening technique of MUC5ac is radioimmunoassay (RIA).For example, in a kind of form of RIA, under the existence through radiolabeled MUC5ac, test substances is mixed with PAM4MAb.In the method, the amount through marking MUC5ac that the concentration of test substances is combined with MAb is inversely proportional to, and directly related with the amount of the free MUC5ac through marking.Other suitable screening technique to those skilled in the art will be apparent.

Alternatively, can carry out external test, wherein anti-cancer of pancreas antibody, antibody fragment or fusion are combined with solid phase carrier.For example, MAb can be connected to polymkeric substance, such as glycosaminoglycan, MAb is connected to insoluble carrier, such as, through polymer-coated bead, plate or pipe.

Other suitable external test will be apparent to those skilled in the art.Can change through the certain concentration of the antibody of detectable label and MUC5ac, incubation temperature and time and other condition determination, depend on various factors, comprise the concentration of MUC5ac in sample, the character of sample etc.The binding activities of anti-cancer of pancreas antibody samples can measure according to the method known.Those skilled in the art should be able to utilize normal experiment to determine operating conditions and the optimum determining condition of each mensuration.

The existence of the PAM4 antigen in biological sample can use enzyme linked immunosorbent assay (ELISA) (ELISA) to determine (such as, Gold etc., JClinOncol.24:252-58,2006).In direct competive ELISA, pure or half pure antigen preparation is combined with the solid carrier being insoluble to liquid to be tested or cell extract, and add a certain amount of soluble antibody through detectable label, to allow to detect and/or quantitatively solid phase antigen and the binary complex that formed between labelled antibody.

By contrast, " two determinant " ELISA, also referred to as " dibit point ELISA " or " sandwich assay ", need a small amount of antigen, and this mensuration does not need to carry out large-scale purification to antigen.Therefore, for the antigen detected in clinical sample, two determinant ELISA is better than direct competive ELISA.See the c-myc cancer protein such as used in two quantitative biopsy samples of determinant ELISA.Field etc., Oncogene4:1463 (1989); Spandidos etc., AntiCancerRes.9:821 (1989).

In two determinant ELISA, the MAb of a certain amount of un-marked or antibody fragment (" capture antibody ") are combined with solid carrier, test sample is contacted with capture antibody, and add a certain amount of soluble antibody through detectable label (or antibody fragment), to allow to detect and/or quantitatively capture antibody, antigen and the ternary complex that formed between labelled antibody.In linguistic context of the present invention, antibody fragment is the part of the anti-cancer of pancreas MAb of epi-position in conjunction with MUC5ac.The method of carrying out two determinant ELISA is known.See such as Field etc., the same; Spandidos etc., the same; With Moore etc., " Twin-SiteELISAsforfosandmycOncoproteinsUsingtheAMPAKSyst em ", METHODSINMOLECULARBIOLOGY, the 10th volume, 273-281 page (TheHumanaPress, Inc.1992).

In two determinant ELISA, soluble antibody or antibody fragment must the different MUC5ac epi-positions of epi-position that identify of binding and capturing ability antibody.Two determinant ELISA can be carried out to determine whether there is PAM4 antigen in biopsy samples.Alternatively, can carry out measuring to carry out quantitatively the amount of the MUC5ac existed in body fluid clinical sample.Quantitative measurement can be carried out dilution to carry out to purified MUC5ac by comprising.

Anti-cancer of pancreas MAb, fusion and its fragment are also suitable for formation determination kit.This type of kit can comprise through dividing with the support element receiving one or more container member such as such as bottle, pipe etc. under tight control, and described container member comprises independent immunoassays element separately.

Theme antibody, antibody fragment and fusion can also be used for detecting the existence of PAM4 antigen in the histotomy prepared by pathological sample.This type of in situ detection may be used for determining MUC5ac existence and determine MUC5ac check distribution in tissue.In situ detection can realize by the antibody through detectable label is applied to freezing histotomy.Research shows that PAM4 antigen is kept in paraffin-embedded section.The general technology of in situ detection is that those of ordinary skill is known.See such as Ponder, " CellMarkingTechniquesandTheirApplication ", MAMMALIANDEVELOPMENT:APRACTICALAPPROACH113-38Monk (volume) (IRLPress1987); And Coligan, 5.8.1-5.8.8 page.

Antibody, antibody fragment and fusion can by any suitable label parts, and such as radioactive isotope, enzyme, fluorescence labeling, dyestuff, chromogen, chemiluminescent labeling, bioluminescence marker or spin labeling carry out detectable label.

Label part can be by the radioactive isotope such as using gamma counter or scintillation counter or detected by means such as radioautograph.In preferred embodiments, diagnose conjugate be γ-, β-or Positron emitting isotopes.Label part during the present invention describes refers to and will produce the molecule of signal in predefined conditions.The example of label part comprises radioactive isotope, enzyme, fluorescence labeling, chemiluminescent labeling, bioluminescence marker and spin labeling.

Label part is combined to use with anti-cancer of pancreas antibody and realizes standard technique known in the art.Thus, typical method is described in in Publication about Document: Kennedy etc., ClinChimActa70:1 (1976); Schurs etc., Clin.Chim.Acta81:1 (1977); Shih etc., IntJCancer46:1101 (1990).

Above-mentioned external and in-situ detection method may be used for auxiliary to diagnose pathological condition or by stages.For example, these class methods may be used for detecting the tumour that such as cancer of pancreas etc. expresses PAM4 antigen.

In-vivo diagnostic/detection

Utilize and know through the various diagnostic imaging in vivo methods of radiolabeled MAb.In immunoscintigraphy technology, for example, with γ-transmitting labelled with radioisotope antibody, and introduce in patient.Gammacamera is used to detect γ-launch radioisotopic position and distribution.See such as Srivastava (volume), RADIOLABELEDMONOCLONALANTIBODIESFORIMAGINGANDTHERAPY (PlenumPress1988); Chase, " MedicalApplicationsofRadioisotopes ", REMINGTON'SPHARMACEUTICALSCIENCES, the 18th edition, Gennaro etc. (volume), 624-652 page (MackPublishingCo., 1990); And Brown, " ClinicalUseofMonoclonalAntibodies ", BIOTECHNOLOGYANDPHARMACY227-49, Pezzuto etc. (volume) (Chapman & Hall1993).

For diagnosing image, radioactive isotope can directly or by functional group in the middle of use be combined with antibody indirectly.Available middle functional group comprises sequestrant, such as ethylenediamine tetraacetic acid and diethylene-triamine pentaacetic acid.For example, see Shih etc., the same; With U.S. Patent number 5,057,313.

The radiation dose being delivered to patient is by selecting isotope to realize being detained in minimum half life period, most corpusculum and the permission detection of minimum isotopic mass and the best of breed of Measurement accuracy maintain alap level.Can in conjunction with anti-cancer of pancreas antibody and the radioisotopic example being suitable for diagnosing image comprise ^99mtc, ¹¹¹in and ¹⁸f.

For in-vivo diagnostic object, theme antibody, antibody fragment and fusion can also be marked with paramagnetic ion and multiple radiocontrast medium.The contrast preparation being particularly useful for magnetic resonance imaging comprises gadolinium, manganese, dysprosium, lanthanum or ferric ion.Other reagent comprises chromium, copper, cobalt, nickel, rhenium, europium, terbium, holmium or neodymium.Antibody and its fragment can also be puted together with ultrasonic contrast/reinforcing agent.For example, a kind of acoustic contrast agent is liposome.Further preferably, acoustic contrast agent is inflation liposome.

In a preferred embodiment, bispecific antibody can be puted together with contrast preparation.For example, bispecific antibody can comprise more than a kind of ultrasonic imaging image enhancing agent.In another preferred embodiment, contrast preparation is liposome.Preferably, liposome comprises the divalence DTPA-peptide being covalently attached to outer liposome surface.

Pharmaceutically suitable excipient

Other pharmaceutical methods can be used for controlling the acting duration of anti-cancer of pancreas antibody in treatment use.Controlled release preparation can be prepared by using polymkeric substance to come compound or adsorb antibodies, antibody fragment or fusion.For example, biocompatible polymer comprises the polyanhydride copolymer matrix of vinyl-vinyl acetate copolymer matrix and stearic acid dimer and decanedioic acid.Sherwood etc., Bio/Technology10:1446 (1992).The speed that antibody, antibody fragment or fusion discharge from this type of matrix depends on the molecular weight of antibody, antibody fragment or fusion, the amount of intramatrical antibody and the size of discrete particles.Saltzman etc., Biophys.J.55:163 (1989); Sherwood etc., simultaneously.Other solid dosage forms is described in in Publication about Document: Ansel etc., PHARMACEUTICALDOSAGEFORMSANDDRUGDELIVERYSYSTEMS, the 5th edition (Lea & Febiger1990); With Gennaro (volume), REMINGTON'SPHARMACEUTICALSCIENCES, the 18th edition (MackPublishingCompany1990) and its revised edition.

Antibody, its fragment or the fusion sent to experimenter can comprise one or more pharmaceutically suitable excipient, one or more other compositions or these some combinations.Antibody can be prepared according to known method, with prepare pharmaceutically can composition, thus by immunoconjugates or naked antibody and pharmaceutically suitable vehicle group synthetic mixture.SPBS is an example of pharmaceutically suitable excipient.Other suitable excipient is well known in the art.See such as Ansel etc., PHARMACEUTICALDOSAGEFORMSANDDRUGDELIVERYSYSTEMS, the 5th edition (Lea & Febiger1990); With Gennaro (volume), REMINGTON'SPHARMACEUTICALSCIENCES, the 18th edition (MackPublishingCompany1990) and its revised edition.

Immunoconjugates or naked antibody can through preparation with via such as injecting or continuous infusion and using through intravenous.Ejection preparation can be unit dosage forms, such as, be added with ampoule bottle or the multi-dose container of antiseptic.Composition in suspending liquid, solution or the emulsion form such as in oiliness or aqueous vehicles, and can contain the preparatons such as such as suspending agent, stabilizing agent and/or spreading agent.Alternatively, active component can be before use with suitable mediator, the powder type that such as aseptic apirogen water is restored.

Immunoconjugates, naked antibody, its fragment or fusion can also by subcutaneous or be applied to mammal by other parenteral route.In a preferred embodiment, antibody or its fragment use with the dosage of 20 to 2000 milligrams of albumen/agent.In addition, use and can be undertaken by continuous infusion or injected by single or multiple.In general, age of such as patient, body weight, height, sex, the factor such as the health patient's condition and medical history and changing will be depended on to the dosage of the immunoconjugates of human administration, fusion or naked antibody.Typically, provide in single dose intravenous or the immunoconjugates in about 1mg/kg to 20mg/kg scope of infusion format, antibody fusion protein or naked antibody dosage to experimenter ideally, but also can use lower or higher dosage, optionally determine.This dosage can optionally repeat, such as, weekly and continued for four to ten weeks, preferably weekly and continue eight weeks, and more preferably weekly and lasting surrounding.Can also be reduced it and give frequency, such as week about once and periods of months, or frequently, such as weekly twice or three times.Dosage gives by various parenteral route, and wherein dosage and scheme suitably adjust.

Kit

Various embodiment can relate to the kit of the component containing the pathological tissues being applicable to treatment or diagnosis patient.Exemplary kit can contain at least one antibody as described in this article, Fab or fusion.If do not prepared for such as being sent via alimentary canal by oral delivery etc. containing the composition using component, then can comprise can by the device of some other approach delivery of agents box components.The device of the type used for such as potential delivery etc. is the syringe for composition being injected experimenter's health.Suction apparatus can also be used.In certain embodiments, anti-cancer of pancreas antibody or its Fab can provide (such as in the form containing the sterile liquid formulations of antibody or the Prefilled syringe of lyophilized formulations or automatic injection pen, Kivitz etc., Clin.Ther.2006,28:1619-29).

Reagent constituents can be packaging together or assign in two or more containers.In some embodiments, container can be the bottle containing being suitable for the aseptic freeze-dried composite preparation restored.Kit can also comprise the damping fluid that one or more are suitable for restoring and/or diluting other reagent.Other used vessel includes but not limited to pouch, pallet, box, pipe etc.Reagent constituents can pack and Preservation in sterile condition in container.The another kind of assembly that can comprise is the operation instruction of kit.

Embodiment

Following examples are illustrative embodiment of the present invention, instead of the restriction to Claims scope.Embodiment discusses the research utilizing exemplary anti-cancer of pancreas monoclonal antibody (such as, PAM4).Utilize the clinical research of PAM4MAb display target to most of cancer of pancreas pathology of patient, and in normal structure, have no picked-up sign.Dosimetry shows, likely 10 to 20cGy/mCi is delivered to tumour, and wherein tumour and Red Bone Marrow Dose are than being 3:1 to 10:1.Data display PAM4 can be used for treating cancer of pancreas.

Embodiment 1. humanization PAM4MAb

In preferred embodiments, claimed method and composition utilizes antibody hPAM4, and this is the humanization IgG that muroid PAM4MAb produces for cancer of pancreas mucin.The humanization of muroid PAM4 sequence is for reducing mankind's against murine antibody-like (HAMA) reaction.In order to generating humanized PAM4, transferring to human framework district (FR) antibody sequence by muroid complementarity-determining region (CDR) from the heavy chain of mouse immuning ball protein and variable region of light chain, is its muroid homologue by some mankind FR residue substitutions subsequently.Humanized monoclonal antibodies is applicable in vitro and in vivo Diagnosis and Treat method.

Muroid PAM4MAb variable region framework sequence (Figure 1A and Figure 1B) showed with comparing of the known person antibody-like in Kabat database, PAM4V _kand V _hfR and human antibodies WalkerV _k(Fig. 3 A) and Wil2V _hthe FR of (Fig. 3 B) compares the sequence homology showing top respectively.Therefore, WalkerV is selected _k(Fig. 3 A) and Wil2V _h(Fig. 3 B) FR as human framework, respectively to wherein transplanting PAM4V _kand V _hmuroid CDR.But the FR4 sequence NEWM of end user's antibody-like replaces Wil2FR4 sequence to carry out humanization (Fig. 3 B) to PAM4 heavy chain.Than other FR residue, Ag is combined to the consideration with larger impact based on these residues, side joint has the several amino acid residues in the PAM4FR of presumption CDR to maintain in hPAM4.These residues are V _k21M, 47W, 59P, 60A, 85S, 87F and the 100G of (Fig. 3 A) and V _h27Y, 30P, 38K, 48I, 66K, 67A and the 69L of (Fig. 3 B).HPAM4V _kand V (SEQIDNO:16) _h(SEQIDNO:19) DNA and amino acid sequence are shown in Fig. 4 A and Fig. 4 B.

The modification strategy described by (Leung etc., 1994) such as use Leung, uses long oligonucleotide synthesis to build with the combination of PCR the V designed for hPAM4 _k(Fig. 4 A) and V _h(Fig. 4 B) gene.In order to build hPAM4V _hdomain, at the upper synthesis two kinds of long oligonucleotide hPAM4V of robotization DNA synthesizer (AppliedBiosystems) _ha (173 aggressiveness) and hPAM4V _hb (173 aggressiveness).

HPAM4V _ha represents hPAM4V _hthe nt17 to 189 of domain.

5′-AGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCGAGGCTTCTGGATACACATTCCCTAGCTATGTTTTGCACTGGGTGAAGCAGGCCCCTGGACAAGGGCTTGAGTGGATTGGATATATTAATCCTTACAATGATGGTACTCAGTACAATGAGAAG-3′(SEQIDNO：54)

HPAM4V _hb represents the hPAM4V with nt169 to 341 complementation _hthe minus strand of domain.

5′-AGGGTTCCCTGGCCCCAGTAAGCAAATCCGTAGCTACCACCGAAGCCTCTTGCACAGTAATACACGGCCGTGTCGTCAGATCTCAGCCTGCTCAGCTCCATGTAGGCTGTGTTGATGGACGTGTCCCTGGTCAGTGTGGCCTTGCCTTTGAACTTCTCATTGTACTGAGTACC-3′(SEQIDNO：55)

HPAM4V _ha and V _h3 ' the end sequence (21nt residue) of B is complimentary to one another.Under the PCR condition limited, hPAM4V _ha and V _h3 ' end the annealing of B forms the short double-stranded DNA of all the other long oligonucleotides of side joint.The end of each annealing serves as the primer that single stranded DNA is transcribed, and produces by hPAM4V _hnt17 to 341 form double-stranded DNA.At two kinds of short oligonucleotide hPAM4V _hbACK and hPAM4V _hunder the existence of FOR, amplification is further carried out to form total length hPAM4V to this DNA _h.Underlined part is the restriction site for subclone as shown in Figure 4 B.

hPAM4V _HBACK5′-CAGGTGCAGCTGCAGCAGTCTGGGGCTGAGGTGA-3′(SEQIDNO：56)

hPAM4V _HFOR5′-TGAGGAGACGGTGACCAGGGTTCCCTGGCCCCA-3′(SEQIDNO：57)

At 10 μ L10 × PCR damping fluid (500mMKCl, 100mMTrisHCl pH of buffer 8.3,15mMMgCl ₂), 2 μm of olhPAM4V _hbACK and hPAM4V _khPAM4V to minimum under the existence of FOR and 2.5 units Tag DNA polymerase (PerkinElmerCetus, Norwalk, Conn.) _ha and V _hb (rule of thumb determining) increases.This reaction mixture experience three circulations by 94 DEG C of sex change 1 minute, 45 DEG C of annealing 1 minute be polymerized at 72 DEG C the PCR (PCR) that forms for 1.5 minutes.Carry out after this program 27 circulations by 94 DEG C of sex change 1 minute, 55 DEG C of annealing 1 minute be polymerized at 72 DEG C the PCR (PCR) that forms for 1 minute.To hPAM4V _hdouble-strand pcr amplification product carry out gel-purified, carry out restrictive diges-tion at PstI and BstEII restriction site, and be cloned into heavy chain staging vector V _hin the complementary PstI/BstEII restriction site of pBS2, wherein V _hsequence is assembled completely, and the DNA sequence dna of coding translation initiation codon and secreting signal peptide is connected to 5 ' end with frame and intron sequences is in 3 ' end.V _hpBS2 is modified V _hpBS staging vector (Leung etc., Hybridoma, 13:469,1994), wherein introduces XhoI restriction site, to promote next subcloning steps in 16 bases of translation initiation codon upstream.By the V of assembling _hgene is subcloned in expression vector pdHL2 as XhoI-BamHI restriction fragment, described expression vector contains the Human IgG Heavy Chain and light chain expression box that control by IgH enhancer and MT1 promoter, and as the mouse d/fr gene of selection and amplification label thing.Because the heavy chain district of pdHL2 lacks BamHI restriction site, therefore this connection needs to use connexon to provide the bridge joint between HindIII site existing in the BamHI site of variable chains and pdHL2 carrier.By gained expression vector called after hPAM4V _hpdHL2.

In order to build the total length full length DNA of humanization VK sequence (Fig. 4 A), synthesize hPAM4VKA (157 aggressiveness) and hPAM4VKB (156 aggressiveness) as mentioned above.As described above by two kinds of short oligonucleotide hPAM4V _kbACK and hPAM4VKFOR increases hPAM4V _ka and V _kb.

HPAM4VKA represents the nt16 to 172 of hPAM4VK domain.

5′-CAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTCCAGCTACTTGTACTGGTACCAACAGAAACCAGGGAAAGCCCCCAAACTCTGGATTTATAGCACATCCAACCTGGCTTCTG-3′(SEQIDNO：58)

HPAM4V _kb represents the hPAM4V with nt153 to 308 complementation _kthe minus strand of domain.

5′-GTCCCCCCTCCGAACGTGTACGGGTACCTATTCCACTGATGGCAGAAATAAGAGGCAGAATCTTCAGGTTGCAGACTGCTGATGGTGAGAGTGAAGTCTGTCCCAGATCCACTGCCACTGAAGCGAGCAGGGACTCCAGAAGCCAGGTTGGATGTG-3′(SEQIDNO：59)

HPAM4V _ka and V _k3 ' the end sequence (20nt residue) of B is complimentary to one another.Under the PCR condition limited, hPAM4V _ka and V _k3 ' end the annealing of B forms the short double-stranded DNA of all the other long oligonucleotides of side joint.The end of each annealing serves as the primer that single stranded DNA is transcribed, and produces by hPAM4V _knt16 to 308 form double-stranded DNA.At two kinds of short oligonucleotide hPAM4V _kbACK and hPAM4V _kunder the existence of FOR, amplification is further carried out to form total length hPAM4V to this DNA _k.Underlined part is the restriction site for subclone as described below.

hPAM4V _KBACK5′-GACATCCAGCTGACCCAGTCTCCATCCTCCCTG-3′(SEQIDNO：60)

hPAM4V _KFOR5′-TTAGATCTCCAGTCGTGTCCCCCCTCCGAACGT-3′(SEQIDNO：61)

With PvuII and BglII to hPAM4V _kthe PCR primer through gel-purified carry out restriction digestion, and to be cloned in the complementary PvuII/BclI site of light chain staging vector VKpBR2.VKpBR2 is modified VKpBR staging vector (Leung etc., Hybridoma, 13:469,1994), wherein introduces XbaI restriction site in 16 bases of translation initiation codon upstream.The VK gene of assembling is subcloned into containing V as XbaI-BamHI restricted fragment _hthe expression vector hPAM4V of sequence _hin pdHL2.By gained expression vector called after hPAM4pdHL2.

By digesting about 30 μ ghPAM4pdHL2 linearizations with Sail, and by carrying out electroporation and be transfected in Sp2/0-Ag14 cell under 450V and 25 μ F.Cell through transfection to be inoculated in 96 orifice plates and at CO ₂hatch two days in cell chulture couveuse, and select MTX resistance subsequently.Colony survival is selected to occur within two to three weeks, and measures the secretion of screening human antibodies by ELISA.In brief, the supernatant (~ 100 μ l) from survival colony is added and scribbles Goat anti human IgGF (ab ') in advance ₂in the hole of the ELISA micro plate of fragments specific Ab.At room temperature plate is hatched one hour.By washing three times to remove unconjugated protein with lavation buffer solution (PBS containing 0.05%Tween-20).The Goat anti human IgGFc fragments specific Ab puted together by horseradish peroxidase adds in each hole.After hatching one hour, add containing 4mM o-phenylenediamine dihydrochloride (OPD) and 0.04%H in each hole after washing ₂o ₂the substrate solution (100 μ L/ hole) of/PBS.Make to darken 30 minutes, and by adding 50 μ L4NH ₂sO ₄solution stops reaction.The IgG combined is measured by the absorptance read on ELISA reader under 490nm.Expand and cultivate positive cell clone, and by affinity chromatography purifying hPAM4 from cell culture supernatant on albumin A post.

Measured the Ag binding activities confirming hPAM4 by ELISA in the microtiter plate scribbling pancreatic cancer cell extract.Develop and use the ELISA competition binding of the plate through the coating of PAM4 antigen to measure with the Ag binding affinity of Ag binding affinity compared to the chimeric PAM4 be made up of mouse V domain and mankind's C-structure territory assessing hPAM4.The HRP of constant basis puts together cPAM4 and mixes with cPAM4 or hPAM4 of variable concentrations, adds the hole through coating, and at room temperature hatches 1-2h.Comprise 4mM o-phenylenediamine dihydrochloride and 0.04%H adding ₂o ₂substrate solution after, show by the absorbance read under 490nm the amount that the HRP be combined with CaPan1Ag puts together cPAM4.As shown in the competition assay in Fig. 5, hPAM4 and cPAM4 antibody shows similar binding activities.

Embodiment 2. immunohistochemical staining is studied

The immunohistochemistry display of normal adult tissue, the reactive epi-position of PAM4 is confined to intestines and stomach, wherein dyes more weak, is but positive (table 2).Normal pancreatic tissue, comprises conduit, ductule, acinus and islet cells, is negative for dyeing.Using-system homogenate supports immunohistology data (table 3) usually as the enzyme immunoassay (EIA) based on PAM4 of antigen.PAM4 epi-position is not present in Normal Pancreas and other parenteral tissue.In superfluous natural disposition tissue, ten kinds in 21 kinds of (85%) (table 4 and tables 5) in PAM4 and 25 kind of cancer of pancreas and 26 kinds of colon cancers have reactivity, but only have measured response (table 5) with the tumour of stomach, lung, mammary gland, ovary, prostate, liver or kidney.PAM4 reactivity seems relevant with the Tumor Differentiation stage, breaks up or compared with bad differentiation tumor with moderate, and the dyeing number percent observed in good differentiation cancer of pancreas is higher.In general, what the tumour of bad differentiation accounted for all cancers of pancreas is less than 10%.

These researchs show, and the reactive and Tissue distribution of PAM4 (normally and cancer) is different from for CA19.9, DUPAN2, SPAN1, Nd2 and B72.3 antibody and reports for the antibody of Lewis antigen.Together with some cross-blocks the carried out research used in these MAb, data show that PAM4MAb identifies unique new epi-position.When with CA19.9, DUPAN2 and anti-Le ^awhen the antigen that antibody identifies is compared, PAM4 antigen seems more have limitation in its Tissue distribution, and the pancreatic neoplasm of higher percent has reactivity.In addition, its general reaction intensity under equipotent concentration is larger, and has reactivity with the cell of higher percent in pancreatic neoplasm.Finally, find that PAM4 only has weak reactivity with three kinds in 12 kinds of chronic pancreatitis samples, and CA19.9 and DUPAN2 and all 12 kinds of samples all has strong reactivity.Although depend on type used with having realized that specificity portion and ability, the high response intensity that institute check scope and the number of tissue, PAM4 to distinguish normally and the ability of superfluous natural disposition pancreatic tissue, itself and larger number percent cancer specimen react and distinguish that the ability of the optimum symptom such as Early pancreatic carcinoma and such as pancreatitis is the key character of this exemplary anti-cancer of pancreas antibody.

Table 2. is to the immunoperoxidase staining of normal adult tissue with MAbPAM4

^ait is the number of checked individual samples in-bracket

The reactivity of the monoclonal antibody PAM4 that table 3. is obtained by EIA and normal adult tissue homogenate

A-value is the mean value deriving from two parts of autopsy samples

The immunohistochemical reaction of several monoclonal antibodies of table 4. and pancreatic neoplasm

-: negative; +: the tissue of 5-20% is colored; ++: the tissue of 21-50% is colored;

+++: the tissue of >50% is colored; W, M, P: good, moderate or bad differentiation;

*: transfer tissue; ND: do not carry out

Table 5. is to the immunoperoxidase staining of superfluous natural disposition tissue with MAbPAM4

Embodiment 3. is bio distribution and cancer target in the body of radiolabeled PAM4

The initial biodistribution research of PAM4 carries out in a series of four kinds of different heterograft human pancreatic tumors covering expection differentiation scope.In the tumour that four kinds of tumour systems AsPc1, BxPc3, Hs766T and CaPan1 adopting show separately ¹³¹i-PAM4 concentration (scope: at the 3rd day 21%-48%ID/g) significantly (P<0.01-0.001) mates Ag8 antibody (scope: the 3rd day 3.6%-9.3%ID/g) higher than the non-specific isotype used simultaneously.Biodistribution data is for assessment of respectively with 12, and the dosage of 230,10,684,6,835 and 15,843cGy/mCi is expelled to the potential radiation dose to tumour after AsPc1, BxPc3, Hs766T and CaPan1.Under the actual maximal tolerance dose (MTD) of 0.7mCi, PAM4 can provide fundamental radiation dosage for different kind of neoplasm transplantation.In each tumour system, through radiolabeled PAM4 blood level significantly (P<0.01-0.001) lower than non-specific Ag8.PAM4 to the potential radiation dose of blood than Ag8 low 1.4-4.4 doubly.When PAM4 is carried out standardization to the radiation dose of tumour relative to the blood dosage of PAM4, the dosage that tumour receives is higher than blood 2.2,3.3,3.4 and 13.1 times respectively.Importantly, minimum to the potential radiation dose of nonneoplastic tissue.

CaPan1 tumor model is used to compare the bio distribution of PAM4 and anti-CEA antibody MN-14.In tumour, time point is far away higher than MN-14 in early days for the concentration of PAM4, and for PAM4, the tumour of the 3rd day: blood ratio is 12.7 ± 2.3 by contrast, for MN-14, is then 2.7 ± 1.9.Although PAM4 picked-up is significantly higher than (first day, the P<0.001 of MN-14 in the tumour of earlier time points; 3rd day, P<0.01), but Dosimetric analysis shows that PAM4 is only high than MN-14 3.2 times to the dosage of tumour within fortnight research cycle.This is because PAM4 removes fast from tumour, and these two kinds of antibody are present in tumour with similar concentration at later time point.Be also noted that PAM4 removes fast from tumour in BxPc3 and Hs766T tumor model, but then had no in AsPc1 tumor model.These observationss are different from for other anti-stick protein antibodies, and the situation that such as, G9 and B72.3 in colorectal cancer reports, wherein presents the longer hold-up time separately compared with MN-14 antibody.Show the result of study of the metabolism of PAM4, after being initially incorporated into tumour cell, antibody discharges fast, is likely to there occurs kalabolism or as antigen: antibody complex flows out.Blood is removed also very quick.These data show ¹³¹i may not be the isotopic suitable selection being used for the treatment of application.Such as can frequently use ⁹⁰y or ¹⁸⁸the short-term isotopes such as Re may be more effective reagent.

PAM4 does not demonstrate the evidence of target normal structure, but except CaPan1 tumor model, wherein observes on a small quantity but significant spleen picked-up (scope of the 3rd day: 3.1-7.5%ID/g) statistically.In the clinical practice of anti-stick protein antibodies B72.3 and CC49, observed such spleen target.Importantly, these researchs also report that spleen target does not affect the tumor uptake of antibody, does not disturb the interpretation to Nuclear receptor co repressor yet.These researchs show, spleen target is not due to the cross reaction of antigen in spleen, neither due to by Fc receptors bind, but one or more due in following possibility: the antigen of directly catching in target spleen, or indirectly to be formed in picked-up blood or from the antigen of tumor locus release: antibody complex.The latter will need to there is immune complex in blood.But, do not observe these when early being reached five minutes by gel filtration (HPLC, GF-250 post) inspection and reaching the sample of seven days late; Through radiolabeled antibody as natural materials wash-out.Front one explains that seeming may be more that this is true in view of CaPan1 tumour produces a large amount of PAM4 reactive antigen, higher 100 to 1000 times than other checked tumor cell line.The spleen target lacking PAM4 in these other tumour systems shows that this phenomenon produces relevant with excessive antigen.Spleen target can realize by making albumen dosage be increased to 10 μ g from 2 original μ g dosage.By inference, the PAM4 of more substantial spleen capture antigen and un-marked, instead of through radiolabeled antibody compound.The increase of albumen dosage does not have adverse effect to PAM4 target tumor or nonneoplastic tissue.In fact, albumen dosage increases to more than 100 μ g make to double through the concentration of radiolabeled PAM4 in CaPan1 tumour.

Embodiment 4. develops original position pancreatic tumor model in nude mouse.

In order to the clinical manifestation of cancer of pancreas in simulated animal model more approx, we develop model in situ by tumour cell is injected directly into head of pancreas.Original position CaPan1 tumour grows and non-evident sympton gradually, until produce ascites and death in ten to ten surroundings.Three to surrounding after implanting, animal produces the tangible tumour of about 0.2g.In eight weeks of growth, observe the primary tumor of about 1.2g, with being transferred to liver and spleen (1-3 metastatic tumo(u)r/animal; Each tumour <0.1g).In ten to ten surroundings, barrier film sowing and ascites produce clearly.Ascites is formed and the first obvious sign of accidental jaundice normally tumor growth.Now, tumour is quite large, be 1 to 2g, and animal only has at most three to surrounding to live.

Use through radiolabeled to the animal with surrounding tumor in situ in age (about 0.2g) ¹³¹it is selectively targeted that I-PAM4 shows primary tumor, and first day locator index is 7.9 ± 3.0, and fortnight is increased to 22.8 ± 15.3.Not to be noted to the evidence of other tissue specificity target.Observing metastases in a kind of situation of liver and spleen, two kinds of transfers are all targeted, and have high concentration through radiolabeled antibody.In addition, only about half of animal creates hypodermic tumour at cutting part.Not to be noted significant difference in original position in same animal and hypodermic tumour target, and significant difference is not observed in cancer target aspect in position, no matter whether animal has other hypodermic tumour.The estimation radiation dose of PAM4 to primary tumor and blood is 6,704 and 1,655cGy/mCi respectively.

The Radioimmunotherapy of embodiment 5. cancer of pancreas

Right ¹³¹the preliminary research that I-PAM4 is used for the treatment of is used in athymic mouse to carry out as the CaPan1 tumour of subcutaneous xenograft growth.350 μ Ci are used in an experiment to the animal with 0.25g tumour ¹³¹i-PAM4, this experiment also compares the result for the treatment of of the non-specific Ag8 of similar dosage.Give with 1cm ³the animal of tumour is used ¹³¹the MTD of I-PAM4 is 700 μ Ci.By the 5th week and the 6th week, the animal through PAM4 process demonstrated tumour and significantly degenerates, and even at the 27 week, had five still without tumour in eight.Unprocessed and demonstrate the rapid progress of tumor growth through the animal of Ag8 process, but notice, between these two control groups, there is significant difference.Seven weeks time, the tumour of unprocessed group has been grown to 20.0 ± 14.6 times of initial time point, and warp ¹³¹the tumour of I-Ag8 process only grows 4.9 ± 1.8 times.Now, PAM4 tumour has deteriorated to 0.1 ± 0.1 times of its original size, is significantly different from the animal of unprocessed (P<0.001) and (P<0.01) through non-specific Ag8 process.

These data display CaPan1 tumour to ¹³¹i-PAM4 process is responsive.Result is that tumour regression or progress depend on many factors, comprises initial tumor size.Therefore, with single dose 350 μ Ci ¹³¹i-PAM4 process is with the animal groups of 0.25g, 0.5g, 1.0g or 2.0gCaPan1 tumor load.There is most animals that tumour initial size is 0.25g and the 0.5g animal of 9/10ths (in each group) and within least ten six weeks, demonstrate tumour regression or growth inhibition after treatment.In 1.0g tumor group, in seven, there are five in 16 time-of-week sections, do not show tumor growth, and in 2.0g tumor group, have six in six time-of-week sections, do not demonstrate tumor growth in nine, be in progress subsequently.Although single 350 μ Ci dosage is so ineffective to larger tumour, single dose may not be the scheme being suitable for large tumour.

Toxicity research shows the ability giving multi cycle Radioimmunotherapy, and this therapy may be more effective under larger tumor load.Animal with average 1.0gCaPan1 tumour is given to 350 μ Ci of single dose ¹³¹i-PAM4, gives two dosage at time zero and surrounding, or does not process.The mean survival time that untreated fish group has is that (survival was defined as tumour and reaches 5cm in 3.7 ± 1.0 weeks ³time).Animal is dead after three weeks, does not have animal dis to live through six weeks.350 μ Ci of single dose ¹³¹the time-to-live that I-PAM4 produces significantly increases to 18.8 ± 4.2 weeks (P<0.0001).The scope of animal dead was extended down to twelve-five circulations from 13 weeks.None survival of animal at the end of the research cycle of 26 weeks.

Compared with single dose group, the time-to-live observing two dosage groups significantly increases.Half animal is alive at the time point of 26 weeks, and tumor size is 1.0-2.8cm ³, and average tumor growth rate is 1.6 ± 0.7 times of initial tumor size.For those animals of non-survivors when 26 weeks, mean survival time (17.7 ± 5.3 weeks) is similar to single dose group.

Situ tumor model is also used to carry out the Therapy study of PAM4.Process is not added to the animal groups with surrounding tumor in situ in age (estimation tumor weight is 0.25g), or with 350 μ Ci of single dose ¹³¹i-PAM4 or 350 μ Ci ¹³¹the non-specific Ag8 process of I-.Untreated animal had the mortality ratio of 50%, at the 15 week without survivor by the tenth week.Use non-specific when tumor growth surrounding ¹³¹the animal of I-Ag8 demonstrated the mortality ratio of 50% at the 7th week, in the tenth surrounding without survivor.Although, likely there is radiotoxicity in indifference between (Log rank analysis) these two groups statistically in the animal through Ag8 process.With untreated or through Ag8 process animal compared with, provide remarkable survival advantage (P<0.001) through radiolabeled PAM4,16 weeks experiment at the end of survival rate be 70%.Now, surviving animals is put to death to determine tumor size.All animals have the tumour that average weight is 1.2g, and have four visible one or two little (<0.1g) transfers in seven animals.When growth 16 weeks, these tumours more can represent the tumour in eight week age.

Embodiment 6. chemotherapy with ¹³¹the combined experiment Radioimmunotherapy of I-PAM4

Carry out combinationally using gemcitabine with checkerboard array with ¹³¹the Primary Study of I-PAM4 Radioimmunotherapy; The gemcitabine of single dose (0,100,200,500mg/kg) contrast single dose ¹³¹i-PAM4 (MTD of [MTD=700 μ Ci] 100%, 75%, 50%, 0%).Find that combination MTD is 500mg/kg gemcitabine and 350 μ Ci ¹³¹i-PAM4 (50%MTD).Toxicity, as weighed by body weight loss, being reached and being considered to nontoxic maximal value; That is, body weight loss 20%.Although this combined therapy scheme is significantly more effective than independent gemcitabine, this treatment is more effective unlike independent Radioimmunotherapy.The next item down research carries out under the gemcitabine and Radioimmunotherapy of low dosage, whether observes real synergistic therapeutic effect to check.Give with about 1cm ³the athymic mouse of (being approximately 5% of body weight) tumour uses gemcitabine, the 0th, three, six, nine with give 100mg/kg in 12 days, wherein gives 100 μ Ci at the 0th day ¹³¹i-PAM4.Compared with independent gemcitabine, (two in five tumours are less than 0.1cm to observed statistically the tumour regression of significantly (P<0.0001) ³) and/or growth inhibiting result for the treatment of.Therefore, compared with under the therapeutic agent of low dosage, there is the cooperative effect that gemcitabine and Radioimmunotherapy combine surprisingly.It should be noted that in addition about body weight, do not observe toxicity.If necessary, combined therapy scheme can send multiple circulation, and wherein second treatment circulation starts from 4th week, when above-mentioned independent Radioimmunotherapy has been studied.

Embodiment 7. agent treated is on the immunoreactive impact of PAM4 antigen

Reactivity (the DTT-EC with PAM4 is completely eliminated with DTT process pancreas mucin (15min, room temperature) ₅₀, 0.60+0.00 μM).Unique halfcystine (cystine bridge) in MUC-1 is present in membrane spaning domain, and for DTT by cannot be close.The secreted form of MUC-1 does not contain membrane spaning domain and does not therefore have cystine bridge in molecule.At room temperature with 0.05M sodium periodate, cancer of pancreas mucin is carried out to the immunoreactivity loss (not shown) of the PAM4 antibody of the data generation 40% of 2 hours periodates oxidation processes gained.The research of other periodates has shown loses up to 60% (not shown) with the immunoreactivity of PAM4 antibody.Periodates and DTT result of study show, PAM4 epi-position depends on the glycosylation of some minimum forms in configuration, and may affect by intermolecular disulfide bond formation.

The distribution of embodiment 8.PAM4 antigen and cross reactivity

The expression of PAM4 epi-position in PanIN is atypical for MUC-1.It is similar to the expression reported for MUC5ac, as by commercially available MAb-CLH2-2 detect.But, attempt using PAM4 to catch and obtain negative findings with MAb-CLH2-2 as the sandwich immunoassay of probe.Although this may show PAM4 and CLH2-2 epi-position possibility overlap and therefore block mutually, but it is reported that the cancer of the stomach of CLH2-2 and 42/66 (64%) has reactivity, and PAM4MAb only demonstrates reactivity with 6/40 (15%) cancer of the stomach, and these are only focal reactivities.

Use commercially available anti-MUC5acMAb45M1 to obtain positive findings as the probe reagent (using PAM4 as catches) in EIA, show same antigen molecule to exist two epi-positions.Block research (either direction) and show that epi-position that 45M1 and PAM4 combines is actually two different epi-positions, because do not observe blocking-up.The expression that the mark that the micro-array tissue formed the core by aggressive cancer of pancreas carries out has shown 45M1 and PAM4 epi-position in few patients's sample has significant difference.In 28 samples, 17 examples (61%) are only had to observe consistance.PAM4 and 24/28 example (86%) have reactivity, and 45M1 and 13/28 example (46%) have reactive (not shown).

As the reactive factor of MUC5ac and PAM4 antibody mediated immunity, the result of periodates research is consistent with glycosylation.Therefore, the result of study of apomucin determines it may is indefinite for antigen.

Although catch based on EIA, PAM4 antibody seems MUC5acMAb anti-with 45M1 in conjunction with same antigen albumen, it should be noted that MUC5ac does not have a specificity to cancer of pancreas, and to be found in multiple normal structure (except the gastric mucosa of responding property of PAM4).For example, MUC5ac is found in normal lung, colon and other tissue.PAM4 antibody not in conjunction with normal lung tissue, except reaching limit amount or minimum as indicated above in a few sample.

Relative to the effect of DTT and periodates, likely peptide core disulphide bridges is same, and no matter which kind of tissue produces this protein.Specific amino acid sequence should fold in a specific way, does not rely on tissue-derived.But glycosylation pattern can be depending on tissue-derived and different.

The Phage Display Peptide of embodiment 9.PAM4 antibody combines

Combine with two kinds of different phage display peptide library inspection PAM4 antibody.The first Linear peptide library be made up of 12 amino acid sequences, the cyclic peptide that the second is made up of 7 amino acid sequences by disulphide bridges cyclisation.We replace the indivedual libraries (with anti-CD22 antibody carry out Solid phase) of elutriation for hPAM4 and hLL2, altogether 4 take turns, and screening and both hPAM4 and mPAM4 have reactivity subsequently, and have seldom to the Phage Display Peptide of anergy hLL2.Abandon the bacteriophage combining (that is, in conjunction with epratuzumab [hLL2]) with non specific manner.

For linear Phage Display Peptide, sequence WTWNITKAYPLP (SEQIDNO:7) is by discriminating 30 times (in 35 order-checking bacteriophages), and each display has reactivity with PAM4 antibody.Carry out mutation analysis, wherein build as described above, elutriation and to have screened based on this sequence and each position has the library of 7.5% degeneracy.Notice obtained 19 variability PAM4 being combined to the peptide sequence be positive, wherein 7 same with parental array, 5 have sequence WTWNITKEYPQP (SEQIDNO:62), and all the other uniquely to exist.Table 6 shows the result of this mutation analysis.Lastrow lists the sequence of identification, and next line lists in each amino acid whose frequency of this position discriminating.Parental array frequency the highest (black matrix), secondly the highest variation is that on position 8, E replaces Q replacement L on A and position 11.Seem these displacements to immunoreactivity without any impact greatly.

The phage display amino acid sequence (SEQIDNO:113) of the linear peptides that table 6. is combined with PAM4 antibody makes a variation

The result in phage display ring-type library is significantly different from linear libraries (table 7).Sequence A CPEWWGTTC (SEQIDNO:63) is present in 33 in 35 detected peptide sequences.Following result (position of band asterisk is constant, and does not affect by the selection pressure in library) is occurred to the analysis in ring-type library.

The phage display amino acid sequence (SEQIDNO:114) of the linear peptides that table 7. is combined with PAM4 antibody makes a variation

Two halfcystines (position 2 and 10) form disulphide bridges.T on position 9 can be affected immunoreactivity greatly by any 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.Sequence GTTGTTC (SEQIDNO:64) is present in MUC5ac albumen, and towards amino terminal compared with the cyclic peptide sequences shown in above, described cyclic peptide sequences shows the homology of total polypeptide sequence C end.But cyclic peptide only shows immunoreactive about 10% of linear order and PAM4 antibody.Linear and ring-type consensus sequence and halfcystine associate, and halfcystine is possible or may not relate to DTT to the immunoreactive impact of MUC5ac.

Result reported herein shows that PAM4 epi-position depends on the specified configuration producing and be specific glycosylation pattern by disulphide bridges.

The immunohistology of the cancer of pancreas in embodiment 10. pancreatitis sample

Several pathological condition can make patient be tending towards suffering from cancer of pancreas, such as pancreatitis, diabetes, smoking etc.In this preselected group patient, screening measure is even more important for the neoplastic early detection of pancreas.We examine 9 chronic pancreatitis tissue samples, and it is the patient suffering from this disease that described sample comes from tentative diagnosis.We use anti-CD74MAbLLl to invade the index of profit as inflammatory, and MAb-MA5 is as the positive control of pancreas conduit and acinar cells.In view of two contrast MAb provide the immunohistology evidence consistent with pancreatitis, the pancreatic tissue of PAM4 and inflammation is reacted not have evidence to prove.But, in one case, in tissue sample, also there is the cancer of pancreas of moderate differentiation.In this tumour, PAM4 gives neoplastic cell in tumour to dye by force.In the latter case, although the tissue of inflammation is negative to PAM4, authenticated the small-sized PanIN precancerous lesion through PAM4 mark.It is consistent that the mark of the PanIN in rear a kind of sample and the Vipoma in the patient being diagnosed as nonmalignant disease form early detection.The display of these results can form the detection and/or the diagnosis that have hypersensitivity and optionally carry out use PAM4 antibody in situation to Vipoma relative to benign pancreatic tissue background.

Embodiment 11. suffers from the therapy of unsuitable operation type metastatic Pancreas cancer patients

Patient 118-001CWG is the 63 years old male sex suffering from IV phase cancer of pancreas and many places hepatic metastases made a definite diagnosis in November, 2007.He agrees to take combined radioimmunotherapy treatment and gemcitabine chemotherapy as the first therapeutic strategy, and gives its 6.5mCi/m subsequently ²⁹⁰y-hPAM4 and 200mg/m ²first treatment circulation of gemcitabine combination, wherein gives gemcitabine once in a week in the 1 to 4 week, and the 2 to 4 week weekly-secondaryly to give ⁹⁰y-hPAM4 (3 dosage).After two months, repeat same treatment circulation, because not to be noted larger toxicity after the first cycle.After the first treatment circulation when 4 weeks, notice the CT evidence that the diameter at 2 places in primary tumo(u)r and the hepatic metastases of 3 places reduces surprisingly, and this and the SUV value that FDG-PET scans significantly reduce consistent, 3 now in 4 tumours, are had to get back to normal background SUV level (Fig. 6 and Fig. 7).Before the treatment of this patient, CA-19.9 level 1,297 is down to the further supportive treatment of reduced levels 77 is effective.Table 8 shows utilization ⁹⁰the effect of combination Radioimmunotherapy in this patient of Y-hPAM4 and gemcitabine chemotherapy.Astonishing and unexpectedly, even if the combination of the radioactive nuclide of this type of low dosage and antibody conjugate and this type of low dosage nontoxic gemcitabine dosage also demonstrates such antitumor activity after this therapy of only carrying out single current system journey.

Table 8. utilizes ⁹⁰the effect of combination Radioimmunotherapy in metastatic cancer of pancreas of Y-hPAM4 and gemcitabine chemotherapy

Move

Embodiment 12. suffers from the therapy of the patient of unsuitable operation type metastatic cancer of pancreas

To suffering from extensive unsuitable operation type cancer of pancreas and many places hepatic metastases (diameter is in 1 to 4cm scope), substance lose weight (30 weigh oneself or more), slight jaundice, murky and weak and need 56 years old male sex, 4 infusion gemcitabines weekly of stomachache of medication, each dosage is 200mg/m ².In the end in three gemcitabine infusions, with 10mCi/m ²⁹⁰the dosage of Y and 20mg antibody protein with infusion in two hr iv use through ⁹⁰the radiolabeled humanized antibody of Y-DOTA-hPAM4.After two weeks, by intravenous infusion give a course for the treatment of of patient by 3 600mg/m weekly ²the gemcitabine chemotherapy of dosage composition.Subsequently, patient stands assessment after 4 weeks and has slight leukopenia (2 grades), has no blood or enzyme other Main change relative to baseline, but demonstrate blood CA19.9 titer from 5,700 are improved to 1,200, jaundice alleviates, subjective overall improvement.After this, low dosage gemcitabine (weekly × 4) and 3 dosage are repeated after 3 weeks ⁹⁰the circulation of Y-DOTA-hPAM4.After surrounding, reappraise patient, and CT and PET scanning confirms that total tumor quality (preinvasive cancer and transfer) reduces about 40%, and CA19.9 titer is down to 870 further simultaneously.Patient recovers appetite and vigor, and can get back to more conventional daily routines and without the need to anodyne.He adds 12 pounds after starting this experimental therapy.After multiple scanning and blood values show that this reaction is maintained to 6 weeks.

Embodiment 13. pre-targeting DNL ^tMthe preparation of construct

DDD and AD fusion

DNL ^tMtechnology can be used for manufacturing dimer, tripolymer, the tetramer, six aggressiveness etc., comprises in fact any antibody or its fragment or other effect part.For some preferred embodiment, IgG antibody or Fab antibody fragment can be produced as the fusion containing dimerization and docking structure territory (DDD) or anchoring domain (AD) sequence.Although in preferred embodiments, DDD and AD part is produced as fusion, and technician will be appreciated that and can use other conjugation methods such as such as chemical crosslinking or click chemistry etc. in the scope of claimed method and composition.

Bispecific antibody is formed by the combination Fab-DDD fusion of first antibody and the Fab-AD fusion of second antibody.Alternatively, the construct being combined with IgG-AD fusion and Fab-DDD fusion and combining can be manufactured.This technology not tool is restricted, and any useful protein or peptide can be produced as AD or DDD fusion to be incorporated in DNLTM construct.When using chemical crosslinking, AD and DDD conjugate is not limited to protein or peptide, and can comprise any molecule that any crosslinking technological known in the art and AD or DDD sequence can be used to be cross-linked.In some exemplary, polyglycol (PEG) or other polymer moieties can be incorporated to DNL ^tMin construct, as described in more detail below.

For pre-targeting application, the antibody containing the binding site for the antigen relevant to pathological tissues such as such as tumor associated antigen (TAA) or fragment can with can in conjunction be connected with treat and/or diagnosticum can haptenic second antibody on target construct or fragment combination.Use the bispecific antibody based on DNLTM to experimenter, make circulating antibody remove from blood and be positioned target tissue, and add put together can target construct and make it be combined with located antibody to carry out diagnosing or treating.

Independently transgenic cell line can be developed for each Fab or IgG fusion.After generation, purifying can be carried out to module when needed, or maintain in cell culture supernatant fluid.After a birth, any DDD fusion module all can with any AD fusion block combiner, to produce bispecific DNLTM construct.For dissimilar construct, different AD or DDD sequences can be used.Exemplary DDD and AD sequence is below provided.

DDD1：SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQIDNO：65)

DDD2：CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SFQIDNO：66)

AD1：QIEYLAKQIVDNAIQQA(SEQIDNO：67)

AD2：CGQIEYLAKQIVDNAIQQAGC(SEQIDNO：68)

Technician it should be understood that DDD1 and DDD2 comprises the DDD sequence of the mankind RII alpha form of protein kinase A.But in an alternate embodiment, DDD and AD part may be DDD sequence based on the mankind RI alpha form of protein kinase A and corresponding AKAP sequence, example as shown in following DDD3, DDD3C and AD3.

DDD3

SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK(SEQIDNO：69)

DDD3C

MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK(SEQIDNO：70)

AD3

CGFEELAWKIAKMIWSDVFQQGC(SEQIDNO：71)

Expression vector

Plasmid vector pdHL2 has been used to create much antibody and the construct based on antibody.See Gillies etc., JImmunolMethods (1989), 125:191-202; Losman etc., Cancer (Phila) (1997), 80:2660-6.Bicistronic mRNA mammalian expression vector instructs the synthesis of IgG heavy chain and light chain.For many different IgG-pdHL2 constructs, carrier sequence major part is same, only has variable domains (V _hand V _l) there are differences in sequence.Use biology tool well known by persons skilled in the art, these IgG expression vectors can change into Fab-DDD or Fab-AD expression vector.In order to produce Fab-DDD expression vector, the coded sequence of the hinge of heavy chain, CH2 and CH3 domain is replaced into the sequence (being called DDD1) of front 4 residues of coding hinge, the Gly-Ser connexon of 14 residues and front 44 residues of mankind RII α.In order to produce Fab-AD expression vector, it is the sequence (being called AD1) of front 4 residues of coding hinge, the Gly-Ser connexon of 15 residues and the synthesis AD (being called AKAP-1S) of 17 residues by the sequence substitutions of the hinge of IgG, CH2 and CH3 domain, described AKAP-1S uses bioinformatics and peptide array technique to produce, and demonstrates with high affinity (0.4nM) in conjunction with RII α dimer.See Alto etc., Proc.Natl.Acad.Sci., U.S.A (2003), 100:4445-50.

Design two shuttle vectors and become Fab-DDD1 or Fab-AD1 expression vector to be conducive to IgG-pdHL2 vector, as described below.

The preparation of CH1

Use pdHL2 plasmid vector as template by pcr amplification CH1 domain.The SacII restriction endonuclease site of upstream (5 ') end of left PCR primer by CH1 domain and the 5 ' end as CH1 coded sequence forms.Right primer, by front 4 residues (PKSC (SEQIDNO:115)) of hinge of encoding, is four glycocoll and serine subsequently, is finally the sequence composition of two codons (GS) comprising BamHI restriction site.410bpPCR extension amplification outcome is arrived pCR cloning vector ( inc.) in, and the insetion sequence of screening and cloning on T7 (5 ') direction.

Pass through Sigma (Haverhill, UK) synthesis (G by name ₄s) ₂double chain oligonucleotide (' (the G of DDD1 ₄s) ₂', be disclosed as SEQIDNO:116), to be embroidered with the DDD1 amino acid sequence of 11 residues connecting peptide before encoding, wherein the first two codon comprises BamHI restriction site.Terminator codon and EagI restriction site are connected to 3 ' end.Coded peptide sequence is as follows.

GSGGGGSGGGG SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQIDNO：72)

Synthesis is called two oligonucleotides on RIIA1-44 and under RIIA1-44 and combines with 154, the centre base-pair comprising 174bpDDD1 sequence, and described oligonucleotides has 30 base-pair overlaps on its 3 ' end.Oligonucleotides is annealed, and uses Taq polymerase to carry out primer extension reaction.After primer extends, by pcr amplification double-strand.Extension amplification outcome is arrived in and the insetion sequence of screening on T7 (5 ') direction.

Double stranded synthetic oligonucleotide is with AD1 amino acid sequence of encoding, and have 11 residues connecting peptide before described AD1, wherein the first two codon comprises BamHI restriction site.Terminator codon and EagI restriction site are connected to 3 ' end.Coded peptide sequence is as follows.

GSGGGGSGGGGS QIEYLAKQIVDNAIQQA(SEQIDNO：73)

Two complementary overhangs oligonucleotides of the above-mentioned peptide sequence of composite coding, called after AKAP-ISTop and AKAP-ISBottom, and anneal.By pcr amplification double-strand.Extension amplification outcome is arrived in carrier, and the insetion sequence of screening on T7 (5 ') direction.

DDD1 and CH1 connects

With BamHI and NotI restriction enzyme by the 190bp fragment of encoding D DD1 sequence from in cut, and to be connected to subsequently in same loci, to produce shuttle vector

AD1 and CH1 connects

With BamHI and NotI restriction enzyme by the 110bp fragment that comprises AD1 sequence from in cut, and to be connected to subsequently in same loci, to produce shuttle vector

CH1-DDD1 or CH1-AD1 is cloned in the carrier based on pdHL2

In this modular design, CH1-DDD1 or CH1-AD1 can be incorporated in any IgG construct in pdHL2 carrier.By removing SacII/EagI restricted fragment (CH1-CH3) from pdHL2 and replacing whole CH with the SacII/EagI fragment displacement of CH1-DDD1 or CH1-AD1 cut from corresponding pGemT shuttle vector with one of above construct.

The structure of h679-Fd-AD1-pdHL2

H679-Fd-AD1-pdHL2 is the expression vector being coupled to the h679Fab of the carboxyl terminal of the CH1 domain of Fd for generation of AD1 via the flexible Gly/Ser peptide interval base that 14 amino acid residues form.By replacing SacII/EagI fragment by the vector based on pdHL2 of the variable domains containing h679 for h679-Fd-AD1-pdHL2 with the CH1-AD1 fragment cut from CH1-AD1-SV3 shuttle vector with SacII and EagI.

The structure of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 stablizes dimeric expression vector for generation of what comprise two of fusion C-DDD1-Fab-hMN-14 copies, and wherein DDD1 is connected to hMN-14Fab at the carboxyl terminal of CH1 via flexible peptide interval base.By plasmid vector hMN-14 (the I)-pdHL2 for generation of hMN-14IgG being converted into C-DDD1-Fd-hMN-14-pdHL2 with the digestion of SacII and EagI restriction endonuclease with the CH1-DDD1 fragment removed CH1-CH3 domain and insertion SacII and EagI and cut from CH1-DDD1-SV3 shuttle vector.

Identical technology for generation of plasmid in case carry out multiple known antibodies such as such as hLL1, hLL2, hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 etc. Fab express.In general, antibody variable region coded sequence is presented in pdHL2 expression vector, and transforms expression vector to produce AD or DDD fusion as above.AD and the DDD fusion comprising the Fab fragment of any one in this antibody-like can by the approximate ratio combine of two DDD fusion/mono-AD fusion, to produce the trimerization DNL comprising two Fab fragments of first antibody and a Fab fragment of second antibody ^tMconstruct.

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is the expression vector being connected to the C-DDD2-Fab-hMN-14 of the carboxyl terminal of the Fd of hMN-14 for generation of the dimerization of DDD2 and docking structure territory sequence via the Gly/Ser peptide connexon of 14 amino acid residues.The same hMN-14Fab of secreted fusion two of being kept together by the noncovalent interaction by DDD2 domain copies and forms.

Carry out engineered to expression vector as follows.By the complementary oligonucleotide that synthesis manufacture two is overlapping, described oligonucleotides comprises part and connects the coded sequence of peptide and the residue 1-13 of DDD2.Oligonucleotides is annealed, and uses T4PNK phosphorylation, thus produce compatible with the DNA digested through restriction endonuclease BamHI and PstI respectively so that the pendle be connected on 5' with 3' end.

By double-stranded DNA and by digesting with BamHI and PstI the shuttle vector prepared connect, to produce shuttle vector with SacII and EagI from in cut 507bp fragment, and with digest IgG expression vector hMN-14 (the I)-pdHL2 prepared with SacII and EagI and be connected.Final expression construct called after C-DDD2-Fd-hMN-14-pdHL2.Similar technology has been utilized to create the DDD2 fusion of the Fab fragment of multiple different humanized antibody.

h679-Fd-AD2-pdHL2

Design h679-Fab-AD2 matches as B and the C-DDD2-Fab-hMN-14 as A.H679-Fd-AD2-pdHL2 is the expression vector for generation of h679-Fab-AD2, and described h679-Fab-AD2 has the anchoring domain sequence of AD2, and AD2 is connected to the carboxyl terminal of CH1 domain via the peptide connexon of 14 amino acid residue Gly/Ser.The cysteine residues that AD2 has one is before AD1 anchoring domain sequence, and another after this sequence.

Carry out engineered to expression vector as follows.(AD2Top and AD2Bottom, described oligonucleotides comprises coded sequence and the part catenation sequence of AD2 to the complementary oligonucleotide overlapping by synthesis manufacture two.Two overlaps, complementary oligonucleotides (AD2Top and AD2Bottom) are prepared by synthesis, and described oligonucleotides comprises coded sequence and the part catenation sequence of AD2.Oligonucleotides is annealed, and uses T4PNK phosphorylation, thus produce compatible with the DNA digested through restriction endonuclease BamHI and SpeI respectively so that the pendle be connected on 5' with 3' end.

By double-stranded DNA and by digesting with BamHI and SpeI the shuttle vector prepared connect, to produce shuttle vector from shuttle vector, cut the fragment of 429 base-pairs containing CH1 and AD2 coded sequence with SacII and EagI restriction enzyme, and with carry out digesting with these enzymes and the h679-pdHL2 carrier prepared is connected.Final expression vector is h679-Fd-AD2-pdHL2.

Fab and the IgG fusion that embodiment 14. is connected by Multiple Antibodies generation AD with DDD

IgG and the Fab fusion shown in technique construction table 9 described in embodiment before use, and be incorporated to DNL ^tMin construct.Fusion retains the antigen binding characteristics of parental antibody, and DNL ^tMconstruct shows be incorporated to antibody or the antigen-binding activity of antibody fragment.

Table 9. comprises the fusion of IgG or Fab

Embodiment 15.DNL ^tMsequence variant

In certain preferred aspects, DNL is incorporated to ^tMaD and DDD sequence in construct comprises the amino acid sequence of AD1, AD2, AD3, DDD1, DDD2, DDD3 or DDD3C as discussed above.But, in an alternate embodiment, the sequence variant of AD and/or DDD part can be used to build DNL ^tMcompound.For example, only there are the DDD four kinds of mankind PKADDD sequence variants partly corresponding to PKARI α, RII α, RI β and RII β.RII α DDD sequence is the basis of above disclosed DDD1 and DDD2.Four kinds of mankind PKADDD sequences are as follows.DDD sequence represents the residue 12-61 of residue 1-44, RI α and the residue 13-66 of RI β of residue 1-44, RII β of RII α.(note, the sequence of DDD1 is modified a little by mankind PKARII α DDD part.)

PKARIa

SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK(SEQIDNO：74)

PKARIβ

SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENRQILA(SEQIDNO：75)

PKARIIa

SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ(SEQIDNO：76)

PKARIIβ

SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER(SEQIDNO：77)

The structure-function relationship of AD and DDD domain is the theme of research.(see such as Bums-Hamuro etc., 2005, ProteinSci14:2982-92; Carr etc., 2001, JBiolChem276:17332-38; Alto etc., 2003, ProcNatlAcadSciUSA100:4445-50; Hundsrucker etc., 2006, BiochemJ396:297-306; Stokka etc., 2006, BiochemJ400:493-99; Gold etc., 2006, MolCell24:383-95; Kinderman etc., 2006, MolCell24:397-408, the full text of each document is incorporated herein by reference.)

For example, Kinderman etc. (2006) examine the crystal structure of AD-DDD binding interactions, and infer mankind DDD sequence contain many dimer is formed or AKAP in conjunction with very important conservative amino acid residues, in following SEQIDNO:65, underline expression.(see Kinderman etc., Fig. 1 of 2006, the document is incorporated to herein by reference.) technician it should be understood that when designing the sequence variant of DDD sequence, being avoided by needs changing any underlined residue, can carry out conserved amino acid replacement to dimerization and the not too important residue of AKAP combination simultaneously.

SH IQ IPPG LTE LLQG YT VE VLRQQPPD LVE FA VE YFTR LREARA(SEQIDNO：65)

Alto etc. (2003) the AD sequence to various AKAP albumen has carried out bioinformatic analysis, devises RII selectivity AD sequence, and be called AKAP-IS (SEQIDNO:67), it is 0.4nM to the binding constant of DDD.Design AKAP-IS sequence is as the peptide antagonists of the AKAP be combined with PKA.Residue in AKAP-IS sequence represents with underscore in SEQIDNO:67, wherein replaces and tends to reduce the combination with DDD.Technician it should be understood that when designing the sequence variant of AD sequence, is avoided by needs changing any underlined residue, can combine not too important residue simultaneously carry out conserved amino acid replacement to DDD.

AKAP-IS sequence

QIEYL AKQ IVDN AIQQA(SEQIDNO：67)

Gold (2006) utilizes crystallography and peptide screening to develop SuperAKAP-IS sequence (SEQIDNO:78), and it exceeds five orders of magnitude to the selectivity that the RII isotype of PKA shows compared with RI isotype.Underlined residue represents the position of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor relative to AKAP-IS sequence, which increases the combination with the DDD part of RII α.In the sequence, N-end Q residue is numbered No. 4 residues, and C-terminal A residue is No. 20 residues.Can carry out replacing to affect the residue of the affinity of RII α is residue 8,11,15,16,18,19 and 20 (Gold etc., 2006).Expection is in some alternate embodiment, and SuperAKAP-IS sequence can be substituted by AKAP-ISAD partial sequence, to prepare DNLTM construct.Other alternative sequence that can be substituted by AKAP-ISAD sequence is shown in SEQIDNO:79-81.Represent with underscore relative to the replacement of AKAP-IS sequence.Expection is as the AD2 sequence shown in SEQIDNO:68, and AD part also can comprise other N-terminal residue halfcystine and glycocoll and C-terminal residue glycocoll and halfcystine.

SuperAKAP-IS

QIEY VAKQIVD YAI HQA(SEQIDNO：78)

Substitute AKAP sequence

QIEY KAKQIVD HAI HQA(SEQIDNO：79)

QIEY HAKQIVD HAI HQA(SEQIDNO：80)

QIEY VAKQIVD HAI HQA(SEQIDNO：81)

Fig. 2 of Gold etc. discloses other DDD binding sequence from multiple AKAP albumen shown below, illustrates below.

rII-specificity AKAP

AKAP-KL

PLEYQAGLLVQNAIQQAI(SEQIDNO：82)

AKAP79

LLIETASSLVKNAIQLSI(SEQIDNO：83)

AKAP-Lbc

LIEEAASRIVDAVIEQVK(SEQIDNO：84)

rI-specificity AKAP

AKAPce

ALYQFADRFSELVISEAL(SEQIDNO：85)

RIAD

LEQVANQLADQIIKEAT(SEQIDNO：86)

PV38

FEELAWKIAKMIWSDVF(SEQIDNO：87)

dual specificity AKAP

AKAP7

ELVRLSKRLVENAVLKAV(SEQIDNO：88)

MAP2D

TAEEVSARIVQVVTAEAV(SEQIDNO：89)

DAKAP1

QIKQAAFQLISQVILEAT(SEQIDNO：90)

DAKAP2

LAWKIAKMIVSDVMQQ(SEQIDNO：91)

Stokka etc. (2006) also developed the peptide competitor of AKAP in conjunction with PKA, are shown in SEQIDNO:92-94.Peptide antagonists called after Ht31 (SEQIDNO:92), RIAD (SEQIDNO:93) and PV-38 (SEQIDNO:94).The RII isotype of Ht-31 peptide to PKA represents larger affinity, and RIAD and PV-38 represents more high-affinity to RI.

Ht31

DLIEEAASRIVDAVIEQVKAAGAY(SEQIDNO：92)

RIAD

LEQYANQLADQIIKEATE(SEQIDNO：93)

PV-38

FEELAWKIAKMIWSDVFQQC(SEQIDNO：94)

Hundsrucker etc. (2006) develop AKAP other peptide competitor in conjunction with PKA, and the binding constant of the RII form DDD of itself and PKA is low to moderate 0.4nM.Various AKAP antagonistic peptide sequence is provided in the table 1 of Hundsrucker etc., is reproduced in in following table 10.AKAPIS represents synthesis RII subunit binding peptide.Other peptides all derive from the RII binding structural domain of indicated AKAP.

Table 10.AKAP peptide sequence

peptide sequence

Below with reference to AKAPIS sequence (SEQIDNO:67) by underline indicate different AKAP albumen AD domain in the residue of high conservative.Identical with observed by (2003) such as Alto of described residue, wherein adds C terminal alanine residues.(see the Fig. 4 of (2006) such as Hundsrucker, the document is incorporated herein by reference.) sequence RIIDDD sequence to the peptide antagonists of special high-affinity is those sequences of AKAP-IS, AKAP7 δ-wt-pep, AKAP7 δ-L304T-pep and AKAP7 δ-L308D-pep.

AKAP-IS

QIEYL AKQ IVDN AIQQ A(SEQIDNO：67)

Carr etc. (2001) examine come from the mankind and otherwise non-human proteins different AKAP in conjunction with the sequence homology degree between DDD sequence, and authenticated in DDD sequence and seem the conservative residue of between different DDD parts topnotch.These indicate by underlining with reference to the mankind PKARII α DDD sequence of SEQIDNO:65 below.Also with the residue that italic instruction is conservative especially.Described residue and (2006) such as Kinderman propose to very important those are overlapping but not same with AKAP protein combination.Technician will be appreciated that, when designing the sequence variant of DDD, the most conservative residue (italic) is changed by most preferably avoiding, and also change conserved residues (underscore) by preferably avoiding, and can consider that the residue to both not underlining non-italic carries out conserved amino acid replacement.

S HIQ IPP GLT ELLQGYTV EVLRQ QPP DLVEFAVE YFTR LR EA RA(SEQIDNO：65)

Technician it should be understood that DNL ^tMthese and other 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in the antibody moiety of construct or connexon part can be used for strengthening gained DNL ^tMthe treatment of construct and/or pharmacokinetic property.

Embodiment 16.TF2DNL ^tMthe generation of pre-targeting construct

The trimerization DNL of called after TF2 ^tMconstruct obtains by making C-DDD2-Fab-hMN-14 and h679-Fab-AD2 react.The TF2 of following generation pilot batch, productive rate >90%.By C-DDD2-Fab-hMN-14 (200mg) and the h679-Fab-AD2 (60mg) through albumen L purifying with 1.4:1 mixed in molar ratio.Total protein concentration is in the PBS that is dissolved in containing 1mMEDTA of 1.5mg/ml.Subsequent step relates to TCEP reduction, HIC chromatogram, DMSO oxidation and IMP291 affinity chromatography.Before adding TCEP, SE-HPLC does not demonstrate any a ₂b forms sign.Add 5mMTCEP and cause a fast ₂the formation of b compound, except diadactic structure, this compound is consistent with 157kDa protein.By IMP291 affinity chromatography, TF2 is purified to almost homogeneous (not shown).IMP291 is the haptenic synthetic peptide of HSG (Rossi etc., 2005, ClinCancerRes11:7122s-29s) containing being combined with 679Fab.To IMP291 in conjunction with fraction SE-HPLC analyze display from product, eliminate a ₄, a ₂with free κ chain (not shown).

Non-reduced SDS-PAGE analyzes the most of TF2 of display and there is (not shown) with relative mobility close to the large covalent structure of IgG.Other band shows the incomplete (not shown) of disulfide formation under experimental conditions.Reduction SDS-PAGE display, all relevant to the product (not shown) of other band any occurred in nonreducing gel, only demonstrates the band (not shown) of the composition polypeptide representing TF2.But, in four peptide species the relative mobility of each too close to and cannot split.MALDI-TOF mass spectrum (not shown) display 156,434Da's is unimodal, and it is within 99.5% of TF2 calculated mass (157,319Da).

Pass through measure the functionality determining TF2.By TF2, C-DDD1-hMN-14+h679-AD1 (as non-covalent a ₂the control sample of b compound) or C-DDD2-hMN-14+h679-AD2 (as unreduced a ₂with the control sample of b component) be diluted to 1 μ g/ml (total protein), and the sensor chip by fixing through HSG.About twice of two kinds of control samples to the reaction of TF2, show to only have in control sample h679-Fab-AD component can in conjunction with and be retained on sensor chip.The anti-idiotype WI2IgG display of injecting hMN-14 subsequently only has TF2 to have the DDD-Fab-hMN-14 component with h679-Fab-AD intimate association, as shown in other signal reaction.By WI2 be fixed on the TF2 on sensor chip and be combined produced reacton and increase corresponding to two complete function binding sites in addition, each subunit by C-DDD2-Fab-hMN-14 contributes.This determines (not shown) by TF2 in conjunction with the ability of two Fab fragments of WI2.

Embodiment 17. is for the generation of the TF10 bispecific antibody of pre-targeting

Use similar scheme to produce trimerization TF10DNL ^tMconstruct, it comprises two C-DDD2-Fab-hPAM4 copies and a C-AD2-Fab-679 copy.Cancer target in TF10 derives to antibody component and is studied in great detail humanization MUC5acMAb as through radiolabeled MAb, hPAM4 (such as, Gold etc., Clin.CancerRes.13:7380-7387,2007).Haptens in conjunction with Component Source in humanization antihistamine base-succinyl-glycocoll (HSG) MAb, h679.Use disclosed for generation of (anti-CEA) ₂the method of × anti-HSGbsAbTF2 prepares TF10 bispecific ([hPAM4] as mentioned above ₂× h679) antibody.TF10 construct is with two humanization PAM4Fab and humanization 679Fab.

Two kinds of fusions (hPAM4-DDD and h679-AD2) are expressed independently in the myeloma cell of stable transfection.Merge tissue culture supernatant fluid, obtain the hPAM4-DDD of twice molar excess.Under slight reducing condition, use 1mM reduced glutathione at room temperature reaction mixture to be hatched 24 hours.After reduction, 2mM oxidized form of glutathione is used to complete DNL by mild oxidation ^tMreaction.Use and be separated TF10 in conjunction with the MP291 affinity gel resin of h679Fab by affinity chromatography with high specific.

Complete organization pathology and haemocyte board are for checking hPAM4IgG and the anti-CEA × anti-HSGbsMAb entering clinical testing.In 1/3 sample, hPAM4 combines the pole weak binding (having no combination in body) be confined to bladder and stomach, and is not combined with normal structure owing to anti-CEA × anti-HSGbsMAb.In addition, in vitro study for the clone with H1 and H2 histamine receptor shows the two HSG peptide of IMP288 without antagonism or competition activity, and zooscopy display in the 2 kinds of different species peptide relevant to histamine component is than for becoming image height 20, without pharmacological activity under the dosage of 000 times.Therefore, HSG-histamine derivatives does not have pharmacological activity.

Embodiment 18. uses with TF10 bispecific antibody and warp ¹¹¹the imaging research of the peptide pre-targeting of In mark

Below research display uses pre-targeting technology to carry out the feasibility of in-vivo imaging with the bispecific antibody being incorporated to hPAM4 and the peptide through marking.Prepare the TF10 bispecific antibody comprising two C-DDD2-Fab-hPAM4 copies and a C-AD2-Fab-679 copy as described in the previous embodiment.Use with TF10 and ¹¹¹in-IMP-288 peptide pre-targeting carries out imaging to the nude mice with 0.2 to 0.3g human pancreatic adenocarcinoma xenograft.How Fig. 8 A and the display of the result shown in Fig. 8 B use bsMAb pre-targeting method with warp ¹¹¹two HSG peptide IMP-288 of In mark detect the tumour of clear description in animal model.Six animals above in Fig. 8 A and Fig. 8 B accept the TF10 (be 10:1 and 20:1 with the mol ratio of the molal quantity of given peptide) of 2 various dose, and second day gives their warps ¹¹¹two HSG peptides (IMP288) of In mark.Other 3 animals below in Fig. 8 A and Fig. 8 B only accept ¹¹¹in-IMP-288 (without pre-targeting).Image shown in Fig. 8 B gathers at injection 3h after the peptide of mark, and show the expliciting the position of 0.2-0.3g tumour in pre-targeting animal, do not demonstrate and giving separately ¹¹¹location in the animal of In-peptide.Tumor uptake average out to 20-25%ID/g, tumor/blood ratio is more than 2000:1, and tumour/liver ratio is 170:1, and tumour/kidney ratio is 18/1.

Embodiment 19. for pre-targeting and ¹⁸the generation of the targeting peptides of F mark

In multiple embodiment, prepare warp by new technology ¹⁸the albumen of F mark or peptide, and for diagnosis and/or imaging research, such as PET imaging.For ¹⁸the new technology of F mark relates to preparation ¹⁸f-metal complex, preferably ¹⁸f-aluminium complex, the chelating moiety chelates such as described complex compound and such as DOTA, NOTA or NETA or derivatives thereof.Chelating moiety can use conjugation techniques well known in the art to be connected to protein, peptide or other molecule any.In certain preferred aspects, formed first in the solution ¹⁸f-Al complex compound, and be connected to the chelating moiety puted together with protein or peptide subsequently.But, in an alternate embodiment, can first make aluminium be connected to chelating moiety, and add subsequently ¹⁸f.

Peptide symthesis

Fmoc strategy is used to carry out synthetic peptide by Solid phase peptide synthesis.By using Fmoc/Aloc protecting group that group is added the amino acid whose side chain of diamido, go protection with the tolerance opposite sex.Remove Aloc group by the method for (J.Org.Chem.1987,52:4984-4993) such as Dangles, but add piperidines with the ratio of 1:1 to used acetic acid.The preparation of asymmetric tetra-tert DTPA carries out described in (U.S. Patent Application Publication No. 2005/0002945, embodiment part is incorporated herein by reference) such as McBride.

Tri-tert DOTA, symmetrical tetra-tert DTPA, ITC-benzyl DTPA, p-SCN-Bn-NOTA and TACN be available from (Dallas, TX).DiBocTACN, NODA-GA (tBu) ₃purchased from CheMatech (Dijon, France) with NO2AtBu.Aloc/Fmoc lysine and Dap (diaminopropionic acid derivative (also referred to as Dpr)) be available from (Louisville, KY) or (Torrance, CA).Sieber amide resin be available from (SanDiego, CA).All the other Fmoc amino acid be available from (Burlington, MA), EMD (SanDiego, CA), CHEM (WoodDale, IL) or aluminium chloride hexahydrate be purchased from (Milwaukee, WI).Remaining solvent and reagent are purchased from FISHER (Pittsburgh, PA) or (Milwaukee, WI). ¹⁸f is by IBA (Somerset, NJ) supplies.

IMP272's ¹⁸f marks

Prepared warp ¹⁸the peptide of F mark is IMP272:

DTPA-Gln-Ala-Lys(HSG)-D-Tyr-Lys(HSG)-NH ₂MH1512

IMP272 synthesizes (U.S. Patent number 7,534,431, embodiment part is incorporated herein by reference) as described.

In acetate buffer solution-be diluted in by 1.509g acetic acid ~ 160mL water, and carrying out adjust ph by adding 1MNaOH, being diluted to 250mL subsequently, to produce 0.1M pH value of solution 4.03.

Aluminium acetate buffer solution-pass through 0.1028g six hydration AlCl ₃be dissolved in 42.6mLDI water and prepare aluminum solutions.The 4mL aliquot of aluminum solutions is mixed with 16mL0.1MNaOAc pH value of solution 4, obtains 2mMAl stock solution.

IMP272 acetate buffer solution-by 0.0011g peptide, 7.28 × 10 ^-7molIMP272 is dissolved in 364 μ L0.1MpH4 acetate buffer solutions, obtains 2mM peptide stock solution.

IMP272 F-18 mark-3 μ L aliquots of aluminium stock solution are placed on REACTI-VIAL ^tMin, and with 50 μ L ¹⁸f (according to former state when receiving) and the mixing of 3 μ LIMP272 solution.In heat block, at 110 DEG C, solution is heated 15 minutes, and analyzed by anti-phase HPLC.HPLC analyzes the free of (not shown) display 93% ¹⁸f and 7% is combined with peptide.In reactant, add 10 μ LMP272 solution again add reaction and again heat, and analyzed by anti-phase HPLC (not shown).HPLC trace display 8% ¹⁸f is in voidage and the activity of 92% is connected to peptide.All the other peptide solutions at room temperature hatch together with 150 μ LPBS ~ and 1 hour, and checked by anti-phase HPLC subsequently.HPLC (not shown) display 58% ¹⁸f does not combine, and 42% is still connected to peptide.Data show, when mixing with phosphate, ¹⁸f-Al-DTPA complex compound may be unstable.

By the peptide solution through mark is applied to 1cc (30mg) hLB post (part number: 186001879) go up and carry out the peptide of purifying through mark with 300 μ L water washings to remove unconjugated F-18.By with 2 × 100 μ L1:1EtOH/H ₂o washs pillar by peptide wash-out.In water, at 25 DEG C, hatch purified peptide, analyzed by anti-phase HPLC (not shown).HPLC analyzes display, warp ¹⁸the MP272 of F mark is unstable in water.Hatch 40 minutes in water after, about 17% ¹⁸f discharges from peptide, and 83% is retained (not shown).

With ¹⁸f marks peptide (16 μ L2mMIMP272,48 μ g), and analyzes antibody combination by size exclusion HPLC.Size exclusion HPLC shows, peptide in conjunction with hMN-14 × 679, but not in conjunction with irrelevant bispecific antibody hMN-14 × 734 (not shown).

With other metal marker IMP272 ¹⁸f

By metal stock solution (6 × 10 ^-9mol) ~ 3 μ L aliquots are placed in polypropylene conical flask, and with 75 μ L ¹⁸f (according to receiving former state) mixing, at room temperature hatches ~ 2 minutes, and subsequently with 20 μ L2mM (4 × 10 ^-8mol) the solution mixing of IMP272 in 0.1MNaOAcpH4 damping fluid.In heat block, at 100 DEG C, solution is heated 15 minutes, and analyzed by anti-phase HPLC.With indium (24%), gallium (36%), zirconium (15%), lutetium (37%) and yttrium (2%) (not shown) mark IMP272.These results show, ¹⁸f metal marker technology is not limited to aluminium ligand, but also can utilize other metal.Utilize different metal ligands, different chelating moieties can be utilized to optimize the combination of F-18-metal conjugate.

The warp of serum stable ¹⁸the generation of the peptide MP449 of F mark and use

On Sieber amide resin, prepare peptide IMP448D-Ala-D-Lys (HSG)-D-Tyr-D-Lys (HSG)-NH by following amino acid is added resin with shown order ₂mH ⁺1009:Aloc-D-Lys (Fmoc)-OH; Trt-HSG-OH; Make Aloc cracking; Fmoc-D-Tyr (But)-OH; Aloc-D-Lys (Fmoc)-OH; Trt-HSG-OH; Make Aloc cracking; Fmoc-D-Ala-OH; Finally carry out Fmoc cracking, to produce required peptide.Make peptide from cracking resin subsequently, and by HPLC in addition purifying, produce IMP448, subsequently with ITC-benzyl NOTA coupling.By 0.0757g (7.5 × 10 ^-5mol) peptide IMP448 and 0.0509g (9.09 × 10 ^-5mol) ITC benzyl NOTA mixes, and is dissolved in 1mL water.Subsequently Anhydrous potassium carbonate (0.2171g) is slowly added in the peptide/NOTA solution of stirring.After adding all carbonate, reaction solution is pH10.6.At room temperature reaction stirred is spent the night.With 1MHCl cancellation reaction carefully after 14 hours, and by HPLC in addition purifying, obtain 48mgIMP449.

IMP449's ¹⁸f marks

By peptide IMP449 (0.002g, 1.37 × 10 ^-6mol) be dissolved in 686 μ L (2mM peptide solution) 0.1MNaOAcpH4.02.By the 2mM solution of Al in pH4 acetate buffer (3 μ L) and 15 μ L1.3mCi ¹⁸f mixes.Subsequently solution is mixed with 20 μ L2mMIMP449 solution, and heat 15 minutes at 105 DEG C.Anti-phase HPLC analyzes display, 35% (t _r~ 10min) activity be connected to peptide, and the activity of 65% wash-out (3.1min, not shown) in the voidage of post, show that most of activity is not associated with peptide.The rough potpourri (5 μ L) through mark mixes with the serum human collected, and hatches at 37 DEG C.Take out aliquot after 15 minutes, and analyzed by HPLC.HPLC shows, and the activity of 9.8% is still connected to peptide (declining from 35%).Remove another aliquot after 1 hour, and analyzed by HPLC.HPLC shows, and the activity of 7.6% is still connected to peptide (declining from 35%), this substantially with 15 minutes traces identical (data are not shown).

High dose ¹⁸f marks

Utilize other research display of purified IMP449, warp ¹⁸the peptide highly stable in 37 DEG C of serum humans (91%, not shown) at least one hour of F mark, and partially stabilized in 37 DEG C of serum humans (76%, not shown) at least four hours.Carry out other research preparing IMP449 under the existence of the ascorbic acid as stabilizing agent.(not shown) in those researchs, metal- ¹⁸f-peptide complex compound does not show detectable decomposition after 4 hours in 37 DEG C of serum.At injection warp ¹⁸after the peptide of F mark 30 minutes, find that mouse urine comprises and be combined with peptide ¹⁸f (not shown).These results show, warp disclosed herein ¹⁸f mark peptide for ¹⁸abundant stability is presented under condition in the approximation of F imaging research.

For the research that there is not ascorbic acid, will contain ¹⁸f ~ 21mCi ~ 400 μ L water and 9 μ L are containing 2mMAlCl ₃0.1MpH4NaOAc mixing.Add 60 μ L peptide IMP449 (0.01M, 6 × 10 ^-7mol, is in 0.5NaOHpH4.13), and maintain 15 minutes after solution is heated to 110 DEG C.Subsequently in the following manner purification of crude through mark peptide: reaction solution is placed on 1cc in the cylinder of HLB post, and wash with water to remove unconjugated ¹⁸f, uses 1:1EtOH/H subsequently ₂0 wash-out warp ¹⁸the peptide of F mark.Rough reaction solution enters waste liquid bottle by pillar, washs pillar with 3 × 1mL fraction water (18.97mCi).Subsequently HLB post is placed on new bottle, and with 2 × 200 μ L1:1EtOH/H ₂o wash-out, to collect the peptide (1.83mCi) through mark.After all wash-outs complete, it is active that pillar retains 0.1mCi.By purified warp ¹⁸the aliquot (20 μ L) of the peptide of F mark mixes with the serum human collected by 200 μ L, and heats at 37 DEG C.Aliquot is analyzed by anti-phase HPLC.Result show through ¹⁸the time zero that the purified IMP449 of F mark is hatched at 37 DEG C in serum human, the relative stability (not shown) of a hour (91% through mark peptide), two hours the peptide of mark (77% through) and four hours (76% peptide) through marking.Also observe, warp ¹⁸the IMP449 of F mark is stable in TFA solution, and this solution uses once in a while during anti-phase HPLC chromatogram.For exemplary warp described herein ¹⁸the molecule of F mark, observes and seems to there is general correlativity between stability in TFA and the stability in serum human.These results show, according to warp prepared by method disclosed herein ¹⁸the peptide of F mark demonstrates and is enough to be used successfully to body internal labeling and imaging research in serum human, such as, use PET scanning to detect the stability of the cell or tissue through mark.Finally, because IMP449 peptide contains the thiocarbamide binding to radiolysis sensitivity, so observe several product by RP-HPLC.But when adding ascorbic acid in reaction mixture, the accessory substance produced significantly reduces.

Embodiment 20. utilizes pre-targeting TF10DNL ^tMconstruct and warp ¹⁸the In vivo study of the peptide of F mark

Following preparation warp ¹⁸the IMP449 of F mark.General ~ 0.5mL54.7mCi ¹⁸f mixes containing the 0.1MNaOAcpH4 damping fluid of 2mMAl with 3 μ L.After 3 minutes, add the 0.5MpH4NaOAc damping fluid of 10 μ L containing 0.05MIMP449, and in 96 DEG C of heat blocks, reactant is heated 15 minutes.Reaction content is taken out with syringe.Subsequently by HPLC at C ₁₈on post, purification of crude is through the peptide of mark.Flow velocity is 3mL/min.Buffer A is the water containing 0.1%TFA, and buffer B is in 90% acetonitrile containing in the water of 0.1%TFA.Gradient reached 75/25A:B from 100%A in 15 minutes.First wash-out through mark peptide and the peptide of un-marked between hold-up time (t _r) difference is about 1 minute.HPLC eluent is collected in 0.5 minute (mL) fraction.Peptide through mark has the t between 6 to 9 minutes _r, depend on used pillar.By relevant fraction twice to be diluted in water and solution is placed on 1cc in the cylinder of HLB post, the peptide sample through HPLC purifying is further processed.With 3 × 1mL water elution filter cylinder to remove acetonitrile and TFA, use 400 μ L1:1EtOH/H subsequently ₂0 wash-out warp ¹⁸the peptide of F mark.Purified [Al ¹⁸f] IMP449 is at analytic type HPLCC ₁₈as unimodal wash-out (not shown) on post.

Use with four slow growth subcutaneous CaPan1 xenograft nude mice carries out In vivo study.To three injected in mice TF10 (162 μ g), inject [Al after 18 hours subsequently ¹⁸f] IMP449.TF10 is the humanization bispecific antibody for tumor imaging research, is combined and is combined with HSG monovalence (see such as Gold etc., 2007, J.Clin.Oncol.25 (18S): 4564) with the tumour antigen divalence that PAM-4 limits.A mouse only injects peptide.All mouse carry out autopsy in latter 1 hour in peptide injection.Immediately tissue is counted.To the comparison display be evenly distributed, under the existence of the bispecific antibody of target tumor, be positioned the warp in tumour ¹⁸the level of the peptide of F mark is in fact higher than any normal structure (data are not shown).

Organize picked-up to be similar to and only give [Al ¹⁸f] animal of IMP449 or pre-targeting setting (data are not shown).In pre-targeting animal, human pancreatic adenocarcinoma xenograft CaPan1 increases by 500 (4.6 ± 0.9%ID/g contrasts 0.89%ID/g) the picked-up of 1 hour compared with independent peptide.Now reach specific tumour/non-tumour ratio (such as, tumor/blood and liver are 23.4 ± 2.0 and 23.5 ± 2.8 than respectively).

Result shows, warp ¹⁸the peptide of F mark and such as TF10DNL ^tMconstructs etc. provide containing the antibody construct conbined usage of PAM4 ¹⁸f mark suitable target to carry out in-vivo imaging, such as PET imaging analysis.

Embodiment 21. utilizes other imaging research of TF10

General introduction

Before clinical and clinical research shown through radiolabeled mAb-PAM4 for the application in the nuclear imaging of cancer of pancreas and Radioimmunotherapy.In this article, we examine TF10 construct pre-targeting through radiolabeled peptide to improve the ability of imaging and treatment.The biodistribution research through radiolabeled TF10 and/or TF10 pre-targeting haptens-peptide (IMP-288) and nuclear imaging is carried out in the nude mice with CaPan1 human pancreatic adenocarcinoma xenograft. ¹²⁵i-TF10 removes fast from blood, and by 16 hours, its level was down to <1% injected dose/gram (ID/g).The tumor uptake of this time point is 3.47 ± 0.66%ID/g, and all without gathering in any normal structure.In order to show the practicality of pre-targeting method, within 16 hours after TF10, use ¹¹¹in-IMP-288.After using through radiolabeled peptide 3 hours, the strong picked-up in imaging display tumour, and have no in any normal structure and gather sign.Only giving ¹¹¹target is not observed in the animal of In-peptide.Through TF10 pre-targeting ¹¹¹the tumor uptake of In-IMP-288 is 24.3 ± 1.7%ID/g, and for independent ¹¹¹in-IMP-288, was only 0.12 ± 0.002%ID/g at 16 hours.Pre-targeting group tumor/blood ratio (3 hours, ~ 1,000:1) is significantly greater than ¹¹¹in-PAM4-IgG (24 hours, ~ 5:1; P<0.0003).Radiation dose estimated value shows, TF10/ ⁹⁰y-peptide pre-targeting will provide ratio ⁹⁰the antitumous effect that Y-PAM4-IgG is larger.Therefore, this result support is with compared with direct radiolabeled PAM4-IgG, and TF10 pre-targeting can provide the early detection of imaging for cancer of pancreas, the Diagnosis and Treat of improvement.(Gold etc., CancerRes2008,68 (12): 4819-26)

We authenticated unique biomarker thing existing on the mucin of >85% aggressive cancer of pancreas expression, to comprise in early stage I disease and precursor lesion, pancreas epithelium palilate myxoma in neoplasia and conduit and form (Gold etc., ClinCancerRes2007,13:7380-87).Defined epitope (Gold etc., IntJCancer1994,57:204-10) as detected by mAb-Ρ Α Μ 4 is not present in pancreatic tissue that is normal and inflammation, and in other malignant tissue of great majority.Therefore, the height diagnosis possibility for the neoplastic existence of pancreas is provided to the detection of epi-position.Use warp respectively ¹³¹i-and ^99mthe muroid PAM4IgG of Tc mark or the early clinic of Fab' are studied in 8 in 10 aggressive Pancreas cancer patients and are shown selectively targeted (Mariani etc., CancerRes1995,55:5911s-15s; Gold etc., CritRevOncolHematol2001,39:147-54).In two negative patients, a cancer of pancreas suffering from the bad differentiation not expressing PAM4 epi-position, and another one patient is finally found to suffer from pancreatitis, instead of malignant change.

Therefore, PAM4 can be used for the diagnosis and detection of early stage disease for the high specific of cancer of pancreas.Except improvement detects, ⁹⁰y-PAM4IgG is found the large-scale human pancreatic adenocarcinoma xenograft (Cardillo etc. that can effectively treat in nude mice, ClinCancerRes2001,7:3186-92), and when combining with gemcitabine, observe the further of therapeutic response and improve (Gold etc., ClinCancerRes2004,10:3552-61; Gold etc., IntJCancer2004,109:618-26).Complete I phase therapeutic test in the patient failed to respond to any medical treatment at gemcitabine recently, find ⁹⁰the maximum tolerated dose of Y-humanization PAM4IgG is 20mCi/m ²(Gulec etc., ProcAmerSocClinOnc, the 43rd annual meeting, JClinOncol2007,25 (18S): 636s).Although all patients were at the 8th week or all show progression of disease afterwards, in several cases, observe tumour tentatively shrink.Just carrying out now clinical research with assessment ⁹⁰the divided doses scheme that the gemcitabine of Y-hPAM4IgG and radiosensitizing dosage combines.

In this article, we report and develop novel recombinant humanized bispecific monoclonal antibody (mAb) TF10 based on the targeting specific of PAM4 to cancer of pancreas.This construct also combines unique synthesis haptens histamine-amber and claps acyl-glycocoll (HSG), it is merged in and is availablely applicable to single photon emission computerized tomography (SPECT) and positron emission tomography (PET), and the multiple radioactive nuclide being used for the treatment of object carries out (Karacay etc. in radiolabeled many little peptides, ClinCancerRes2005,11:7879-85; Sharkey etc., Leukemia2005,19:1064-9; Rossi etc., ProcNatlAcadSciUSA2006,103:6841-6; McBride etc., JNuclMed2006,47:1678-88).These researchs illustrate the potentiality of these novel construct targeted pancreatic cancers for imaging or treatment use.

Method and material

Preparation TF2 described above and TF10 bispecific DNL ^tMconstruct and IMP288 targeting peptides.Sodium iodide ( ¹²⁵i) and inidum chloride ( ¹¹¹in) be available from tF10 uses ¹²⁵i is by iodide process method.Used by iodide process method ¹²⁵i carries out conventional labels to TF10, uses size exclusion column spinner to carry out purifying.With ¹¹¹it is as discussed previouslyly carry out (Rossi etc., ProcNatlAcadSciUSA2006,103:6841-6 that InCl carries out radioactive label to DOTA-peptide and DOTA-PAM4-IgG; McBride etc., JNuclMed2006,47:1678-88).By the inspection of size exclusion high performance liquid chromatography through the purity of radiolabeled product, wherein determine that dissociate, unconjugated isotopic amount by instantaneous TLC.

For TF10 distribution research, give with subcutaneous CaPan1 human pancreatic adenocarcinoma xenograft female athymic nude mice ~ 20g ( farms) inject ¹²⁵i-TF10 (10 μ Ci; 40 μ g, 2.50 × 10 ^-10mol).At different time points, carry out autopsy to organizing mouse (n=5) more, take out tumour and nonneoplastic tissue, count with gamma counter, to determine the number percent (%ID/g) of every gram of injected dose organized, these values are for calculating blood clearance and tumour/non-tumour ratio.

For pre-targeting biodistribution research, use bispecific mAb/ through radiolabeled peptide mol ratio 10:1.For example, use TF10 (80 μ g, 5.07 × 10 to the nude mouse group with subcutaneous CaPan1 human pancreatic adenocarcinoma xenograft ^-10and second group of maintenance is untreated mol).Latter 16 hours of TF10 injection, uses ¹¹¹in-IMP-288 haptens-peptide (30 μ Ci, 5.07 × 10 ^-11mol).At several time points, autopsy is carried out to mouse, take out tumour and nonneoplastic tissue, and count with gamma counter, to determine %ID/g.Tumour/non-tumour ratio is calculated by these data.In an independent research, for the object comparing bio distribution, nuclear imaging and potential therapeutic activity, give each group of mouse ¹¹¹in-DOTA-PAM4-IgG (20 μ C, 50 μ g, 3.13 × 10 ^-10mol).By time m-activity curve calculation of radiation dose estimated value, wherein suppose at time zero non-activity.Use Si Shi t inspection assessment significant difference.

In order to carry out core immunoscintigraphy, at after the injection of radiolabeled peptide 3 hours, or after radiolabeled hPAM4-IgG injection 24 hours, be furnished with for ¹¹¹the double end Solus gammacamera (ADACLaboratories) of the middle energy collimating apparatus of In carries out imaging to the mouse with tumour.To mouse imaging altogether 100,000cpm or 10min, be as the criterion with first comer.

Result

the vitro characterization of two special mAbTF10.by the combination (Fig. 9) of elisa assay TF10 and target mucin antigen.Result display divalence TF10, PAM4-IgG and PAM4-F (ab') ₂binding curve (half maximum combined is calculated as 1.42 ± 0.10,1.31 ± 0.12 and 1.83 ± 0.16nmol/L respectively close to identical; All P>0.05), and the chemically conjugated thing of monovalence bsPAM4 (PAM4-Fab' × anti-DTPA-Fab') have significantly lower affinity (compared with TF10, half maximum combined 30.61+2.05nmol/L; P=0.0379), show that TF10 combines in divalence mode.Be combined with MUC5ac ¹²⁵the immunoreactivity mark of I-TF10 is 87%, find that 9% in unconjugated TF10 form, and 3% is free iodide form (not shown).90% ¹¹¹in-IMP-288 and TF10 is in conjunction with (not shown).Total what be combined with TF10 ¹¹¹in In-IMP-288, when adding excessive mucin (200 μ g), 92% under high molecular wash-out, only 3% with Nonviscous protein reactive TF10 fraction wash-out.In addition 5% through radiolabeled peptide wash-out in free peptide volume.When there is not TF10, in radiolabeled peptide, none and mucin antigen are in conjunction with (not shown).

¹²⁵ the ox thing distribution of I-TF10 in the nude mice with CaPan1 tumour.tF10 display is removed fast from blood, starts, be down to the only 0.13 ± 0.02%ID/g of 16 hours with the 21.03 ± 1.93%ID/g of 1 hour.Be calculated as 2.19 hours biological half-life [95% fiducial interval (95%CI), 2.11-2.27 hour].Increased activity when tissue picked-up display 1 hour in liver, spleen and kidney, only just removes [for liver, spleen and kidney T respectively for 16 hours fast _1/2=2.09 hours (95%CI, 2.08-2.10), 2.84 hours (95%CI, 2.49-3.29) and 2.44 hours (95%CI, 2.28-2.63)].Gathering and secreting of active most probable reflection radioiodine under one's belt, shows probably in liver and spleen, have catabolic activity through radioiodinated TF10, thus explains it and remove fast from blood.However, by 16 hours, the concentration of radioiodine in stomach is at below 1%ID/g.Give ¹²⁵i-TF10 and demonstrate similar Tissue distribution without tumour nude mice 16 hours five of carrying out in Autopsied a group, shows that tumour does not affect bispecific mAb and distributes and remove (data are not shown) from normal structure.Certainly, likely before the initial time point of inspection, there is difference.The tumor uptake of TF10 reaches peak value (7.16 ± 1.10%ID/g) for 6 hours after injection, and is down to half maximum combined (3.47 ± 0.66%ID/g) at 16 hours.In ensuing 32 hours, tumor uptake reduces nearly 2 times again, but stable in ensuing 24 hours subsequently.

the warp of TF10 pre-targeting ¹¹¹ the bio distribution of the peptide of In mark.although the maximum tumor uptake of TF10 occurred at 6 hours, previous experiences shows, gives needing the time point being scavenged into <1%ID/g (that is, 16 hours) in the blood level of TF10 through radiolabeled peptide.Unacceptable height combination is there is (namely through radiolabeled peptide in higher the causing of the TF10 level in blood in blood, low tumor/blood ratio), and will mean that the concentration of TF10 in tumour reduces, and therefore reduces through the concentration of radiolabeled peptide in tumour at time administration for peptides after a while.Therefore, 16 h apart are used to carry out preliminary pre-targeting research.? ¹¹¹the amount of In-IMP-288 keeps constant (30 μ Ci, 5.07 × 10 ^-11mol), when, the TF10 giving recruitment makes the application dosage of TF10 and IMP-288 represent from 5:1 with mol ratio to become 20:1 (table 11).

Table 11. is (without TF10) or the TF10 pre-targeting through difference amount separately ¹¹¹the bio distribution of In-IMP-288

At 3 hours, in blood ¹¹¹the amount of In-IMP-288 almost can't detect (0.01%).Amount of application along with bispecific mAb increases by four times, and tumor uptake is increased to 28.55 ± 0.73%ID/g from 19.0 ± 3.49%ID/g and (observes significant in fact difference when comparing one group of each TF10/ peptide ratio relative to another group; P<0.03 or better), but normal structure picked-up is without any obvious increase.The tumor uptake ratio given in the animal of TF10 gives independent ¹¹¹during In-IMP-288, height >100 doubly.In the normal structure of the animal received before using TF10 or do not receive ¹¹¹the comparison sheet of In activity reveals similar absolute value, in most of the cases without significant difference.This shows, fully removes from all normal structures by 16 hours bispecific mAb, thus avoids the obvious peptide picked-up in these tissues.Tumor/blood is than >2,000:1, and other tissue ratio is more than 100:1.Even if tumour/kidney ratio is also more than 10:1.The highest radioisotopic tumor uptake of minimum target nonneoplastic tissue is produced by the ratio of 20:1; But arbitrary TF10/ peptide ratio all can be used for realizing specific cancer target, and the two all relates to signal intensity and contrast-ratio.10:1 ratio is selected to study further, because through absolute difference no significant difference between 10:1 (24.3 ± 1.71%ID/g) and 20:1 (28.6 ± 0.73%ID/g) ratio of the tumor uptake of radiolabeled peptide.

Give bispecific mAb/ peptide ratio 10:1 through TF10 pre-targeting ¹¹¹in-IMP-288 or independent ¹¹¹the image of the animal of In-IMP-288 peptide is shown in Figure 10 A, Figure 10 B and Figure 10 C.Most diameter≤the 0.5cm of these tumours, weight is ~ 0.25g.Described image shows high strength picked-up (Figure 10 A) in the tumour of the animal of TF10 pre-targeting.Increase the image background intensity of the animal of TF10 pre-targeting, so that with independent for giving ¹¹¹the image intensity that the animal of In-IMP-288 gathers matches (Figure 10 B).But, when mouse optimized image for TF10 pre-targeting, signal intensity and contrast too high so that do not observe in body that other is active.Give separately ¹¹¹tumor-localizing is had no, even if when strengthening image intensity (Figure 10 C) in the animal of In-IMP-288.

Carry out other experiment to assess target ¹¹¹full IgG and the TF10 pre-targeting of In-hPAM4 ¹¹¹the dynamics that In-IMP-288 peptide is compared. ¹¹¹the tumor uptake of In-peptide is at the initial time point (3 hours) the highest (15.99 ± 4.11%ID/g) of inspection, and be only 0.02 ± 0.01%ID/g through the haemoconcentration of radiolabeled peptide, thus provide the average tumor/blood ratio of 946.3 ± 383.0.Pass in time, remove through radiolabeled peptide from tumour, biological half-life is 76.04 hours.In nonneoplastic tissue, absorbing in kidney the highest, is 1.89 ± 0.42%ID/ in 3 hourly averages, passes steady decrease (33.6 hours biological half-lifes) in time.Liver picked-up starts from 0.15 ± 0.06%ID/g, and passing remains basically unchanged in time.With TF10 pre-targeting ¹¹¹in-IMP-288 compares, ¹¹¹in-hPAM4-IgG removes comparatively slow from blood, even if substantially removed in first 24 hours, is down to the only 11.5 ± 1.7%ID/g of 24 hours from the 30.1%ID/g of 3 hours.Variable rising picked-up in spleen shows that antibody can be removed from blood by the mucinous target of secretion be trapped in spleen.Tumor uptake reached peak value 80.4 ± 6.1%ID/g at 48 hours, and kept high level monitoring periods duration.High tumor uptake is along with than expecting that IgG blood faster removes the tumor/blood ratio producing 5.2 ± 1.0 when 24 is little.Figure 10 C shows ¹¹¹in-PAM4-IgG uses the animal painting of latter 24 hours, illustrates that tumour in described early stage range estimation, but can still have suitable activity in abdominal cavity.Tumour/non-tumour ratio in most of the cases TF10 pre-targeting ¹¹¹haptens-peptide the ratio of In mark ¹¹¹in-hPAM4-IgG is higher, but except kidney, wherein ¹¹¹in-IMP-288 and ¹¹¹in-hPAM4-IgG at the tumour/kidney of time subsequently than similar.But, TF10 pre-targeting ¹¹¹tumour/kidney the ratio of In-IMP-288 is enough to (such as, ~ 7:1) and easily distinguished tumour and normal structure at 3 hours.

Figure 11 A to Figure 11 D describe send radioactive nuclide ( ⁹⁰the potential treatment ability of direct and pre-targeting method Y).Although respective maximum tolerated dose (for ⁹⁰y-hPAM4 is 0.15mCi, and for TF10-pre-targeting ⁹⁰y-IMP-288 is 0.9mCi), compared with the TF10 of pre-targeting, when being sent by PAM4-IgG, in tumour, radioisotopic concentration (%ID/g) seems higher (Figure 11 A), but the radiation dose of tumour should be similar (for ⁹⁰y-PAM4-IgG and through TF10 pre-targeting ⁹⁰y-IMP-288 is 10,080 and 9,229cGy respectively) (Figure 11 C).The advantage of pre-targeting method is the activity abnormal low (9cGy) in blood, almost than ⁹⁰y-hPAM4IgG (1,623cGy) low 200 times (Figure 11 C).It is equally important that notice to the radiation dose of liver and other non-Neoplastic organ than TF10 pre-targeting ⁹⁰y-IMP-288 much lower (Figure 11 B, Figure 11 D).Exception be kidney, wherein respective maximum dose (for ⁹⁰y-PAM4-IgG and TF10- ⁹⁰y-IMP-288 be respectively 612 and 784cGy) under the radiation dose similar (Figure 11 B, Figure 11 D) of two schemes.Data show, for ⁹⁰y-PAM4-IgG, as great majority other through radiolabeled full IgGmAb, dose-limiting toxicity will be blood; But for TF10 pre-targeting scheme, dose-limiting toxicity will be kidney.

Discuss

Find current diagnostic methods, all if provide the PET imaging of ultrasonic, the computer tomography (CT) of anatomic image and magnetic resonance imaging (MRI) technology and metabolism environment through being usually used in providing hypersensitivity in the detection at pancreatic mass.But in most cases, these data are based on the detection to the pathology of >2cm in the colony occurring clinical symptoms.Now, in the progress of cancer of pancreas, prognosis is on duty mutually.In order to improve the result of patient, be necessary in asymptomatic patient, detect little early stage pancreas neoplasm.

Use the imaging of mAb target method, the imaging of all use mAb-PAM4 as described herein, can provide the diagnosis of these little early-stage cancers.The most important thing is the specificity of mAb.We provide considerable data, comprise immunohistochemistry research (Gold etc., the ClinCancerRes2007 of tissue sample; 13:7380-7; Gold etc., IntJCancer1994; 57:204-10) and immunoassays (Gold etc., the JClinOncol2006 of patients serum; 24:252-8), demonstrate the high response of mAb-PAM4 and biomarker, its existence provides pancreas neoplastic height diagnosis possibility.In addition, although we determine that PAM4 does not neither also have reactivity with active pancreatitis with Normal adult pancreatic tissue, but with the very early time in pancreas go to live in the household of one's in-laws on getting married natural disposition be in progress (in pancreas epithelium, in neoplasia 1 and conduit, palilate myxoma is formed) there is reactivity, and biomarker is keeping high level expression (Gold etc., ClinCancerRes2007 in the whole process of aggressive Pancreatic Carcinoma; 13:7380-7).Use with the preclinical study of the nude mouse of human pancreatic tumors xenograft demonstrate PAM4 through radiolabeled muroid, chimeric and humanization pattern selectively targeted.

In current research, we examine the construct TF10 based on restructuring bispecific PAM4 of future generation, and its PAM4 arm is divalence, and anti-HSG haptens arm is monovalence.The construct of this pre-targeting system, i.e. DOCK-AND-LOCK ^tM, there is several key characters, comprise its general applicability and synthesis easily.But, for consideration of the present invention, be quantivalency with the Main Differences of the chemical building body previously reported, which improve the combination with tumour antigen, and importantly improve its pharmacokinetics.TF10 removes than viewed quicker to chemically conjugated thing from nonneoplastic tissue.For chemical building body, the time that the blood level of bispecific construct is less than 1%ID/g is latter 40 hours of injection, and TF10 is then 16 hours.Removing more fast of pre-targeting agent substantially improves tumor/blood ratio, maintains high signal intensity (%ID/g) at tumor locus simultaneously.

Except providing early detection and diagnostic means, result also supports that TF10 pre-targeting system is for cancer therapy.With compared with direct radiolabeled PAM4-IgG, pre-targeting method is conducive to the consideration of the Net long wave radiation dosage of tumour and nonneoplastic tissue.Dosage estimated value shows, with kidney for compared with TF10 pre-targeting system, for through direct radiolabeled PAM4, two delivery systems have different dose-limiting toxicities: bone marrow toxicity.This is significant through radiolabeled PAM4 as therapeutic agent for future clinical exploitation.

Select the first-line drug gemcitabine being used for cancer of pancreas can provide the significantly radiosensitizing of tumour cell.In previous research, we show compared with the arm independent with any one, gemcitabine and the combination through direct radiolabeled PAM4-IgG provide synergistic antitumor effect (Gold etc., ClinCancerRes2004,10:3552-61; Gold etc., IntJCancer2004,109:618-26).The dose-limiting factor of this combination is overlapping blood toxicity.But due to dose-limiting organ seemingly kidney instead of the blood tissues of TF10 pre-targeting, therefore the toxicity combined with gemcitabine should be more weak, thus allow increase to use radioactive isotope, therefore there is larger antitumor efficacy.

By the photo-quality imaging that TF10 pre-targeting realizes in preclinical models, with compared with direct radiolabeled DOTA-PAM4-IgG, carry out clinical testing for this imaging system and provide compellent reason.Cancer target mAb to the excrescent specificity of pancreas with can make various imaging compounds with for SPECT ( ¹¹¹in), PET ( ⁶⁸ga), the HSG-haptens-peptide of ultrasonic (Au), or other contrast preparation, or be used for the treatment of on this point ⁹⁰the bispecific antibody platform technology coupling that Y or other radioactive nuclide are puted together provides the high potentiality (Goldenberg etc., JNuclMed2008,49:158-63) improving general patients prognosis.Specifically, we believe that the immune PET program based on TF10 will have important clinical value, individuality under the excessive risk suffering from cancer of pancreas is in (such as with screening, genetic predisposition, chronic pancreatitis, smoker etc.), and to there is according to routine techniques suspicious abdomen images and/or finding and means that the patient with indication follows up owing to there is biomarker-specific thing or aberrant biological chemistry.When being used as sustained medical plan a part of of these patients of follow-up, the early detection of cancer of pancreas can be realized.Finally, with compared with direct radiolabeled PAM4-IgG, TF10 pre-targeting and gemcitabine combine the opportunity that can provide and better control tumor growth.

Embodiment 22. utilizes gemcitabine and uses the warp of TF10 pre-targeting ⁹⁰the therapy of the pancreatic cancer xenografts of the peptide of Y mark

General introduction

⁹⁰y-hPAM4IgG is just combining with gemcitabine at present and is checking III/IV phase Pancreas cancer patients in the I/II phase tests.We disclose the new method of pre-targeting radioactive nuclide, the radioactivity of similar quantity can be delivered to pancreatic cancer xenografts by the method, but hematotoxicity is less, is more suitable for combining with gemcitabine.Give with ~ 0.4cm ³the nude mice administered recombinant bsMAbTF10 of subcutaneous CaPan1 human pancreatic adenocarcinoma, use after 1 day through ⁹⁰haptens-the peptide (IMP-288) of Y mark.The various dosage of gemcitabine and scheme all add in this treatment, and monitor that tumour progression reaches 28 weeks.In, independent 0.7mCiPT-RAIT only produces 60% instantaneous blood count loss, gives the animal (mankind equivalent ~ 1000mg/m of independent 0.9mCiPT-RAIT and 0.7mCiPT-RAIT+6mg gemcitabine ²) after 9 months, have no the Histological Evidence of Toxicity of Kidney.Single dose 0.25 or the independent PT-RAIT of 0.5mCi can eliminate the tumour of 20% and 80% respectively completely.Monthly gradation PT-RAIT (giving 0.25mCi/ dosage when each gemcitabine circulation starts) adds standard gemcitabine scheme (6mg × 3 weekly; Stop 1 week; Repeat 3 times) significantly increase tumour relative to independent PT-RAIT and reach 3.0cm ³median Time.Other treatment plan checking gemcitabine to add the non-cell toxicity radiosensitizing dosage scheme of PT-RAIT also show and significantly improves therapeutic response relative to independent PT-RAIT.Result display PT-RAIT is the new method of more promising treatment cancer of pancreas.Current data display combination PT-RAIT and gemcitabine will strengthen therapeutic response.

Method

Preparation TF10 bispecific antibody described above.For pre-targeting, there is the nude mice TF10 of human pancreatic cancer cell system CaPan1.After permission TF10 is enough to the time (16h) of removing from blood, use through radiolabeled divalence HSG-peptide.Small-molecular-weight HSG-peptide (~ 1.4kD) was removed in several minutes from blood, the intravasation external space, can in conjunction with the anti-HSG arm of the TF10bsMAb through pre-targeting at this place.Within a few hours, >80% secretes in urine through radiolabeled HSG-peptide, leaves tumor-localizing peptide and trace is in normal structure.

Result

Figure 12 describes to derive from and uses 0.15mCi ⁹⁰y-hPAM4IgG or 0.25 or 0.50mCi is through TF10 pre-targeting ⁹⁰y-IMP-288 is to the (~ 0.4cm determined ³) CaPan1 tumour carries out the therapeutic activity of single therapy.0.5mCi pre-targeting dosage contrast 0.15mCi observes similar antitumor activity through direct radiolabeled IgG dosage, but hematotoxicity is very serious (not shown) under the direct conjugate of this level, and pre-targeting dosage only has moderate toxicity (not shown).In fact, the MTD of pre-targeting of 90Y-IMP-288 is used to be at least 0.9mCi in nude mice.

The combination that Figure 13 shows gemcitabine and PT-RAIT has cooperative effect to antitumor therapy.Mouse 1000mg/m is given weekly in peritonaeum ²(6mg) gemcitabine (GEM) of mankind's dose,equivalent, continues 3 weeks, after 1 week, repeats the program 2 times subsequently in stopping.3 circulation treatments each in, (0.25mCi is through TF10 pre-targeting within 1 day after a GEM dosage, to give PT-RAIT ⁹⁰y-IMP-288).Independent GEM does not make significant difference to tumour progression, and (survival rate is to advance to 3.0cm ³time meter).Compared with untreated animal, independent PT-RAIT improves survival rate, but by close to 10 weeks, GEM and the PT-RAIT scheme of combination adds median survival rate.Because hematotoxicity is to PT-RAIT, tool is not dose-limiting, but it is one of restriction of gemcitabine therapy, so these researchs show that PT-RAIT can add standard GEM therapy, thus has the potentiality of intensified response.The remarkable cooperative effect that gemcitabine adds PT-RAIT is astonishing and unexpected.

Another research examines time of application adds the Graft Versus Tumor of PT-RAIT effect to enhancing gemcitabine.The GEM of single 6mg dosage is in 0.25mCiTF10 pre-targeting ⁹⁰within before Y-IMP-288 1 day or 1 day afterwards, give (not shown).This study demonstrates and know viewpoint about GEM, namely radiosensitizingly preferably to give before irradiation.Through the percentage survival display of the mouse for the treatment of, gave the time-to-live difference that independent PT-RAIT and PT-RAIT adds between gemcitabine at 22 hours after radiolabeled peptide very little.But, within 19 hours before PT-RAIT, use gemcitabine and make the time-to-live significantly increase (not shown).

(1mg weekly, in peritonaeum with Cetuximab for single PT-RAIT (0.25mCi); 7 weeks) or demonstrate GEM+ Cetuximab and PT-RAIT in being combined in CaPan1 animal with Cetuximab+GEM (weekly 6mg × 3) and combine and provide better initial reaction (Figure 14), but add the relevant reaction of PT-RAIT encouraging (Figure 14) to independent Cetuximab, because it is same with PT-RAIT+GEM good or better.Because the overall survival rate in this research is excellent, after research in 24 weeks terminates, only have 2 tumour progressions in each group to >2.0cm3, these results show the latent effect of Cetuximab when adding PT-RAIT.

Embodiment 23. gradation pre-targeting Radioimmunotherapy (PT-RAIT) is to the effect of pancreas cancers therapy

We have evaluated use ⁹⁰the fournier's treatment of the two HSG peptide (IMP-288) of Y-DOTA-and TF10.With 0.32-0.54cm ³carry out in the nude mice of subcutaneous CaPan1 human pancreatic adenocarcinoma xenograft using TF10 and the research through radiolabeled IMP-288.For treatment, give through TF10 pre-targeting ⁹⁰y-IMP-288:[A] once (the 0th week, 0.6mCi); Or [B] gradation (the 0th week and the 1st week, 0.3mCi); [C] (the 0th week, the 1st week and the 2nd week, 0.2mCi); Or [D] (the 0th week, the 1st week and the 4th week, 0.2mCi).

Most of mouse, be respectively in 9/10,10/10,9/10 and 8/10 in [A], [B], [C] and [D] group and observe tumour regression (>90%).In [A] group, there is maximum tumour regression at 3.7 weeks in 50% mouse, is 6.1,8.1 and 7.1 weeks respectively by contrast in [B], [C] and [D].Some tumours demonstrate regrowth.At the 14th week, in gradation group (2 × 0.3mCi), observe optimum therapeutic response, wherein 6/10 mouse is without tumour (NT), by contrast, is 3/10 and is 1/10 in 1 × 0.6mCi group in 3 × 0.2mCi group.Do not observe obvious body weight loss.Gradation PT-RAIT provides another replacement scheme with minimum toxicity being used for the treatment of cancer of pancreas.

Embodiment 24. ⁹⁰y-hPAM4 Radioimmunotherapy (RAIT) adds radiosensitizing gemcitabine (GEM) and treats advanced pancreatic cancer (PC)

PC is had to the humanized antibody of high specific ⁹⁰y-hPAM4 late in Disease and the RAIT that strengthens of GEM in preclinical study, demonstrate instantaneous activity.This research have evaluated ⁹⁰y-hPAM4 add GEM suffer from untreated and in the patient of unresectable PC repetitive therapy circulation. ⁹⁰y dose cohort increases, and carries out repetition 4 weeks circulation (weekly 200mg/m to patient ²gEM, once in a week ⁹⁰y-hPAM42-4 week) until there is progress or unacceptable toxicity.CT, FDG-PET and CA19.9 serum levels is used to carry out reaction assessment.

At front 2 dosage levels (6.5 and 9.0mCi/m ²⁹⁰y-hPAM4 × 3) 8 patients (3F/5M, 56-72 year) in, hematotoxicity is instantaneous 1-2 level.Two patients respond the initial therapy using FDGSUV, and CA19.9 reduces, and CT lesions showed is degenerated.Behind 9 and 11 months and after altogether distinguishing 3 and 4 circulations, two patients continue to keep good performance state, without other toxicity.After initial therapy, PET and CT display has stable reaction and the 3rd patient that CA19.9 level reduces carries out the 2nd circulation now.Other four patients have early stage progression of disease, and all the other patients are still standing assessment.In use ⁹⁰after Y-hPAM4 adds the gradation RAIT of low dosage gemcitabine, dosage continues to increase, and shows initially ⁹⁰therapeutic activity under Y dosage level, hematotoxicity is minimum, even if after 4 treatment circulations.

Embodiment 25. uses the cancer of pancreas early detection of Mab-PAM4 and in vitroimmunoassay

Immunohistochemistry research is carried out with PAM4 antibody.The result display PAM4 obtained with the histotomy of dyeing does not react (not shown) with normal pancreas conduit, ductule and acinar tissue.By contrast, the use being applied to the MA5 antibody of homologue's sample demonstrates the diffusivity positive staining (not shown) of normal pancreatic duct and acinar tissue.Break up in the histotomy of cancer of pancreas in good differentiation or moderate, PAM4 dyeing is for positive, and major part is tenuigenin dyeing, but is strengthened at cell surface.Normal pancreatic tissue in homologue's section is not colored.

Table 12 shows the result of the immunohistochemical analysis of PAM4MAb in each differential period pancreatic cancer samples.In general, for all pancreatic cancer samples, recall rate is 87%, 100% good differentiation cancer of pancreas detected and almost 90% moderate differentiation cancer of pancreas detected.

Table 13 shows the cancer of pancreas precursor lesion that PAM4 immunohistochemical staining also detects high number percent, comprises PanIn-1A to PanIN-3, IPMN (in conduit palilate myxoma) and MCN (mucus cystic neoplasm).In general, PAM4 staining examine is to 89% of all pancreas precursor lesions.The display of these results can detect cancer of pancreas and the precursor lesion of almost 90% by analyzed in vitro based on the immune detection of PAM4 antibody.Observe PAM4 in very early time PanIN development to express.The (not shown) that dyes by force is observed in IPMN and MCN sample.PAM4 epi-position is present in most of cancer of pancreas with high concentration (spreading dyeing by force).PAM4 demonstrates and very early time cancer of pancreas precursor lesion, comprises the diffusivity strong reactivity of PanIN-1, IPMN and MCN, but with normal pancreatic tissue anergy.Generally speaking, these result display PAM4 antibody diagnosis and/or detection can with high specific detection very early time cancer of pancreas development.

Develop the immunoassay based on enzyme for the PAM4 antigen in blood serum sample.Figure 15 shows the result that will PAM4 immunoassays used to come antidiastole cancer of pancreas contrast normal structure and other types of cancer.Relatively cancer of pancreas (n=53) and all other samples (n=233), comprise pancreatitis and breast cancer, oophoroma and colorectal cancer and lymthoma, result display cancer of pancreas detection sensitivity is 77.4%, and detection specificity is 94.3%.The data of Figure 15 are presented in table 14 in a tabular form.

The reactive MUC5ac of PAM4 in table 14. patients serum

Use the data construct ROC curve (not shown) from table 14.Check 283 patients altogether, comprise 53 Pancreas cancer patients, and the existence of comparison loop MUC5ac in Pancreas cancer patients and other samples all, ROC curve provides 0.88 ± 0.03 (95%ci, AUC 0.84-0.92), P value <0.0001, shows that cancer of pancreas and non-pancreatic cancer samples have the difference of highly significant for distinguishing.Relatively pancreas CA and other tumour and normal structure, the determination of serum based on PAM4 shows the susceptibility of 77% and the specificity of 95%.

MUC5ac concentration in normal patient serum sample, " in early days " (1 phase) cancer of pancreas and all pancreatic cancer samples is compared.Sample comprises the serum of the serum of 13 healthy volunteers, the serum of 12 1 phase patients, the serum of 13 2 phase patients and 25 3/4 phase (late period) Pancreas cancer patients.Use the cutoff (horizontal line) of 8.8 units/ml, as by the analysis of ROC curve statistical determined.The frequency distribution of PAM4 antigen concentration is shown in Figure 16, and this figure shows " in early days " 1 phase cancer of pancreas of 92% more than the dead line of diagnosis of pancreatic cancer.The ROC curve measured based on PAM4 is shown in Figure 17, and its display measures for the PAM4 detecting cancer of pancreas has 81.6% susceptibility and 84.6% specificity.

These results confirm that the enzyme immunoassay (EIA) combined based on PAM4 antibody can detect and the PAM4 reactive antigen in quantitative Pancreas cancer patients serum.Immunoassay demonstrates high specific to cancer of pancreas and susceptibility.Major part 1 phase Disease can use PAM4 immunoassay to detect.

In a word, the immunohistology program of PAM4 antibody is adopted to authenticated aggressive cancer of pancreas and its precursor lesion PanIN, IPMN and MCN of about 90%.The enzyme immunoassay (EIA) based on PAM4 of MUC5ac quantitatively in human patients serum can show the high sensitivity and specificity that Early pancreatic carcinoma detects.Because PAM4 is to the high specific of cancer of pancreas, the target of target in the body that mucin biomarker can also serve as imaging and therapeutic agent.When can more effectively treat, the immune PET imaging for detecting " in early days " cancer of pancreas can be used for the early diagnosis of cancer of pancreas.Use the Radioimmunotherapy of humanization PAM4 antibody construct, preferably combine with Radiosensitizers, be used for the treatment of the U of cancer of pancreas.

Other research that in embodiment 26. serum human, PAM4 antigen in vitro detects

In certain embodiments, in experimenter, the existence of MUC5ac and/or the existence of diagnosis of pancreatic cancer is detected preferably by carrying out analyzed in vitro to the sample that such as blood, blood plasma or blood serum sample etc. can utilize non-invasive techniques to obtain.This type of ex vivo analyses may be preferred in such as screening sequence, wherein believes that individuality has pancreatic neoplasm at ad-hoc location without priori reason.The target of this research be exploitation reliable, accurately, based on the determination method of serum for detecting this disease in the very early time of cancer of pancreas.

General introduction

Immunoassays based on PAM4 are used for quantitative healthy volunteer (N=19), the known diagnosis antigen that to be the patient (N=68) of cancer of pancreas and tentative diagnosis be in the serum of the patient (N=29) of chronic pancreatitis.The susceptibility that cancer of pancreas detects is 82%, and the false positive rate of normal healthy controls is 5%.Terminal illness patient has the antigen levels (P<0.01) significantly higher than those early stage Diseases, and for 3/4 phase terminal illness, 2 phases and 1 phase, diagnostic sensitivity is respectively 91%, 86% and 62%.We also have evaluated chronic pancreatitis serum, find 38% pair of antigen positive.But this observation and immunohistochemistry find inconsistent, this immunohistochemistry finds to show that PAM4 antigen is not produced by the pancreatic tissue of inflammation.In addition, the seropositivity Pancreatitis Patients that several tissue samples can be used for Pathological interpretation has the evidence of superfluous natural disposition precursor lesion.The immunohistochemistry display of other pancreatitis sample, 90% is that PAM4 is negative, and all the other are only the weak positive.This shows that intraserous PAM4 antigen positive level not derives from the pancreatic tissue of inflammation, but can be the early stage index of cancer of pancreas.

These results show, and PAM4 determination of serum may be used for detecting Early pancreatic carcinoma, and the positive serum level of PAM4 antigen not derives from the pancreatic tissue of inflammation, but can provide subclinical pancreas neoplastic evidence.

Materials and methods

human sampleserum (N=68) is available from being diagnosed as cancer of pancreas and the patient treated at JohnsHopkinsMedicalCenter (Baltimore, MD), and freezer storage <5.These patients experienced by pancreatic surgery excision separately, thus are Accurate Diagnosis and by stages offering an opportunity.For 1 phase disease, do not observe neoplastic cell in pancreas outside.But the micrometastasis disease be not detected may appear in Pancreas cancer patients, comprise those patients being reported as 1 phase disease.For this reason, we have evaluated follow-up survival data.Be described at least 1 year (time of following up a case by regular visits to by last record) of all patients survive suffering from 1 phase disease, Median survival time is 2.70 (25%=1.32), by contrast, nearest SEER data (2002-2006) report, the Median survival time of the 1 phase Disease of being treated by excision is 1.42 (N=68).

29 parts of serum deriving from the patient being diagnosed as chronic pancreatitis are altogether obtained from JohnsHopkinsMedicalCenter and ZeptometrixCorp. (Franklin, MA).Healthy volunteer (N=19) provides blood for control sample at CenterforMolecularMedicineandImmunology.All samples all identifies through the past, is only the clinical data that researcher provides diagnosis, staging, follow-up time-to-live and primary tumo(u)r size.

reagenthuman pancreatic's mucin preparation is separated from the human pancreatic adenocarcinoma CaPan1 grown nude mouse as xenograft.In brief, make 1g be organized in 10mL to homogenize containing in the 0.1M ammonium bicarbonate of 0.5M sodium chloride.Subsequently sample is carried out centrifugal to obtain supernatant, column fractionation, hydroxyapatite carries out stratographic analysis to voidage material.Make the fraction of not adsorbing relative to deionized water enough hemodialysis, and freeze-drying subsequently.In containing the 0.01M sodium phosphate buffer (pH7.2) (phosphate buffered saline (PBS) [PBS]) of 0.15M sodium chloride, prepare 1mg/mL solution, and be used as the stock solution of immunoassays reference material.Anti-TNF-α mucin antiserum is prepared, as described above (Gold etc., CancerRes43:235-38,1983) by carrying out immunity to rabbit.IgG purification fraction, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and molecular sieve high-efficient liquid chromatography assessment purity.Having reactive muroid MA5 antibody with MUC1 protein core is available from Immunomedics, Inc. (MorrisPlains, NJ).The control antibodies Ag8 of non-binding homotype coupling is from P3X63-Ag8 murine myeloma purifying.

sample preparationall mensuration is all carried out in the blind mode of covering.In order to the sample for the preparation of immunoassays, 300 μ L serum are placed in 2.0mL microcentrifugal tube, and extract with equal-volume n-butyl alcohol.By violent for this pipe vortex 2 minutes, now add 300 μ L chloroforms, and by this pipe vortex 2 minutes again; This rear step comprises in a program to put upside down water layer and organic layer.Lower centrifugal 5 minutes are arranged at 12,000rpm subsequently in microcentrifuge.Water-based upper strata is moved in clean pipe, and sample 1:2 is diluted in 2.0% (w/v) casein-sodium salt in containing in the solution in the 0.1M sodium phosphate buffer pH7.2 (PBS) of 0.15M sodium chloride, to carry out immunoassays.

enzyme immunoassay (EIA)be coated with 100 μ L containing 20 μ g/mL humanization PAM4IgG PBS 96 hole polyethylene boards in carry out immunoassays, wherein overnight incubation at 4 DEG C.Block each hole by adding 200 μ L2.0% (w/v) solution of casein in PBS and hatch 1.5 hours at 37 DEG C subsequently.From hole, remove blocking solution, with the 250 μ LPBS containing 0.1% (v/v) Tween-20, plate is washed 5 times.100 μ L reference materials or unknown sample to be added in triplicate in suitable hole and hatch 1.5 hours at 37 DEG C.As above plate is washed 5 times with PBS-Tween-20 subsequently.

Anti-stick for multi-clone rabbit protein antibodies is being diluted to 5 μ g/mL containing 1.0% (w/v) caseic containing in the PBS of the non-specific IgG of 50 μ g/mL, is adding in each hole and hatch 1 hour at 37 DEG C.As mentioned above subsequently from hole, wash out polyclonal antibody, and through the donkey anti-rabbit IgG (JacksonImmunoResearchLaboratories of peroxidase labelling, WestGrove, PA) be diluted in containing 1.0% (w/v) casein, also containing in the PBS of 50 μ g/mL IgG with 1:2000, add in each hole, and hatch 1 hour at 37 DEG C.After as above washing plate, by 100 μ L3,3 ', 5,5 '-tetramethyl benzidine substrate solution to add in each hole and at room temperature hatches 30 minutes.Stop reaction by adding 50 μ L4.0N sulfuric acid, and use 250 spectrophotometers (MolecularDevices, Sunnyvale, CA) read optical density under 450nm wavelength.Due to the obvious microheterogeneity of PAM4 antigen, we select based on the initial reference reference material from heteroplastic CaPan-1 human pancreatic tumors purifying, report our result with arbitrary unit/mL.

immunohistochemistrythe sample through the embedding of stone mistake available from CooperativeHumanTissueNetwork is cut into 4 micron sections on positive charge anticreep microslide (ThermoScientific, Waltham, MA).Maintain 20 minutes after subsequently histotomy being heated to 95 DEG C in pH9.0Tris damping fluid target reparation solution (Dako, Carpinteria, CA), allow it to be cooled to room temperature, at room temperature use 3%H subsequently ₂o ₂cancellation 15 minutes.Subsequently by primary antibodie with 10 μ g/mL and ABC kit (VectorLaboratories, Burlingame, CA) one is used from tagged tissue.Used by two virologists and to report that with the early stage research institute for biomarker in cancer of pancreas consistent specification to be marked (Gold etc. to microslide independently, 2007, ClinCancerRes13:7380-87): 0-is negative, the tissue of label L EssT.LTssT.LT1%; Tissue between the weak focal mark 1% and 25% of 1-; Tissue between the strong focal mark 1%-25% of 2-; The tissue of 3-weak diffusivity mark >25%; The tissue of 4-strong diffusivity mark >25%.Only consider structural constituent's (such as pancreatic cancer cell, normal pancreatic duct etc.) that assessment is suitable.

statistical studyproduce typical curve by immunoassays data, carry out regretional analysis to insert the concentration (Prism4.0 software, GraphPad, LaJolla, CA) of unknown sample.Recipient's operating characteristics (ROC) curve is produced by using Med-Calc statistics software package (version 7.5) (Med-Calc, MariakerkeBelgium).Use the variable in more any two groups of Si Shi t inspection.Cochran-Armitage is used to check the trend detected between recall rate and disease stage.

Result

The accuracy and precision of immunoassays assesses the reference standard thing that one group of nominal concentration is 15.60,6.20,2.50 and 1.00 units/mL, to determine accuracy and precision in several discontinuous days (N=7).The curve of reference material generally provides r ²the result fit value of >0.990.Accuracy is calculated as in 8% of the nominal value of first three concentration, but for 1.00 units/mL reference material, is then down to about 22%.The linear regression of the nominal in this control series and measuring unit/mL gives Trendline, and slope is 0.965, and y intercept is 0.174 (r ²=0.999), wherein slope 1.00 and y intercept 0.00 by formation 100% accuracy (Figure 18).For two least concentration reference materials, nominal and the mean absolute difference of replying between quality equal 0.190 ± 0.173 unit/mL, show that the least absolute error of EIA is about 0.2 unit/mL.For 4 reference standard things, the coefficient of variation (CV) value is 6.40%, 4.85%, 12.0% and 66.4% respectively.In a word, these data show that PAM4 immunoassays provide to be in and analyze accuracy in guidance that the immunoassays measured value of thing advises and repeatability level; For the concentration being greater than cutoff (2.40 units/mL), accuracy and precision is in 15%, and for being in the concentration of cutoff, accuracy and precision is in 20%.In order to check further this, we have detected 3 parts of serum, wherein within 3 days, take from normal healthy controls independent for two parts.Two parts of normal healthy controls provide the average result of 0.27 ± 0.06 and 0.30 ± 0.27 unit/mL, separately close to the least absolute error of EIA, have the result height CV of 21.65% and 88.19% respectively.Other patients serum provides the mean value of 19.45 ± 2.51 units/mL, and CV is 12.9%.

antigen in patients serum quantitativein Primary Study reported in the embodiment above, the immunoassays based on PAM4 serum have the apparent susceptibility of 77% and the specificity of 94% to cancer of pancreas.Should note, the cancer specimen in most pancreas and non-pancreas source is available from participating in standing by high Dengzhou the patient that IRB that Cancer center (GardenStateCancerCenter) carries out ratifies clinical testing, and at-80 DEG C freezen protective more than 10 years.But, the time that pancreatitis sample freezen protective is significantly shorter.We have evaluated one group new 24 parts from the serum being diagnosed as Pancreas cancer patients.Only two parts of serum have the PAM4 reactive antigen level being regarded as the positive.Therefore, we consider and have evaluated immunoassays not by expecting the reason of carrying out, and comprise the quality of immunoassay reagent, antigen degradation and/or the possibility removed from serum, it is that immune complex form exists or is blocked Binding Capacity.We find, the substrate in the sample of fresh serum human and/or freezen protective short cycle (<5) obviously blocks it be combined with PAM4 antibody in conjunction with the reactive epi-position of PAM4, therefore prevents from being detected by immunoassay.The recovery number percent reclaiming antigen from the fresh normal human serum (N=2) of the PAM4-antigen strengthening taking concentration as 5-20 unit/mL is about 33% or less.

In a series of report, Slomiany and colleague disclose gastric mucin covalent bond and/or associated lipids and fatty acid (Slomiany etc., 1984, ArchBiochemBiophys229:560-67; Slomiany etc., 1986, BiochemBiophysResCommun141:387-93; Zalesna etc., 1989, BiochemInt18:775-84), and these lipids and fatty acid have certain effects to mucinous physicochemical property.In addition, it is reported, fatty acid synthetase level and activity significantly raise in cancer of pancreas, in the cancer and other pathological condition of other form also so (Walter etc., 2009, CancerEpidemiolBiomarkersPrev19:2380-85).Because blocking substrate may be lipid in essence, so we carry out organic extraction to the serum of the freezen protective <5 deriving from this group of 24 Pancreas cancer patients.As mentioned above, do not extract in advance, only have 2 parts (8.3%) to have in 24 increment product and be regarded as positive PAM4-antigen levels, and after organic extraction, in 24 increment product, have 22 parts (92%) to have positive PAM4-antigen levels.

According to the research reported in above embodiment, we can also reappraise 10 parts of Pancreas cancer patients serum of freezen protective >15 to confirm previous result.Whether no matter extract, all 10 increment product all have the antigen levels being regarded as the positive.Relatively to hang oneself extraction or to give slope without the regretional analysis of paired result of the serum of extraction be 1.10 (r ²=0.94) Trendline, shows no matter whether extract these long-term frozen serum, result is all similar.It is generally acknowledged, the standing storage of sample can be led inhibition degradation of substrates or combine and reduce and epi-position exposure.The all further test of serum was all carried out when carrying out organic extraction to sample before immunoassays.

The sample of assessment PAM4 reactive antigen comprises the patient that 68 confirmations have cancer of pancreas, to divide by stages: 21 1 phases, 14 2 phases, and 33 3 phases and 4 phases (late period).In addition, comprise 19 parts and be diagnosed as the serum of patients' collection of chronic pancreatitis from healthy adult volunteer and 29 as a control group.Cmax shown in point diagram (Figure 19) is 80 units/mL, because serum volume is not enough to carry out other dilution research.Although report the cutoff of 10.2 units/mL in above embodiment, but owing to using the difference (comprising IgG in reagent concentration, damping fluid) of organic extraction program and EIA scheme, we select to process the current data set irrelevant with Previous results.In order to more all pancreatic cancer samples contrast healthy adult, calculated the positive cutoff of 2.4 units/mL by ROC curve statistical (Figure 20).The overall detection sensitivity of cancer of pancreas is 82%, and area under curve is 0.92 ± 0.03 (95%CI=0.84-0.97).Under this sensitivity levels, for normal healthy controls group, observe the false positive rate of 5%, single positive case has the circulating antigen of 3.65 units/mL, is just in time greater than cutoff.In order to compare PAM4 immunoassay result, insufficient serum volume hampers CA19-9 immunoassays.

As shown in table 15, the detection sensitivity of early stage 1 phase cancer of pancreas is relatively high, has 13 parts (62%) to be greater than cutoff in 21 increment product.Can predict, this recall rate is lower than observed by organizing 2 phases (86%) and late period 3 and 4 phase (91%) patient.For recall rate and the stage of disease, have recorded statistically significant trend (P<0.01).We think this most likely caused by tumor size or burden.The average tumor size of 1 phase, 2 phases and 3/4 phase group is 2.14 ± 1.02cm respectively ³, 3.36 ± 1.18cm ³with 3.45 ± 1.06cm ³.Although significant difference (P>0.41) on the tumor size no statistical significance between 2 phases and 3/4 phase group, but time compared with 1 phase tumor size, observe statistically significant difference (P<0.004 or better) separately for these two groups.But, it should be noted that the irrelevant (r of antigen concentration in individual tumor size and serum ²=0.0065).

The sample being reported as 1 phase can be divided into 1A phase (N=13) and 1B phase (N=8) subgroup based on tumor size, and recall rate is respectively 54% and 75%; But, because in each subgroup, the number of patient is less, should be noted.The average tumor size of 1A phase is 1.41 ± 0.58cm ³(scope: 0.4cm ³-2.0cm ³), and that the 1B phase is 3.15 ± 0.44cm ³(scope: 2.5cm ³-4cm ³); For the comparison of two groups, P<0.001.Although little more interim than 1B of the tumor size on the whole in 1A phase disease, in individual tumor size and blood between PAM4 antigen concentration without obvious statistic correlation (r ²=0.03).In addition, it should be noted that in 13 parts of 1A phase samples, have 4 to have in 7 positive cases to be significantly higher than the PAM4 antigen levels of cutoff, scope is 17.65-32.65 unit/mL.

PAM4 reactive antigen in table 15. patients serum

We also have evaluated one group of serum that 29 tentative diagnosis are the patient of chronic pancreatitis.Under 2.4 units/mL cutoff that the ROC assessment by normal and Pancreas cancer patients confirms, 11 Pancreatitis Patients (38%) are positive.The ROC tracing analysis of directly comparing pancreatitis serum and pancreatic cancer samples gives the area under curve (95%CI=0.68-0.85) of 0.77 ± 0.05.The intermediate value of pancreatitis group is 1.28 units/mL, suitable with healthy volunteer's group (1.18 units/mL), but significantly lower than (3.5 times) 1 phase cancer of pancreas group (4.53 units/mL).It should be noted that our early stage pancreatitis sample result demonstrates significantly lower false positive rate, be only 5%.But those pancreatitis sample freezen protective are less than 5 years, and before analysis without organic extractant phase.

Biopsy and/or surgical samples derive from 14 parts of chronic pancreatitis samples, and wherein 6 parts from the patient being regarded as the circulation MUC5ac positive.In 3 in these 6 positive cases, in histotomy, authenticated precursor lesion.Consider that positive serum test is due to pancreatitis or there is superfluous natural disposition precursor lesion subsequently.We derive to other 30 parts the biopsy samples being diagnosed with pancreatitic patient and have carried out Immunohistochemistry.In 30 increment product, authenticated portion by using PAM4 dyeing is obvious Non-Invasive cancer of pancreas, portion has large PanIN-2-3 pathology (in independent sample), and acinus around-tracheal tissue transforms (ADM) and normal structure is negative (data is not shown).In remaining 28 increment product, 19 parts have the parenchyma being enough to assess, wherein the 16 parts evidences with ADM.Be all that PAM4 is negative except two examples in all these cases, and in these cases sample separately, only obtain very focal weak ADM mark (data are not shown).

checking research-we start one group of blood serum sample group of complete annotation that has from the patient with known diagnosis result to put together.To comprising healthy individuals and aggressive Pancreas cancer patients (cancer knurl, neuroendocrine and other form), first group of patients serum (N ~ 450) of optimum pancreatic disease (adenomatous pathology, pancreatitis etc.) and non-cancer of pancreas and benign disease (bile, duodenum, carcinoma of ampulla, cholecystitis, gastritis etc.) assess (blind cover research), to confirm in much bigger patient group and to promote about the specific Previous results of PAM4.Table 16 presents the interim analysis based on completed research so far; In general, these data are significantly similar to our early time data.Utilize and analyze by ROC PC being contrasted to healthy adult the cutoff measured, the overall detection sensitivity of cancer of pancreas is 80% under the specificity of 96%.2 parts are only had to be positive in 16 parts of neuroendocrine tumors, just above cutoff.

Up to now, 53 tentative diagnosis are have 14 (26%) to be identified as the PAM4 positive in pancreatitic patient, reported lower than in the announcement that we are nearest.We attempt the result of clinical data with this pancreatitis group to be associated now, and follow up with laboratory for these patients provide clinical.2 (being cystadenoma) are only had to be regarded as the positive in 11 optimum gland cancer pathology patients.Other cystadenoma has the PAM4 antigen levels being greater than 200 units/mL.Biopsy is described as by pathologists report " highly doubtful cancer ".

PAM4 reactive antigen in table 16. patients serum

The value of a-intermediate value peace mean value ± SD represents with unit/mL.

B – recipient operating characteristic curve (ROC); The value of 95% fiducial interval that what area under curve (AUC) presented is.

Discuss

The reactive epi-position of research display PAM4-of the immunohistology of employing tissue sample reported in above embodiment and the EIA of circulating antigen is the biomarker of aggressive cancer of pancreas, and express (that is, PanIN-1) in the neoplastic very early time of pancreas.It can not detect in normal pancreatic tissue (conduit, acinus and islet cells), can not detect in checked most of non-cancer of pancreas (thymus gland, lung, stomach etc.).Therefore, for cancer of pancreas, in serum, the rising of PAM4 epi-position concentration provides the positive likelihood ratio of 16.8.What previous research lacked is clinical information about disease stage.Therefore, up to now, we still can not assess can the value of immunoassays of curable early stage disease for detecting.

We report in this article and use the EIA based on PAM4 of blood serum sample to detect Early pancreatic carcinoma patient, and can the anosis individuality of accurate discrimination.For 1 phase patient, being 62% for detecting the determination susceptibility of Early pancreatic carcinoma, is then 86% for 2 phase Diseases, and serum levels generally advances with disease stage and increases.The high number percent of 1 phase and 2 phase Diseases is asymptomatic clinically.We infer, use PAM4 determination of serum method to detect tumor growth can provide for survivor the prospect made moderate progress at these commitments.

Cancer patient in this research carried out excision, thus provided and carry out accurately possibility by stages to each patient.But many Pancreas cancer patients are under a cloud occurs micrometastasis disease, even if they do not involve the obvious regional lymph nodes of tissue.This highlights the general considerations in early detection research, especially low incidence of disease disease, such as cancer of pancreas.The accumulation of the sample fully determined is problematic.In addition, make problem again further hydridization be have in these cancers of pancreas manyly to occur under the existence of chronic pancreatitis, cholecystitis and superfluous natural disposition precursor lesion and other symptom.

Be in the serum of chronic pancreatitis 29 parts of tentative diagnosis, 38% is identified as PAM4-antigen positive.But have multidigit to have the evidence of superfluous natural disposition precursor lesion in these seropositivities patient, the tissue sample of described patient can be used for pathology to be explained.In addition, comparing the tissue reactive that obtained by immunohistology and observing contradiction at ordinary times by the antigenemia clear water that immunoassays obtain.Immunohistochemistry shows, the sample assessed of 10% is only had to demonstrate the evidence of PAM4 dyeing in ADM, but this be remarkable lower than most pancreatic cancer samples under observed intensity (Gold etc., 2007, ClinCancerRes13:7380-87).Therefore, result shows that the Positive Level of PAM4 antigen in serum not derives from the pancreatic tissue of inflammation, but can provide the neoplastic evidences of subclinical pancreas such as such as PanIN pathology, and on minimum level, positive findings provides the ultimate principle of these patient clinicals follow-up.

The discovery through genetic engineering modified animal model deriving from cancer of pancreas shows, human pancreatic adenocarcinoma can produce (Leach, 2004, CancerCell5:7-11) before PanIN-1 pathology.ADM viewedly in the sudden change KRAS target model described by (2007, AmJPathol171:263-73) such as Zhu changes the earliest.In yet another aspect, Shi etc. (2009, MolCancerRes7:230-36) report, although KRAS gene mutation can occur in ADM, they mainly occur in the ADM relevant to PanIN pathology.Author shows that this drives in the wrong direction by PanIN and extends to ADM and occurring around.Up to now, there is not the conclusive evidence that ADM is in progress into PanIN.Interested is in two Pancreatitis Patients, and PAM4 and ADM has this fact reactive.

At present, screen general cancer of pancreas colony and be regarded as medically or economics be unworthy, because this disease is simply too rare.But the patient increased to some extent the screening expectation risk that can develop cancer of pancreas is quite interested.Some research displays, screen pancreas precursor lesion (Canto etc., 2006, ClinGastroenterolHepatol4:766-81 that the individuality with strong cancer of pancreas family history can differentiate applicable excision; Canto, 2005, ClinGastroenterolHepatol3:S46-58; Brentnall etc., 1999, AnnInternMed131:247-55).For example, the relative of Pancreas cancer patients develop the risk remarkable higher (Shi etc., 2009, ArchPatholLabMed133:365-74) compared with general groups of cancer of pancreas.The familial pancreatic cancer patient of little number percent has cancer of pancreas tumor susceptibility gene PALB2 (gametophyte of BRCA2 and positioning body) sudden change (Tischkowitz etc., 2009, Gastroenterology137:1183-86).Similarly, the risk of long-term chronic film adenositis patient evolution cancer of pancreas increases to some extent, and in Early onset (teenager) hereditary pancreatitis patient, risk is (Lowenfels etc., 1993, NewEngJMed328:1433-37 more than 30%; Lowenfels etc., 1997, JNatlCancerInst89:442-46).The risk (Rutter etc., 2004, Cancer101:2809-16) exceeding 20 to 34 times is observed in the many moles of familial atypical (FAMMM) syndrome individuality.And multinomial research display, in the diabetic individual meeting some standard, the risk of development cancer of pancreas significantly increases (Pannala etc., 2009, LancetOncol10:88-95).Use these patients of PAM4 immunoassays Longitudinal Surveillance can provide neoplastic early detection.Second potential use of immunoassays can be used as after treatment and the means that detect of the palindromia of those patients after excision that particularly tumour is confined to pancreas by inference.

The relatively high specificity of PAM4 antibody provides with high tumor uptake and the high tumour/non-tumour means than both targeted imaging agent and therapeutic agent.We have demonstrated PAM4 as through the directly radiolabeled or nuclear imaging of bispecific pre-targeting reagent for cancer of pancreas and the potentiality of Radioimmunotherapy.And, be recently reported and give for assessment of gradation ⁹⁰the PRELIMINARY RESULTS (Pennington etc., 2009, JClinOncol27:15s, summary 4620) that the full IgG of Y-PAM4 (Ke Liwo monoclonal antibody) and the clinical 1b phase of the combination of the radiosensitizing scheme of gemcitabine test.Based on RECIST standard, in 22 3/4 phase Diseases (major part was 4 phases), 68% evidence demonstrating Disease epizootic, the patient of 23% has partial reaction.Therefore, the positive findings based on the immunoassays of PAM4 provides the imaging and treatment that continue PAM4 target, thus provides the ultimate principle of personalized treatment.

Immunoassays based on PAM4 can differentiate most of Pancreas cancer patients in all stages.Although directly can not compare with CA19.9 in current research, but in one group of limited pancreatopathy cancer serum (N=41), statistically significant difference (P<0.01) is demonstrated to previously comparing of two biomarkers, wherein in 71% Patient Sample A, PAM4-antigen levels is positive, and in 59% sample, CA19.9 antigen levels is positive.In general, think that CA19.9 lacks the Sensitivity and Specificity providing cancer of pancreas early detection and/or diagnosis.But, this mensuration for control indicate poor prognosis treatment after CA19.9 lasting serum levels raise.Similarly, we report with the form of summary (Pennington etc., 2009, JClinOncol27:15s, summary 4620) recently and use cycle P AM4-antigen levels to predict antitumor reaction.

The condition (such as, keeping freezing time span) that sample is preserved in the display of these results can have remarkable result to the accessibility of epi-position in research.For the immunoassays based on PAM4, fatty acid or lipid matter can in conjunction with defined epitopes and interference immunoassays.But, likely this material other material of being low molecular weight peptide or dissolving in organic solvent.The ability removing this material by carrying out organic extraction to serum makes PAM4 immunoassays reproduce.In addition, also proposed the problem about the interactional biological significance of cyclic inhibition thing: MUC5ac.But, when PAM4 antibody to be used as in body target agent (such as, Radioimmunotherapy) time, the existence of cycle P AM4-antigen is not factor, because observed in assessed most of patient through radiolabeled PAM4 target tumor growth site up to now.Therefore, the PAM4-antigen seemed in tumour is containing blocking material.

Embodiment 27. warp ⁹⁰the hPAM4 antibody of Y mark and gemcitabine late in cancer of pancreas the IB/II phase study

Carry out warp ⁹⁰the IB/II phase of hPAM4 antibody (Ke Liwo monoclonal antibody) late in Pancreas cancer patients of Y mark is studied.Always have 100 previous untreated III or IV phase Pancreas cancer patients and participate in the disclosure Labeling Study, accept weekly × 4 gemcitabines, and within the 2nd, 3 and 4 week, accept (treatment circulation) ⁹⁰fertile monoclonal antibody in Y-gram.Can repetitive therapy circulation until progression of disease or until patient demonstrates unacceptable toxicity.10 patients exit in early days, and 90 patients, wherein 82 have IV phase (metastatic) disease, accept 1-4 treatment circulation.Tumor response is assessed by the change of serum C A19.9 of (at first every 4 weeks) after CT, FDG/PET and each circulation.

In the part i of this research, with 6.5,9,12 or 15mCi/m ²× 3 ⁹⁰in Y-gram, fertile monoclonal antibody treats 38 patients, and 200mg/m ²the low fixed dosage gemcitabine of × 4 is used for radiosensitizing.13 patients are treated again 1-3 time with same loop.According to the RECIST standard based on CT, total disease control rate is 58%, comprise complete reaction (CR), partial reaction (PR) and stable disease (SD), optimum response comprises 6 (16%) PR patients and 16 (42%) SD patients.

38 median overall time-to-live (OS) through the patient for the treatment of are 7.7 months, can match in excellence or beauty with other scheme of advanced pancreatic cancer.At higher therapeutic dose (12 and 15mCi/m ²⁹⁰fertile monoclonal antibody × 3 in Y-gram) under, the intermediate value OS of record is 8.0 months.13 are accepted to the patient of the repetitive cycling of combination treatment, intermediate value OS is improved to 11.8 months.After observing treatment, the extension time-to-live of outbreak mostly is 14.8 months most, and wherein 8 patients realize time-to-live >6 month (3 patient >1).Anecdote report shows that this treatment improves performance state and pain degree.

52 patients treated in this research part ii accept 3 12mCi/m weekly ²'s ⁹⁰y dosage and 200,600 or 1000mg/m ²the gemcitabine dosage of × 4, wherein 14 patients accept gemcitabine dosage identical but ⁹⁰y dosage is 6.5,9 or 12mCi/m ²repetitive therapy circulation.Result derives from 47 in 52 patients.200mg/m ²the disease control rate of group is 72%, has 19%PR and 53%SD.For 600 and 1000mg/m ²group, disease control rate is 63% (0%PR) and 68% (18%PR) respectively.Comparatively Homogemcitabines dosage and 200mg/m ²lowest dose level compare, seem not provide treatment repercussion to answer advantage.When reporting, survival data is unavailable for this group patient.Treat well tolerable, to through radiolabeled mg/m ²without infusion reaction, and seldom there is non-blood spinoff.It is instantaneous that blood after the 1st and the 2nd circulation suppresses.

These results show, through Yttrium-90 ( ⁹⁰y) mark and the repetitive cycling combining the fractionated dose of the Ke Liwo monoclonal antibody given with gemcitabine show therapeutic activity in inoperable Pancreas cancer patients late.The repetitive cycling therapy that Ke Liwo monoclonal antibody adds low dosage gemcitabine improves the overall survival time of Locally Advanced or metastatic Pancreas cancer patients compared with single cycle therapy.

The detection of the early stage pancreas duct adenocarcinoma (PDAC) of embodiment 28.: based on the PAM4 immunoassay of serum susceptibility, specificity and distinguish character

Disclosed in embodiment 26, utilize the enzyme immunoassay (EIA) based on serum of PAM4 antibody can use the known PDAC patient correctly differentiating 81%, and this mensuration have potential sensitivity for the early stage disease of detection.These discoveries extend to larger PATIENT POPULATION, comprise the serum from pernicious and optimum pancreas and surrounding tissue disease more than 600 parts.Cover in analysis blind, assess self-confirmation has the PAM4-antigen concentration level of the patient of PDAC (N=298), other cancer (N=99), pancreas benign disease (N=126) and the serum of healthy adult (N=79) by enzyme immunoassay (EIA).

The overall sensitivity that PDAC detects is 76%, and the 1 phase patient test of 64% is for positive, and the susceptibility of terminal illness higher (85%).In most of the cases, the blood-serum P AM4-antigen levels of Pancreatic Neuroendocrine Tumors or other source (squamous cell, GIST etc.) cancer patient has no rising.Only about half of ampulla (48%) and extrahepatic bile ducts (50%) adenocarcinoma patients have the Positive Level of cycle P AM4-antigen.Be diagnosed as in the patient of optimum pancreas symptom at 126, only 24 (19%) is positive, and exactly, has 18 (23%) to be positive in 80 chronic pancreatitis (CP) patients.ROC tracing analysis to demonstrate between PDAC and CP group statistically significant difference (P<0.0001), and area under curve is 0.84 ± 0.02 (95%CI:0.79-0.89).The positive and the negative likelihood of distinguishing PDAC and the optimum symptom of pancreas are 4.00 and 0.30 respectively.

In a word, PAM4 immunoassays have detected the 1 phase PDAC patient close to 2/3rds, and do like this and have fine resolution for optimum pancreatic disease.Result provides Longitudinal Surveillance is measured the patient being considered as being under high PDAC (such as, familial pancreatic cancer, new outbreak diabetes etc.) risk ultimate principle by PAM4.

Embodiment 29. measures based on PAM4 and distinguishes ductal adenocarcinoma of pancreas (PDAC) and chronic pancreatitis and optimum non-mucus pancreatic cyst

We examine the expression of the reactive MUC5ac of PAM4 in chronic pancreatitis and optimum non-mucus pancreas cystic pancreatic disorders.By the micro-array tissue (N=14) of immunohistochemical evaluation PDAC and reactive MUC5ac and MUC1 (mAb-MA5) of PAM4 of surgical samples, the expression of MUC4 (mAb-8G7) and CEACAM6 (mAb-MN-15) that derive from chronic pancreatitis (N=32) and optimum non-mucus cystic pancreatic disease (N=19).

PAM4 reactive MUC5ac, MUC1, MUC4 and CEACAM6 are expressed in the aggressive cancer of pancreas of 79% (11/14), 100% (14/14), 86% (12/14) and 100% (14/14).PAM4 only the optimum non-mucus cystic lesion of weak mark 6% (1/19), the serous cystadenoma (SCA) of 1/15 and 0/4 scaly epithelium be coated to tumour (2 routine Lymphoepithelial cysts and 2 examples have the retention cyst of squamous metaplasia).But, in the SCA of 53% (8/15), 0% (0/15) and 13% (2/15), and be coated to expression MUC1, MUC4 and CEACAM6 being detected in 4 in tumour, 3 and 3 examples respectively at 4 routine scaly epitheliums.PAM4 marked the chronic pancreatitis sample of 19% (6/32); But this PAM4 reactivity is confined to the PanIN precursor lesion relevant to chronic pancreatitis.The tissue of inflammation is negative.The expression of MUC1, MUC4 and CEACAM6 is detected in the chronic pancreatitis of 90% (27/30), 78% (25/32) and 97% (31/32).All in the sample of positive mark, except PanIN, reactivity is present in non-superfluous natural disposition inflammation pancreatic tissue.

In a word, only 6% optimum non-mucus cystic lesion in and the expression of PAM4 detected in the precursor lesion relevant to chronic pancreatitis.These results show, contrary with MUC1, MUC4 and CEACAM6, and PAM4 can be used for distinguishing the optimum non-mucus cystic lesion of pancreas and chronic pancreatitis and PDAC.

Embodiment 30.PAM4 and CA19-9 biomarker combine and detect for improving cancer of pancreas

Ductal adenocarcinoma of pancreas (PDAC) is almost general lethal factor, mainly owing to can not detect early stage disease.Therefore, can differentiate that the discriminating of the biomarker of early stage PDAC patient can improve the overall survival time.Cover in research blind, PAM4 and CA19-9 immunoassays are carried out to the serum of 480 patients, comprises those patients and healthy adult (N=50) that confirmation has PDAC (N=234), other cancer (N=84), pancreas benign disease (N=89).

The overall sensitivity of PDAC is similar, is 74% and 77% respectively to PAM4 and CA19-9.The detection sensitivity of early stage 1 phase disease (N=26), although slightly high for PAM4 antigen, also similar statistically, be 65% and 58% (P=0.5775) respectively to PAM4 and CA19-9.But, significantly lower to the specificity of CA19-9, particularly for chronic pancreatitis (CP): be determined as 68% contrast 86% (P=0.014) to PAM4.In addition, the display of CA19-9 result, to the neoplastic significantly higher recall rate of non-PDAC, comprises other cancer patient transferring to pancreas.Therefore, positive likelihood ratio (+LR) (+LR=2.41) of CA19-9 measures (+LR=5.29) lower than PAM4.

PAM4 and CA19-9 antigen levels in PDAC (r independent of one another ²=0.003, P=0.410); But positive and negative explanation is consistent with the case of 68%.Therefore, the analysis of combination biomarker improves overall PDAC recall rate (84%), and specificity does not significantly reduce (83%).For combination immunoassays, compared with independent arbitrary mensuration, PDAC contrasts relatively demonstrating of the ROC curve of CP and PDAC contrast benign disease statistically to be improved (two relatively middle P<0.0001), significantly to detect and to distinguish PDAC and benign disease.

Although PAM4 immunoassays are for the diagnosis and detection of PDAC, provide hypersensitivity and specificity, comprise CA19-9 biomarker and make the positive of PDAC patient differentiate significantly to be increased to 84% from 74%.

Embodiment 31. uses PAM4 immunoassays as the correlativity of tumor response

Whether the particular tendency that we have studied reactive MUC5ac concentration (in few patients) of PAM4 can be used as treating the instruction of rear tumor response.Have evaluated the some positions be in progress now ⁹⁰y-hPAM41b/II clinical trial phase patient.When treatment terminates latter 4 weeks assess patient (treatment circulation is 4 weeks), serum antigen level reduces >40% and indicates reaction.The PAM4 antigen levels that all patients suffering from PD have continues to raise.The trend of two patients is provided in Figure 21 A and Figure 21 B.In these two cases, the trend of circulation MUC5ac level is consistent with the gross tumor volume trend determined by CT.These results show that blood-serum P AM4 level can be used for the reactivity of the anti-cancer therapeutic regimen of monitoring cancer of pancreas.

The discriminating of the target antigen of embodiment 32.PAM4 antibody

We conducted one group block and catch/Probe pairings enzyme immunoassay (EIA), with assess PAM4 antibody and and MUC1 (MA5, KC4, HMFG1, SM3, H23), MUC2 (G9), MUC4 (8G7) and MUC5ac (45M1) there is relation between reactive antibody.The mucin reference material deriving from CaPan1 human tumor xenograft demonstrates containing the reactive mucin material for all these antibody, except having those antibody reactive with G9 (MUC2).In checked all MAb, it is reported and only have 1 example (45M1) and MUC5ac to have reactivity, when PAM4 is used as capture agent, provide the positive reaction in sandwich EIA.Compared with PAM4, the cancer of pancreas of 45M1 antibody and much lower number percent has reactivity (IHC by TMA), and therefore can not be used as the single probe based on the PAM4 immunoassays of serum.

As mentioned above, we carry out peptide-phage display research by carrying out continuous Biopanning by the muroid of PAM4-IgG and humanization pattern.Produce consensus sequence (12-aggressiveness-WTWNITKAYPLP (SEQIDNO:7)), when its input BLAST protein search frame, under the query context being located at 100%, differentiate that MUC5ac and MUC16 has 7/12 and 5/12 same amino acid respectively in 12-oligomeric sequences.

Mass spectroscopy is used to carry out the PAM4 immunoprecipitating antigen studying to differentiate to come from titular tumour fluid (these fluids of previously passed analytical reagent composition, to differentiate specific MUC existing in potpourri).By analyzing the PAGE of the PAM4 immunoprecipitation material from 3 parts of indivedual tumour fluid samples, in each sample, only there are two identical bands of a spectrum (not shown).These bands of a spectrum all containing MUC5ac as main mucin material.

We have studied the character of the material be combined with PAM4 epi-position in human blood, its needs carried out organic extraction before immunoassays.As discussed above, Slomiany and colleague have observed gastric mucin covalent bond and/or associated lipids and fatty acid.In addition, fatty acid synthetase level and active significantly to raise in cancer of pancreas, also like this in other form cancer and other pathological condition.Infer that blocking material may be lipid in itself, we have carried out EIA (Figure 22) when existing and there is not 100 μMs of palmitic acids, and under the OD450 being equivalent to 1.0, observe statistically significant 69% reactive minimizing (P<0.0001).It should be noted that the Normal adult serum levels of palmitic acid is in the scope of Isosorbide-5-Nitrae 80 to 3,730 μMs, be significantly higher than the concentration used in this EIA experiment.

Embodiment 33.PAM4 distinguishes pancreas duct adenocarcinoma (PDAC) and chronic pancreatitis (CP)

The patient of symptom and/or symptom that the suggestion of current implement directions presents doubtful cancer of pancreas carries out pancreas draft CT imaging research to detect the tumor mass in pancreas.Carry out follow-up imaging by endoscopic technique (such as, EUS, ERCP) and high disease detection susceptibility can be provided, and when sucking with fine needle/biopsy can provide good diagnostic accuracy when combining.But great majority carry out terminal illness patient in these programs; That is, tumour is greater than 2cm.Early pancreatic carcinoma detects and is still a problem, especially when there is pancreatitis background.Thus, realities of the day in all detection cases 7% are only early stage diseases.When not carrying out effectively treatment to PC in late period, the prognosis of these patients is troubling.

Reliably can distinguish cancer and optimum symptom and/or provide the biomarker of the means paying the utmost attention to the patient assessed that follows up will have important clinical and be worth, especially when biomarker can detect early stage disease.We have developed monoclonal antibody PAM4, it shows high degree of specificity to pancreas duct adenocarcinoma (PDAC).

Capture assessment and several mucin material (comprising MUC1, MUC2, MUC3, MUC4, MUC5ac etc.) in sandwich EIA at allos PAM4 and there is the signal reaction of reactive MAb of restriction.Known only have MAb (45M1,2-11M1) and the mucinous ad hoc structure territory of MUC5ac of signal reaction of can providing is reacted.In addition, three kinds of other anti-MUC5acMAb (21M1,62M1 and 463M1) can suppress the interaction between PAM4 and its mucin antigen separately.These data show that MUC5ac has reactive antigen with PAM4.Be different from other anti-MUC5acMAb (45M1,2-11M1, CLH2 etc.), the specificity that PAM4 is larger than the cancer rising in other organ to PDAC display, and the biomarker that can be used for PDAC and the target being oriented picture and therapy for antibody can be served as.

Table 17. suffers from the PAM4 antigen in the serum of the patient of known disease

PDAC group is made up of 40% early stage patient and 60% patients with terminal.Recall rate is 64% and 85% respectively.Determine that PDAC contrasts the Sensitivity and Specificity that CP (Figure 23 A) and PDAC contrasts the PAM4 calibrating of all benign tissue samples (Figure 23 B).AUC calculated value is 0.84 and 0.85 respectively.

By using the immunoassays based on serum, chronic pancreatitis (CP) patient of about 20% is positive.This problem is that false positive maybe may find that hidden neoplastic result is extremely important to the positive CP patient of explanation PAM4.Therefore, we have carried out extensive immunohistochemical evaluation to the PAM4 reactivity in CP tissue sample.

Figure 24 is shown and is marked non-superfluous the comparing property of natural disposition prostata tissue of PDAC contrast by the antibody of PAM4 antibody contrast for MUC1, MUC4, CEACAM6 and CA19-9.Each antibody and PDAC react.PAM4 display and normal structure anergy.The PanIN-2 pathology produced in display CP background, the acinus-tracheal tissue relevant with acinar cells partial loss, certain fiberization and PanIN is out of shape antibody (not shown) more identical in the sample of (ADM).Utilize any tissue of PAM4 in CP, comprise in the ADM of separation and do not observe mark (not shown).It is certain for (not shown) that other antibody shows with non-superfluous natural disposition tissue separately.Table 18 and table 19 display utilize PAM4 to contrast to carry out for the antibody of MUC1, MUC4, CEACAM6 and CA19-9 the comparative result that marks.

The expression of table 18. biomarker in pancreas duct adenocarcinoma

The focal mark of a-, the suitable structural constituent of 5% to 25% marks through indicated MAb; Diffusivity, the suitable structural constituent of >25% marks through indicated MAb; Always, focal+diffusivity.The value provided in b-bracket is the number percent accounting for assessed total N number of PDAC sample.

The expression of table 19. biomarker in chronic pancreatitis

We infer that the non-superfluous natural disposition of PAM4 and chronic pancreatitis (CP) patient organizes not tool reactive, but have reactivity with the known PDAC developed in the soft tissue of inflammation and its superfluous natural disposition precursor lesion (such as PanIN).Together with the result previously studied, we have evaluated 51 parts of CP samples altogether, find the soft tissue tool reactivity in no instance of PAM4 and inflammation.On the other hand, other biomarker each studied, MUC1, MUC4, CEACAM6 and CA19-9, can not distinguish PDAC and optimum non-superfluous natural disposition tissue.Below these biomarkers in CP is correlated with PanIN pathology and in non-superfluous natural disposition conduit with the ADM be separated to express in various degree.The EIA based on PAM4 for the antigen in quantitative patient's serum shows hypersensitivity and the specificity of PDAC detection.The 1 phase Disease of about 2/3 is to cycle P AM4 antigen positive.We infer, the CP patient (other patient of the disease higher with the risk may suffering from development PDAC) being found the PAM4 reactive antigen in the circulating cycle with Positive Level may have hidden PDAC and/or bulk precursor lesion, thus produces PAM4 biomarker.

The atlas analysis of the PAM4 epi-position on embodiment 34.MUC5ac

General introduction

Carry out indirectly and sandwich enzyme immune measure (EIA) to compare and contrast PAM4 reactivity and several anti-stick protein antibodies specific mucin kind (such as, MUC1, MUC4, MUC5ac etc.) to known response.Also carry out the reactive research being intended to block PAM4 and its specific antigen.We demonstrate separately and MUC5ac to have reactive MAb2-11M1 and 45M1 can be provide signal in the allos sandwich immunoassay of trapping antibody at PAM4.In addition, we authenticated separately and MUC5ac has the reaction that reactive MAb21M1,62M1 and 463M1 suppress PAM4 and its specificity epitope.MAb for MUC1, MUC3, MUC4, MUC16 and CEACAM6 does not capture antigen-reactive with PAM4, and they can not block the reaction of PAM4 and its antigen.We infer that MUC5ac is PAM4 antibody response mucin kind.

Background

Mucin glycoprotein is the serious glycosylated protein of high molecular, and it comprises at least 19 kinds of materials that the protein core based on uniqueness is classified.Find that they can as the cross-film component of cell or as secretory product.Mucinous unconventionality expression often betides in many cancer forms (see Hollingsworth and Swanson, 2004, NatRevCancer4:45-60; Kufe, 2009, NatRevCancer9:874-85; Rachagani etc., 2009, Biofactors35:509-27), comprise pancreas duct adenocarcinoma (PDAC) (Ringel and Lohr, 2003, Molancer2:9-13; Andrianifahanana etc., 2001, ClinCancerRes7:4033-40; Torres etc., 2012, CurrPharmDes18:2472-81).The new expression of specific mucin kind when producing or not producing the splicing variants newly transcribed and translate and/or rise/downward (Schmid, 2003, OncolRep10:1981-85) has fully been have recorded in document.Also observe and changed carbohydrate portions (Brockhausen, 2006, EMBORep7:599-604 by adding new end sugar (such as, neuraminic acid), not enough glycosylation and other aberrant biological chemistry route; Yue etc., 2009, MolCellProteomics8:1697-707; Haab etc., 2010, AnnSurg251:937-45).These modifications may cause configured construction to change and/or defined epitope occurs or disappears.In addition, the change distributed in the cell of considered mucin kind can be observed, such as MUC1, it is transmembrane glycoprotein in the normal tissue, but in tenuigenin, its (Jass etc., 1995, JPathol176:143-49 is have also discovered when superfluous natural disposition transforms; Cao etc., 1997, VirchowsArch431:159-66).These events can prove biology in neoplasia development and progression and clinical meaning and be provided for the neoformation label/target of cancer early detection and targeted therapies.

Our laboratory reports at first and uses polyclonal antiserum to differentiate pancreas conduit mucin, under its sensitivity levels provided in indirect immunohistochemistry (IHC), display is containing the epi-position (Gold etc. pancreas to relative specificity, 1983, CancerRes43:235-38), and finally cause producing monoclonal antibody (MAb) PAM4 (Gold etc., 1994, IntJCancer57:204-10), its humanization form also referred to as gram in lie prostrate monoclonal antibody.PAM4 shows high specific to PDAC, have minimum to anergy to normal and optimum non-superfluous natural disposition pancreatic tissue, but it really shows and originates from some other organ (such as, stomach, colon, lung) gland cancer there are measured response (at about 10% of all check samples) (Gold etc., 1994, IntJCancer57:204-10; Gold etc., 2007, ClinCancerRes13:7380-87; Gold etc., 2010, CancerEpidemiolBiomarkersPrev19:2786-94).PAM4 differentiates biomarker (Gold etc., 2010, the CancerEpidemiolBiomarkersPrev19:2786-94 of the height diagnosis possibility that can provide the neoplastic existence of pancreas if present; Gold etc., 2006, JClinOncol24:252-58; Gold etc., 2013, Cancer119:522-28).Therefore, the clinical practice (Gold etc., 2010, the CancerEpidemiolBiomarkersPrev19:2786-94 that detect early stage disease is being continued on for; Gold etc., 2013, Cancer119:522-28) and antibody target imaging and therapy (Gulec etc., 2011, ClinCancerRes17:4091-4100; Ocean etc., 2012, Cancer118:5497-5506).Except PDAC, PAM4 biomarker is also expressed in neoplasia (PanIN in precursor lesion, pancreas epithelium, comprise and develop pathology the earliest, PanIN-1A) and in conduit in papillary-mucinous neoplasia (IPMN), show that its expression may have carcinogenic meaning (Gold etc., 2007, ClinCancerRes13:7380-87).In current research, we have studied the consistance with this clinical associated antibodies with reactive mucin kind, to understand this mucin can play what effect in cancer of pancreas development and progress.

Method

antigen and antibody-be separated the mucin (Gold etc., 2006, JClinOncol24:252-58) containing the fraction being named as CPM1 from the Capan-1 mankind PDAC xenograft nude mouse as described previously.In brief, this forms by homogenizing to dissection tumour in the 0.1M ammonium bicarbonate containing 0.5M sodium chloride.After high speed centrifugation (20,000g × 45min), 4B-CL post carries out stratographic analysis to solable matter, and subsequently with identical ammonium bicarbonate-sodium chloride solution wash-out.Collect void volume material, dialyse for 0.01M sodium phosphate pH7.2, and subsequently by hydroxyapatite to remove nucleic acid and protein.Containing unconjugated mucinous fraction by enough hemodialysis again to remove salt and to be used as antigenic source.

Antibody for current research is listed in table 20 together with source-information with clone.For sandwich and blocking-up research, the muroid (mPAM4) that PAM4 can provide in Immunomedics, Inc. (MorrisPlains, NJ) and humanization (hPAM4; Ke Li lies prostrate monoclonal antibody) pattern acquisition.Other MAb all are muroid IgG.Mouse ascites liquid containing MAb21M1,45M1,62M1 and 463M1 is provided by doctor's J.Bara friendship of Paris, FRA INSERM.The PAM4 antibody being used as the negative contrast blocking research and the ascites fluid (having reactivity with Hep-G2 hepatocellular carcinoma cells) containing anti-alpha-fetoprotein antibody are provided by Immunomedics, Inc. (MorrisPlains, NJ).The anti-CPM1 of rabbit polyclonal (Gold etc., 1994, IntJCancer57:204-210; Gold etc., 2010, CancerEpidemiolBiomarkersPrev19:2786-94) the IgG donkey anti-rabbit IgG (JacksonImmunoResearch, WestGrove, PA) served as by marking through horseradish peroxidase (HRP) carries out the positive control that detects.

The monoclonal antibody that table 20. uses

Antigen	Clone name	Source
			MUC1	MA5	Immunomedics
MUC1	KC4	Immunomedics
			MUC1	CM1	Gene Tex
MUC2	994/152	Abcam
			MUC3	M3.1	Abcam
MUC3	M3A	LifeSpan Bio
			MUC4	8G7	Santa Cruz Biotech
MUC5ac	2-11M1	Santa Cruz Biotech
			MUC5ac	45M1	Santa Cruz Biotech
MUC5ac	CLH2	Santa Cruz Biotech
			MUC16	X306	Novus Bio
MUC16	X325	Abcam
			CEACAM5	MN14	Immunomedics
CEACAM6	MN15	Immunomedics
			CA 19-9	CA 19-9	Santa Cruz Biotech

Immunomedics，Inc.-MorrisPlains，NJ；GcncTcx-Irvinc，CA；Abeam-Cambridge，MA；LifeSpanBiosciences，Inc.-Seattle，WA；SantaCruzBiotechnology，Inc.-SantaCruz，CA；NovusBiologicals-Littleton，CO。

enzyme immunoassay (EIA)-describe the program (Gold etc., 1994, the IntJCancer57:204-210 that measure for indirect and sandwich enzyme immune; Gold etc., 2010, CancerEpidemiolBiomarkersPrev19:2786-94).For indirect immunoassays, elementary MAb uses with the concentration of 10 μ g/mL, to provide the hypersensitivity to input.For sandwich immunoassay, coating on hole by capturing MAb with the concentration of 10 μ g/mL, adding CPM1 antigen with the various concentration of 10 μ g/mL at the most subsequently.MAb probe is added to detect the reaction of the antigen of capturing subsequently with the high concentration of 10 μ g/mL.The anti-species specific IgG (JacksonImmunoResearch, WestGrove, PA) that entry evaluation secondary marks through HRP is to determine the optium concentration (normally 1: 1000 or 1: 2000) measured.Carrying out MAb suppression research by adding inhibition MAb in the hole be coated with through CPM1 antigen, starting with the pure MAb high concentration of 100 μ g/mL or the dilution of 1:10 ascites fluid, and being titrated to lower amount.Hatch 1h at 37 DEG C together with inhibiting antibody after, this plate is washed, and hPAM4 is added in hand-hole with the concentration of 0.25 μ g/mL.Use second probe subsequently, the anti-human IgG conjugate namely through HRP mark detects hPAM4 combination.

sDS-PAGE and Western blotting-under non reducing conditions, use 4-20%Tris-Gly gel under 125V, carry out SDS-PAGE be about 2h.Use Mini protein transduction through splitting moves on nitrocellulose membrane and continues 1h by cell system (Bio-RadLaboratories, Hercules, CA) under 100V.In order to check the consistance of recombinant protein, triplicate sample runs glue on same gel and film, wherein the sample through transfer is cut into three to add HRP-GAM detection with HRP-anti-Myc, HRP-hPAM4 and 45M1 respectively.Use SUPERSIGNAL ^tMwestDura chemical luminous substrate (ThermoFisherScientific, Waltham, MA) produces signal.

Result

Several MAb and the reactivity (Figure 25) of plate scribbling the high-molecular weight mucins fraction CPM1 be separated from Capan-1 human pancreatic adenocarcinoma xenograft is have evaluated by indirect EIA.Muroid PAM4 and have the reactivity that specific reaction MAb provides rising in these indirect immunoassays with MUC1 and MUC5ac mucin, wherein also been observed comparatively small reactivity for the MAb for MUC3 and CEACAM6.Have no the reaction of MAb and MUC2, MUC4, MUC16 and CEACAM5 glycoprotein or CA19-9 carbohydrate epitope in essence.

Defined epitope structure it should be noted that negative EIA reaction may not indicate and there is not mucin-antigen, because may exist but can not enter (that is, hiding).This may be the situation (Reis etc., 1997, IntJCancer74:112-21) of the anti-MUC5ac of MAb-CLH2 for the peptide generation deriving from mucin tandem sequence repeats, because another two anti-MUC5acMAb have highly reactive.Similarly, the reactivity of the anti-MUC1 of CM1 is significantly lower than the anti-MUC1 antibody of MA5 and KC4.Capan-1 cell produces the tumour with the mucinous good differentiation of high glycosylation.Therefore, may there is reactive CLH2 and CM1 can not have reactivity with CPM1, because this tandem sequence repeats epi-position can not enter by mucinous tandem repeat domains corresponding to it.

We have evaluated the mucin whether anti-stick albumen MAb capture with PAM4 subsequently and have reactivity.Use the plate be coated with through humanization PAM4 (hPAM4) to capture the specific mucin antigen of CPM1 fraction, detect with various anti-stick albumen MAb subsequently.The muroid MAb (mMAb) with MUC1, MUC3, MUC4, MUC16 and CEACAM6 with specific reaction does not provide signal (not shown) in these allos sandwich immunoassay.On the other hand, anti-both MUC5acmMAb45M1 and 2-11M1 tested provide the positive reaction (Figure 26) of the antigen of capturing with hPAM4, wherein 45M1 display be significantly greater than 2-11M1 reaction (for MAb45M1 and 2-11M1, respectively Kd=14.32 ± 1.08 μ g/mL and 24.4 ± 7.83 μ g/mL; P<0.001).But none can provide CPM1 polyclone IgG fraction anti-with rabbit same strong signal intensity in these indivedual anti-MUC5acMAb.Importantly, mPAM4 is not in conjunction with the antigen that hPAM4 captures, and hPAM4, also not in conjunction with the antigen that mPAM4 captures, shows that PAM4 epi-position exists with low-density, only has Single locus in possible mucin antigen.

Design follow-up research is to suppress hPAM4 and to close (Figure 27 A to Figure 27 B) through hardening of being coated with of CPM1.Although the anti-MUC5ac of 2-11M1 can not suppress hPAM4-CPM1 to combine, 45M1 anti-MUC5ac can provide limited inhibition, wherein IC _max=25.5% suppresses (Figure 27 A).The mPAM4 included as positive control provides IC under concentration 0.1 μ g/mL _max=92.4% from suppressing, and the anti-MUC1 antibody of MA5 and KC4 does not provide suppression, even if under assessed maximum concentration (10 μ g/mL) (Figure 27 A).HPAM4 can not block mPAM4 and CPM1 antigen completely and combine (IC _max=52.8%) (not shown), this is the discovery of it is as expected, since it is known the humanization pattern of PAM4 has the affinity lower than muroid parent.Using containing for MUC5ac the ascites fluid serial dilution with the mMAb of known collection of illustrative plates as suppression reagent, result is shown in Figure 27 B.MMAb21M1,62M1 and 463M1 provide separately and are similar to for the suppression of mPAM4 from the result shown in blocking-up, and wherein 45M1 ascites provides limited suppression, is similar to (Figure 27 B) observed when commercially available 45M1-IgG.Comprising the ascites fluid containing muroid anti-alpha-fetoprotein (AFP) in this as negative contrast does not suppress hPAM4 and CPM1 to combine (Figure 27 B).Unfortunately, ascites volume deficiency hinders and measures MAb concentration, makes to calculate relatively to block efficiency.

Discuss

Current embodiment shows, PAM4 and MUC5ac mucin glycoprotein has reactivity.Figure 28 presents in the research had for us collection of illustrative plates (Nollet etc., 2002, the IntJCancer99:336-43 of the MUC5ac mucin domain of the reactive epi-position indicated by several anti-MUC5acMAb adopted; Nollet etc., 2004, HybridHybridomics23:93-99; Lidell etc., 2008, FEBSJ275:481-89).The peptide core of CLH2 and tandem repeat domains has reactivity (Reis etc., 1997, IntJCancer74:112-21), and may be the hiding epi-position that Capan-1 tumour derives in MUC5ac.2-11M1 and mucin N end (Nollet etc., 2004, HybridHyridomics23:93-99) and at the 45M1 (Lidell etc., 2008, FEBSJ275:481-89) at the end region of the N farthest place of rich halfcystine C end there is reactivity.The mucin that these MAb capture with PAM4 has reactivity, and the MAb for MUC1, MUC3, MUC4 and MUC16 is not then.We observe 45M1 and provide larger signal reaction more remarkable in 2-11M1, show that the 45M1 epitope density in CPM1 is greater than 2-11M1 epi-position.But, this may merely due to the 2-11M1 epi-position loss caused by the relative proteolytic digestion without glycosylated N end and/or during purifying to the molecule shearing of this very big glycoprotein.Under any circumstance, 2-11M1 antibody does not suppress hPAM4-CPM1 to interact, and shows that this epi-position is positioned at PAM4 epi-position at a distance.

On the other hand, 45M1 suppresses hPAM4-CPM1 to interact really, although only partly, shows that the interaction of PAM4 epi-position in mucinous C end region or by this antibody and mucin molecule changes configuration.MAb21M1,62M1 and 463M1 are also located in the mucinous C end region of MUC5ac (Nollet etc., 2002IntJCancer99:336-43; Deng, 2004, HybridHybridomics23:93-99; Lidell etc., 2008, FEBSJ275:481-89), and the remarkable suppression to the reaction of PAM4 mucin is provided separately.In a word, our data provide PAM4 and same mucin (MUC5ac) and have reactivity and the PAM4 epi-position positive evidence that directly blocked by the interaction of these MAb and MUC5ac antigens or modify through configuration.

Our Preliminary report PAM4 and MUC1 mucin material has reactivity (Gold etc., 2007 a, ClinCancerRes13:7380-87; Gold etc., 2006, JClinOncol24:252-58).This is based on MUC1 gene transfection research, wherein observes PAM4 and the MUC1 through gene transfection ⁺clone instead of MUC1 ^-parental cell line or vector control cell line reaction.But other evidence obtained since then queries this explanation, propose MUC1 transfection and also may raise other mucin.The Previous results in our laboratory supports current discovery.Find that PAM4 epi-position is to dithiothreitol (DTT) (0.02M, 15min, 20 DEG C) or heat (100 DEG C, 2min) carry out appropriateness reduction and there is hypersensitivity, show that this epi-position is peptide in itself, and highly depend on the specified configuration (Gold etc., 1994, IntJCancer57:204-10) being kept complete protein core by disulfide bond.This can not be the MUC1 that all halfcystines are all positioned at mucin membrane spaning domain, but the loss of reactivity shown after mucin antigen reduces with several anti-MUC5acMAb is consistent.In addition, adopt immunohistochemical method, we report expression frequency and the fractions distribution of the PAM4 epi-position in PDAC and its precursor lesion and are greater than for (the Gold etc. described by MUC1 for those the similarity described by MUC5ac, 2007, ClinCancerRes13:7380-87).

In a word, the antibody be combined with the PAM4 epi-position of MUC5ac can be used for detection and the antidiastole of cancer of pancreas.The immunoconjugates of this antibody-like can be used for pancreas cancers therapy.

The DOTA conjugate of embodiment 35.PAM4

HPAM4 antibody is prepared described in embodiment.By the CDR grafting V of hPAM4 _hinsert based in the increased expression system pdHL2 plasmid vector of DHFR with V κ chain gene.By plasmids in murine myeloma cell line Sp2/0-Ag14 (ATCC, Manassas, VA), to produce the cell clone that can produce hPAM4.Complete mature amino acid sequence is as follows.

HPAM4 heavy chain

QVQLQQSGAEVKKPGASVKVSCEASGYTFPSYVLHWVKQAPGQGLEWIGYINPYNDGTQYNEKFKGKATLTRDTSINTAYMELSRLRSDDTAVYYCARGFGGSYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO：117)

HPAM4 light chain

DIQLTQSPSSLSASVGDRVTMTCSASSSVSSSYLYWYQQKPGKAPKLWIYSTSNLASGVPARFSGSGSGTDFTLTISSLQPEDSASYFCHQWNRYPYTFGGGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQIDNO：118)

HPAM4V κ and V _hdNA and amino acid sequence be shown in Fig. 4 A and Fig. 4 B, wherein CDR differentiates with runic and underscore.

Current cell clone title is hPAM4-2E3, and is produce in Sp2/0 host cell DHFR expression system.Antibody is humanization IgG _{1 κ}glycoprotein.Glycosylation site (Asn299) every mole of hPAM4DOTA on heavy chain has following composition: 0.5Fuc, 6.3GlcNAc, 6.3Man, 0.3Gal and 0.15Neu5Gc; Glycosylation species: G0F70%, G1F23%, G2F2%, G1FS14%, G2FS11%.Authenticated 16 S-S keys (32SH) and the theoretical prediction of pressing completely based on above sequence is located.

HPAM4-DOTA product be by with 12 yuan of macrocyclic chelants Isosorbide-5-Nitraes, the purified hPAM4IgG preparation of 7,10-tetraazacyclododecanand-N, N ', N ", N " '-tetraacethyl (DOTA) coupling.

DOTA puts together via one of carboxy moiety and the reactive site on hPAM4 antibody, to produce stable conjugate.Assuming that via the lysine side chain amino groups coupling of stable amido link and antibody.

By first 1-ethyl-3-(3-dimethylaminopropyl) carbodiimides (EDC) exist under make DOTA and N-hydroxyl sulfoacid succinimide (sulfo-NHS) react with produce activate DOTA, subsequently the DOTA of activation is hatched together with purified hPAM4 antibody carry out chemically conjugated.Condition is optimized to produce to the replacement ratio of each antibody molecule 4-7 DOTA part, as by mass spectroscopy determined.

***

Those skilled in the art should be apparent, can carry out various modifications and variations to product of the present invention, composition, Method and process.Therefore, wish that this type of modifications and variations are contained in the present invention, condition is that they are in the scope of appended claims with its equivalent.

Claims (40)

1. treat the method for cancer of pancreas for one kind, it comprises uses to the individuality suffering from cancer of pancreas the antibody combining and be positioned at the epi-position of second to the 4th rich Cysteine domains (amino acid residue 1575-2052) of MUC5ac, and wherein said antibody and therapeutic agent are puted together.

2. the method for claim 1, wherein said antibody or its fragment with comprise variable region of light chain CDR sequence C DR1 (SASSSVSSSYLY, SEQIDNO:1), CDR2 (STSNLAS, and CDR3 (HQWNRYPYT SEQIDNO:2), and variable region of heavy chain CDR sequence C DR1 (SYVLH SEQIDNO:3), SEQIDNO:4), CDR2 (YINPYNDGTQYNEKFKG, SEQIDNO:5) and the antibody of CDR3 (GFGGSYGFAY, SEQIDNO:6) in conjunction with identical epi-position or competition binding MUC5ac.

3. the method for claim 1, wherein said therapeutic agent is selected from radioactive nuclide, immunomodulator, hormone, hormone antagonist, enzyme, antisense oligonucleotides, siRNA, enzyme inhibitor, photosensitivity therapeutic agent, cytotoxic agent, medicine, toxin, AI and short Apoptosis agent.

4. method as claimed in claim 3, wherein said radioactive nuclide is selected from ¹⁴c, ¹³n, ¹⁵o, ³²p, ³³p, ⁴⁷sc, ⁵¹cr, ⁵⁷co, ⁵⁸co, ⁵⁹fe, ⁶²cu, ⁶⁷cu, ⁶⁷ga, ⁶⁷ga, ⁷⁵br, ⁷⁵se, ⁷⁵se, ⁷⁶br, ⁷⁷as, ⁷⁷br, ^80mbr, ⁸⁹sr, ⁹⁰y, ⁹⁵ru, ⁹⁷ru, ⁹⁹mo, ^99mtc, ^103mrh, ¹⁰³ru, ¹⁰⁵rh, ¹⁰⁵ru, ¹⁰⁷hg, ¹⁰⁹pd, ¹⁰⁹pt, ¹¹¹ag, ¹¹¹in, ^113min, ¹¹⁹sb, ^121mte, ^122mte, ¹²⁵i, ^125mte, ¹²⁶i, ¹³¹i, ¹³³i, ¹⁴²pr, ¹⁴³pr, ¹⁴⁹pm, ¹⁵²dy, ¹⁵³sm, ¹⁶¹ho, ¹⁶¹tb, ¹⁶⁵tm, ¹⁶⁶dy, ¹⁶⁶ho, ¹⁶⁷tm, ¹⁶⁸tm, ¹⁶⁹er, ¹⁶⁹yb, ¹⁷⁷lu, ¹⁸⁶re, ¹⁸⁸re, ^189mos, ¹⁸⁹re, ¹⁹²ir, ¹⁹⁴ir, ¹⁹⁷pt, ¹⁹⁸au, ¹⁹⁹au, ¹⁹⁹au, ²⁰¹tl, ²⁰³hg, ²¹¹at, ²¹¹bi, ²¹¹pb, ²¹²bi, ²¹²pb, ²¹³bi, ²¹⁵po, ²¹⁷at, ²¹⁹rn, ²²¹fr, ²²³ra, ²²⁴ac, ²²⁵ac, ²⁵⁵fm and Th ²²⁷.

5. method as claimed in claim 4, wherein said radioactive nuclide is ⁹⁰y.

6. method as claimed in claim 3, wherein said medicine is selected from 5 FU 5 fluorouracil, Ah method is for Buddhist nun, aplidin, azaribine, Anastrozole, anthracycline, Axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, Bryostatin-1, busulfan, calicheamycin, camptothecine, carboplatin, 10-hydroxycamptothecine, carmustine, Celebrex, Chlorambucil, neoplatin (CDDP), Cox-2 inhibitor, Irinotecan (CPT-11), SN-38, carboplatin, Cladribine, camptothecine, gram azoles is for Buddhist nun, endoxan, cytarabine, Dacarbazine, Dasatinib, dinaciclib, docetaxel, dactinomycin D, daunorubicin, Doxorubicin, 2-pyrrolin Doxorubicin (2PDOX), pro-2PDOX, cyano group morpholinyl Doxorubicin, Doxorubicin glucosiduronic acid, epirubicin glucuronide, Erlotinib, estramustine, epipodophyllotoxin, Erlotinib, grace is for Nuo Te, estrogen receptor binding agents, Etoposide (VP16), Etoposide glucosiduronic acid, etoposide phosphate, Exemestane, FTY720, floxuridine (FUdR), 3', 5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, Flutamide, farnesyl protein transferase inhibitor, Flavopiridol, good fortune he for Buddhist nun, ganetespib, GDC-0834, GS-1101, Gefitinib, gemcitabine, hydroxycarbamide, according to Shandong for Buddhist nun, idarubicin, Ai Dailalisi, ifosfamide, Imatinib, leunase, Lapatinib, lenalidomide, formyl tetrahydrofolic acid, LFM-A13, lomustine, mustargen, melphalan, purinethol, Ismipur, amethopterin, mitoxantrone, mithramycin, mitomycin, mitotane, NVB, HKI-272, nilotinib, nitroso ureas, Aura handkerchief Buddhist nun, plicamycin, procarbazine, taxol, PCI-32765, Pentostatin, PSI-341, Raloxifene, Semustine, Sorafenib, streptozotocin, SU11248, Sutent, Tamoxifen, Temozolomide (aqueous form of DTIC), anti-platinum, Thalidomide, thioguanine, thiotepa, Teniposide, topotecan, uracil mastard, PTK787, vinorelbine, vinblastine, vincristine, vinca alkaloids and ZD1839.

7. method as claimed in claim 3, wherein said toxin is selected from ricin (WA), abrin, alpha toxin, saporin, ribonuclease (RNA enzyme), DNA enzymatic I, staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin and pseudomonad endotoxin.

8. method as claimed in claim 3, wherein said immunomodulator is selected from cell factor, stem cell factor, lymphocytotoxin, Hemopoietic factor, colony stimulating factor (CSF), interferon (IFN), hematopoietin, thrombopoietin TNF (TNF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), interferon-' alpha ', interferon beta, interferon gamma, interferon lambda, human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxain, relaxation precipitinogen, follicle-stimulating hormone (FSH) (FSH), thyrotropic hormone (TSH), luteinizing principle (LH), hepatocyte growth factor, prostaglandin, fibroblast growth factor, prolactin, galactagogin, OB albumen, tumor necrosis factor α, TNF, Miao Le Shi pipe inhibiting substances, mouse promoting sexual gland hormone related peptide, inhibin, activin, vascular endothelial growth factor, integrin, TPO (TPO), NGF-, PDGF, TGF-α, TGF-, insulin-like growth factor I, insulin-like growth factor II, hematopoietin (EPO), macrophage-CSF (M-CSF), IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, IL-25, LIF, FLT-3, angiostatin, thrombospondin, Endostatin and lymphotoxin.

9. method as claimed in claim 3, wherein said tyrosine kinase inhibitor how is selected from card for Buddhist nun, Dasatinib, Erlotinib, Gefitinib, Imatinib, Lapatinib, leflunomide, nilotinib, pazopanib, SU5416, Sorafenib, Sutent, SU11248, PTK787, PCI-32765 (according to Shandong for Buddhist nun), PCI-45292, GDC-0834, LFM-A13 and RN486.

10. the method for claim 1, it also comprises uses other therapeutic agent of at least one to described individuality, and other therapeutic agent of wherein said at least one is selected from second antibody, the second antigen binding antibody fragment, immunoconjugates, immunomodulator, hormone, hormone antagonist, enzyme, antisense oligonucleotides, siRNA, enzyme inhibitor, photosensitivity therapeutic agent, cytotoxic agent, medicine, toxin, AI and short Apoptosis agent.

11. method as claimed in claim 10, wherein said second antibody, the second antigen binding antibody fragment or immunoconjugates combine and are selected from following antigen: CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, Le ^a, Le (y), CEACAM5, CEACAM6, CSAp, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC16, MUC17, HLA-DR, CD40, CD74, CD138, HER2/neu, EGFR, EGP-1, EGP-2, VEGF, PlGF, IGF, tenascin, platelet derived growth factor, IL-6, bcl-2, K-ras, p53 and cMET.

12. methods as claimed in claim 10, wherein said second antibody, second antigen binding antibody fragment or immunoconjugates are selected from hR1 (anti-IGF-1R), hPAM4 (anti-MUC5ac), hIMMU-31 (anti-AFP), hLL1 (anti-CD74), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hL243IgG4P (anti-HLA-DR), hMN-14 (anti-CEACAM5), hMN-15 (anti-CEACAM6), hRS7 (anti-EGP-1 or anti-TROP-2), hMN-3 (anti-CEACAM6), Ab124 (anti-CXCR4) and Ab125 (anti-CXCR4).

The method of 13. 1 kinds of detections or diagnosis of pancreatic cancer, it comprises:

A) blood, serum, blood plasma or tissue sample is obtained from individuality; With

B) the anti-stick protein antibodies of the epi-position of second to the 4th rich Cysteine domains (amino acid residue 1575-2052) of MUC5ac is positioned at or its Fab carries out immunoassays with combining;

The mucinous cancer of pancreas existed in the described individuality of instruction of wherein said cancer of pancreas, and described immunoassays can detect Early pancreatic carcinoma.

14. methods as claimed in claim 13, wherein said antibody or its fragment with comprise variable region of light chain CDR sequence C DR1 (SASSSVSSSYLY, SEQIDNO:1), CDR2 (STSNLAS, and CDR3 (HQWNRYPYT SEQIDNO:2), and variable region of heavy chain CDR sequence C DR1 (SYVLH SEQIDNO:3), SEQIDNO:4), CDR2 (YINPYNDGTQYNEKFKG, SEQIDNO:5) and the antibody of CDR3 (GFGGSYGFAY, SEQIDNO:6) in conjunction with identical epi-position or competition binding MUC5ac.

15. methods as claimed in claim 13, it also comprises and carries out immunoassays with one or more Additional antibodies in conjunction with the pancreatic cancer cell in described sample.

16. method as claimed in claim 15, wherein said Additional antibodies combines and is selected from following antigen: CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, CEACAM5, CEACAM6, Le ^a, Le (y), CSAp, IGF (IGF), Glycoproteins in Epithelial-1 (EGP-1), Glycoproteins in Epithelial-2 (EGP-2), TROP2, CD80, placenta growth factor (PlGF), carbonic anhydrase IX, tenascin, IL-6, HLA-DR, CD40, CD74, CD138, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC16, MUC17, TAG-72, EGFR, platelet derived growth factor (PDGF), VEGF, PlGF, bcl-2, Kras, p53, cMET and HER2/neu.

17. methods as claimed in claim 16, wherein said Additional antibodies is in conjunction with CA19.9.

18. methods as claimed in claim 17, wherein saidly utilize the immunoassays of anti-MUC5ac antibody and anti-CA19.9 antibody to have the susceptibility of 84% and the specificity of 83% for the detection of cancer of pancreas.

19. methods as claimed in claim 13, wherein said immunoassays can the non-mucus cystic pancreatic disease individuality of distinguishing benign and 1A phase, 1B phase and 2 phase cancers of pancreas individualities.

20. methods as claimed in claim 13, wherein said immunoassays have the false positive rate of 6% or lower for optimum pancreatic disorders individuality.

21. methods as claimed in claim 13, wherein said sero-immunity measures the cancer of pancreas that can detect in asymptomatic individuality.

22. methods as claimed in claim 13, wherein said sample be blood serum sample and described method be also included in carry out described immunoassays before organic extractant phase is carried out to described blood serum sample.

23. methods as claimed in claim 22, wherein said organic phase is butanols.

24. method as claimed in claim 13, wherein said immunoassays detect the existence of mucinous tumors (IPMN) and Intraductal papillary mucinous tumors in PanIN-1A, PanIN-1B, PanIN-2, aggressive cancer of pancreas, cancer of pancreas, mucus cystoma (MCN), pancreas.

25. methods as claimed in claim 13, wherein said anti-MUC5ac antibody comprises variable region of light chain CDR sequence C DR1 (SASSSVSSSYLY, SEQIDNO:1), CDR2 (STSNLAS, and CDR3 (HQWNRYPYT SEQIDNO:2), and variable region of heavy chain CDR sequence C DR1 (SYVLH SEQIDNO:3), SEQIDNO:4), CDR2 (YINPYNDGTQYNEKFKG, and CDR3 (GFGGSYGFAY, SEQIDNO:6) SEQIDNO:5).

26. method as claimed in claim 13, wherein said anti-MUC5ac antibody or its fragment can in conjunction with comprising the linear peptides of amino acid sequence WTWNITKAYPLP (SEQIDNO:7) or comprising the cyclic peptide of amino acid sequence ACPEWWGTTC (SEQIDNO:8).

27. methods as claimed in claim 13, its serum levels also comprised by monitoring MUC5ac determines the reactivity of cancer of pancreas to therapy.

The method of 28. 1 kinds of detections or diagnosis of pancreatic cancer, it comprises:

A) the anti-cancer of pancreas mucin antibody or its Fab that combine and be positioned at the epi-position of second to the 4th rich Cysteine domains (amino acid residue 1575-2052) of MUC5ac is used to individuality; With

B) the described antibody be combined with pancreatic cancer cell or its fragment is detected;

Wherein said anti-MUC5ac antibody at least one diagnosticum is marked.

29. methods as claimed in claim 28, wherein said antibody with comprise variable region of light chain CDR sequence C DR1 (SASSSVSSSYLY, SEQIDNO:1), CDR2 (STSNLAS, and CDR3 (HQWNRYPYT SEQIDNO:2), and variable region of heavy chain CDR sequence C DR1 (SYVLH SEQIDNO:3), SEQIDNO:4), CDR2 (YINPYNDGTQYNEKFKG, SEQIDNO:5) and the antibody of CDR3 (GFGGSYGFAY, SEQIDNO:6) in conjunction with identical epi-position or competition binding MUC5ac.

30. methods as claimed in claim 28, wherein said anti-MUC5ac antibody is connected to that combine can haptenic antibody on target construct or antigen binding antibody fragment; And described method also comprise use be connected at least one diagnosticum can target construct.

31. methods as claimed in claim 28, wherein said anti-MUC5ac antibody or its Fab and at least one diagnosticum are puted together.

32. methods as claimed in claim 30, wherein said diagnosticum is selected from radioactive nuclide, contrast preparation, fluorescer, chemiluminescence agent, luminescent biological agent, paramagnetic ion, enzyme and photosensitivity diagnosticum.

33. method as claimed in claim 32, wherein said diagnosticum is selected from ¹¹⁰in, ¹¹¹in, ¹⁷⁷lu, ¹⁸f, ⁵²fe, ⁶²cu, ⁶⁴cu, ⁶⁷cu, ⁶⁷ga, ⁶⁸ga, ⁸⁶y, ⁹⁰y, ⁸⁹zr, ^94mtc, ⁹⁴tc, ^99mtc, ¹²⁰i, ¹²³i, ¹²⁴i, ¹²⁵i, ¹³¹i, ^154-158gd, ³²p, ¹¹c, ¹³n, ¹⁵o, ¹⁸⁶re, ¹⁸⁸re, ⁵¹mn, ^52mmn, ⁵⁵co, ⁷²as, ⁷⁵br, ⁷⁶br, ^82mrb, ⁸³the radioactive nuclide of Sr or other γ, β or positron emitter.

34. methods as claimed in claim 33, wherein said radioactive nuclide is ¹⁸f and described method also comprises PET imaging.

35. methods as claimed in claim 32, wherein said paramagnetic ion is selected from chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III).

36. methods as claimed in claim 32, wherein said diagnosticum is the fluorescent labelling compound being selected from fluorescein isothiocynate, rhodamine, rhodophyll, phycocyanobilin, allophycocyanin, o-phthalaldehyde(OPA) and fluorescamine, or be selected from the chemiluminescent labeling compound of luminol, different luminol, aromatics acridinium ester, imidazoles, acridinium salt and oxalate, or be selected from the bioluminescent compound of fluorescein, luciferase and aequorin.

37. methods as claimed in claim 30, wherein said method is used in operation, in endoscope or Ink vessel transfusing program.

38. methods as claimed in claim 30, wherein said haptens binding antibody is in conjunction with HSG or In-DTPA.

39. 1 kinds of anti-cancer of pancreas antibody, it comprises:

A) heavy chain amino acid sequence SEQIDNO:117; With

B) light-chain amino acid sequence SEQIDNO:118.

40. antibody as claimed in claim 39, it also comprises DOTA (DOTA) chelating moiety of one or more and described antibody conjugate.