Antibody-drug conjugate (ADC) binding to 158P1D7 protein

Abstract

Antibody drug conjugates (ADCs) that bind to 158P1D7 protein and variants thereof are described herein. 158P1D7 shows tissue-specific expression in normal adult tissues and is aberrantly expressed in the cancers listed in Table 1. Therefore, the ADCs of the present invention provide a therapeutic composition for treating cancer.

Description

[0001] The present application is a divisional application of an invention application filed on August 23, 2013, with application number 201380055601.6 and titled “Antibody-drug conjugate (ADC) binding to 158P1D7 protein”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 61 / 692,448, filed on August 23, 2012, the contents of which are incorporated herein by reference in their entirety.

[0004] STATEMENT OF RIGHTS TO INVENTS MADE UNDER FEDERALLY SPONSORED RESEARCH

[0005] none.

[0006] Submit sequence listing as ASCII text file

[0007] The following is incorporated herein by reference in its entirety by submitting the content in an ASCII text file: Computer Readable Form (CRF) of Sequence Listing (File Name: 511582005088SeqList.txt, Record Date: August 21, 2013, Size: 40,154 bytes). Technical Field

[0008] The present invention relates to antibodies, binding fragments and antibody drug conjugates (ADCs) thereof that bind to 158P1D7 protein. The present invention further relates to prognostic, preventive and therapeutic methods and compositions for treating cancers expressing 158P1D7. Technical Background

[0009] Cancer is the second leading cause of death in the world after coronary artery disease. Millions of people die from cancer each year. According to the American Cancer Society, in the United States alone, cancer causes more than 500,000 deaths each year, and more than 1.2 million new cases are diagnosed each year. Although deaths from heart disease have declined significantly, deaths from cancer are generally on the rise. It is expected that by the early next century, cancer will become the leading cause of death.

[0010] Several cancers are clearly major killers worldwide. In particular, lung, prostate, breast, colon, pancreatic, ovarian and bladder cancers represent the leading causes of cancer death. These cancers, and almost all others, share a common lethal characteristic. Metastatic disease of cancer is fatal with very few exceptions. Furthermore, even for those patients who initially survive the primary cancer, general experience shows that their lives are greatly altered. Many cancer patients experience severe anxiety due to concerns about possible recurrence or treatment failure. Many patients experience physical weakness following treatment. Furthermore, many cancer patients experience recurrence.

[0011] Prostate cancer ranks fourth among the most common cancers in men worldwide. In North America and Northern Europe, prostate cancer is undoubtedly the most common cancer in men and the second leading cause of cancer death in men. In the United States alone, more than 30,000 men die from this disease each year, second only to lung cancer. Although these data are amazing, there is still no effective treatment for metastatic prostate cancer. Prostate surgery, radiotherapy, hormone removal therapy, surgical castration and chemotherapy are still the main treatment methods. Unfortunately, these treatments are ineffective for many people and are often associated with adverse consequences.

[0012] On the diagnostic front, the lack of prostate cancer markers that can reliably detect early localized tumors remains a significant limitation in the diagnosis and management of this disease. Although the serum prostate-specific antigen (PSA) test has been a useful tool, it is widely considered to lack specificity and overall utility in several important respects.

[0013] Progress in identifying other specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts in mice that reproduce different disease stages. LAPC (Los Angeles prostate cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immunodeficient (SCID) mice and have been shown to mimic the ability to switch from androgen dependence to androgen independence (Klein et al., 1997, Nat. Med. 3:402). Recently identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93:7252), prostate-specific membrane antigen (PSMA) (Pinto et al., Clin Cancer Res 1996 Sep 2(9):1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci USA. 1999 Dec 7;96(25):14523-8), and prostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95:1735).

[0014] While previously identified markers such as PSA have facilitated efforts to diagnose and treat prostate cancer, there remains a need to identify additional markers and therapeutic targets for prostate cancer and related cancers to further improve diagnosis and treatment. An estimated 130,200 cases of colorectal cancer occurred in the United States in 2000, including 93,800 cases of colon cancer and 36,400 cases of rectal cancer.

[0015] Colorectal cancer is the third most common cancer in both men and women. Incidence rates declined significantly between 1992 and 1996 (-2.1% per year). Studies show that these declines are due to increased screening and polyp removal, which prevent polyps from developing into invasive cancers. An estimated 56,300 deaths occurred in 2000 (47,700 from colon cancer, 8,600 from rectal cancer), accounting for about 11% of all U.S. cancer deaths.

[0016] Currently, surgery is the most common form of treatment for colorectal cancer and is often curative for cancer that has not spread. Most people whose cancer has severely perforated the bowel wall or has spread to the lymph nodes receive chemotherapy or chemotherapy plus radiation therapy before or after surgery. A colostomy (making an opening in the abdomen to remove waste from the body) is sometimes needed for colon cancer but rarely for rectal cancer. There remains a need for effective diagnostic and treatment methods for colorectal cancer.

[0017] Bladder cancer accounts for approximately 5% of all new cancer cases in the United States in men (the fifth most common cancer) and approximately 3% in women (the eighth most common cancer). The incidence rate is slowly increasing, consistent with the increase in the older population. In 1998, there were an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence rate in the United States is 32 per 100,000 for men and 8 per 100,000 for women. The historical 3:1 male / female ratio may be declining, a decline that is related to smoking behavior in women. In 1998, there were an estimated 11,000 deaths from bladder cancer (7,800 in men and 3,900 in women). The incidence and mortality rates of bladder cancer increase substantially with age, and the problem will become more severe as the population ages.

[0018] Most bladder cancers recur in the bladder. The approach to managing bladder cancer is a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer indicates the limitations of TUR. Most muscle-invasive cancers cannot be cured by TUR alone. Radical cystectomy and urinary diversion are the most effective methods to remove the cancer but have an undeniable impact on urinary and sexual function. There is still a great need for treatments that benefit patients with bladder cancer.

[0019] In 2000, there were an estimated 164,100 new cases of lung and bronchial cancer, accounting for 14% of all cancer diagnoses in the United States. The incidence of lung and bronchial cancer has declined significantly among men, from a high of 86.5 cases per 100,000 in 1984 to 70.0 in 1996. The rate of increase in women began to slow in the 1990s. In 1996, the incidence in women was 42.3 cases per 100,000.

[0020] Lung and bronchial cancer caused an estimated 156,900 deaths in 2000, accounting for 28% of all cancer deaths. Between 1992 and 1996, the mortality rate from lung cancer decreased significantly among men (-1.7% per year) while it continued to increase significantly among women (0.9% per year). Since 1987, more women have died each year from lung cancer than from breast cancer, which has been the leading cause of cancer death in women for 40 years. The most likely explanation for the decline in lung cancer incidence and mortality is the decline in smoking rates over the past 30 years; however, the decline in smoking behavior among women has lagged behind that among men. Importantly, although the decline in tobacco use among adults has slowed, tobacco use among young people is increasing again.

[0021] Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice. Because the disease has often spread by the time it is discovered, radiation therapy and chemotherapy are often used in conjunction with surgery. The treatment of choice for small cell lung cancer is chemotherapy alone or in combination with chemotherapy and radiation therapy; with this regimen, a larger proportion of patients achieve remission, which in some cases lasts a long time. However, there is an ongoing need for effective treatments and diagnostic methods for lung and bronchial cancer.

[0022] An estimated 182,800 new cases of invasive breast cancer are expected to occur in U.S. women during the year 2000. In addition, approximately 1,400 new cases of breast cancer are expected to be diagnosed in men in 2000. After rising by approximately 4% per year during the 1980s, the incidence of breast cancer in women stabilized in the 1990s to approximately 110.6 cases per 100,000 people.

[0023] In the United States alone, there were an estimated 41,200 deaths from breast cancer in 2000 (40,800 in women and 400 in men). Breast cancer ranks second among cancer deaths in women. According to the most recent data, mortality rates declined significantly between 1992 and 1996, with the greatest declines among younger women (both white and black). These declines likely occurred because of earlier detection and improved treatment.

[0024] Depending on the medical setting and patient preference, treatment for breast cancer may include lumpectomy (local removal of the tumor) and removal of lymph nodes under the arm, mastectomy (surgical removal of the breast) and removal of lymph nodes under the arm, radiation therapy, chemotherapy, or hormone therapy. Two or more approaches are often used together. Many studies have shown that for early-stage disease, long-term survival rates after lumpectomy plus radiation therapy are similar to those after a modified mastectomy. Significant advances in reconstructive techniques have provided several options for breast reconstruction after mastectomy. More recently, this reconstruction has been done at the same time as the mastectomy.

[0025] Local excision of ductal carcinoma in situ (DCIS) and an appropriate amount of surrounding normal breast tissue can prevent local recurrence of DCIS. Radiation therapy to the breast and / or tamoxifen can reduce the chance of DCIS developing in the remaining breast tissue. This is important because DCIS can develop into invasive breast cancer if left untreated. However, these treatments have serious side effects or sequelae. Therefore, there is a need for effective breast cancer treatments.

[0026] In 2000, there were an estimated 23,100 new cases of ovarian cancer in the United States. It accounts for 4% of all female cancers and ranks second among gynecological cancers. The incidence of ovarian cancer decreased significantly between 1992 and 1996. In 2000, there were an estimated 14,000 deaths from ovarian cancer. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.

[0027] Treatment options for ovarian cancer are surgery, radiation therapy, and chemotherapy. Surgery usually involves removal of one or both ovaries, fallopian tubes (salpingo-oophorectomy), and uterus (hysterectomy). In some very early stage tumors, only the involved ovary is removed, especially in young women who wish to have children. In advanced disease, attempts are made to remove all intra-abdominal disease to improve the effectiveness of chemotherapy. There remains a great need for effective treatment options for ovarian cancer.

[0028] There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past 20 years, pancreatic cancer rates have declined in men. Disease rates in women have remained roughly constant but may be beginning to decline. Pancreatic cancer caused an estimated 28,200 deaths in the United States in 2000. Over the past 20 years, the death rate has declined slightly in men (about -0.9% per year) and increased slightly in women.

[0029] The treatment options for pancreatic cancer are surgery, radiation therapy, and chemotherapy. These treatment options can prolong survival and / or relieve symptoms in many patients but may not result in a cure for most. There is a great need for other treatment and diagnostic options for cancer. These include the use of antibodies, vaccines, and small molecules as therapeutic approaches. In addition, there is a need to use these approaches as research tools to diagnose, detect, monitor, and advance the state of the art in all areas of cancer treatment and research.

[0030] The therapeutic utility of monoclonal antibodies (mAbs) (G. Kohler and C. Milstein, Nature 256:495-497 (1975)) is being realized. Monoclonal antibodies are now approved as therapies for transplantation, cancer, infectious diseases, cardiovascular disease, and inflammation. Different isotypes have different effector functions. This functional difference is reflected in the different three-dimensional structures of the various immunoglobulin isotypes (PM Alzari et al., Annual Rev. Immunol., 6:555-580 (1988)).

[0031] Because mice are easy to immunize and recognize most human antigens as foreign objects, anti-human target mAbs with therapeutic potential generally have mouse sources. However, mouse mAbs have inherent disadvantages as human therapy. Since the circulating half-life of mAbs in humans is shorter than that of human antibodies, they require more frequent administration. More seriously, repeated administration of mouse antibodies to the human immune system can cause the human immune system to respond, which is caused by recognizing mouse proteins as foreign objects and producing human anti-mouse antibodies (HAMA) responses. This type of HAMA response can cause allergic reactions and quickly remove mouse antibodies from the system, thereby making mouse antibody treatment invalid. To avoid this effect, attempts have been made to establish the human immune system in mice.

[0032] Initial attempts were made to create transgenic mice that would respond to antigens with antibodies that had human sequences (see Bruggemann et al., Proc. Nat'l. Acad. Sci. USA 86:6709-6713 (1989)), but this was limited by the amount of DNA that could be stably maintained by the cloning vector. The use of yeast artificial chromosome (YAC) cloning vectors enabled the introduction of large germline fragments of the human Ig loci into transgenic mammals. YACs were used to introduce essentially most of the human V, D, and J region genes into mice with the same spacing as seen in the human genome, along with human constant regions. One such transgenic mouse line is called Mice are commercially available from Amgen Fremont, Inc. (Fremont, CA). Summary of the invention

[0033] The present invention provides antibodies, binding fragments and antibody drug conjugates (ADCs) thereof that can bind to 158P1D7 protein and 158P1D7 protein polypeptide fragments. In some embodiments, the present invention includes fully human antibodies coupled to therapeutic agents. In certain embodiments, the premise is that the complete nucleic acid sequence in Figure 3 is not encoded and / or the complete amino acid sequence in Figure 2 is not prepared. In certain embodiments, the complete nucleic acid sequence in Figure 3 is encoded and / or the complete amino acid sequence in Figure 2 is prepared, and either of the two adopts a corresponding human unit dosage form.

[0034] The present invention also provides various immunogenic or therapeutic compositions (eg, antibody drug conjugates) and therapeutic strategies for cancers expressing 158P1D7 (eg, cancers of the tissues listed in Table I, particularly bladder cancer). BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The cDNA and amino acid sequences of .158P1D7 are shown in Figure 1 The initiator methionine is underlined. The open reading frame extends from nucleotides 23-2548 and includes the stop codon.

[0036] Figure 2. Nucleic acid and amino acid sequences of the 158P1D7 antibody. Figure 2 (A) shows the cDNA and amino acid sequence of the Ha15-10ac12 heavy chain. The double underline indicates the heavy chain variable region, and the underline indicates the heavy chain human IgG2 constant region. Figure 2 (B) shows the cDNA and amino acid sequence of the Ha15-10ac12 light chain. The double underline indicates the light chain variable region, and the underline indicates the human kappa constant region.

[0037] Figure 3. Amino acid sequence of 158P1D7 antibody. Figure 3 (A) shows the amino acid sequence of the Ha15-10ac12 heavy chain. The double underline indicates the heavy chain variable region, and the underline indicates the human IgG2 constant region. Figure 3 (B) shows the amino acid sequence of the Ha15-10ac12 light chain. The double underline indicates the light chain variable region, and the underline indicates the human kappa constant region.

[0038] Figure 4. Alignment of the Ha15-10ac12 antibody with human Ig germlines. Figure 4 (A) Alignment of the Ha15-10ac12 heavy chain (SEQ ID NO: 3, positions 1-360; SEQ ID NO: 4, positions 1-120) with human Ig germlines. Figure 4 (B) Alignment of the Ha15-10ac12 light chain (SEQ ID NO: 5, positions 1-240; SEQ ID NO: 6, positions 1-80) with human Ig germline IGKV2D-28*01 (SEQ ID NO: 10).

[0039] Figure 5.Efficacy of Ha15-10ac12vcMMAE in the human bladder cancer AG-B7 xenograft model established subcutaneously in SCID mice.

[0040] Figure 6 .Efficacy of Ha15-10ac12vcMMAE in subcutaneously established human bladder cancer RT-4-XCL implanted in SCID mice.

[0041] Figure 7 .Efficacy of Ha15-10ac12vcMMAE in subcutaneously established human lung cancer NCI-H322M-XCL implanted in SCID mice.

[0042] Figure 8 .Efficacy of Ha15-10ac12vcMMAE in the human bladder cancer AG-B7 xenograft model established subcutaneously in SCID mice.

[0043] Figure 9. Detection of 158P1D7 protein in cancer patient samples by IHC. Figures 9(A) and 9(B) show bladder cancer samples. Figures 9(C) and 9(D) show breast cancer samples. Figures 9(E) and 9(F) show lung cancer samples. Figures 9(G) and 9(H) show glioblastoma cancer samples.

[0044] Fig.10 .Efficacy of Ha15-10ac12vcMMAE in the human bladder cancer SW780 xenograft model established subcutaneously in SCID mice.

[0045] Fig.11 . In vitro efficacy of Ha15-10ac12vcMMAE in CHP-212 cytotoxicity assay compared with M15-68(2)18 (also known as 68(18)1.1) Mab.

[0046] Fig.12 .Efficacy of Ha15-10ac12vcMMAE in the patient-derived human bladder cancer AG-B8 xenograft model established subcutaneously in SCID mice.

[0047] Fig.13 .Saturation curve of Ha15-10ac12vcMMAE.

[0048] Fig.14 . Bar graph showing the mean fluorescence intensity (MFI) of Ha15-10ac12vcMMAE.

[0049] Figure 15. Figure 15 (A) CHP-212 cells and Figure 15 (B) IGROV-1 cells

[0050] Evaluation of in vitro cytotoxicity of Ha15-10ac12vcMMAE. DETAILED DESCRIPTION OF THE INVENTION

[0051] Chapter Overview

[0052] I.) Definition

[0053] II.) 158P1D7 Antibody

[0054] III.) Overview of Antibody-Drug Conjugates

[0055] III(A).Maytansinoid

[0056] III(B). Auristatin and dolostatin

[0057] III(C).Caliphaticum

[0058] III(D). Other cytotoxic agents

[0059] IV.) Antibody Drug Conjugates Binding 158P1D7

[0060] V.) Connector unit

[0061] VI.) Extension Unit

[0062] VII.) Amino Acid Unit

[0063] VIII.) Spacer Unit

[0064] IX.) Drug Unit

[0065] X.) Drug loading

[0066] XI.) Method for determining ADC cytotoxicity

[0067] XII.) Treatment of cancers expressing 158P1D7

[0068] XIII.) Targeting 158P1D7 for Antibody-Based Therapy

[0069] XIV.) 158P1D7 ADC mixture

[0070] XV.) Combination therapy

[0071] XVI.) Kits / Products

[0072] I.) Definition:

[0073] Unless otherwise defined, all technical terms, symbols and other scientific terms or special words used herein are intended to have the meanings commonly understood by those skilled in the art to which the present invention belongs. In some cases, the terms with conventional understanding meanings are defined herein for the purpose of illustrating and / or facilitating quoting, and such definitions are included herein and are not necessarily understood to represent significant differences with conventional understandings in the art. Many techniques and methods described herein or cited are fully understood by those skilled in the art and are usually adopted by conventional methods, for example, Sambrook et al. in Molecular Cloning: A Laboratory Manual ("Molecular Cloning: Laboratory Manual") Second Edition (1989), Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York) describes the widely used molecular cloning methods. When appropriate, unless otherwise stated, the process involving the use of commercially available kits and reagents is usually carried out according to the scheme and / or parameters given by the manufacturer.

[0074] When a trade name is used herein, reference to the trade name also refers to the product preparation, generic drug, and active pharmaceutical ingredient of the trade name product, unless otherwise indicated.

[0075] The terms "advanced cancer," "locally advanced cancer," "advanced disease," and "locally advanced disease" refer to cancer that has extended through the associated tissue capsule and include C-stage disease under the American Urological Association (AUA) system, C1-C2 stage disease under the Whitmore-Jewett system, and T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and these patients have significantly less favorable outcomes than those with clinically localized (organ-confined) cancer.

[0076] The abbreviation "AFP" refers to dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylenediamine (see Formula XVI below).

[0077] The abbreviation "MMAE" refers to monomethyl auristatin E (see Formula XI below).

[0078] The abbreviation "AEB" refers to the ester formed by the reaction of auristatin E with p-acetylbenzoic acid (see Formula XX below).

[0079] The abbreviation "AEVB" refers to the ester formed by the reaction of auristatin E with benzoylvaleric acid (see Formula XXI below).

[0080] The abbreviation "MMAF" refers to dovaline-valine-dolaisoleunine-dolaproine-phenylalanine (see Formula XVIV below).

[0081] Unless otherwise indicated, the term "alkyl" refers to a saturated straight or branched hydrocarbon having about 1 to 20 carbon atoms (and all combinations and subcombinations of ranges of carbon atoms and specific numbers of carbon atoms therebetween), preferably about 1 to 8 carbon atoms. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl and 3,3-dimethyl-2-butyl.

[0082] Alkyl, alone or as part of another group, may be optionally substituted by one or more groups, preferably 1-3 groups (and any additional substituents selected from halogen), including but not limited to: -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C( O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, =O, -N3, -NH2, -NH(R'), -N(R')2 and -CN, wherein R' is each independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -aryl, wherein said -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), - O-(C2-C8 alkynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl may be optionally further substituted with one or more groups including, but not limited to, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R", -OC(O) R", -C(O)OR", -C(O)NH2, -C(O)NHR", -C(O)N(R")2, -NHC(O)R", -SR", -SO3R", -S(O)2R", -S(O)R", -OH, -N3, -NH2, -NH(R"), -N(R")2 and -CN, wherein R" is each independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -aryl.

[0083] Unless otherwise indicated, the terms "alkenyl" and "alkynyl" refer to straight and branched carbon chains having about 2-20 carbon atoms (and all combinations and subcombinations of carbon atom ranges and specific numbers of carbon atoms therebetween), preferably about 2-8 carbon atoms. The alkenyl chain has at least one double bond in the chain, and the alkynyl chain has at least one triple bond in the chain. Examples of alkenyl include, but are not limited to, ethylene or vinyl, allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, and -2,3-dimethyl-2-butenyl. Examples of alkynyl include, but are not limited to, acetylenic, propargyl, ethynyl, propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1-butynyl.

[0084] Alkenyl and alkynyl, either alone or as part of another group, may be optionally substituted by one or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogen), including but not limited to: -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, =O, -N3, -NH2, -NH(R'), -N(R')2 and -CN, wherein R' is each independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -aryl, wherein said -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl) , -O-(C2-C8 alkynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl and -C2-C8 alkynyl may be further optionally substituted by one or more substituents, including but not limited to: -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R", -OC(O )R", -C(O)OR", -C(O)NH2, -C(O)NHR", -C(O)N(R"), -NHC(O)R", -SR", -S03R", -S(O)2R", -S(O)R", -OH, -N3, -NH2, -NH(R"), -N(R")2 and -CN, wherein R" is each independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -aryl.

[0085] Unless otherwise indicated, the term "alkylene" refers to a saturated branched or straight chain hydrocarbon radical having about 1-20 carbon atoms (and all combinations and subcombinations of carbon atom ranges and specific numbers of carbon atoms therebetween), preferably about 1-8 carbon atoms, and having two monovalent free radical centers derived by removing two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, 1,4-cyclohexylene, and the like. Alkylene, occurring alone or as part of another group, may be optionally substituted by one or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogen), including but not limited to: -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C -(O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, =O, -N3, -NH2, -NH(R'), -N(R')2 and -CN, wherein R' is each independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -aryl, wherein said -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), - O-(C2-C8 alkynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl may be further optionally substituted with one or more substituents, including but not limited to: -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R", -OC(O )R", -C(O)OR", -C(O)NH2, -C(O)NHR", -C(O)N(R"), -NHC(O)R", -SR", -S03R", -S(O)2R", -S(O)R", -OH, -N3, -NH2, -NH(R"), -N(R")2 and -CN, wherein R" is each independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -aryl.

[0086] Unless otherwise indicated, the term "alkenylene" refers to an optionally substituted alkylene group containing at least one carbon-carbon double bond. Exemplary alkenylene groups include, for example, ethenylene (-CH=CH-) and propenylene (-CH=CHCH2-).

[0087] Unless otherwise indicated, the term "alkynylene" refers to an optionally substituted alkylene group containing at least one carbon-carbon triple bond. Exemplary alkynylene groups include, for example, ethynylene (-C≡C-), propargyl (-CH2C≡C-) and 4-pentynyl (-CH2CH2CH2C≡CH-).

[0088] Unless otherwise indicated, the term "aryl" refers to a monovalent aromatic hydrocarbon radical of 6-20 carbon atoms (and all combinations and subcombinations of carbon atom ranges and specific numbers of carbon atoms therebetween) obtained by removing a hydrogen atom from a carbon atom in a parent aromatic ring system. In the exemplary structures, certain aryl groups are represented by "Ar". Typical aryl groups include, but are not limited to, groups derived from benzene, substituted benzene, phenyl, naphthalene, anthracene, biphenyl, and the like.

[0089] Aryl, either alone or as part of another group, may be optionally substituted by one or more groups, preferably 1-5 groups, or even 1-2 groups, including but not limited to: -halogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O) NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, -NO2, -N3, -NH2, -NH(R'), -N(R')2, and -CN, wherein R' is each independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl, wherein the -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl The alkyl radicals may be further optionally substituted by one or more groups, including but not limited to: -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R", -OC(O)R", -C(O)OR", -C(O)NH2, -C(O)NHR", -C(O)N(R")2, -NHC(O)R", -SR", -S03R", -S(O)2R", -S(O)R", -OH, -N3, -NH2, -NH(R"), -N(R")2 and -CN, wherein R" is each independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -aryl.

[0090] Unless otherwise indicated, the term "arylene" refers to an optionally substituted divalent aromatic radical (i.e., obtained by removing two hydrogen atoms from the same or two different carbon atoms of a parent aromatic ring system) which can be in the ortho, meta or para configuration, as shown in the following structure (using phenyl as an exemplary aromatic radical):

[0091]

[0092] Typical "-(C1-C8 alkylene) aryl", "-(C2-C8 alkenylene) aryl" and "-(C2-C8 alkynylene) aryl" include, but are not limited to, benzyl, 2-phenyleth-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthyleth-1-yl,

[0093] yl, 2-naphthylvin-1-yl, naphthobenzyl, 2-naphthylphenylethyl-1-yl and the like.

[0094] Unless otherwise indicated, the term "heterocycle" refers to a monocyclic, bicyclic or polycyclic system having 3-14 ring atoms (also referred to as ring members), wherein at least one ring atom in at least one ring is a heteroatom selected from N, O, P or S (including all combinations and recombinations of the range and specific number of carbon atoms and heteroatoms therebetween). The heterocycle may have 1-4 ring heteroatoms independently selected from N, O, P or S. One or more N, C or S atoms in the heterocycle may be oxidized. The monocyclic heterocycle preferably has 3-7 ring atoms (such as 2-6 carbon atoms and 1-3 heteroatoms independently selected from N, O, P or S), and the bicyclic heterocycle preferably has 5-10 ring members (such as 4-9 carbon atoms and 1-3 heteroatoms independently selected from N, O, P or S). The ring containing the heteroatom may be an aromatic ring or a non-aromatic ring. Unless otherwise indicated, the heterocycle is attached to its side group at any heteroatom or carbon atom that can produce a stable structure.

[0095] For a description of heterocycles, see Paquette, "Principles of Modern Heterocyclic Chemistr" (WA Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds, A series of Monographs (John Wiley & Sons, New York, 1950 to present), particularly Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. 82:5566 (1960).

[0096] Examples of "heterocyclic" groups include, for example, but are not limited to, pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thionapthyl, indolyl, indolenolyl, quinolyl, isoquinolyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidinonyl, pyrrolinyl, tetrahydrofuranyl, bistetrahydrofuranyl, tetrahydropyranyl, bistetrahydropyranyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, azinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl, isobenzofuranyl, , 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4H-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl,

[0097] Preferred "heterocyclic" groups include, but are not limited to, benzofuranyl, benzothiophenyl, indolyl, benzopyrazolyl, coumarinyl, isoquinolyl, pyrrolyl, thienyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolyl, pyrimidinyl, pyridyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl and tetrazolyl.

[0098] The heterocyclic group, alone or as part of another group, may be optionally substituted by one or more groups, preferably 1-2 groups, including but not limited to: -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C -(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, -N3, -NH2, -NH(R'), -N(R')2, and -CN, wherein R' is each independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl, wherein the -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl The alkyl radicals may be further optionally substituted by one or more groups, including but not limited to: -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R", -OC -C(O)R", -C(O)OR", -C(O)NH2, -C(O)NHR", -C(O)N(R")2, -NHC(O)R", -SR", -S03R", -S(O)2R", -S(O)R", -OH, -N3, -NH2, -NH(R"), -N(R")2 and -CN, wherein R" is each independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or aryl.

[0099] By way of example and not limitation, the carbon-bonded heterocycle may be bonded at the 2, 3, 4, 5, or 6 position of a pyridine; the 3, 4, 5, or 6 position of a pyridazine; the 2, 4, 5, or 6 position of a pyrimidine; the 2, 3, 5, or 6 position of a pyrazine; the 2, 3, 4, or 5 position of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole; the 2, 4, or 5 position of an oxazole, imidazole, or thiazole; the 3, 4, or 5 position of an isoxazole, pyrazole, or isothiazole; the 2 or 3 position of an aziridine; the 2, 3, or 4 position of an azetidine; the 2, 3, 4, 5, 6, 7, or 8 position of a quinoline; or the 1, 3, 4, 5, 6, 7, or 8 position of an isoquinoline. More typically, the carbon-bonded heterocycle includes 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl or 5-thiazolyl.

[0100] By way of example and not limitation, the nitrogen-bonded heterocycle can be bonded at the 1 position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, dihydroindole, or 1H-indazole; the 2 position of isoindole or isoindoline; the 4 position of morpholine; and the 9 position of carbazole or β-carboline. More typically, the nitrogen-bonded heterocycle includes 1-aziridinyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0101] Unless otherwise indicated, the term "carbocycle" refers to a saturated or unsaturated non-aromatic monocyclic, bicyclic or polycyclic ring system having 3-14 ring atoms (and all combinations and subcombinations of ranges of carbon atoms and specific numbers of carbon atoms therebetween) and all ring atoms being carbon atoms. Monocyclic carbocycles preferably have 3-6 ring atoms, more preferably 5 or 6 ring atoms. Bicyclic carbocycles preferably have 7-12 ring atoms, such as arranged as a bicyclic [4,5], [5,5], [5,6] or [6,6] system, or have 9 or 10 ring atoms, arranged as a bicyclic [5,6] or [6,6] system. The term "carbocycle" includes, for example, a monocyclic carbocycle fused to an aromatic ring (e.g., a monocyclic carbocycle fused to a benzene ring). Carbocycles preferably have 3-8 carbon ring atoms.

[0102] The carbocyclic group, occurring alone or as part of another group, may be optionally substituted by one or more groups, preferably 1-2 groups (and any additional substituents selected from halogen), including but not limited to: -halogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR' , -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, =O, -N3, -NH2, -NH(R'), -N(R')2 and -CN, wherein R' is each independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -aryl, wherein the -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -aryl -C8 alkenyl, -C2-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), and -aryl may be further optionally substituted with one or more substituents, including but not limited to: -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R ", -OC(O)R", -C(O)OR", -C(O)NH2, -C(O)NHR", -C(O)N(R")2, -NHC(O)R", -SR", -S03R", -S(O)2R", -S(O)R", -OH, -N3, -NH2, -NH(R"), -N(R")2 and -CN, wherein R" is each independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl or -aryl.

[0103] Examples of monocyclic carbocyclic substituents include -cyclopropyl, -cyclobutyl, -cyclopentyl, -1-cyclopent-1-enyl, -1-cyclopent-2-enyl, -1-cyclopent-3-enyl, cyclohexyl, -1-cyclohex-1-enyl, -1-cyclohex-2-enyl, -1-cyclohex-3-enyl, -cycloheptyl, -cyclooctyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, and -cyclooctadienyl.

[0104] The term "carbocyclo" used alone or as part of another group refers to an optionally substituted divalent carbocyclic group as defined above (ie, obtained by removing two hydrogen atoms from the same or two different carbon atoms of the parent carbocyclic ring system).

[0105] Unless otherwise indicated herein, the hyphen (-) refers to the point of attachment of the side chain molecule. Therefore, the term "-(C1-C8 alkylene)aryl" or "-C1-C8 alkylene (aryl)" refers to a C1-C8 alkylene as defined herein, wherein the alkylene is attached to the side chain molecule at any carbon atom of the alkylene and one hydrogen atom attached to the alkylene carbon atom is replaced by an aryl as defined herein.

[0106] When a particular group is "substituted", the group may have one or more substituents, preferably 1-5 substituents, more preferably 1-3 substituents, most preferably 1-2 substituents, independently selected from the list of substituents. However, the group may generally have any number of substituents selected from halogen. A substituted group is thus indicated.

[0107] According to the inventor's intention, the definition of any substituent or variable at a particular molecular position should be independent of its definition at other positions of the molecule. It should be understood that one of ordinary skill in the art can select substituents and substitution patterns on the compounds of the present invention to provide compounds that are chemically stable and easily synthesized using techniques known in the art and methods described herein.

[0108] The protecting group used herein refers to a group that selectively blocks (temporarily or permanently) a reactive site in a multifunctional compound. Suitable hydroxy protecting groups used in the present invention are pharmaceutically acceptable and may or may not need to be cut off from the parent compound after administration to the subject to make the compound active. In vivo, cleavage is carried out by normal metabolic processes. Hydroxyl protecting groups are well known in the art, see Protective Groups in Organic Synthesis, TW Greene and PGM Wuts (John Wiley & Sons, 3rd Edition), incorporated herein by reference in their entirety for all purposes, such protecting groups include, for example, ethers (e.g., alkyl ethers and silyl ethers, including, for example, dialkyl silyl ethers, trialkyl silyl ethers, dialkyl alkoxy silyl ethers), esters, carbonates, carbamates, sulfonates and phosphate protecting groups.Examples of hydroxy protecting groups include, but are not limited to, methyl ether; methoxymethyl ether, methylthiomethyl ether, (phenyldimethylsilyl)methoxymethyl ether, benzyloxymethyl ether, p-methoxybenzyloxymethyl ether, p-nitrobenzyloxymethyl ether, o-nitrobenzyloxymethyl ether, (4-methoxyphenoxy)methyl ether, guaiacol methyl ether, tert-butoxymethyl ether, 4-pentenyloxymethyl ether, siloxymethyl ether, 2-methoxyethoxymethyl ether, 2,2,2-trichloroethoxymethyl ether, bis(2-chloroethoxy)methyl ether, 2-(trimethylsilyl)ethoxymethyl ether, menthoxymethyl ether ( menthoxymethylether), tetrahydropyranyl ether, 1-methoxycyclohexyl ether, 4-methoxytetrahydrothiopyranyl ether, 4-methoxytetrahydrothiopyranyl ether S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl ether, 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl ether, 1,4-dioxane-2-yl ether, tetrahydrofuranyl ether, tetrahydrothiophenyl ether; substituted ethers such as 1-ethoxyethyl ether, 1-(2-chloroethoxy)ethyl ether, 1-[2-(trimethylsilyl)- )ethoxy]ethyl ether, 1-methyl-1-methoxyethyl ether, 1-methyl-1-benzyloxyethyl ether, 1-methyl-1-benzyloxy-2-fluoroethyl ether, 1-methyl-1-phenoxyethyl ether, 2-trimethylsilyl ether, tert-butyl ether, allyl ether, propargyl ether, p-chlorophenyl ether, p-methoxyphenyl ether, benzyl ether, p-methoxybenzyl ether, 3,4-dimethoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, tripropylsilyl ether, dimethylisopropylsilyl ether, diethylisopropylsilyl ether, dimethylhexylsilyl ether, Tert-butyldimethylsilyl ether, diphenylmethylsilyl ether, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenylacetate, benzoate, methyl carbonate, 9-fluorenylmethyl carbonate, ethyl carbonate, 2,2,2-trichloroethyl carbonate, 1,1,-dimethyl-2,2,2-trichloroethyl carbonate, alkyl sulfonate, methanesulfonate, benzylsulfonate, toluenesulfonate, methylene acetal, ethylidene acetal and t-butylmethylidene ketal. Preferred protecting groups are represented by the formula: -R. a 、-Si(R a )(R a )(R a )、-C(O)R a 、-C(O)OR a 、-C(O)NH(R a )、-S(O)2R a, -S(O)2OH, P(O)(OH)2 and -P(O)(OH)OR a , where R a It is C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, -C1-C 20 Alkylene (Carbocyclic), -C2-C 20 Alkenylene (Carbocyclic), -C2-C 20 Alkyne (Carbocyclic), -C6-C 10 Aryl, -C1-C 20 Alkylene (aryl), -C2-C 20 Alkenylene (aryl), -C2-C 20 Alkyne (aryl), -C1-C 20 Alkylene (heterocyclic), -C2-C 20 Alkenylene (heterocyclic) or -C2-C 20 Alkyne(heterocycle), wherein the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocyclic and heterocyclic groups, either alone or as part of another group, are optionally substituted.

[0109] "Altering the native glycosylation pattern" for the purposes of this article is intended to mean deleting one or more carbohydrate moieties found in the native sequence 158P1D7 (by removing potential glycosylation sites or by removing glycosylation by chemical and / or enzymatic methods), and / or adding one or more glycosylation sites that are not present in the native sequence 158P1D7. In addition, the phrase includes quantitative changes in the glycosylation of the native protein, involving changes in the nature and proportions of the various carbohydrate moieties present.

[0110] The term "analog" refers to a molecule that is structurally similar to another molecule or has similar or corresponding properties (eg, a 158P1D7-related protein). For example, a 158P1D7 protein analog can be specifically bound by an antibody or T cell that specifically binds to 158P1D7.

[0111] Unless otherwise indicated, the term "antibody" is used in the broadest sense. Therefore, an "antibody" can be naturally produced or artificial, such as a monoclonal antibody produced by conventional hybridoma technology. 158P1D7 antibodies include monoclonal antibodies and polyclonal antibodies and fragments containing the antigen binding domain and / or one or more complementary determining regions in these antibodies. The term "antibody" used herein refers to any form of antibody or fragment thereof, which specifically binds to 158P1D7 and / or exhibits the desired biological activity and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies) and antibody fragments, the precursor of which is that it can specifically bind to 158P1D7 and / or exhibit the desired biological activity. The methods and compositions provided herein can use any specific antibody. Therefore, in one embodiment, the term "antibody" includes a molecule comprising at least one variable region from a light chain immunoglobulin molecule and at least one variable region from a heavy chain molecule, which combine to form a specific binding site for a target antigen. In one embodiment, the antibody is an IgG antibody. For example, the antibody is an IgG1, IgG2, IgG3 or IgG4 antibody. Antibodies for use in the present methods and compositions can be produced in cell culture, phage or various animals, including but not limited to cattle, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees and apes. Therefore, in one embodiment, the antibodies of the present invention are mammalian antibodies. Phage technology can be used to separate starting antibodies or to produce variants with altered specificity or affinity characteristics. Such techniques are conventional and well known in the art. In one embodiment, the antibodies are recombinantly produced by methods known in the art. For example, recombinant antibodies can be produced by transfecting host cells with a vector comprising a DNA sequence encoding the antibody. One or more vectors can be used to transfect a DNA sequence expressing at least one VL region and one VH region into a host cell.Exemplary descriptions of recombinant methods for antibody generation and production include Delves, ANTIBODY PRODUCTION: ESSENTIAL TECHNIQUES (Wiley, 1997); Shephard et al., MONOCLONAL ANTIBODIES (Oxford University Press, 2000); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (Academic Press, 1993); and CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons, latest edition). The antibodies of the present invention may be modified by recombinant methods to improve the efficacy of the antibodies in mediating the desired function. Therefore, antibodies that can be modified by substitution using recombinant methods are encompassed within the scope of the present invention. Typically, the substitution is a conservative substitution. For example, an amino acid in at least one antibody constant region may be substituted with a different residue. See, for example, U.S. Patent No. 5,624,821, U.S. Patent No. 6,194,551, Application No. WO9958572 and Angal et al., Mol.Immunol.30:105-08 (1993). Amino acid modifications include deletions, additions and substitutions of amino acids. In some cases, the changes are made to reduce unwanted activity, such as complement-dependent cytotoxicity. The antibody is often labeled by covalently or non-covalently binding a substance that provides a detection signal. Various labels and coupling techniques are known and widely reported in scientific and patent literature. These antibodies can be screened based on binding to normal or defective 158P1D7. See, for example, ANTIBODY ENGINEERING:A PRACTICAL APPROACH ("Antibody Engineering: Practical Methods") (Cambridge University Press (Oxford University Press), 1996). Suitable antibodies with the desired biological activity can be identified by the following in vitro and in vivo tests, including but not limited to: proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and in vivo tests such as inhibition of tumor growth. The antibodies provided herein can also be used for diagnostic applications. As capture or non-neutralizing antibodies, the antibodies can be screened based on the ability to bind to a specific antigen but not inhibit receptor binding or biological activity of the antigen. As neutralizing antibodies, the antibodies can be used in competitive binding assays. They can also be used for quantitative analysis of 158P1D7 or its receptors.

[0112] As used herein, the term "antigen-binding portion" or "antibody fragment" of an antibody (or simply "antibody portion") refers to an antibody that retains specific binding to an antigen (e.g., 158P1D7 and variants; Figure 1 ) ability of the 158P1D7 antibody. It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is composed of V L 、V H , C L and C H1 (ii) F(ab')2 fragment, a bivalent fragment consisting of two Fab fragments connected by a disulfide bridge at the hinge region; (iii) V H and C H1 (iv) an antibody single-arm V L and V H Fv fragment composed of V H dAb fragments composed of domains (Ward et al., (1989) Nature

[0113] 341:544-546); and (vi) separate complementarity determining regions (CDRs). Moreover, although the two domains V L and V H They are encoded by different genes, but can be connected into a protein chain using synthetic linkers through recombinant methods. L and V H The domains pair to form monovalent molecules (called single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA

[0114] 85:5879-5883). Such single-chain antibodies should also be included in the term "antigen-binding portion" of an antibody. These antibody fragments are obtained by conventional methods known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies.

[0115] As used herein, any form of "antigen" can be used to generate antibodies specific for 158P1D7. Therefore, the triggering antigen can be a single epitope, multiple epitopes or complete protein alone or in conjunction with one or more immunogenic promoters known in the art. The triggering antigen can be a separated full-length protein, a cell surface protein (e.g., immunized with cells transfected with at least part of the antigen) or a soluble protein (e.g., immunized with only the extracellular domain of the protein). The antigen can be produced in genetically modified cells. The DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and encodes at least a portion of the extracellular domain. The term "part" as used herein refers to the minimum number of amino acids or nucleic acids, when appropriate, constituting the immunogenic epitope of the antigen of interest. Any genetic vector suitable for transforming cells of interest can be used, including but not limited to adenovirus vectors, plasmids, and non-viral vectors (e.g., cationic lipids). In one embodiment, the antibody in the method and composition of the present invention specifically binds to at least a portion of the extracellular domain of 158P1D7 of interest.

[0116] The antibodies or antigen-binding fragments thereof provided herein can be coupled to a "biologically active agent". The term "biologically active agent" as used herein refers to any synthetic or naturally occurring compound that binds to an antigen and / or enhances or modulates a desired biological effect to enhance cell-killing toxins. In one embodiment, the binding fragment used in the present invention is a biologically active fragment. The term "biologically active" as used herein refers to an antibody or antibody fragment that can bind to a desired antigen epitope and exert a biological effect directly or indirectly. Direct effects include, but are not limited to: regulating, stimulating and / or inhibiting growth signals; regulating, stimulating and / or inhibiting anti-apoptotic signals; regulating, stimulating and / or inhibiting apoptotic or necrotic signals; regulating, stimulating and / or inhibiting ADCC cascades; and regulating, stimulating and / or inhibiting CDC cascades.

[0117] "Bispecific" antibodies are also used in the present methods and compositions. As used herein, the term "bispecific antibody" refers to an antibody, typically a monoclonal antibody, that has binding specificity for at least two different antigenic epitopes. In one embodiment, the epitopes are from the same antigen. In another embodiment, the epitopes are from two different antigens. Methods for preparing bispecific antibodies are known in the art. For example, bispecific antibodies can be recombinantly produced by co-expressing two immunoglobulin heavy / light chain pairs. See, e.g., Milstein et al., Nature 305:537-39 (1983). Alternatively, chemical linkage can be used to prepare bispecific antibodies. See, e.g., Brennan et al., Science 229:81 (1985). Bispecific antibodies include bispecific antibody fragments. See, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993), Gruber et al., J. Immunol. 152:5368 (1994).

[0118] The monoclonal antibodies described herein specifically include "chimeric" antibodies and fragments of such antibodies, in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody type or subclass, while the remainder of the chain(s) is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody type or subclass, provided that it specifically binds to the target antigen and / or exhibits the desired biological activity (U.S. Pat. No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA

[0119] 81:6851-6855(1984)).

[0120] The term "chemotherapeutic agent" refers to all chemical compounds that are effective in inhibiting tumor growth. Non-limiting examples of chemotherapeutic agents include alkylating agents, such as nitrogen mustards, vinylmethylamine compounds, and alkylthiosulfonates; antimetabolites, such as folic acid, purine or pyrimidine antagonists; mitotic inhibitors, such as anti-tubulin agents (e.g., vinca alkaloids, auristatins, and podophyllotoxin derivatives); cytotoxic antibiotics; compounds that disrupt or interfere with DNA expression or replication, such as DNA minor groove binders; growth factor receptor antagonists. In addition, chemotherapeutic agents include cytotoxic agents (as defined herein), antibodies, biomolecules, and small molecules.

[0121] Whether expressly stated or not, the term "compound" refers to and encompasses the chemical compound itself and its various forms (unless the following is expressly excluded in the text): amorphous and crystalline forms of the compound, including polymorphs, wherein these forms may be part of a mixture or in isolated states; free acid and free base forms of the compound, which are generally in the form shown in the structure provided herein; isomers of the compound, referring to optical isomers and tautomers, wherein optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, optical isomers include isolated optical isomers and optical isomer mixtures, including racemic and non-racemic mixtures; wherein isomers may be in isolated form or in the form of a mixture with one or more other isomers; isotopic compounds, including deuterated and deuterated isomers; Tritium compounds, and including compounds containing radioactive isotopes, the radioactive isotopes including therapeutically and diagnostically effective radioactive isotopes; polymer forms of compounds, including dimers, trimers, etc.; salts of compounds, preferably pharmaceutically acceptable salts, including acid addition salts and base addition salts, including salts with organic counterions and inorganic counterions, including zwitterionic forms, if the compound is associated with two or more counterions, then the two or more counterions can be the same or different; and compound solvates, including hemisolvates, monosolvates, disolvates, etc., including organic solvates and inorganic solvates, the inorganic solvates including hydrates; if the compound is associated with two or more solvent molecules, then the two or more solvent molecules can be the same or different. In some cases, the reference to the compounds of the present invention herein includes explicit reference to one or more of the above forms, such as salts and / or solvates, however, this reference is only for emphasis and is not intended to exclude other forms identified above.

[0122] The terms "complementarity determining region" and "CDR" are known in the art and refer to non-contiguous amino acid sequences within an antibody variable region that confer specificity and binding affinity to the antibody. Typically, there are three (3) CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three (3) CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region.

[0123] The precise amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including the following: Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Maryland ("Kabat" numbering scheme), Al-Lazikani et al. (1997) JMB 273, 927-948 ("Chothia" numbering scheme), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography", J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme), Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and IMGT unique numbering for immunoglobulin and T-cell receptor variable domains and Ig superfamily V-like domains”, Dev Comp Immunol, 2003 Jan;27(1):55-77 (“IMGT” numbering scheme) and Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool”, J Mol Biol, 2001 Jun 8

[0124] ; 309(3):657-70 (AHo numbering scheme).

[0125] The boundaries of a given CDR may vary with the scheme used for identification. For example, the Kabat scheme is based on structural alignment, and the Chothia scheme is based on structural information. The numbering of the Kabat and Chothia schemes is based on the most common antibody region sequence length, wherein insertion is indicated by inserting letters, such as "30a", and deletion occurs in some antibodies. These two schemes place certain insertions and deletions ("insertion and deletion markers (indel)") at different sites, resulting in different numbering. The contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many respects. Table V hereinafter enumerates the positions of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 identified by Kabat, Chothia and contact schemes, respectively. For CDR-H1, the residue numbering using the Kabat and Chothia numbering schemes is provided.

[0126] Therefore, unless otherwise indicated, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (such as a variable region) and a single CDR (such as CDR-H1, CDR-H2) of an antibody or region thereof should be understood to include a complementary determining region defined by any of the known schemes described above. In some cases, the scheme used to identify a particular CDR or CDRs is indicated, such as CDRs defined by Kabat, Chothia or contact methods. In other cases, the specific amino acid sequence of the CDR is given.

[0127] The term "conservative substitution" as used herein refers to amino acid substitutions known to those skilled in the art and generally does not change the biological activity of the resulting molecule. It is recognized by those skilled in the art that single amino acid substitutions in non-essential regions of polypeptides generally do not significantly change biological activity (see, for example, Watson et al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin / Cummings Pub. Co., p. 224 (4th edition 1987)). Such exemplary substitutions are preferably substitutions made according to those shown in Table II and Table III. For example, such changes include substitutions of any other amino acid in these hydrophobic amino acids with any isoleucine (I), valine (V) and leucine (L); aspartic acid (D) replaces glutamic acid (E) and vice versa; glutamine (Q) replaces aspartic acid (N) and vice versa; and serine (S) replaces threonine (T) and vice versa. Other substitutions may also be considered conservative substitutions, depending on the environment of the specific amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) are often interchangeable, as are alanine (A) and glycine (G). Methionine (M) is relatively hydrophobic and is often interchangeable with leucine and isoleucine and sometimes with valine. Lysine (K) and arginine (R) are often interchangeable at positions where the significant characteristic of an amino acid residue is its charge and the pK difference between the two amino acid residues is not important. Other changes may also be considered "conservative" in a particular context (see, e.g., Table III herein, "Biochemistry" 2nd edition, pp. 13-15, edited by LubertStryer (Stanford University); Henikoff et al., PNAS 1992, vol. 89, 10915-10919; Lei et al., J Biol Chem 1995 May 19; 270(20): 11882-6). Other substitutions are also permissible and may be determined empirically or based on known conservative substitutions.

[0128] The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression activity of a cell, a cell function, and / or causes cell destruction. The term is intended to include radioisotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof. Examples of cytotoxic agents include, but are not limited to, auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, maytansinoids, ricin, ricin A chain, combrestatins, duocarmycin, dolastatin, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, dihydroxyanthracndione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha sarcin, gelonin, metoptoxin, limitocin, phenomycin, enomycin, curcin, anticrotin, calicheamicin, saponin, officinalis inhibitors and glucocorticoids as well as other chemotherapeutic agents and radioisotopes such as At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 Or Bi 213 , P 32 and including Lu 177 The antibody can also be coupled to an activating enzyme of an anticancer prodrug, which converts the prodrug into its active form.

[0129] The term "diabodies" as used herein refers to small antibody fragments with two antigen-binding sites, which are comprised of the same polypeptide chain (V H -V L ) in the light chain variable region (V L )'s heavy chain variable region (V H ). When a linker that is too short to allow pairing between the two domains on the same chain is used, each domain is forced to pair with the complementary domain of another chain, creating two antigen-binding sites. Diabodies are described in more detail in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-48 (1993).

[0130] In the context of the effect of a 158P1D7 binding agent on 158P1D7 expressing cells, the term "depletion" refers to a decrease or elimination in the number of 158P1D7 expressing cells.

[0131] As used herein, the term "gene product" refers to a peptide / protein or mRNA. For example, "a gene product of the invention" is sometimes referred to herein as "an oncoamino acid sequence," "an oncoprotein," "an oncoprotein listed in Table I," "an oncomRNA," "an oncomRNA listed in Table I," and the like. In one embodiment, an oncoprotein is a peptide / protein or mRNA. Figure 1 The oncoprotein may be Figure 1 In one embodiment, the amino acid sequence is used to determine sequence identity and similarity. In another embodiment, the sequence is Figure 1 In another embodiment, the sequence is a sequence variant further described herein.

[0132] "Heteroconjugate" antibodies can be used in the methods and compositions of the present invention. As used herein, the term "heteroconjugate antibody" refers to two covalently linked antibodies. Such antibodies can be prepared by known methods of synthetic protein chemistry, including the use of cross-linking agents. See, e.g., U.S. Pat. No. 4,676,980.

[0133] The term "homolog" refers to a molecule that exhibits homology to another molecule by, for example, having the same or similar chemical residue sequences at corresponding positions.

[0134] In one embodiment, the antibodies provided herein are "human antibodies". The term "human antibody" as used herein refers to an antibody in which the complete sequence of the light chain and heavy chain sequences, including the complementary determining regions (CDRs), is substantially derived from human genes. In one embodiment, human monoclonal antibodies are produced by trioma technology, human B cell technology (see, e.g., Kozbor et al., Immunol. Today 4:72 (1983)), EBV transformation technology (see, e.g., Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY

[0135] 77-96 (1985)) or using phage display (see, e.g., Marks et al., J. Mol. Biol. 222:581 (1991)). In a specific embodiment, the human antibody is produced in a transgenic mouse. Techniques for preparing partial to full human antibodies are known in the art and any such techniques can be used. According to a particularly preferred embodiment, full human antibody sequences are prepared in transgenic mice engineered to express human heavy and light chain antibody genes. Exemplary descriptions of preparing transgenic mice that produce human antibodies are described in Application No. WO 02 / 43478 and U.S. Patent No. 6,657,103 (Abgenix) and its family of patents. B cells from mice producing the desired antibodies can then be fused to prepare hybridoma cell lines for continuous production of antibodies. See, for example, U.S. Pat. Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016 and 5,545,806; and Jakobovits, Adv. Drug Del. Rev. 31:33-42 (1998); Green et al., J. Exp. Med. 188:483-95 (1998).

[0136] The term "humanized antibody" as used herein refers to an antibody form containing sequences from non-human (e.g., mouse) antibodies and human antibodies. Such antibodies are chimeric antibodies, which contain very few sequences derived from non-human immunoglobulins. Typically, the humanized antibody comprises almost all of at least one and usually two variable regions, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the FR regions are FR regions of human immunoglobulin sequences. The humanized antibody also optionally comprises at least a portion of an immunoglobulin constant region (Fc), generally an Fc of a human immunoglobulin. See, e.g., Cabilly U.S. Pat. No. 4,816,567; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86: 10029-10033; and ANTIBODY ENGINEERING: A PRACTICAL APPROAC ("Antibody Engineering: Practical Methods") (Oxford University Press 1996).

[0137] As used herein, the term "inhibits" or "inhibits" means to reduce by a detectable amount, or to completely prevent.

[0138] The phrases "isolated" or "biologically purified" refer to a material that is substantially or essentially free of components that normally accompany the material in its native state. Therefore, the isolated peptides described herein are preferably free of substances that are normally associated with the peptide in its native environment. For example, a nucleotide can be referred to as "isolated" when it is substantially separated from contaminating polynucleotides that correspond to or are complementary to a non-158P1D7 gene or a polypeptide encoding a non-158P1D7 gene product or fragment thereof. A person skilled in the art can readily employ nucleic acid isolation methods to obtain isolated 158P1D7 polynucleotides. For example, when a 158P1D7 protein is removed from a cellular component that is normally associated with the protein by physical, mechanical or chemical methods, it is referred to as an "isolated" protein. A person skilled in the art can readily employ standard purification methods to obtain isolated 158P1D7 proteins. Alternatively, isolated proteins can be prepared chemically.

[0139] Suitable "labels" include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents that teach the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. In addition, the antibodies provided herein can be used as antigen-binding components of fluorobodies. See, e.g., Zeytun et al., Nat. Biotechnol. 21: 1473-79 (2003).

[0140] The term "mammal" refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.

[0141] The terms "metastatic cancer" and "metastatic disease" refer to cancer that has spread to regional lymph nodes or distant sites and are intended to include stage D disease in the AUA system and stage TxNxM+ in the TNM system.

[0142] As used herein, the term "modulator" or "test compound" or "drug candidate" or its grammatical equivalent describes certain molecules to be tested, such as proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, etc., and the test is to test its ability to directly or indirectly change the cancer phenotype or express cancer sequences (such as nucleic acid or protein sequences) and the effects on cancer sequences (e.g., signal transduction, gene expression, protein interaction, etc.). On the one hand, the modulator neutralizes the effects of the cancer protein of the present invention. "Neutralization" means that the activity of the protein and the resulting effects on the cell are inhibited or blocked. On the other hand, the modulator neutralizes the effects of the gene of the present invention and its corresponding protein by neutralizing the protein level. In a preferred embodiment, the modulator changes the expression profile, or the expression profile of the nucleic acid or protein provided herein, or the downstream effect pathway. In one embodiment, the modulator inhibits the cancer phenotype, for example, inhibits it into a normal tissue fingerprint. In another embodiment, the modulator induces a cancer phenotype. Typically, multiple test mixtures are run in parallel with different substance concentrations to obtain differential responses to different concentrations. Typically, one of these concentrations is used as a negative control, i.e., a concentration of zero or a concentration below the detection level.

[0143] Modulators, drug candidates or test compounds include a large number of chemical species, but they are generally organic molecules, preferably small organic compounds with a molecular weight greater than 100 and less than about 2500 Daltons. Preferred small molecules are less than 2000 or less than 1500 or less than 1000 or less than 500 Daltons. Candidate substances contain functional groups necessary for structural interactions with proteins, especially hydrogen bond formation, and usually contain at least amine, carbonyl, hydroxyl or carboxyl, preferably at least two chemical functional groups. Candidate substances often contain cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Modulators also include biomolecules, such as peptides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Peptides are particularly preferred. One type of modulator is a peptide, such as a peptide containing about 5 to about 35 amino acids, preferably a peptide containing about 5 to about 20 amino acids, and particularly preferably a peptide containing about 7 to about 15 amino acids. Preferably, the cancer regulatory protein is a soluble protein, comprising a non-transmembrane region and / or having an N-terminal Cys to assist in dissolution. In one embodiment, the C-terminus of the fragment remains a free acid and the N-terminus is a free amine to assist in coupling, i.e., coupling cysteine. In one embodiment, the cancer protein of the present invention is coupled to an immunogenic agent as described herein. In one embodiment, the cancer protein is coupled to BSA. The peptides of the present invention (e.g., peptides of a preferred length) can be connected to each other or to other amino acids to form longer peptides / proteins. The regulatory peptide can be a digest of the above-mentioned naturally occurring protein, a random peptide or a "biased" random peptide. In a preferred embodiment, the peptide / protein-based regulator is an antibody and a fragment thereof as defined herein.

[0144] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., except for a possible naturally occurring mutation that may be present in a small amount, the individual antibodies contained in the population are identical. Monoclonal antibodies have a high degree of specificity for a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations generally contain a large number of antibodies for (or specific for) different epitopes. In one embodiment, polyclonal antibodies contain a variety of monoclonal antibodies with different epitope specificities, affinities or avidities in a single antigen containing multiple antigenic epitopes. The attributive "monoclonal" indicates that the antibody is characterized in that it is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the production of antibodies by any ad hoc method. For example, the monoclonal antibody used according to the present invention can be prepared by first by Kohler et al., Nature 256:495 (1975) described hybridoma method, or by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries by techniques such as those described in Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991). These monoclonal antibodies typically bind with a Kd of at least about 1 μM, more typically at least about 300 nM, generally at least about 30 nM, preferably at least about 10 nM, more preferably at least about 3 nM or better, which is typically determined by ELISA.

[0145] "Pharmaceutical excipients" include materials such as adjuvants, carriers, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives and the like.

[0146] "Pharmaceutically acceptable" refers to compositions that are non-toxic, inert, and / or physiologically compatible with humans or other mammals.

[0147] The term "polynucleotide" refers to a polymeric form of nucleotides of at least 10 bases or base pairs in length, which may be ribonucleotides or deoxynucleotides or modified forms of any nucleotide type, and is intended to include single-stranded and double-stranded forms of DNA and / or RNA. In the art, the term is often used interchangeably with "oligonucleotide". A polynucleotide may include a nucleotide sequence disclosed herein, wherein Figure 1 The thymidine (T) shown may also be uracil (U); this definition relates to the differences between the chemical structures of DNA and RNA, specifically the observation that one of the four major bases of RNA is uracil (U) rather than adenosine (T).

[0148] The term "polypeptide" refers to a polymer containing at least about 4, 5, 6, 7 or 8 amino acids. The standard three-letter (see Table II) or single-letter names of amino acids are used throughout the specification. In the art, the term is often used interchangeably with "peptide" or "protein".

[0149] A "recombinant" DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro

[0150] As used herein, the term "single-chain Fv" or "scFv" or "single-chain" antibody refers to an antibody comprising a V H and V L Antibody fragments that contain multiple domains, wherein these domains are present in a single polypeptide chain. H and V L The regions also contain polypeptide linkers, which allow the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0151] The terms "specificity", "specific binding" and "specific binding" as used herein refer to the selective binding of an antibody to a target antigen epitope. The binding specificity of an antibody can be detected by comparing binding to a suitable antigen and binding to an unrelated antigen or antigen mixture under a set of specified conditions. If the antibody binds to a suitable antigen at least 2, 5, 7 and preferably 10 times higher than to an unrelated antigen or antigen mixture, the antibody is considered a specific antibody. In one embodiment, a specific antibody is an antibody that binds only to the 158P1D7 antigen but not to unrelated antigens. In another embodiment, a specific antibody is an antibody that binds to a human 158P1D7 antigen but not to a non-human 158P1D7 antigen, and the non-human 158P1D7 antigen has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid homology relative to the 158P1D7 antigen. In another embodiment, the specific antibody is an antibody that binds to the human 158P1D7 antigen and to the murine 158P1D7 antigen, but has a higher degree of binding to the human antigen. In another embodiment, the specific antibody is an antibody that binds to the human 158P1D7 antigen and to the primate 158P1D7 antigen, but has a higher degree of binding to the human antigen. In another embodiment, the specific antibody binds to the human 158P1D7 antigen and any non-human 158P1D7 antigen, but has a higher degree of binding to the human antigen or any combination thereof.

[0152] As used herein, "treat" or "therapeutic" and their grammatically related terms refer to any improvement in the outcome of any disease, such as prolonged survival, lower morbidity and / or reduction of side effects (byproducts of alternative treatment modalities); as will be readily appreciated by those skilled in the art, complete cure of the disease is preferred, even though this is not a requirement for therapeutic effect.

[0153] The term "variant" refers to a molecule that has changes from a stated type or standard, such as in a particular stated protein (e.g., Figure 1 A protein having one or more different amino acid residues at corresponding positions of a 158P1D7 protein (e.g., a 158P1D7 protein). An analog is an example of a variant protein. Splicing isoforms and single nucleotide polymorphisms (SNPs) are other examples of variants.

[0154] The "158P1D7 protein" and / or "158P1D7-related protein" of the present invention include those proteins specifically identified herein (see Figure 1 ), and allelic variants, conservative substitution variants, analogs and homologs that can be isolated / generated and identified without undue experimentation according to the methods outlined herein or methods readily available in the art. Also included are fusion proteins that combine multiple parts of different 158P1D7 proteins or fragments thereof, as well as fusion proteins of 158P1D7 proteins and heterologous polypeptides. Such 158P1D7 proteins are collectively referred to as 158P1D7-related proteins, proteins of the present invention, or 158P1D7. The term "158P1D7-related protein" refers to a polypeptide fragment or 158P1D7 protein sequence having 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids; or a polypeptide fragment or 158P1D7 protein sequence having at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 16 75, 800, 825, 830, 835, 840, 841 or more amino acids or a polypeptide fragment of the 158P1D7 protein sequence.

[0155] II.) 158P1D7 Antibody

[0156] Another aspect of the present invention provides antibodies that bind to 158P1D7-related proteins (see Figure 1 ). In one embodiment, the antibody that binds to the 158P1D7-related protein is an antibody that specifically binds to the 158P1D7 protein comprising the amino acid sequence SEQ ID NO.: 2. The antibody that specifically binds to the 158P1D7 protein comprising the amino acid sequence SEQ ID NO.: 2 includes antibodies that can bind to other 158P1D7-related proteins. For example, the antibody that binds to the 158P1D7 protein comprising the amino acid sequence SEQ ID NO.: 2 can bind to 158P1D7-related proteins, such as 158P1D7 variants and homologs or analogs thereof.

[0157] The 158P1D7 antibodies of the present invention are particularly useful in cancer (see, e.g., Table I) prognostic tests, imaging, diagnosis, and treatment methods. Similarly, such antibodies can be used for the treatment and / or prognosis of bladder cancer and other cancers in which 158P1D7 is also expressed or overexpressed to a certain extent. In addition, when the antibodies are coupled to monomethyl auristatin E (MMAE) as described herein, the 158P1D7 antibodies of the present invention can be therapeutically used to treat cancers involving 158P1D7 expression (particularly bladder cancer, such as advanced or metastatic bladder cancer).

[0158] Various methods for preparing antibodies, particularly monoclonal antibodies, are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host with a 158P1D7-related protein, peptide or fragment in an isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Harlow and Lane, eds. (1988); Harlow, Antibodies, Cold Spring Harbor Press, New York (1989)). In addition, 158P1D7 fusion proteins, such as 158P1D7 GST-fusion proteins, can also be used. In a specific embodiment, the production comprises Figure 1 In another embodiment, a GST fusion protein containing all or most of the amino acid sequence in 158P1D7 is synthesized and used as an immunogen.

[0159] Alternatively, an immune response to the encoded immunogen is generated using naked DNA immunization techniques (with or without purified 158P1D7-related protein or 158P1D7-expressing cells) known in the art (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15:617-648).

[0160] Can be analyzed as Figure 1 The 158P1D7 protein amino acid sequence shown is used to select the 158P1D7 protein specific region to generate antibodies. For example, the hydrophobicity and hydrophilicity analysis of the 158P1D7 amino acid sequence is used to identify the hydrophilic region of the 158P1D7 structure. Various other methods known in the art can be used to identify the 158P1D7 protein region that displays the immunogenic structure and other regions and domains, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. The hydrophilicity profile can be generated using the method of Hopp, TP and Woods, KR, 1981, Proc. Natl. Acad. Sci. USA 78: 3824-3828. Kyte, J. and Doolittle, RF, 1982, J. Mol. Biol.

[0161] 157:105-132 method to generate hydrophilicity (Hydropathicity) profile. Janin J., 1979, Nature 277:491-492 method can be used to generate accessible residue percentage (%) profile. Bhaskaran R., Ponnuswamy PK, 1988, Int. J. Pept. Protein Res. 32:242-255 method can be used to generate average flexibility (Average Flexibility) profile. Deleage, G., Roux B., 1987, Protein Engineering 1:289-294 method can be used to generate β-turn (Beta-turn) profile. Therefore, each region identified by any of these schemes or methods is within the scope of the present invention. The preferred method for producing 158P1D7 antibodies is further illustrated by the examples provided herein. Methods for preparing proteins or polypeptides used as immunogens are well known in the art. Methods for preparing immunogenic conjugates of proteins with carriers (e.g., BSA, KLH or other carrier proteins) are also well known in the art. In some cases, direct coupling is performed using, for example, carbodiimide reagents; in other cases, linking reagents (e.g., reagents provided by Pierce Chemical Co., Rockford, Illinois) are also effective. As is understood in the art, the 158P1D7 immunogen is often administered by injection over a suitable period of time and using a suitable adjuvant. During the immunization protocol, antibody titers may be used to determine the adequacy of antibody formation.

[0162] 158P1D7 monoclonal antibodies can be produced by various methods well known in the art. For example, immortalized cell lines secreting the desired monoclonal antibodies are prepared using the commonly known standard hybridoma technology of Kohler and Milstein or modified methods to immortalize antibody-producing B cells. Immortalized cell lines secreting the desired antibodies are screened by immunoassays in which the antigen is a 158P1D7-related protein. When suitable immortalized cell cultures are identified, cells can be expanded from in vitro cultures or ascites and antibodies produced.

[0163] The antibodies or fragments of the present invention may also be produced by recombinant methods. Regions that specifically bind to the desired regions of the 158P1D7 protein may also be produced in the context of chimeric or complementary determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 158P1D7 antibodies may also be produced and preferably used in therapeutic situations. Methods for humanizing mouse and other non-human antibodies by replacing the corresponding human antibody sequence with one or more non-human antibody CDRs are well known (see, e.g., Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536). See also Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89:4285 and Sims et al., 1993, J. Immunol. 151:2296.

[0164] In a preferred embodiment, the antibodies of the present invention include fully human 158P1D7 antibodies (158P1D7 MAbs). Methods for producing fully human 158P1D7 MAbs are provided in various methods in the art. For example, a preferred embodiment provides a technique for using transgenic mice, inactivating antibody production, and engineering with human heavy and light chain loci, also known as Xenomouse (Amgen Fremont, Inc.). Exemplary descriptions of preparing transgenic mice that produce human antibodies can be found in U.S. Patent No. 6,657,103. See also U.S. Patent Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; and Mendez et al., Nature Genetics, 15: 146-156 (1998); Kellerman, SA and Green, LL, Curr. Opin. Biotechnol 13, 593-597 (2002).

[0165] In addition, human antibodies of the invention can be produced by HuMAb mice (Medarex, Inc.), which contain a mini-locus of human immunoglobulin genes encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, as well as targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg et al., (1994) Nature 368(6474):856-859).

[0166] In another embodiment, mice carrying human immunoglobulin sequences on transgenic and transchromosomes (e.g., mice carrying human heavy chain transgenic and human light chain transchromosomes) can be used to produce fully human antibodies of the present invention. Such mice are referred to herein as "KM mice," which are described in Tomizuka et al., (2000) Proc. Natl. Acad. Sci. USA 97: 722-727 and Tomizuka et al. PCT WO 02 / 43478.

[0167] Human monoclonal antibodies of the present invention can also be prepared using phage display methods to screen human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies have been established in the art. See, for example: Ladner et al., 5,223,409; U.S. Pat. Nos. 5,403,484; and 5,571,698; Dower et al., U.S. Pat. Nos. 5,427,908 and 5,580,717; McCafferty et al., U.S. Pat. Nos. 5,969,108 and 6,172,197; and Griffiths et al., U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081.

[0168] Human monoclonal antibodies of the invention can also be prepared using SCID mice, in which human immune cells have been reconstituted so that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.

[0169] Human monoclonal antibodies of the invention can also be prepared using mice in which the genomic sequences carrying endogenous mouse variable segments for immunoglobulin heavy chains (VH, DH, and JH segments) and / or kappa light chains (VK and JK) are replaced in whole or in part with human genomic sequences carrying unrearranged germline variable segments of human immunoglobulin heavy chains (VH, DH, and JH segments) and / or kappa light chains (VK and JK) (Regeneron, Tarrytown, New York). See, e.g., U.S. Pat. Nos. 6,586,251, 6,596,541, 7,105,348, 6,528,313, 6,638,768, and 6,528,314.

[0170] In one embodiment, the 158P1D7 MAb of the present invention comprises the heavy chain and light chain variable regions of the antibody called Hal5-10ac12, which is produced by Chinese hamster ovary (CHO) cells deposited with American Type Culture Collection (ATCC) Accession No.: PTA-13102 (see Figure 3), or its heavy chain and light chain variable regions contain amino acid sequences homologous to the amino acid sequences of the heavy chain and light chain variable regions of Hal5-10ac12 (such as functional fragments thereof), and these antibodies retain the desired functional properties of the 158P1D7 MAb of the present invention. The heavy chain variable region of Hal5-10ac12 has an amino acid sequence from the 1st Q residue to the 120th S residue region of SEQ ID NO: 7, and the light chain variable region of Hal5-10ac12 has an amino acid sequence from the 1st D residue to the 113th R residue region of SEQ ID NO: 8.

[0171] In one embodiment, the 158P1D7 antibody contains the heavy chain CDRs of the heavy chain variable region of HA15-10ac12, such as the heavy chain CDRs 1, 2 and / or 3 of the heavy chain variable region of HA15-10ac12, such as the CDRs 1, 2 and / or 3 of the heavy chain variable region of HA15-10ac12, such as the CDRs 1, 2 and / or 3 of the amino acid sequence set forth in SEQ ID NO:7, which are determined by any known numbering scheme for identifying CDRs, such as those described herein. In one embodiment, the 158P1D7 antibody contains the light chain CDRs of the light chain variable region of HA15-10ac12, such as the light chain CDRs 1, 2 and / or 3 of the light chain variable region of HA15-10ac12, such as the CDRs 1, 2 and / or 3 of the amino acid sequence set forth in SEQ ID NO:8, which are determined by any known numbering scheme for identifying CDRs, such as those described herein. In one aspect, the CDRs 1-3 of the heavy chain variable region of Ha15-10ac12 contain the amino acid sequences of residues 31-35, residues 50-66, and residues 99-109 of SEQ ID NO:7, respectively. In one aspect, the CDRs 1-3 of the light chain variable region of Ha15-10ac12 contain the amino acid sequences of residues 24-39, residues 55-61, and residues 94-102 of SEQ ID NO:8, respectively. Thus, in some aspects, the 158P1D7 antibody contains a heavy chain CDR1 having residues 31-35 of SEQ ID NO:7, a heavy chain CDR2 having residues 50-66 of SEQ ID NO:7, and / or a heavy chain CDR2 having residues 99-109 of SEQ ID NO:7.

[0172] CDR3, and / or a light chain CDR1 with residues 24-39 of SEQ ID NO:8, a light chain CDR2 with residues 55-61 of SEQ ID NO:8, and / or a light chain CDR3 with residues 94-102 of SEQ ID NO:8. The embodiments provided also include antibodies that compete with antibodies having such variable regions and / or CDR sequences for binding to antigens. In some embodiments, the antibodies provided include a constant region. The constant region can be any constant region subclass. In one embodiment, a human IgG2 constant region can be used as a heavy chain constant region and a human Igκ constant region as a light chain constant region.

[0173] For example, the invention provides an isolated monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein:

[0174] (a) the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to the heavy chain variable region amino acid sequence shown in Figure 3; and

[0175] (b) the light chain variable region comprises an amino acid sequence that is at least 80% homologous to the light chain variable region amino acid sequence shown in FIG. 3 .

[0176] In other embodiments, V H and / or V L The amino acid sequence can be the same as that shown in FIG3 H and V L The sequences are 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous.

[0177] In another embodiment, the present invention provides an isolated monoclonal antibody, or an antigen binding portion thereof, comprising a humanized heavy chain variable region and a humanized light chain variable region, wherein:

[0178] (a) the heavy chain variable region comprises one, two or three complementarity determining regions (CDRs) having the amino acid sequence of heavy chain variable region CDR 1, 2 and / or 3 of the heavy chain variable region shown in Figure 3, which are determined by any known numbering scheme, such as any method described herein;

[0179] (b) the light chain variable region comprises one, two or three CDRs having the amino acid sequence of light chain variable region CDR 1, 2 and / or 3 of the light chain variable region shown in Figure 3, which are determined by any known numbering scheme, such as any method described herein.

[0180] In another embodiment, the antibody, or antigen binding portion thereof, competes for binding with an antibody having such heavy and / or light chain CDRs.

[0181] The engineered antibodies of the present invention include V H and / or V L Those antibodies in which the framework residues in the antibody are modified (e.g., to improve the antibody characteristics). Such framework modifications are generally performed to reduce the immunogenicity of the antibody. For example, one method is to "backmutate" the framework residues of one or more corresponding germline sequences. More specifically, the antibody that has undergone somatic mutation may contain framework residues that are different from the germline sequence from which the antibody is derived. The residue can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is derived. In order to convert the framework region sequence back to its germline configuration, the somatic mutation can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis (e.g., leucine "backmutation" is methionine). The present invention is also intended to cover these "backmutation" antibodies.

[0182] Another type of framework modification involves mutating one or more residues in the framework region, or even one or more residues in the CDR region, to remove T cell epitopes and thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization," and is further described in detail in U.S. Patent Publication No. 2003 / 0153043 by Carr et al.

[0183] In addition to or in place of modifications in the framework or CDR regions, the antibodies of the invention can be engineered to contain modifications in the Fc region, generally to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular toxicity. Additionally, the 158P1D7 MAb of the invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation, again altering one or more functional properties of the MAb. Various of these embodiments are described in detail below.

[0184] In one embodiment, the hinge region of CH1 is modified so that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This method is further described in U.S. Pat. No. 5,677,425 to Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1 is altered to, for example, facilitate light chain and heavy chain assembly or to increase or decrease the stability of 158P1D7 MAb.

[0185] In another embodiment, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the 158P1D7 MAb. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment so that the antibody has impaired staphylococcal protein A (SpA) binding relative to native Fc hinge domain SpA binding. This method is further described in U.S. Pat. No. 6,165,745 to Ward et al.

[0186] In another embodiment, the 158P1D7 Mab is modified to increase its biological half-life. Various methods can be used. For example, mutations can be introduced as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to increase the biological half-life, the antibody CH1 or CL region can be changed to contain rescue receptor binding epitopes taken from two loops in the CH2 domain of the IgG Fc region, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 to Presta et al.

[0187] In another embodiment, the effector function of 158P1D7 MAb is altered by replacing at least one amino acid residue with a different amino acid residue to alter the Fc region. For example, one or more amino acids selected from amino acid specific residues can be replaced by a different amino acid residue so that the affinity of the antibody for the effector ligand is changed but the antigen binding ability of the parent antibody is retained. The effector ligand to which the affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This method is further described in U.S. Pat. Nos. 5,624,821 and 5,648,260 to Winter et al.

[0188] The reactivity of 158P1D7 antibody and 158P1D7 related protein can be established by many well-known methods, including Western blot, immunoprecipitation, ELISA and FACS analysis, using appropriate 158P1D7 related protein, 158P1D7 expression type cell or its extract. 158P1D7 antibody or its fragment can be marked with detection marker or coupled to a second molecule. Suitable detection markers include but are not limited to radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelates or enzymes. In addition, bispecific antibodies specific for two or more 158P1D7 epitopes are produced using methods generally known in the art. Homodimeric antibodies can also be produced by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res.53:2560-2565).

[0189] In another preferred embodiment, the 158P1D7 MAb of the present invention is an antibody containing a heavy chain and a light chain called the Ha15-10ac12 antibody. The heavy chain of Ha15-10ac12 consists of an amino acid sequence ranging from the 1st Q residue to the 466th K residue of SEQ ID NO:7, and the light chain of Ha15-10ac12 consists of the 1st D residue to the 219th C residue of SEQ ID NO:8. Its sequence is listed in Figures 2 and 3. In a preferred embodiment, Ha15-10ac12 is coupled to a cytotoxic agent.

[0190] Chinese hamster ovary (CHO) cells producing the antibody designated Ha15-10ac12 were sent (via FedEx) to the American Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108 on July 25, 2012 and assigned accession number: PTA-13102.

[0191] III.) Overview of Antibody-Drug Conjugates

[0192] In another aspect, the present invention provides an antibody drug conjugate (ADC), which comprises an antibody coupled to a cytotoxic agent (e.g., a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatic toxin or a fragment thereof of bacterial, fungal, plant or animal origin) or a radioactive isotope (i.e., a radioactive conjugate). In another aspect, the present invention also provides a method of using the ADC. In one aspect, the ADC comprises any of the 158P1D7 MAbs described above covalently linked to a cytotoxic agent or a detection agent.

[0193] The use of antibody-drug conjugates for local delivery of cytotoxic or cytostatic agents, i.e., drugs that kill or inhibit tumor cells in cancer therapy (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) allows for targeted delivery of the drug moiety to tumors, where it accumulates intracellularly, whereas systemic administration of these unconjugated drug agents may produce unacceptable levels of toxicity to normal cells as well as to the tumor cells being eliminated (Baldwin et al., (1986) Lancet (March 15, 1986) pp. 603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review"). Review) in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al., eds., pp. 475-506). Therefore, the maximum efficacy with the least toxicity is sought. Both polyclonal antibodies and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother., 21: 183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate and vindesine (Rowland et al., (1986) supra). Toxins used in antibody-toxin conjugates include bacterial toxins (e.g., diphtheria toxoid), plant toxins (e.g., ricin), small molecule toxins (e.g., geldanamycin) (Mandler et al. (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicinomycin (Lode et al. (1998) Cancer Res. 58:2928; Hinman et al. (1993) Cancer Res. 58:2928). Res. 53:3336-3342).The toxin can achieve its cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.Some cytotoxic drugs tend to be inactivated or have reduced activity when coupled to large antibodies or protein receptor ligands.

[0194] Examples of antibody drug conjugates are (ibritumomab tiuxetan, Biogen / Idec), It is an antibody isotope conjugate that contains a mouse IgG1κ monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes and a thiourea linker-chelator linked to 111 In or 90 Y radioisotope (Wiseman et al. (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al. (2002) Blood 99(12):4336-42; Witzig et al. (2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al. (2002) J. Clin. Oncol. 20(15):3262-69).

[0195] In addition, MYLOTARG TM (gemtuzumab ozogamicin, Wyeth Pharmaceutical) is an antibody-drug conjugate comprising a human CD33 antibody linked to calicinomycin and was approved in 2000 for injection to treat acute myeloid leukemia (Drugs of the Future (2000) 25(7):686; U.S. Patent Nos. 4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001).

[0196] In addition, Cantuzumab mertansine (Immunogen, Inc.), an antibody-drug conjugate comprising the huC242 antibody linked to the maytansine drug moiety DM1 via a disulfide linker SPP, is entering Phase II trials for the treatment of cancers that express CanAg (e.g., colon, pancreatic, gastric, and other cancers).

[0197] Additionally, MLN-2704 (Millennium Pharm., BZLBiologics, Immunogen Inc.) is an antibody-drug conjugate comprising an anti-prostate-specific membrane antigen (PSMA) monoclonal antibody linked to the maytansinoid drug moiety DM1 that is being developed for the potential treatment of prostate tumors.

[0198] Finally, auristatin peptides (such as monomethyl auristatin (MMAE), a synthetic analog of Aplysia) have been conjugated to chimeric monoclonal antibodies cBR96 (specific for Lewis Y in carcinomas) and cAC10 (specific for CD30 in hematological malignancies) (Doronina et al. (2003) Nature Biotechnology 21(7):778-784). cAC10 is under therapeutic development.

[0199] In addition, described herein are chemotherapeutic agents for generating ADCs. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotonin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and tricothecenes. See, for example, WO93 / 21232 published on October 28, 1993. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 I. 131 In, 90 Y and 186Re. The conjugation of the antibody to the cytotoxic agent can be accomplished using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidates (such as dimethyl adipic acid imidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazido compounds (such as bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described by Vitetta et al. (1987) Science, 238:1098. Carbon-14-labeled 1-isothiocyanatobenzyl-3-methylenedioxytriaminepentaacetic acid (MX-DTPA) is an exemplary chelator for coupling radionuclides to antibodies (WO 94 / 11026).

[0200] Also contemplated herein are conjugates of antibodies with one or more small molecule toxins, such as calicheamicin, maytansinoids, dolastatin, auristatins, crescent toxins, and CC1065, and derivatives of such toxins that have toxin activity.

[0201] III(A).Maytansinoid

[0202] Maytansine compounds suitable for use as maytansine drug moieties are well known to those skilled in the art and can be isolated from natural sources according to known methods, produced by genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973), or synthesized according to known methods to prepare maytansine and maytansine analogs.

[0203] Maytansinoid drug moieties include those with modified aromatic rings, for example: C-19-dechloro (US4256746) (prepared by lithium aluminum hydride reduction of ansamytocin P2); C-20-hydroxy (or C-20-demethyl) + / -C-19-dechloro (US Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomycetes or by dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (-OCOR), + / -dechloro (US Pat. No. 4,294,757) (prepared by acylation using acyl chlorides) as well as those with modifications at other positions.

[0204] Exemplary maytansinoid drug moieties also include those with modifications, such as: C-9-SH (US 4,424,219) (prepared by reaction of maytansinol with H2S or P2S5); C-14-alkoxymethyl (demethoxy / CH2OR) (US4331598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2Oac) (US

[0205] 4450254) (prepared by Norcardia); C-15-hydroxy / acyloxy (US 4,364,866) (prepared by transformation of maytansinol by Streptomyces); C-15-methoxy (US Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudlflora); C-18-N-demethyl (US Pat. Nos. 4,362,663 and 4,322,348) (prepared by demethylation of maytansinol by Streptomyces); and 4,5-deoxy (US 4,371,533) (prepared by reduction of maytansinol with titanium trichloride / LAH).

[0206] ADC containing maytansinoid, its preparation method and its therapeutic application are disclosed in, for example, U.S. Patent Nos. 5,208,020, 5,416,064, 6,441,163 and European Patent No. EP 0 425 235 B1, the disclosure of which is expressly incorporated herein by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93: 8618-8623 (1996) described an ADC containing maytansinoid, which is called DM1 and is linked to the monoclonal antibody C242 for human colorectal cancer. The conjugate was found to be highly cytotoxic to cultured colon cancer cells and exhibited antitumor activity in an in vivo tumor growth assay. Chari et al., Cancer Research 52:127-131 (1992) described ADCs in which maytansinoids were conjugated to mouse antibody A7, which binds to an antigen on a human colon cancer cell line, or to another mouse monoclonal antibody TA.1, which binds to the HER-2 / neu oncogene, via a disulfide linker. The cytotoxicity of the TA.1-maytansinoid conjugate was tested in vitro in the human breast cancer cell line SK-BR-3, which expresses 3 x 10 5 The drug conjugate achieved a level of cytotoxicity similar to that of the free maytansinoid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule. The A7 maytansinoid conjugate exhibited low systemic cytotoxicity in mice.

[0207] III(B). Auristatin and Aplysia

[0208] In some embodiments, the ADC comprises an antibody of the invention coupled to dorostatin or a peptide analog and derivative of Aplysia, auristatin (U.S. Pat. Nos. 5,635,483; 5,780,588). Aplysia and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584) and have anticancer (US 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42: 2961-2965). Aplysia and auristatin drug moieties can be linked to the antibody via the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (WO 02 / 088172).

[0209] Exemplary auristatin embodiments include N-terminally linked monomethyl auristatin drug moieties DE and DF as disclosed in Senter et al., Proceedings of the American Association for Cancer Research, Volume 45, Abstract No. 623, published March 28, 2004, and described in U.S. Patent Publication No. 2005 / 0238649, the disclosures of which are expressly incorporated herein by reference in their entireties.

[0210] An exemplary auristatin embodiment is MMAE (wherein the wavy line represents a linker (L) covalently attached to the antibody drug conjugate).

[0211]

[0212] Another exemplary auristatin embodiment is MMAF, wherein the wavy line represents a linker (L) covalently attached to the antibody drug conjugate (US2005 / 0238649):

[0213]

[0214] Other exemplary embodiments comprising MMAE or MMAF and various linker components (described further herein) have the following structures and abbreviations (wherein Ab means antibody and p is 1 to about 8):

[0215]

[0216]

[0217] Generally, peptide-based drug moieties can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. For example, such peptide bonds can be prepared according to liquid phase synthesis methods (see and K. Lübke, "The Peptides", Vol. 1, pp. 76-136, 1965, Academic Press), which is well known in the field of peptide chemistry technology. The auristatin / aplysia drug moiety can be prepared according to the following methods: US 5635483; US 5780588; Pettit et al. (1989) J. Am. Chem. Soc. 111: 5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13: 243-277; Pettit, GR et al. Synthesis, 1996, 719-725; Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 15: 859-863; and Doronina (2003) Nat Biotechnol 21 (7): 778-784.

[0218] III(C).Caliphaticum

[0219] In other embodiments, the ADC comprises an antibody of the invention conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics is capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of calicheamicin family conjugates, see U.S. Patents 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296 (all belonging to American Cyanamid Company). Structural analogs of calicheamicin that can be used include, but are not limited to: γ1 I , α2 I , α3 I , N-acetyl-γ1 I , PSAG and θ I 1 (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the aforementioned patent of American Cyanamid Corporation). Another anti-tumor drug that can be coupled to an antibody is QFA, which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not easily cross the plasma membrane. Therefore, antibody-mediated internalization allows cells to take up these agents, which significantly enhances their cytotoxic effects.

[0220] III(D). Other cytotoxic agents

[0221] Other anti-tumor agents that can be conjugated to the antibodies of the invention include BCNU, streptozotocin, vincristine and 5-fluorouracil, agents collectively referred to as LL-E33288 complexes described in US Pat. Nos. 5,053,394 and 5,770,710, and esperamicins (US Pat. No. 5,877,296).

[0222] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, delta-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, e.g., WO 93 / 21232 published Oct. 28, 1993.

[0223] The present invention also includes ADCs formed between antibodies and compounds having nucleolytic activity (eg, ribonucleases or DNA endonucleases, such as deoxyribonucleases; DNases).

[0224] To selectively destroy tumors, the antibody may contain a highly radioactive atom. Various radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , Pb 212 When the conjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, such as tc 99m or I 123 , or spin labels for nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0225] Radioactive or other labels can be introduced into the conjugate by known methods. For example, the peptide can be synthesized biosynthetically or by chemical amino acid synthesis using suitable amino acid precursors containing, for example, fluorine-19 in place of hydrogen. Labels such as tc can be attached via the cysteine ​​residues of the peptide. 99m or I 123 、Re 186 、Re 188 andIn 111 Yttrium-90 can be attached via lysine residues. Iodine-123 can be introduced using the IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80:49-57. Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

[0226] IV.) Antibody Drug Conjugate Compounds Binding to 158P1D7

[0227] The present invention provides antibody drug conjugate compounds for drug targeted delivery. The inventors have found that the antibody drug conjugate compounds have potent cytotoxicity and / or cell growth inhibition activity for cells expressing 158P1D7. The antibody drug conjugate compounds include an antibody unit covalently linked to at least one drug unit. The drug unit can be directly connected or connected by a linker unit (-LU-).

[0228] In some embodiments, the antibody drug conjugate compound is a compound of the following formula or a pharmaceutically acceptable salt or solvate thereof:

[0229] L-(LU-D) p (I)

[0230] in:

[0231] L is an antibody unit, such as 158P1D7 Mab of the present invention, and

[0232] (LU-D) is a Linker Unit-Drug Unit moiety, wherein:

[0233] LU- is the joint unit, and

[0234] -D is a drug unit having cytostatic or cytotoxic activity on target cells; and

[0235] p is an integer from 1 to 20.

[0236] In some embodiments, p ranges from 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2. In some embodiments, p ranges from 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3. In other embodiments, p is 1, 2, 3, 4, 5, or 6. In some embodiments, p is 2 or 4.

[0237] In some embodiments, the antibody drug conjugate compound is a compound of the following formula or a pharmaceutically acceptable salt or solvate thereof:

[0238] L-(A a -W w -Y y -D) p (II)

[0239] in:

[0240] L is an antibody unit, such as 158P1D7 MAb; and

[0241] -A a -W w -Y y - is a joint unit (LU), wherein:

[0242] -A- is the extension unit,

[0243] a is 0 or 1,

[0244] -W- are each independently an amino acid unit,

[0245] w is an integer from 0 to 12,

[0246] -Y- is a self-degradable spacer unit,

[0247] y is 0, 1, or 2;

[0248] -D is a drug unit having cytostatic or cytotoxic activity on target cells; and

[0249] p is an integer from 1 to 20.

[0250] In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1 or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, p ranges from 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2. In some embodiments, p ranges from 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3. In other embodiments, p is 1, 2, 3, 4, 5, or 6. In some embodiments, p is 2 or 4. In some embodiments, if w is not 0, y is 1 or 2. In some embodiments, if w is 1-12, y is 1 or 2. In some embodiments, w is 2-12 and y is 1 or 2. In some embodiments, a is 1, w and y are 0.

[0251] For compositions comprising multiple antibodies, p represents drug loading, i.e., the average number of drug molecules per antibody. The range of drug loading can be 1-20 drugs (D) per antibody. The average number of drugs per antibody in the preparation of the coupling reaction can be identified by conventional methods such as mass spectrometry, ELISA experiments and HPLC. The quantitative distribution of antibody-drug conjugates can also be determined in the form of p. In some cases, homogeneous antibody-drug conjugates with a certain determined p value can be separated, purified, and identified from antibody-drug conjugates of other drug loadings by methods such as reversed-phase HPLC or electrophoresis. In an exemplary embodiment, p is 2-8.

[0252] Antibody drug conjugate compounds can be generated by any technology known to those skilled in the art. In short, the antibody drug conjugate compound comprises 158P1D7 MAb as an antibody unit, a drug and an optional linker for connecting the drug to the binding agent. In a preferred embodiment, the antibody is 158P1D7 MAb, which contains the heavy chain and light chain variable regions of the above-mentioned Hal5-10ac12 antibody. In a more preferred embodiment, the antibody is 158P1D7 MAb, which contains the heavy chain and light chain of the above-mentioned Hal5-10ac12 antibody. A variety of different reactions can be used for the covalent connection of drugs and / or linkers to binding agents. It is often achieved by the reaction of amino acid residues of binding agents (such as antibody molecules), and the amino acid residues include the amino group of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl group of cysteine ​​and different parts of aromatic amino acids. One of the most commonly used non-specific covalent bonding methods is the carbodiimide reaction of the carboxyl (or amino) group of the connecting compound and the amino (or carboxyl) group of the antibody. In addition, bifunctional reagents (e.g., dialdehydes or imidoesters) have been used to connect the amino groups of the compound to the amino groups of the antibody molecule. Schiff base reactions can also be used to connect drugs to binding agents. The method involves periodate oxidation of drugs containing ethylene glycol or hydroxyl groups to form an aldehyde group that then reacts with the binding agent. The connection occurs by forming a Schiff base with the amino group of the binding agent. Isothiocyanates can also be used as coupling agents to covalently connect drugs and binding agents. Other techniques are known to those skilled in the art and are within the scope of the present invention.

[0253] In certain embodiments, the intermediate as a joint precursor reacts with the drug under suitable conditions. In certain embodiments, the reactive group is used on the drug and / or the intermediate. The reaction product (or derivative drug) between the drug and the intermediate then reacts with the 158P1D7 MAb under suitable conditions.

[0254] Each specific unit of antibody drug conjugate compound will be further described in detail herein. The synthesis and structure of exemplary linker unit, extension unit, amino acid unit, self-degradable spacer unit and drug unit are also described in U.S. Patent Publication Nos. 2003-0083263, 2005-0238649 and 2005-0009751, each of which is incorporated herein by reference in its entirety for all purposes.

[0255] V.) Connector unit

[0256] Typically, the antibody-drug conjugate compound comprises a linker unit between the drug unit and the antibody unit. In some embodiments, the linker is cleavable under intracellular conditions, and the drug unit is released from the antibody after the linker is cleaved under intracellular conditions. In other embodiments, the linker unit is not cleavable and the drug is released, for example, by antibody degradation.

[0257] In some embodiments, the linker can be cut by a cleavage agent present in the intracellular environment (e.g., in a lysosome or endosome or cell membrane caveolea). The linker can be, for example, a peptidyl linker cut by an intracellular peptidase or protease, the enzyme including but not limited to a lysosomal or endosome protease. In some embodiments, the length of the peptidyl linker is at least two amino acids or at least three amino acids. The cleavage agent can include cathepsins B and D and plasmin, which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs in target cells (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). The most common is a peptidyl linker that can be cut by an enzyme present in 158P1D7 expressing cells. For example, a peptidyl linker (such as a Phe-Leu or Gly-Phe-Leu-Gly (SEQ ID NO: 9) linker) cut by a thiol-dependent protease cathepsin-B can be used, and the protease is highly expressed in cancer tissues. Other examples of such linkers are described in, for example, U.S. Patent No. 6,214,345, which is incorporated herein by reference in its entirety and for all purposes. In a particular embodiment, the peptidyl linker that can be cleaved by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin with a val-cit linker). An advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is generally attenuated during conjugation and the serum stability of the conjugate is generally high.

[0258] In other embodiments, the cleavable linker has pH sensitivity, i.e., is sensitive to hydrolysis at certain pH values. Typically, pH-sensitive linkers can be hydrolyzed under acidic conditions. For example, acid-labile linkers (such as hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) that can be hydrolyzed in lysosomes can be used. (See, e.g., U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123; Neville et al., 1989, Biol. Chem. 264: 14653-14661.) Such linkers are relatively stable under neutral pH conditions (e.g., in blood), but are unstable under low pH conditions of 5.5 or 5.0 (close to the pH of lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker (such as a thioether that is attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).

[0259] In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, linkers that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio) toluene), SPDB, and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel, ed., Oxford U. Press, 1987). See also U.S. Pat. No. 4,880,935.)

[0260] In other specific embodiments, the linker is a malonic acid linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem.

[0261] 3(10):1299-1304) or 3'-N-amide analogs (Lau et al., 1995, Bioorg-Med-Chem.

[0262] 3(10):1305-12).

[0263] In other embodiments, the linker unit is not cleavable and releases the drug by degrading the antibody. (See U.S. Publication No. 2005 / 0238649, which is incorporated herein by reference in its entirety and for all purposes).

[0264] Typically, the linker is substantially insensitive to the extracellular environment. For the linker, the term "substantially insensitive to the extracellular environment" as used herein means that when the antibody-drug conjugate compound appears in the extracellular environment (such as in plasma), no more than about 20%, usually no more than about 15%, more usually no more than about 10%, even more usually no more than about 5%, no more than about 3%, or no more than about 1% of the linker in the antibody-drug conjugate compound sample is cleaved. Whether the linker is substantially insensitive to the extracellular environment can be determined, for example, by the following method: the antibody-drug conjugate compound is incubated with plasma for a period of time (such as 2, 4, 8, 16 or 24 hours), and then the amount of free drug present in the plasma is quantitatively determined.

[0265] In other non-mutually exclusive embodiments, the linker promotes cellular internalization. In certain embodiments, the linker promotes cellular internalization when coupled to a therapeutic agent (i.e., in the case of the linker-therapeutic agent portion of an antibody-drug conjugate compound described herein). In other embodiments, the linker promotes cellular internalization when coupled to an auristatin compound and 158P1D7 Mab.

[0266] Various exemplary linkers useful in the present compositions and methods are described in WO 2004-010957, US Publication No. 2006 / 0074008, US Publication No. 20050238649, and US Publication No. 2006 / 0024317 (each of which is incorporated herein by reference in its entirety for all purposes).

[0267] A "Linker Unit" (LU) is a bifunctional compound that can be used to connect a Drug unit and an Antibody unit to form an Antibody-Drug Conjugate compound. In some embodiments, the Linker Unit has the following formula:

[0268] -A a -W w -Y y -

[0269] Where: -A- is the extension unit,

[0270] a is 0 or 1,

[0271] -W- are each independently an amino acid unit,

[0272] w is an integer from 0 to 12,

[0273] -Y- is a self-decomposable spacer unit, and

[0274] y is 0, 1, or 2.

[0275] In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1 or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, if w is 1-12, y is 1 or 2. In some embodiments, w is 2-12 and y is 1 or 2. In some embodiments, a is 1, w and y are 0.

[0276] VI.) Extension Unit

[0277] When the extension unit (A) is present, it is capable of connecting the antibody unit to the amino acid unit (-W-) (if present), the spacer unit (-Y-) (if present); or to the drug unit (-D). Useful functional groups that may be present on 158P1D7 Mab (e.g., Ha15-10ac12) (naturally occurring or added by chemical manipulation) include, but are not limited to: sulfhydryl, amino, hydroxyl, anomeric hydroxyl of sugars, and carboxyl. Suitable functional groups are sulfhydryl and amino. In one example, the sulfhydryl group can be generated by reducing the intramolecular disulfide bonds of 158P1D7 Mab. In another embodiment, the sulfhydryl group can be generated by reacting the amino group of the lysine portion of the 158P1D7 MAb with 2-iminothiolane (Traut's reagent) or other sulfhydryl generating reagents. In certain embodiments, the 158P1D7 MAb is a recombinant antibody that is engineered to carry one or more lysines. In certain other embodiments, the recombinant 158P1D7 MAb is engineered to carry additional sulfhydryls, such as additional cysteine.

[0278] In one embodiment, the Stretcher unit forms a bond with a sulfur atom of the Antibody unit. The sulfur atom may be derived from a thiol group of the antibody. The square brackets of Formulas IIIa and IIIb represent representative Stretcher units of this embodiment, wherein L-, -W-, -Y-, -D, w and y are as defined above, and R 17 Selected from -C1-C 10 Alkylene-, -C1-C 10 Alkenylene-, -C1-C 10 Alkyne-, carbocyclic-, -O-(C1-C8 alkylene)-, O-(C1-C8 alkenylene)-, -O-(C1-C8 alkynylene)-, -arylene-, -C1-C 10 Alkylene-arylene-, -C2-C 10 Alkenylene-arylene, -C2-C 10 Alkyne-arylene, -arylene-C1-C 10 Alkylene-, -arylene-C2-C 10 Alkenylene-, -arylene-C2-C 10 Alkynylidene-, -C1-C 10 Alkylene-(carbocyclylene)-, -C2-C 10 Alkenylene-(carbocyclylene)-, -C2-C 10 Alkynylidene-(carbocyclylene)-, -(carbocyclylene)-C1-C 10 Alkylene-, -(Carbocyclylene)-C2-C 10 Alkenylene-, -(Carbocyclyl)-C2-C 10 Alkyne, -heterocyclylene-, -C1-C 10Alkylene-(heterocyclylene)-, -C2-C 10 Alkenylene-(heterocyclylene)-, -C2-C 10 Alkyne-(heterocyclylene)-, -(heterocyclylene)-C1-C 10 Alkylene-, -(heterocyclylene)-C2-C 10 Alkenylene-, -(heterocyclylene)-C1-C 10 Alkynylidene-、-(CH2CH2O) r -or-(CH2CH2O) r -CH2-, r is an integer from 1 to 10, wherein the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, carbocyclylene, heterocyclylene, and arylene groups, alone or as part of another group, are optionally substituted. In some embodiments, the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, carbocyclylene, heterocyclylene, and arylene groups, alone or as part of another group, are unsubstituted. In some embodiments, R 17 Selected from -C1-C 10 Alkylene-, -Carbocyclylene-, -O-(C1-C8 alkylene)-, -Arylene-, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(carbocyclylene)-, -(carbocyclylene)-C1-C 10 Alkylene-, -C3-C8 heterocyclylene-, -C1-C 10 Alkylene-(heterocyclylene)-, -(heterocyclylene)-C1-C 10 Alkylene-, -(CH2CH2O) r and -(CH2CH2O) r -CH2-, r is an integer from 1 to 10, wherein the alkylene group is unsubstituted and the remaining groups are optionally substituted.

[0279] It should be understood from all exemplary embodiments that, even if not explicitly stated, 1-20 drug moieties may be attached to the antibody (p=1-20).

[0280]

[0281] An illustrative Stretcher unit is a Stretcher unit of Formula IIIa, wherein R 17 is -(CH2)5-:

[0282]

[0283] Another illustrative Stretcher unit is a Stretcher unit of Formula IIIa, wherein R 17Yes -(CH2CH2O) r -CH2-; r is 2:

[0284]

[0285] An illustrative Stretcher unit is a Stretcher unit of Formula IIIa, wherein R 17 is -arylene- or arylene-C1-C 10 Alkylene-. In some embodiments, aryl is unsubstituted phenyl.

[0286] Another illustrative Stretcher unit is a Stretcher unit of Formula IIIb, wherein R 17 is -(CH2)5-:

[0287]

[0288] In certain embodiments, the Stretcher unit is linked to the Antibody unit via a disulfide bond between a sulfur atom of the Antibody unit and a sulfur atom of the Stretcher unit. A representative Stretcher unit of this embodiment is shown in the square brackets of Formula IV, wherein R 17 , -L-, -W-, -Y-, -D, w and y are as defined above.

[0289]

[0290] It should be noted in this application that, unless otherwise specified herein, the S portion of the following formula refers to the sulfur atom of the antibody unit.

[0291]

[0292] In other embodiments, the stretcher unit contains an active site that can form a bond with a primary or secondary amino group of an antibody. Examples of these active sites include, but are not limited to, active esters such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. The square brackets of formulas Va and Vb represent representative stretchers of this embodiment, wherein -R 17 -, L-, -W-, -Y-, -D, w and y are as defined above;

[0293]

[0294] In some embodiments, the stretch unit contains an active site that can react with a (-CHO) group of a modified sugar that may be present on the antibody. For example, the sugar can be mildly oxidized using a reagent such as sodium periodate, and the resulting oxidized sugar (-CHO) unit can be condensed with a stretch unit containing a functional group such as a hydrazide, an oxime, a primary or secondary amine, a hydrazine, a thiosemicarbazone, a carboxylic acid hydrazine, and an aryl hydrazide, as described by Kaneko et al., 1991, Bioconjugate Chem. 2:133-41. 2:133-41. The square brackets of Formulas VIa, VIb, and VIc represent representative stretch units of this embodiment, wherein -R 17 -, L-, -W-, -Y-, -D, w and y are as defined above.

[0295]

[0296] VII.) Amino Acid Unit

[0297] When present, the Amino Acid unit (-W-) links the Stretcher unit to the Spacer unit (if the Spacer unit is present), links the Stretcher unit to the Drug moiety (if the Spacer unit is not present), and links the Antibody unit to the Drug unit (if the Stretcher unit and Spacer unit are not present).

[0298] W w - can be, for example, a monopeptide, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit. -W- units each independently have the general formula shown in the square brackets below, w being an integer from 0 to 12:

[0299]

[0300] Where R 19 is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2,

[0301] -CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,

[0302]

[0303] In some embodiments, the Amino Acid unit can be enzymatically cleaved by one or more enzymes, including cancer or tumor associated proteases, to release the Drug unit (-D), which in one embodiment is protonated in vivo upon release to provide Drug (D).

[0304] In certain embodiments, the amino acid unit may comprise a natural amino acid. In other embodiments, the amino acid unit may comprise a non-natural amino acid. Formulas (VII)-(IX) represent illustrative Ww units:

[0305]

[0306] Where R 20 and R 21 as follows:

[0307]

[0308]

[0309]

[0310] Where R 20 , R 21 and R 22 as follows:

[0311]

[0312]

[0313] Where R 20 , R 21 , R 22 and R 23 as follows:

[0314]

[0315] Exemplary amino acid units include, but are not limited to, units of Formula VII, wherein: R 20 is benzyl and R 21 is -(CH2)4NH2; R 20 is isopropyl and R 21 is -(CH2)4NH2; or R 20 is isopropyl and R 21 Another exemplary amino acid unit is a unit of formula VIII, wherein R 20 is benzyl, R 21 is benzyl and R 22 It is -(CH2)4NH2.

[0316] Useful -W w -unit can be designed and optimized for its selectivity for specific enzymes such as tumor-associated proteases. In one embodiment, -W w -units are units cleaved catalytically by cathepsins B, C and D, or plasmin proteases.

[0317] In one embodiment, -W w - is a dipeptide, tripeptide, tetrapeptide or pentapeptide. 19 , R 20 , R 21 , R 22 or R 23 When it is not hydrogen, 19 , R 20 , R 21 , R 22 or R 23 The attached carbon atom is a chiral carbon.

[0318] With R 19 , R 20 , R 21 , R 22 or R 23 The attached carbon atoms are each independently of the other in the (S) or (R) configuration.

[0319] In one aspect of the amino acid unit, the amino acid unit is valine-citrulline (vc or val-cit). In another aspect, the amino acid unit is phenylalanine-lysine (i.e., fk). In another aspect of the amino acid unit, the amino acid unit is N-methyl valine-citrulline. In another aspect, the amino acid unit is 5-aminovaleric acid, homophenylalanine lysine, tetraisoquinoline carboxylic acid lysine, cyclohexylalanine lysine, isopipecotic acid (isonepecotic acid) lysine, β-alanine lysine, glycine serine valine glutamine and isopipecotic acid.

[0320] VIII.) Spacer Unit

[0321] When the spacer unit (-Y-) is present, it connects the amino acid unit and the drug unit (when the amino acid unit is present). Alternatively, when the amino acid unit is not present, the spacer unit connects the stretcher unit and the drug unit. When the amino acid unit and the stretcher unit are not present, the spacer unit also connects the drug unit and the antibody unit.

[0322] There are two main types of spacer units: non-self-degradable or self-degradable. A non-self-degradable spacer unit is a spacer unit in which a portion or all of the spacer unit remains bound to the drug portion after the amino acid unit of the antibody drug conjugate is cleaved, especially after enzymatic cleavage. Examples of non-self-degradable spacer units include, but are not limited to, (glycine-glycine) spacer units and glycine spacer units (both shown in Scheme 1) (see below). When a conjugate containing a glycine-glycine spacer unit or a glycine spacer unit is enzymatically cleaved by an enzyme (such as a tumor cell-associated protease, a cancer cell-associated protease, or a lymphocyte-associated protease), a glycine-glycine-drug portion or a glycine-drug portion is cleaved from L-Aa-Ww-. In one embodiment, an independent hydrolysis reaction occurs in the target cell, cleaving the glycine-drug portion bond and releasing the drug.

[0323] Solution 1

[0324]

[0325] In some embodiments, the non-self-immolative spacer unit (-Y-) is -Gly-. In some embodiments, the non-self-immolative spacer unit (-Y-) is -Gly-Gly-.

[0326] In one embodiment, a Drug-Linker Conjugate is provided in which there is no Spacer unit (y=0), or a pharmaceutically acceptable salt or solvate thereof.

[0327] Alternatively, a conjugate containing a self-immolative spacer unit can release -D. As used herein, the term "self-immolative spacer" refers to a bifunctional chemical moiety that can covalently link two spaced chemical moieties into a stable tripartite molecule, and spontaneously separate from the second chemical moiety if the bond to the first moiety is cleaved.

[0328] In some embodiments, -Y y - is a p-aminobenzyl alcohol (PAB) unit (see Schemes 2 and 3), the phenylene portion of which is replaced by Q m Substituted, wherein Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro or -cyano; m is an integer from 0 to 4.

[0329] No matter it exists alone or as part of another group, the alkyl, alkenyl and alkynyl groups can be optionally substituted. In some embodiments, -Y- is connected to -W through the amino nitrogen atom of the PAB group. w-PAB group and directly linked to -D via a carbonate, carbamate or ether group. Without wishing to be bound by any particular theory or mechanism, Scheme 2 describes a possible mechanism of drug release from a PAB group directly linked to -D via a carbamate or carbonate group, as described in Toki et al., 2002, J. Org. Chem. 67: 1866-1872.

[0330] Solution 2

[0331]

[0332] In Scheme 2, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro or -cyano; m is an integer from 0 to 4; and p ranges from 1 to about 20. The alkyl, alkenyl and alkynyl groups, whether present alone or as part of another group, may be optionally substituted.

[0333] Without wishing to be bound by any particular theory or mechanism, Scheme 3 depicts a possible mechanism for drug release from a PAB group directly attached to -D via an ether or amine linkage, wherein D contains an oxygen or nitrogen group that forms part of the drug unit.

[0334] Solution 3

[0335]

[0336] In Scheme 3, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro or -cyano; m is an integer from 0 to 4; and p ranges from 1 to about 20. The alkyl, alkenyl and alkynyl groups may be optionally substituted, whether present alone or as part of another group.

[0337] Other examples of self-immolative spacers include, but are not limited to, aromatic compounds with electrical properties similar to those of the PAB group, such as 2-aminoimidazole-5-methanol derivatives (Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and o- or p-aminobenzyl acetals. Spacers that undergo cyclization upon hydrolysis of the amide bond may be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., 1972, J. Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acid amide (Amsberry et al., 1990, J. Org. Chem. 55:5867). The elimination of amine-containing drugs substituted at the α-position to glycine (Kingsbury et al., 1984, J. Med. Chem. 27:1447) is also an example of a self-immolative spacer.

[0338] In one embodiment, the spacer unit is a branched bis(hydroxymethyl)-styrene (BHMS) unit, as shown in Scheme 4, which can be used to incorporate and release a variety of drugs.

[0339] Solution 4

[0340]

[0341] In Scheme 4, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro or -cyano; m is an integer from 0 to 4; n is 0 or 1; and p ranges from 1 to about 20. The alkyl, alkenyl and alkynyl groups, whether present alone or as part of another group, may be optionally substituted.

[0342] In some embodiments, the -D moieties are the same. In another embodiment, the -D moieties are different.

[0343] In one aspect, the Spacer unit (-Y y -) is represented by formula (X)-(XII):

[0344]

[0345] Wherein Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro or -cyano; m is an integer from 0 to 4. The alkyl, alkenyl and alkynyl groups may be optionally substituted, whether present alone or as part of another group.

[0346]

[0347] Embodiments of Formulas I and II comprising antibody-drug conjugate compounds may include:

[0348]

[0349] where w and y are each 0, 1, or 2, and

[0350]

[0351] where w and y are each 0,

[0352]

[0353] IX.) Drug Unit

[0354] The drug moiety (D) can be any cytotoxic, cytostatic or immunomodulatory (e.g., immunosuppressive) drug. D is a drug unit (part) having an atom that can form a bond with a spacer unit, an amino acid unit, a stretcher unit, or an antibody unit. In some embodiments, the drug unit D has a nitrogen atom that can form a bond with a spacer unit. The terms "drug unit" and "drug moiety" used herein have the same meaning and can be used interchangeably.

[0355] Useful classes of cytotoxic or immunomodulatory agents include, for example, tubulin inhibitors, DNA minor groove binders, DNA replication inhibitors, and alkylating agents.

[0356] In some embodiments, the drug is an auristatin, such as auristatin E (also known in the art as a derivative of Aplysia-10) or a derivative thereof. For example, the auristatin can be an ester formed between auristatin E and a ketoacid. For example, auristatin E can be reacted with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. The synthesis and structure of exemplary auristatins are described in U.S. Patent Application Nos. 2003-0083263, 2005-0238649, and 2005-0009751; International Patent Publication Nos. WO 04 / 010957, WO 02 / 088172 and U.S. Patent Nos. 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, each of which is incorporated herein by reference in its entirety and for all purposes.

[0357] Auristatins have been shown to interfere with microtubule dynamics as well as nuclear and cell division and have anticancer activity. Auristatins bind to tubulin and can produce cytotoxic or cytostatic effects on 158P1D7 expressing cells. There are many different assays known in the art that can be used to determine whether auristatins or the resulting antibody-drug conjugates produce cytostatic or cytotoxic effects on a desired cell line.

[0358] Methods for determining whether a compound binds to tubulin are known in the art. See, for example, Muller et al., Anal. Chem 2006, 78, 4390-4397; Hamel et al., Molecular Pharmacology, 1995 47:965-976; and Hamel et al., The Journal of Biological Chemistry, 1990 265:28, 17141-17149. For the purposes of the present invention, the relative affinity of a compound for tubulin can be determined. Some preferred auristatins of the present invention have an affinity for tubulin ranging from 10 times lower than the binding affinity of MMAE to tubulin (weaker affinity) to 10 times, 20 times, or 100 times higher than the binding affinity of MMAE to tubulin (stronger affinity).

[0359] In some embodiments, -D is of formula D Eor D F Auristatin or a pharmaceutically acceptable salt or solvate thereof:

[0360]

[0361] In which, the following description is given independently at each position:

[0362] The wavy lines indicate bonds;

[0363] R 2 Yes-C1-C 20 Alkyl, -C2-C 20 Alkenyl or -C2-C 20 Alkynyl;

[0364] R 3 Yes -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl, -C2-C 20 Alkynyl, -Carbocycle, -C1-C 20 Alkylene (Carbocyclic), -C2-C 20 Alkenylene (Carbocyclic), -C2-C 20 Alkyne (Carbocyclic), -Aryl, -C1-C 20 Alkylene (aryl), -C2-C 20 Alkenylene (aryl), -C2-C 20 Alkyne (aryl), -heterocyclic, -C1-C 20 Alkylene (heterocyclic), -C2-C 20 Alkenylene (heterocyclic) or -C2-C 20 Alkynylidene (heterocyclic);

[0365] R 4 Yes -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl, -C2-C 20 Alkynyl, carbocyclic, -C1-C 20 Alkylene (Carbocyclic), -C2-C 20 Alkenylene (Carbocyclic), -C2-C 20 Alkyne (Carbocyclic), -Aryl, -C1-C 20 Alkylene (aryl), -C2-C 20 Alkenylene (aryl), -C2-C 20 Alkyne (aryl), -heterocyclic, -C1-C 20 Alkylene (heterocyclic), -C2-C 20 Alkenylene (heterocyclic) or -C2-C 20 Alkynylidene (heterocyclic);

[0366] R5 is -H or -C1C8 alkyl;

[0367] or R 4 and R 5 together form a carbocyclic ring having the formula -(CR a R b ) s -, where R a and R b are independently -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl, -C2-C 20 alkynyl or carbocyclic, and s is 2, 3, 4, 5 or 6,

[0368] R 6 Yes -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl or -C2-C 20 Alkynyl;

[0369] R 7 Yes -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl, -C2-C 20 Alkynyl, carbocyclic, -C1-C 20 Alkylene (Carbocyclic), -C2-C 20 Alkenylene (Carbocyclic), -C2-C 20 Alkyne (Carbocyclic), -Aryl, -C1-C 20 Alkylene (aryl), -C2-C 20 Alkenylene (aryl), -C2-C 20 Alkyne (aryl), -heterocyclic, -C1-C 20 Alkylene (heterocyclic), -C2-C 20 Alkenylene (heterocyclic) or -C2-C 20 Alkynylidene (heterocyclic);

[0370] R 8 are independently -H, -OH, -C1-C 20 Alkyl, -C2-C 20 Alkenyl, -C2-C 20 Alkynyl, -O-(C1-C 20 Alkyl), -O-(C2-C 20 alkenyl), -O-(C1-C 20 Alkynyl) or -carbocycle;

[0371] R 9 Yes -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl or -C2-C 20Alkynyl;

[0372] R 24 is -aryl, -heterocyclic or -carbocyclic;

[0373] R 25 Yes -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl, -C2-C 20 Alkynyl, -Carbocyclic, -O-(C1-C 20 Alkyl), -O-(C2-C 20 alkenyl), -O-(C2-C 20 Alkynyl) or OR 18 , where R 18 is -H, a hydroxyl protecting group or in OR 18 When it represents =O, it is a direct bond;

[0374] R 26 Yes -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl or -C2-C 20 alkynyl, -aryl, -heterocycle or -carbocycle;

[0375] R 10 is -aryl or -heterocycle;

[0376] Z is -O-, -S-, -NH- or -NR 12 -, where R 12 Yes-C1-C 20 Alkyl, -C2-C 20 Alkenyl or -C2-C 20 Alkynyl;

[0377] R 11 Yes -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl, -C2-C 20 Alkynyl, -aryl, -heterocyclic, -(R 13 O) m -R 14 or -(R 13 O) m -CH(R 15 )2;

[0378] m is an integer from 1 to 1000;

[0379] R 13 Yes-C2-C 20 Alkylene, -C2-C 20 Alkenylene or -C2-C 20 Alkynylidene;

[0380] R 14 Yes -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl or -C2-C 20 Alkynyl;

[0381] Each time it appears, R 15 are independently -H, -COOH, -(CH2) n -N(R 16 )2, -(CH2) n -SO3H, -(CH2) n -SO3-C1-C 20 Alkyl, -(CH2) n -SO3-C2-C 20 Alkenyl or -(CH2) n -SO3-C2-C 20 Alkynyl;

[0382] Each time it appears, R 16 are independently -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl, -C2-C 20 Alkynyl or -(CH2) n -COOH; and

[0383] n is an integer from 0 to 6;

[0384] The alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocyclic and heterocyclic groups mentioned herein, either alone or as part of another group, are optionally substituted.

[0385] Formula D E Auristatins include those wherein the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocyclic and heterocyclic groups are unsubstituted.

[0386] Formula D E Auristatins include R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 The group is unsubstituted and R 19 , R 20 and R 21 Groups are those optionally substituted as described herein.

[0387] Formula D E Auristatins include those compounds that meet the following definition or pharmaceutically acceptable salt or solvate forms thereof:

[0388] R 2 is -C1-C8 alkyl;

[0389] R 3 , R 4 and R 7 Independently selected from -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl, -C2-C 20 Alkynyl, monocyclic C3-C6 carbocycle, -C1-C 20 Alkylene (monocyclic C3-C6 carbocyclic ring), -C2-C 20 Alkenylene (monocyclic C3-C6 carbocyclic ring), -C2-C 20 Alkyne (monocyclic C3-C6 carbocycle), C6-C 10 Aryl, -C1-C 20 Alkylene (C6-C 10 Aryl), -C2-C 20 Alkenylene (C6-C 10 Aryl), -C2-C 20 Alkynylidene (C6-C 10 Aryl), heterocyclic, -C1-C 20 Alkylene (heterocyclic), -C2-C 20 Alkenylene (heterocyclic) or -C2-C 20 Alkyne(heterocycle); wherein the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, carbocycle, aryl and heterocycle groups are optionally substituted;

[0390] R 5 Yes - H;

[0391] R 6 is -C1-C8 alkyl;

[0392] R 8 Each independently selected from -OH, -O-(C1-C 20 Alkyl), -O-(C2-C 20 alkenyl) or -O-(C2-C 20 alkynyl), wherein the alkyl, alkenyl and alkynyl groups are optionally substituted;

[0393] R 9 is -H or -C1-C8 alkyl;

[0394] R 24 is an optionally substituted phenyl group;

[0395] R 25 Yes-OR 18 , where R 18 is H, a hydroxyl protecting group or in OR18 When it represents =O, it is a direct bond;

[0396] R 26 Selected from -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl, -C2-C 20 alkynyl or carbocyclic, wherein the alkyl, alkenyl, alkynyl and carbocyclic groups are optionally substituted.

[0397] Formula D E Auristatins include those compounds that meet the following definition or a pharmaceutically acceptable salt form thereof:

[0398] R 2 It is methyl;

[0399] R 3 is -H, -C1-C8 alkyl, -C2-C8 alkenyl or -C2-C8 alkynyl, wherein the alkyl, alkenyl and alkynyl are optionally substituted;

[0400] R 4 is -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, monocyclic C3-C6 carbocyclic, -C6-C 10 Aryl, -C1-C8 alkylene (C6-C 10 Aryl), -C2-C8 alkenyl (C6-C 10 Aryl), -C2-C8 alkynylene (C6-C 10 alkylene (monocyclic C3-C6 carbocycle), -C2-C8 alkenylene (monocyclic C3-C6 carbocycle), -C2-C8 alkynylene (monocyclic C3-C6 carbocycle); wherein the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl and carbocyclic groups appearing alone or as part of another group are optionally substituted;

[0401] R 5 Yes - H;

[0402] R 6 It is methyl;

[0403] R 7 is -C1-C8 alkyl, -C2-C8 alkenyl or -C2-C8 alkynyl;

[0404] R 8 each is a methoxy group;

[0405] R 9 is -H or -C1-C8 alkyl;

[0406] R 24 It is phenyl;

[0407] R 25 Yes-OR 18 , where R 18 is H, a hydroxyl protecting group or in OR 18 When it represents =O, it is a direct bond;

[0408] R 26 It's methyl.

[0409] Formula D E Auristatins include those compounds meeting the following definition or pharmaceutically acceptable salts thereof:

[0410] R 2 is methyl; R 3 is -H or -C1-C3 alkyl; R 4 is -C1-C5 alkyl; R 5 Yes – H; R 6 is methyl; R 7 is isopropyl or sec-butyl; R 8 is methoxy; R 9 is -H or -C1-C8 alkyl; R 24 is phenyl; R 25 Yes-OR 18 ; where R 18 is -H, a hydroxyl protecting group or in OR 18 When it represents =O, it is a direct bond; and R 26 It's methyl.

[0411] Formula D E Auristatins include those compounds that meet the following definition or pharmaceutically acceptable salt or solvate forms thereof:

[0412] R 2 is methyl or C1-C3 alkyl,

[0413] R 3 is -H or -C1-C3 alkyl;

[0414] R 4 is -C1-C5 alkyl;

[0415] R 5 It is H;

[0416] R 6 is -C1-C3 alkyl;

[0417] R 7 is -C1-C5 alkyl;

[0418] R 8 is -C1-C3 alkoxy;

[0419] R 9 is -H or -C1-C8 alkyl;

[0420] R 24 It is phenyl;

[0421] R 25 Yes-OR 18 , where R 18 is H, a hydroxyl protecting group or in OR 18 represents =O when it is a direct bond; and

[0422] R 26 It is a -C1-C3 alkyl group.

[0423] Formula D F Auristatins include those compounds meeting the following definition or pharmaceutically acceptable salts thereof:

[0424] R 2 It is methyl;

[0425] R 3 , R 4 and R 7 Independently selected from -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl, -C2-C 20 Alkynyl, monocyclic C3-C6 carbocycle, -C1-C 20 Alkylene (monocyclic C3-C6 carbocyclic ring), -C2-C 20 Alkenylene (monocyclic C3-C6 carbocyclic ring), -C2-C 20 Alkyne (monocyclic C3-C6 carbocycle), C6-C 10 Aryl, -C1-C 20 Alkylene (C6-C 10 Aryl), -C2-C 20 Alkenylene (C6-C 10 Aryl), -C2-C 20 Alkynylidene (C6-C 10 aryl), heterocyclic group, -C1-C 20 Alkylene (heterocyclic), -C2-C 20 Alkenylene (heterocyclic) or -C2-C 20 wherein said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, carbocyclic, aryl and heterocyclic groups, whether alone or as part of another group, are optionally substituted;

[0426] R 5 Yes - H;

[0427] R 6 It is methyl;

[0428] R 8 each is a methoxy group;

[0429] R 9 Yes -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl or -C2-C 20 Alkynyl; wherein the alkyl, alkenyl and alkynyl groups are optionally substituted;

[0430] R 10 is an optionally substituted aryl group or an optionally substituted heterocyclic group;

[0431] Z is -O-, -S-, -NH- or -NR 12 -, where R 12 Yes-C1-C 20 Alkyl, -C2-C 20 Alkenyl or -C2-C 20 alkynyl, each optionally substituted;

[0432] R 11 Yes -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl, -C2-C 20 Alkynyl, aryl, heterocyclic group, -(R 13 O) m -R 14 or -(R 13 O) m -CH(R 15 )2, wherein the alkyl, alkenyl, alkynyl, aryl and heterocyclic groups are optionally substituted;

[0433] m is an integer from 1 to 1000 or m=0;

[0434] R 13 Yes-C2-C 20 Alkylene, -C2-C 20 Alkenylene or -C2-C 20 alkynylene, each optionally substituted;

[0435] R 14 Yes -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl or -C2-C 20 Alkynyl, wherein the alkyl, alkenyl and alkynyl groups are optionally substituted;

[0436] Each time it appears, R 15 are independently -H, -COOH, -(CH2) n -N(R 16 )2, -(CH2) n -SO3H, -(CH2) n-SO3-C1-C 20 Alkyl, -(CH2) n -SO3-C2-C 20 Alkenyl or -(CH2) n -SO3-C2-C 20 Alkynyl, wherein the alkyl, alkenyl and alkynyl groups are optionally substituted;

[0437] Each time it appears, R 16 are independently -H, -C1-C 20 Alkyl, -C2-C 20 Alkenyl, -C2-C 20 Alkynyl or -(CH2) n -COOH, wherein the alkyl, alkenyl and alkynyl groups are optionally substituted;

[0438] n is an integer from 0 to 6.

[0439] In certain embodiments, R 10 is an optionally substituted phenyl group.

[0440] Formula D F Auristatins include R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 The group is unsubstituted and R 10 and R 11 The compounds are as described herein.

[0441] Formula D F Auristatins include those compounds wherein the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocyclic and heterocyclic groups are unsubstituted.

[0442] Formula D F Auristatins include those compounds meeting the following definition or pharmaceutically acceptable salts thereof:

[0443] R 2 is -C1-C3 alkyl; R 3 is -H or -C1-C3 alkyl; R 4 is -C1-C5 alkyl; R 5 Yes – H; R 6 is -C1-C3 alkyl; R 7 is -C1-C5 alkyl; R 8 is -C1-C3 alkoxy; R 9 is -H or -C1-C8 alkyl; R 10is optionally substituted phenyl; Z is –O-, –S- or –NH-; R 11 As defined above.

[0444] Formula D F Auristatins include those compounds meeting the following definition or pharmaceutically acceptable salts thereof:

[0445] R 2 is methyl; R 3 is -H or -C1-C3 alkyl; R 4 is -C1-C5 alkyl; R 5 Yes – H; R 6 is methyl; R 7 is isopropyl or sec-butyl; R 8 is methoxy; R 9 is -H or -C1-C8 alkyl; R 10 is optionally substituted phenyl; Z is –O-, –S- or –NH-; R 11 As defined above.

[0446] Formula D F Auristatins include those compounds that meet the following definition or a pharmaceutically acceptable salt form thereof:

[0447] R 2 is methyl; R 3 is -H or -C1-C3 alkyl; R 4 is -C1-C5 alkyl; R 5 Yes – H; R 6 is methyl; R 7 is isopropyl or sec-butyl; R 8 is methoxy; R 9 is -H or -C1-C8 alkyl; R 10 is phenyl; Z is –O- or –NH-; R 11 As defined above, hydrogen is preferred.

[0448] Formula D F Auristatins include those compounds that meet the following definition or a pharmaceutically acceptable salt form thereof:

[0449] R 2 is -C1-C3 alkyl; R 3 is -H or -C1-C3 alkyl; R 4 is -C1-C5 alkyl; R 5 Yes – H; R 6 is -C1-C3 alkyl; R 7 is -C1-C5 alkyl; R 8 is -C1-C3 alkoxy; R 9 is -H or -C1-C8 alkyl; R10 is phenyl; Z is –O- or –NH-; R 11 As defined above, hydrogen is preferred.

[0450] Formula D E or D F Auristatins include R 3 , R 4 and R 7 are independently isopropyl or sec-butyl and R 5 In one exemplary embodiment, R 3 and R 4 Each is isopropyl, R 5 Yes H, R 7 The remaining substituents are as defined herein.

[0451] Formula D E or D F Auristatins include R 2 and R 6 Each is methyl and R 9 Those compounds where is H. The remaining substituents are as defined herein.

[0452] Formula D E or D F Auristatins include R 8 Those compounds wherein each occurrence is -OCH3. The remaining substituents are as defined herein.

[0453] Formula D E or D F Auristatins include R 3 and R 4 are isopropyl, R 2 and R 6 Each is methyl, R 5 It is H, R 7 is sec-butyl, R 8 Each occurrence is -OCH3 and R 9 Those compounds where is H. The remaining substituents are as defined herein.

[0454] Formula D F Auristatins include those compounds wherein Z is -O- or -NH-. The remaining substituents are as defined herein.

[0455] Formula D F Auristatins include R 10 The remaining substituents are as defined herein.

[0456] Formula D F Auristatins include R 10The remaining substituents are as defined herein.

[0457] Formula D F The auristatins include those wherein Z is -O- and R 11 The remaining substituents are as defined herein.

[0458] Formula D F The auristatin includes when Z is -NH, R 11 Yes-(R 13 O) m -CH(R 15 )2, wherein R 15 Yes - (CH2) n -N(R 16 )2 and R 16 is -C1-C8 alkyl or -(CH2) n -COOH. The remaining substituents are as defined herein.

[0459] Formula D F The auristatin includes when Z is -NH, R 11 Yes-(R 13 O) m -CH(R 15 )2, wherein R 15 Yes - (CH2) n -SO3H. The remaining substituents are as defined herein.

[0460] In a preferred embodiment, when D is of formula D E When auristatin is used, w is an integer of 1-12, preferably 2-12, y is 1 or 2, and a is preferably 1.

[0461] In some embodiments, when D is of formula D F When auristatin is used, a is 1 and w and y are 0.

[0462] Illustrative Drug units (-D) include Drug units having the following structures or pharmaceutically acceptable salts or solvates thereof:

[0463]

[0464]

[0465]

[0466] In one aspect, a hydrophilic group, such as but not limited to triethylene glycol (TEG), may be formed by R 11 Attached to the drug unit. Without being limited by theory, the hydrophilic group helps internalization and non-agglomeration of the drug unit.

[0467] In some embodiments, the Drug unit is not TZT- 1027. In some embodiments, the Drug unit is not auristatin E, Aplysia 10, or auristatin PE.

[0468] An exemplary antibody drug conjugate compound has the following structure or a pharmaceutically acceptable salt thereof, wherein "L" or "mAb-s-" represents the 158P1D7 Mab referred to herein as Ha15-10ac12:

[0469] L-MC-vc-PAB-MMAE

[0470]

[0471] or

[0472] L-MC-vc-PAB-MMAF

[0473]

[0474] or

[0475] L-MC-MMAF

[0476]

[0477] In some embodiments, the drug unit is calicheamicin, camptothecin, maytansinoid or anthracycline antibiotics. In some embodiments, the drug is a taxane, a topoisomerase inhibitor, a vinca alkaloid, etc.

[0478] In some typical embodiments, suitable cytotoxic agents include, for example, DNA minor groove binders (e.g., enediynes and lexitropsins, CBI compounds; see also U.S. Pat. No. 6,130,237), duocarmycin, taxanes (e.g., paclitaxel and docetaxel), purine toxins, and vinca alkaloids. Other cytotoxic agents include, for example, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, fusobactin, epothilone A and B, estramustine, cryptophysin, cymadotin, maytansinoids, picrocelotide, elasinolide, and mitoxantrone.

[0479] In some embodiments, the drug is an anti-tubulin agent. Examples of anti-tubulin agents include: auristatins, taxanes (e.g. (paclitaxel), (docetaxel), T67 (Tularik), and vinca alkaloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine). Other anti-tubulin drugs include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophycin, cymadotin, maytansinoids, combretastatins, scutellarin, and eleutherobin.

[0480] In certain embodiments, the cytotoxic agent is a maytansine, another group of anti-tubulin agents. For example, in a specific embodiment, the maytansine is maytansine or DM-1 (IMGN Company (ImmunoGen, Inc.); see also Chari et al., 1992, Cancer Res 52: 127-131).

[0481] In certain embodiments, the cytotoxic or cytostatic agent is a dolastatin. In certain embodiments, the cytotoxic or cytostatic agent is an auristatin. Thus, in a specific embodiment, the cytotoxic or cytostatic agent is MMAE (Formula XI). In another specific embodiment, the cytotoxic or cytostatic agent is AFP (Formula XVI).

[0482]

[0483] In certain embodiments, the cytotoxic or cytostatic agent is a compound of Formula XII-XXI or a pharmaceutically acceptable salt thereof:

[0484]

[0485]

[0486]

[0487] X.) Drug Loading

[0488] p represents drug loading, i.e., the average number of drug moieties per antibody in the molecule. The drug loading can range from 1 to 20 drug moieties (D) per antibody. The ADC of the present invention includes a collection of antibodies coupled to drug moieties in the range of 1 to 20. The average number of drug moieties per antibody in the ADC preparation from the coupling reaction can be identified by conventional methods such as mass spectrometry and ELISA experiments. The quantitative distribution of the ADC can also be determined in the form of p. In some cases, a uniform ADC with a certain determined p value can be separated, purified, and identified from ADCs of other drug loadings by means such as electrophoresis.

[0489] For some antibody drug conjugates, p may be limited by the number of antibody binding sites. For example, when the connection is a cysteine ​​thiol group in the above exemplary embodiment, the antibody may have only one or more cysteine ​​thiol groups, or may have only one or more sufficient reactive thiol groups that can be connected to the joint through it. In some embodiments, higher drug loading (e.g., p>5) may cause some drug antibody conjugates to aggregate, insoluble, toxic, or lose cell permeability. In some embodiments, the drug loading range of ADC of the present invention is 1-about 8, about 2-about 6, about 3-about 5, about 3-about 4, about 3.1-about 3.9, about 3.2-about 3.8, about 3.2-about 3.7, about 3.2-about 3.6, about 3.3-about 3.8, or about 3.3-about 3.7. In fact, it has been shown that for some ADCs, the optimal ratio of each antibody drug portion may be less than 8 and may be about 2-about 5. See US

[0490] 2005-0238649A1 (incorporated herein by reference in its entirety).

[0491] In certain embodiments, the drug moiety coupled to the antibody in the coupling reaction is less than the theoretical maximum. For example, the antibody may contain lysine residues that do not react with the drug linker intermediate or linker reagent, as described below. Typically, antibodies do not contain a large number of free and reactive cysteine ​​thiol groups that can be connected to the drug moiety; in fact, most cysteine ​​thiol residues in antibodies exist in the form of disulfide bridges. In certain embodiments, the antibody can be reduced with a reducing agent (e.g., dithiothreose (DTT) or tricarbonylethylphosphine (TCEP)) under partial or full reducing conditions to produce reactive cysteine ​​thiol groups. In certain embodiments, the antibody undergoes denaturing conditions to display reactive nucleophilic groups, such as lysine or cysteine.

[0492] The loading capacity (drug / antibody ratio) of ADC can be controlled by various methods, such as: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the coupling reaction time or temperature, (iii) partial or limited cysteine ​​thiol modification reducing conditions, (iv) engineering the amino acid sequence of the antibody by recombinant technology to modify the number and site of cysteine ​​to control the number and / or site of linker-drug attachment (e.g., thiol MAbs or thiol Fabs prepared as described herein and in WO2006 / 034488 (which are incorporated herein by reference in their entirety)).

[0493] It should be understood that when more than one nucleophilic group reacts with a drug linker intermediate or linker reagent and then reacts with a drug moiety reagent, the resulting product is a mixture of ADC compounds in which one or more drug moieties attached to the antibody are distributed. The average number of drugs per antibody can be calculated from the mixture by a dual ELISA antibody assay that is specific for the antibody and specific for the drug. Individual ADC molecules can be identified in the mixture using mass spectrometry and separated by HPLC, such as hydrophobic interaction chromatography (see, e.g., Hamblett, KJ et al., "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate," Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, AACR Proceedings, Vol. 45, March 2004; Alley, SC et al., "Controlling the location of drug attachment in antibody-drug conjugates," Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, AACR Proceedings, Vol. 45, March 2004). In certain embodiments, a homogeneous ADC having a single loading value can be isolated from the conjugation mixture by electrophoresis or chromatography.

[0494] XI.) Method for determining ADC cytotoxicity

[0495] Methods for determining whether a drug or antibody drug conjugate produces a cytostatic and / or cytotoxic effect on a cell are known. Typically, the cytotoxic or cytostatic activity of an antibody drug conjugate can be determined by exposing mammalian cells expressing the target protein of the antibody drug conjugate to a cell culture medium; culturing the cells for a period of time, about 6 hours to about 5 days; and determining cell viability. Cell-based in vitro tests can be used to determine the viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activity) of an antibody drug conjugate.

[0496] To determine whether the antibody drug conjugate produces cytostatic effects, a thymidine incorporation assay can be used. For example, target antigen-expressing cancer cells seeded at a density of 5,000 cells / well in a 96-well plate can be cultured for 72 hours and exposed to 0.5 μCi of thymidine for the last 8 hours of the 72-hour period. 3 H-thymidine. The expression of H-thymidine in cultured cells was measured in the presence and absence of antibody drug conjugates. 3 Incorporation of H-thymidine.

[0497] To determine cytotoxicity, necrosis or apoptosis (programmed cell death) can be determined. Necrosis is usually accompanied by increased plasma membrane permeability; cell swelling and plasma membrane rupture. Apoptosis is usually characterized by membrane blebbing, cytoplasmic condensation and activation of endogenous nucleases. Detection of any of these effects on cancer cells indicates that antibody drug conjugates can be used for cancer treatment.

[0498] The uptake of dyes such as neutral red, trypan blue or ALAMAR TM Blue) to measure cell viability (see, e.g., Page et al., 1993, Intl. J. Oncology 3:473-476). In such assays, cells are incubated in a medium containing the dye, washed, and the remaining dye is measured spectrophotometrically to reflect the uptake of the dye by the cells. Cytotoxicity can also be measured using the protein-bound dye sulforhodamine B (SRB) (Skehan et al., 1990, J. Nat'l Cancer Inst. 82:1107-12).

[0499] Alternatively, tetrazolium salts (eg, MTT) are used in a quantitative colorimetric assay that measures the survival and proliferation of mammalian cells by detecting viable but not dead cells (see, eg, Mosmann, 1983, J. Immunol. Methods 65:55-63).

[0500] Apoptosis can be quantitatively analyzed by measuring, for example, DNA fragmentation. Spectrophotometric methods for quantitative in vitro detection of DNA fragmentation are commercially available. Examples of such assays include TUNEL (detection of labeled nucleotides incorporated into fragmented DNA) and ELISA-based assays, described in Biochemica, 1999, No. 2, pp. 34-37 (Roche Molecular Biochemicals).

[0501] Apoptosis can also be determined by detecting changes in cell morphology. For example, with respect to necrosis, the loss of plasma membrane integrity can be determined by detecting the uptake of certain dyes (e.g., fluorescent dyes such as acridine orange or ethidium bromide). Methods for determining the number of apoptotic cells have been described in Duke and Cohen's Current Protocols in Immunology (Coligan et al., 1992, pp. 3.17.1-3.17.16). Cells can also be labeled with DNA dyes (e.g., acridine orange, ethidium bromide, or propidium iodide) and the chromatin condensation of the cells and the margination along the inner nuclear membrane can be observed. Other morphological changes that can be detected to determine apoptosis include, for example, cytoplasmic condensation, increased membrane blebbing, and cell shrinkage.

[0502] The presence of apoptotic cells can be determined from the attached and "floating" compartments of the culture. For example, the two compartments can be collected by removing the supernatant, trypsinizing the attached cells, mixing the preparations after a centrifugation wash step (e.g., 2000 rpm for 10 minutes) and detecting apoptosis (e.g., by measuring DNA fragmentation). (See, e.g., Piazza et al., 1995, Cancer Research 55:3110-16).

[0503] The effect of 158P1D7 therapeutic composition can be evaluated in vivo in a suitable animal model. For example, a xenogeneic cancer model can be used, wherein cancer explants or xenograft tissues passaged are introduced into immunocompromised animals, such as nude mice or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-408). For example, PCT patent application WO98 / 16628 and U.S. Patent No. 6,107,540 describe various human prostate cancer xenograft models, which can reproduce the development of primary tumors, micrometastasis and the formation of osteoblast metastasis characteristic of late disease. Efficacy can be predicted by tests that measure tumor formation inhibition, tumor regression or metastasis, etc.

[0504] In vivo tests to assess the promotion of apoptosis can be used to assess therapeutic compositions. In one embodiment, the presence of apoptotic foci in xenografts of tumor-bearing mice treated with the therapeutic composition is detected and compared with untreated control xenograft-bearing mice. The extent of apoptotic foci found in the tumors of treated mice provides an indication of the therapeutic efficacy of the composition.

[0505] The therapeutic composition for implementing the above method can be formulated into a pharmaceutical composition comprising a carrier suitable for the desired delivery method. Suitable carriers include any substance that maintains the anti-tumor function of the therapeutic composition when combined with the therapeutic composition, and are generally unresponsive to the patient's immune system. Examples include, but are not limited to, any number of standard pharmaceutical carriers, such as sterile phosphate buffered saline solutions, bacteriostatic water, etc. (see, Remington's Pharmaceutical Sciences (" Leimington's Pharmaceutical Sciences") 16th edition, A. Osal. ed., 1980).

[0506] The therapeutic preparation can be dissolved and can be administered by any route that can deliver the therapeutic composition to the tumor site. Possibly effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intraorgan, in situ, etc. Preferred formulations for intravenous injection include: preservative-containing bacteriostatic water, preservative-free sterile water, and / or a solution of the therapeutic composition diluted in a polyvinyl chloride or polyethylene bag (USP) containing 0.9% sterile sodium chloride for injection. The therapeutic protein preparation can be lyophilized and stored in sterile powder form, preferably under vacuum conditions, and then reconstituted in bacteriostatic water (containing, for example, benzyl alcohol preservative) or sterile water before injection.

[0507] Dosages and administration regimens for treating cancer by the methods described above can vary depending on the method and the target cancer, and generally depend on a number of other factors as understood in the art.

[0508] XII.) Treatment of cancers expressing 158P1D7

[0509] The identification of 158P1D7 as a protein that is normally expressed in a defined set of tissues, but is also expressed in cancers (such as those listed in Table I), expanding therapeutic approaches to treat these cancers.

[0510] It is noteworthy that targeted anti-tumor therapy is useful even when the target protein is expressed on normal tissues, even on vital normal organ tissues. Vital organs are organs that are essential for maintaining life, such as the heart or colon. Non-vital organs are organs that can be removed and the individual can still survive. Examples of non-vital organs are ovaries, breasts, and prostates.

[0511] Expression of the target protein in normal tissues, even in critical normal tissues, does not undermine the effectiveness of the targeted agent for that protein in treating certain tumors that also overexpress that protein. For example, expression in critical organs does not cause damage nor does it cause damage itself. In addition, organs that are considered non-essential (such as the prostate and ovaries) can be removed without affecting mortality. Finally, some critical organs are not affected by expression in normal organs due to immunoprivilege. Immunoprivileged organs are organs that are protected from blood by the blood-organ barrier and therefore cannot be immunotherapy. Examples of immunoprivileged organs are the brain and testicles.

[0512] Therefore, the therapeutic methods of inhibiting the activity of 158P1D7 protein are useful for patients with 158P1D7 expressing cancer. These therapeutic methods are generally divided into three categories. The first category regulates the function of 158P1D7 (because the protein is involved in tumor cell growth) to inhibit or block tumor cell growth or induce tumor cell death. The second category includes various methods for inhibiting the binding or association of 158P1D7 protein with its binding partners or other proteins. The third category includes various methods for inhibiting 158P1D7 gene transcription or 158P1D7 mRNA translation.

[0513] Therefore, it is possible to assess whether a cancer patient has 158P1D7 expression and its level, preferably using the immunohistochemical evaluation of tumor tissue, quantitative 158P1D7 imaging or other techniques that reliably indicate whether 158P1D7 expression exists or the level. For this reason, immunohistochemical analysis of tumor biopsy or surgical specimens is preferred. The method of immunohistochemical analysis of tumor tissue is well known in the art.

[0514] XIII.) Targeting 158P1D7 for Antibody-Based Therapy

[0515] 158P1D7 is an attractive target in antibody-based therapeutic strategies. Many antibody strategies targeting extracellular and intracellular molecules are well known in the art (see, for example, complement and ADCC-mediated killing and the use of intracellular antibodies). Since 158P1D7 is expressed by various cancer cell lines relative to corresponding normal cells, 158P1D7 immunoreactive compositions are prepared for systemic administration, which exhibit excellent sensitivity but do not appear due to toxicity, nonspecificity and / or non-targeted effects caused by the combination of immunoreactive compositions with non-target organs and tissues. Antibodies that specifically react with the 158P1D7 domain can be used for systemic treatment of cancers expressing 158P1D7, preferably as antibody drug conjugates (i.e., ADCs) in which the conjugates carry toxins or therapeutic agents.

[0516] It will be appreciated by those skilled in the art that antibodies can be used to specifically target and bind to immunogenic molecules, e.g. Figure 1The immunogenic region of the 158P1D7 sequence shown in Figure 158P1D7. In addition, it will be appreciated by those skilled in the art that coupling antibodies to cytotoxic agents is routine (see, e.g., Slevers et al., Blood 93:11 3678-3684 (June 1, 1999)). When cytotoxic agents and / or therapeutic agents are delivered directly to cells (e.g., by coupling them to antibodies specific to cell-expressed molecules such as 158P1D7), the cytotoxic agents can produce their known biological effects (i.e., cytotoxicity) on those cells.

[0517] A variety of compositions and methods for killing cells with antibody cytotoxic conjugates are known in the art. In the case of cancer, the general method includes administering a biologically effective amount of a conjugate to a mammal having a tumor, the conjugate comprising a selected cytotoxic and / or therapeutic agent linked to a targeting agent (e.g., 158P1D7 MAb, preferably Ha15-10ac12), which is bound to an antigen (e.g., 158P1D7) expressed for binding or localization on the cell surface. A typical embodiment is a method for delivering a cytotoxic and / or therapeutic agent to a 158P1D7-expressing cell, the method comprising conjugating a cytotoxic agent to an antibody that immunospecifically binds to a 158P1D7 epitope, and contacting the cell with the antibody drug conjugate (ADC). Another illustrative embodiment is a method for treating an individual suspected of having metastatic cancer, the method comprising administering a pharmaceutical composition to the individual parenterally, the pharmaceutical composition containing a therapeutically effective amount of an antibody coupled to a cytotoxic and / or therapeutic agent.

[0518] The 158P1D7 antibody can be used for cancer immunotherapy according to various methods that have been successfully used to treat other types of cancer, including but not limited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18: 133-138), multiple myeloma (Ozak et al., 1997, Blood 90: 3179-3186, Tsunenari et al., 1997, Blood 90: 2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res. 52: 2771-2776), B cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol. 19: 93-101), leukemia (Zhon et al., 1996, Leuk. Res. 20: 581-589), colorectal cancer (Moun et al., 1994, Cancer Res. 54: 6160-6166; Velders et al., 1995, Cancer Res. 55: 4398-4403) and breast cancer (Shepard et al., 1991, J. Clin. Immunol. 11: 117-127). Some treatment methods include coupling naked antibodies to toxins or radioactive isotopes, such as Y 91 or I 131 Separately coupled to an anti-CD20 antibody (e.g., Zevalin™, IDEC Pharmaceuticals Corp. or Bexxar TM , Coulter Pharmaceuticals), while other approaches involve co-administering antibodies with other therapeutic agents, such as Herceptin TM (trastuzumab (trastuzu MAb)) and paclitaxel (Genentech, Inc.) In a preferred embodiment, the antibody will be conjugated to a cytotoxic agent, as above, preferably the auristatin derivative known as MMAE (Seattle Genetics, Inc).

[0519] Although 158P1D7 antibody therapy is useful for all cancer stages, antibody therapy is particularly suitable for advanced or metastatic cancer. The antibody therapy described in the present invention is symptomatic treatment for patients who have received one or more rounds of chemotherapy. Alternatively, for patients who have not yet received chemotherapy, the antibody therapy described in the present invention is combined with a chemotherapy or radiotherapy regimen. In addition, antibody therapy can reduce the dose of concomitant chemotherapy, especially for patients who cannot tolerate the toxicity of chemotherapeutic agents well. Fan et al. (Cancer Res.53:4637-4642, 1993), Prewett et al. (International J.of Onco.9:217-224, 1996) and Hancock et al. (Cancer Res.51:4575-4580, 1991) describe the co-application of various antibodies and chemotherapeutic agents.

[0520] Therefore, the preferred monoclonal antibodies used in the treatment methods of the present invention are fully human antibodies and specifically bind to the target 158P1D7 antigen with high affinity. Although the antibody drug conjugates of the present invention can be used to treat cancers in which 158P1D7 is expressed, the antibody drug conjugates of the present invention are particularly useful for treating bladder cancer.

[0521] XIV.) 158P1D7 ADC mixture

[0522] The treatment methods of the present invention include administering a single 158P1D7 ADC and a combination or mixture of different MAbs (i.e., 158P1D7 MAb or MAb that binds to another protein). Such MAb mixtures may have certain advantages because they contain MAbs that target different epitopes, utilize different effector mechanisms, or combine direct cytotoxic MAbs with MAbs that rely on immune effector functions. Such combination MAbs may exhibit synergistic therapeutic effects. In addition, the administration of 158P1D7 MAb may be accompanied by other treatment methods, including but not limited to various chemotherapy and biological factors, androgen blockers, immunomodulators (e.g., IL-2, GM-CSF), surgery, or radiotherapy. In a preferred embodiment, a coupled form of 158P1D7 MAb is administered.

[0523] 158P1D7 ADC preparations are administered by any route capable of delivering antibodies to tumor cells. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumoral, intradermal, etc. Treatment typically includes repeated administration of 158P1D7 ADC preparations by acceptable routes of administration (e.g., intravenous injection (IV)), and typically dosage ranges include, but are not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg / kg body weight. Typically, doses in the range of 10-1000 mg MAb per week are effective and well tolerated.

[0524] Based on (trastuzumab) clinical experience in the treatment of metastatic breast cancer, an initial loading dose of about 4 mg / kg of patient weight IV of a MAb formulation and a weekly dose of about 2 mg / kg IV thereafter represent an acceptable dosing regimen. Preferably, the initial loading dose is given as an infusion of 90 minutes or longer. If the initial dose is well tolerated, periodic maintenance doses are given as infusions of 30 minutes or longer. It will be appreciated by those skilled in the art that various factors can affect the ideal dosage regimen in a particular case. These factors include, for example, the binding affinity and half-life of the MAb used, the patient's 158P1D7 expression level, the degree of 158P1D7 antigen excretion from the circulation, the desired steady-state antibody concentration level, the frequency of treatment, and the effects of chemotherapy or other agents used in conjunction with the treatment methods of the present invention, as well as the health status of a particular patient.

[0525] Optionally, the patient's 158P1D7 levels (e.g., levels of circulating 158P1D7 antigen and / or 158P1D7 expressing cells) should be assessed in a given sample to assist in determining the most effective dosing regimen, etc. Such assessments are also used for monitoring purposes during treatment and in conjunction with other parameter assessments (e.g., urine cytology and / or immune cell levels in bladder cancer treatment, or by analogy, serum PSA levels in prostate cancer treatment) to assess treatment success.

[0526] The object of the present invention is to provide 158P1D7 ADCs that inhibit or delay the growth of tumor cells expressing 158P1D7. A further object of the present invention is to use such 158P1D7 ADCs, particularly to combine such 158P1D7 ADCs with other drugs or immunologically active treatments, to provide a method for inhibiting angiogenesis and other biological functions and thereby reducing the growth of tumors in mammals, preferably humans.

[0527] XV.) Combination therapy

[0528] In one embodiment, when 158P1D7 ADC is used in combination with a chemotherapeutic agent or radiotherapy or a combination thereof to treat a tumor (including a human tumor), there is a synergistic effect. In other words, when used in combination with a chemotherapeutic agent or radiotherapy or a combination thereof, the 158P1D7 ADC enhances the inhibition of tumor growth beyond the expected level. Synergy can be shown, for example, by the following means: the inhibition of tumor growth by the combined treatment is greater than the expectation of the superposition of the effect of the 158P1D7 ADC treatment alone or the 158P1D7 ADC and the chemotherapeutic agent or radiotherapy treatment. Preferably, the synergistic effect is confirmed by the remission of cancer, wherein the treatment with 158P1D7 ADC or the superposition of the combined treatment with 158P1D7 ADC and the chemotherapeutic agent or radiotherapy is not expected to remit cancer.

[0529] The method of using 158P1D7 ADC in combination with chemotherapy or radiotherapy or both to inhibit tumor cell growth includes that 158P1D7 ADC is administered before, during or after chemotherapy or radiotherapy and any combination thereof (i.e. before and during, before and after, during and after, or before, during and after chemotherapy and / or radiotherapy). For example, 158P1D7 ADC is generally administered 1-60 days, preferably 3-40 days, and more preferably within 5-12 days before radiotherapy and / or chemotherapy. However, according to the treatment regimen and specific patient needs, the method is implemented in a manner that provides the most effective treatment and ultimately prolongs the patient's life.

[0530] Administration of chemotherapeutic agents can be achieved in various ways, including systemic administration by parenteral and enteral routes. In one embodiment, 158P1D7 ADC and chemotherapeutic agents are administered in the form of separate molecules. Specific examples of chemotherapeutic agents or chemotherapy include: cisplatin, dacarbazine (DTIC), dactinomycin, chloramphenicol (nitrogen mustard), streptozotocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (docetaxel), Aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha, leuprolide, megestrol acetate, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozotocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, gemcitabine, chlorambucil, taxol, and combinations thereof.

[0531] The radiation source used with the 158P1D7 ADC can be external or internal to the patient being treated. When the radiation source is external to the patient, the treatment is called external beam radiation therapy (EBRT). When the radiation source is internal to the patient, the treatment is called brachytherapy (BT).

[0532] The above treatment regimens may be further combined with other cancer therapeutic agents and / or regimens, such as additional chemotherapies, cancer vaccines, signal transduction inhibitors, agents for treating abnormal cell growth or cancer, antibodies (e.g., anti-CTLA-4 antibodies (Pfizer) as described in WO / 2005 / 092380) or other ligands that inhibit tumor growth by binding to IGF-1R, and cytokines.

[0533] When mammals undergo additional chemotherapy, the above-mentioned chemotherapeutic agents can be used. In addition, growth factor inhibitors, biological response modifiers, anti-hormone therapy, selective estrogen receptor modulators (SERMs), angiogenesis inhibitors and anti-androgens can be used. For example, anti-hormones can be used, such as anti-estrogens such as Nolvadex (tamoxifen), or anti-androgens such as Casodex (4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3-'-(trifluoromethyl) diampromide).

[0534] The above treatment methods can be combined with any of a variety of surgical, chemotherapeutic or radiotherapeutic methods. The treatment methods described in the present invention can allow chemotherapy (or other treatments) to be administered at reduced doses and / or with less frequency, which is advantageous for all patients and particularly for patients who cannot tolerate the toxicity of chemotherapeutic agents well.

[0535] XVI.) Kits / Products

[0536] For laboratory, prognostic, preventive, diagnostic and therapeutic uses described herein, kits are within the scope of the invention. Such kits may comprise carriers, packages or containers that are compartmentalized to accommodate one or more containers such as vials, tubes, etc., each container comprising a separate element for the method, and a label or insert containing instructions for use (e.g., the uses described herein). For example, the container may contain an antibody with a detectable label or capable of being detectably labeled. The kit may comprise a container containing a drug unit. The kit may include all or part of the amino acid sequences or their analogs in Figures 2 or 3, or nucleic acid molecules encoding these amino acid sequences.

[0537] The kit of the present invention generally comprises the above-mentioned container and one or more containers related thereto, which contain materials required from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carriers, packaging, containers, vials and / or tubes, labels listing the contents and / or instructions for use, and package inserts with instructions for use.

[0538] The label may be present on or with the container to indicate that the composition is used for a specific therapeutic or non-therapeutic application, such as prognosis, prevention, diagnosis or laboratory application, and may also indicate instructions for in vivo or in vitro use (e.g., the applications described herein). Instructions and / or other information may also be included in instructions or labels with or on the kit. The label may be on or attached to the container. When the letters, numbers or other characters forming the label are molded or etched into the container itself, the label may be on the container; when the container is present in a receiving container or carrier that holds the container, the label may be attached to the container, such as as a package insert. The label may indicate that the composition is used for diagnosis, treatment, prevention or prognosis of a disease, such as cancer of the tissues shown in Table I.

[0539] The terms "kit" and "article of manufacture" may be used synonymously.

[0540] In another embodiment of the present invention, an article comprising a composition (e.g., an antibody or an antibody drug conjugate (ADC)) is provided, such as a material for diagnosing, prognosing, preventing and / or treating tissue cancer (such as those shown in Table I). The article generally comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The container can be made of various materials (such as glass, metal or plastic). The container can hold amino acid sequences, small molecules, nucleic acid sequences, cell populations and / or antibodies. In another embodiment, the container comprises antibodies, binding fragments thereof or specific binding proteins for assessing 158P1D7 protein expression in cells and tissues, or for related laboratory, prognostic, diagnostic, preventive and therapeutic purposes; instructions and / or guidelines for such applications can be placed on or with the container, as can reagents and other compositions or tools for this purpose.

[0541] Alternatively, the container may contain a composition effective for treating, diagnosing, prognosing or preventing a condition and have a sterile access port (e.g., the container may be an IV bag or vial with a stopper pierced by a hypodermic needle). The active agent in the composition may be an antibody that specifically binds to 158P1D7 or an antibody drug conjugate that specifically binds to 158P1D7.

[0542] The article of manufacture may also include a second container containing a pharmaceutically acceptable buffer (e.g., phosphate-buffered saline, Ringer's solution, and / or dextrose solution). Other materials desirable from a commercial and user standpoint may also be included, including other buffers, diluents, filters, stirrers, needles, syringes, and / or package inserts with instructions and / or directions for use. Example

[0543] Various aspects of the present invention are further described and illustrated by the following embodiments, which are not intended to limit the scope of the present invention.

[0544] Example 1

[0545] 158P1D7 antigen

[0546] The 158P1D7 gene sequence was discovered using suppression subtractive hybridization (SSH) methods known in the art. A 223 bp 158P1D7 SSH sequence was identified from a bladder cancer pool subtracted from normal bladder cDNA. A full-length 158P1D7 cDNA clone was isolated from a bladder cancer tissue pool. The cDNA was 2,555 bp in length and encoded an ORF of 841 amino acids (see Figure 1 ). The 158P1D7 gene is shown to be homologous to the SLITRK6 gene. For further references, see US 6,863,892 (Agensys, Inc., Santa Monica, California), US 7,358,353 (Agensys, Inc., Santa Monica, California), and EP 1,311,675 (Agensys, Inc., Santa Monica, California). For exemplary embodiments of the 158P1D7 antigen, see Figure 1 .

[0547] Example 2

[0548] Preparation of 158P1D7 monoclonal antibody (MAb)

[0549] In one embodiment, therapeutic monoclonal antibodies ("MAbs") to 158P1D7 and 158P1D7 variants include those antibodies that interact with each protein-specific epitope or common sequence-specific epitopes between variants, and the antibodies can bind, internalize, disrupt or modulate the biological functions of 158P1D7 or 158P1D7 variants, such as those that can disrupt interactions with ligands, substrates and binding partners. Immunogens used to generate these MAbs include those designed to encode or contain extracellular domains or complete 158P1D7 protein sequences, regions predicted to contain functional motifs, and regions of 158P1D7 protein variants predicted to be antigenic by computer analysis of amino acid sequences. Immunogens include peptides and recombinant proteins such as tag5-158P1D7, a purified mammalian cell-derived histidine-tagged protein. Alternatively, cells engineered to express high levels of 158P1D7 (eg, UMUC-158P1D7 or 3T3-158P1D7) are used to immunize mice.

[0550] Using XenoMouse (Amgen Freemont, Inc.) produced a MAb against 158P1D7 in which the murine heavy and kappa light chain loci had been inactivated and the majority of the human heavy and kappa light chain immunoglobulin loci had been inserted. XenoMice producing human gamma 2 were immunized with recombinant 3T3 cells expressing 158P1D7, generating a MAb called Ha15-10ac12.

[0551] ELISA demonstrated that the 158P1D7 MAb Ha15-10ac12 specifically bound to cells expressing 158P1D7 (recombinant and endogenous) as well as recombinant 158P1D7 protein.

[0552] After isolating mRNA from each hybridoma cell using Trizol reagent (Life Technologies, Gibco BRL), the DNA coding sequence of 158P1D7 MAb Ha15-10ac12 was determined.

[0553] The following scheme is adopted to sequence the variable nucleic acid sequences of anti-158P1D7 Ha15-10ac12 heavy chain and light chain from hybridoma cells. Hybridoma cells secreting Ha15-10ac12 were lysed with Trizol reagent (Life Technologies, Gibco BRL). Total RNA was purified and quantitatively analyzed. First-strand cDNA was obtained from total RNA using oligo (dT) 12-18 primers by Gibco-BRL Superscript preamplification system. First-strand cDNA was amplified using human immunoglobulin variable heavy chain primers and human immunoglobulin variable light chain primers. PCR products were sequenced and variable heavy and light chain regions were measured.

[0554] The nucleic acid and amino acid sequences of the variable heavy and light chain regions are listed in Figures 2 and 3. The alignment of the Ha15-10ac12 MAb with human Ig germlines is shown in Figure 4A-4B .

[0555] Example 3

[0556] Expression of Ha15-10ac12 using recombinant DNA methods

[0557] To recombinantly express the Ha15-10ac12 MAb in transfected cells, the Ha15-10ac12 MAb variable heavy chain and light chain sequences were cloned upstream of the human heavy chain IgG2 and human light chain Igκ constant regions, respectively. The complete Ha15-10ac12 MAb human heavy chain and light chain cassettes were cloned downstream of the CMV promoter / enhancer in the cloning vector. A polyadenylation site was included downstream of the MAb coding sequence. The recombinant Ha15-10ac12 MAb-expressing construct was transfected into CHO-K1SV cells. The binding of Ha15-10ac12-related MAbs secreted from recombinant CHO cells to cell surface 158P1D7 was evaluated by flow cytometry. UMUC-control and UMUC-158P1D7 cells were stained using Ha15-10ac12 MAbs from hybridomas or from CHO cells transfected with Ha15-10ac12 heavy and light chain vector constructs. Binding was detected by flow cytometry.

[0558] The results showed that the binding of Ha15-10ac12 recombinantly expressed in CHO cells to 158P1D7 was similar to that of Ha15-10ac12 purified from hybridomas. Binding of Ha15-10ac12 MAbs secreted from recombinant cells to 158P1D7 recombinant protein was also evaluated by ELISA. Binding of Ha15-10ac12 to 158P1D7 protein was identical between CHO-derived and hybridoma-derived MAb material.

[0559] Chinese hamster ovary (CHO) cells producing the antibody designated Ha15-10ac12 were sent (via FedEx) to the American Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108 on July 25, 2012 and assigned accession number: PTA-13102.

[0560] Example 4

[0561] Antibody-drug conjugate of Ha15-10ac12 Mab

[0562] The following protocol was used to conjugate the Hal5-10ac12 MAb ( FIG. 2 ) to an auristatin derivative referred to as MMAE (Formula XI) via a vc (Val-Cit) linker as described herein to produce an antibody drug conjugate (ADC) referred to as Hal5-10ac12vcMMAE of the present invention. Conjugation of the vc (Val-Cit) linker to MMAE (Seattle Genetics, Inc., Seattle, WA) was accomplished using the general method shown in Table IV to produce cytotoxic vcMMAE (see US / 2006 / 0074008).

[0563] Then, the antibody drug conjugate (ADC) of the present invention, referred to as Ha15-10ac12vcMMAE, was prepared using the following protocol.

[0564] Briefly, the Hal5-10ac12 MAb in formulation buffer (10 mM acetate pH 5.0 with 5% sorbitol) was buffer exchanged against a reducing buffer (25 mM sodium borate, 300 mM sodium chloride, pH 9.0 ± 0.1). The Hal5-10ac12 MAb was then partially reduced by the addition of 5 mM EDTA and 2.65 molar equivalents of TCEP (relative to the moles of the Hal5-10ac12 MAb). The mixture was then stirred at 37°C for three (3) hours. After disulfide reduction, the mixture was cooled to a target temperature of 15-17°C and five (5) drug equivalents of vcMMAE per mole were added in the form of a 4% (v / v) DMSO solution. After 60-75 minutes, 1 mole of N-acetyl-L-cysteine ​​was added for each mole of vcMMAE added at the beginning of the coupling to quench excess unreacted vc. After 15 minutes, the Ha15-10ac12vcMMAE was adjusted to a target pH of 6.0-6.4 using a concentrated histidine pH 5.2 buffer stock and filtered through a 0.5 / 0.2 μm PES membrane to remove aggregated antibody drug conjugates. After filtration, tangential flow filtration was immediately performed to remove DMSO and quenched drug linkers and exchange the antibody drug conjugates into a buffer (20 mM histidine (pH 6.0 ± 0.1) containing 5.5% dehydrated trehalose (trehalose dehydrate). After tangential flow filtration, the Ha15-10ac12vcMMAE was diluted to a final concentration of 6 ± 1 mg / mL and polysorbate 20 was added to a final concentration of 0.01%.

[0565] The resulting antibody drug conjugate (ADC) was named Ha15-10ac12vcMMAE and had the following formula:

[0566]

[0567] Wherein, MAb is Ha15-10ac12 (Figure 2 and Figure 3), and p is 1 to 12. The preferred p value of the antibody drug conjugate in this embodiment is about 3.5 to 3.7.

[0568] Example 5

[0569] Characterization of Ha15-10ac12vcMMAE

[0570] Antibody drug conjugates that bind 158P1D7 were generated using the methods described in the Example entitled "Antibody Drug Conjugates of Ha15-10ac12 MAb" and screened, identified, and characterized using a combination of assays known in the art.

[0571] A. Affinity determination by FACS

[0572] The binding affinity of Ha15-10ac12vcMMAE to 158P1D7 expressed on the surface of SW780 cells was tested. Briefly, eleven (11) dilutions of Ha15-10ac12vcMMAE were incubated with SW780 cells at a final concentration of 6.67nM-0.0001nM overnight at 4°C (50,000 cells per well). At the end of the incubation, the cells were washed and incubated with anti-hIgG-PE detection antibody at 4°C for 45 minutes. After washing the unbound detection antibody, the cells were analyzed by FACS. The mean fluorescence intensity (MFI) values ​​were obtained and listed in (Table VI). The MFI values ​​were entered into Graphpad Prisim software and analyzed using the one-site binding (hyperbolic) equation Y=Bmax*X / (Kd+X) to generate the Ha15-10ac12vcMMAE saturation curve, shown in ( Fig.13 ). Bmax is the MFI value when Ha15-10ac12vcMMAE binds 158P1D to the maximum extent; Kd represents the binding affinity of Ha15-10ac12vcMMAE, which is the concentration of Ha15-10ac12vcMMAE required to achieve half-maximal binding.

[0573] The calculated affinity (Kd) of Ha15-10ac12vcMMAE to 158P1D7 expressed on the surface of SW780 cells was 0.005 nM.

[0574] B. FACS binding

[0575] The binding of Ha15-10ac12vcMMAE to 158P1D7 expressed on the surface of UMUC, SW780 and CHP-212 cells was tested by FACS. In brief, cells were harvested and inoculated with a concentration of 50,000 cells / well in a well plate. Ha15-10ac12vcMMAE was diluted to 3 μg / mL (for UMUC and SW780 cells) or 10 μg / mL (for CHP-212 cells) and incubated with cells (1 hour at 4°C). At the end of the incubation, the cells were washed and incubated with anti-hIgG-PE detection antibodies at 4°C for 1 hour. After washing away the unbound detection antibodies, the cells were analyzed by FACS. The mean fluorescence intensity (MFI) values ​​(Table VII) were obtained and histograms ( Fig.14 ).

[0576] Example 6

[0577] Ha15-10ac12vcMMAE-mediated cytotoxicity in vitro

[0578] The ability of Ha15-10ac12vcMMAE to mediate SLITRK-6-dependent cytotoxicity was assessed using the human neuroblastoma CHP-212 cell line, which endogenously expresses SLITRK-6, and the human ovarian cancer cell line IGROV-1, which does not express SLITRK-6.

[0579] In brief, CHP-212 and IGROV-1 cells were seeded at a concentration of 1000 cells / well in 50 μl complete medium on a 96-well plate and placed in a tissue culture incubator at 37 degrees Celsius, 5% CO2. The next day, 2x stock solutions of Ha15-10ac12vcMMAE and isotype control antibodies coupled to vcMMAE were prepared in complete medium and 50 μl of ADC serial dilutions were added to appropriate wells. Cells were treated with Ha15-10ac12vcMMAE and isotype control antibodies coupled to vcMMAE in a tissue culture incubator at 37 degrees Celsius, 5% CO2 for six (6) days. At the end of the incubation, 12 μl of Alamar Blue or Presto Blue was added to each well and incubated for four (4) hours. The plates were read using a BioTek Synergy H4 microplate reader using 540 excitation and 590 emission wavelengths.

[0580] These data show that the ADC named Ha15-10ac12vcMMAE can selectively induce cytotoxicity to the SLITRK-6-expressing CHP-212 cell line, while it cannot induce cytotoxicity to the IGROV-1 cell line that does not express SLITRK-6. Therefore, these results show that Ha15-10ac12vcMMAE can selectively deliver cytotoxic drugs to cells expressing 158P1D7 and kill them (Figure 15).

[0581] In another experiment, human neuroblastoma cells CHP-212 expressing SLITRK6 (the target of the Ha15-10ac12 MAb and M15-68(2)18 (also known as 68(18)1.1, see WO 2004 / 072263) MAb) were incubated with the test formulations Ha15-10ac12vcMMAE and M15-68(2)18 to demonstrate any in vitro cell killing (cytotoxic) activity of these substances. Briefly, ninety-six (96) well plates were seeded with 4000 CHP-212 cells / ml and the test antibodies were added in a dilution gradient of nine (9) points from 10,000 ng / ml down six (6) fold to 0.006 ng / ml plus a final test well of 0.0 ng / ml. Each well was tested in triplicate. In addition, control antibodies and control cells (IGROV-1) were tested in the same manner for comparison with the test substances. The test was run for four (4) days, and then Presto Blue (a pigment for staining live cells) was added to each well to obtain a colorimetric reading of cell viability. A colorimetric reading of cell viability was obtained. The cell survival percentage in each well was then calculated and the data were analyzed using a nonlinear fit of a sigmoidal dose response. IC50 values ​​were calculated using Prism graphics software and the cytotoxic effects of Ha15-10ac12vcMMAE were compared to M15-68(2)18 and other controls used in the experiment.

[0582] The results showed that Ha15-10ac12vcMMAE had excellent in vitro cytotoxicity compared with M15-68(2)18 antibody and other controls ( Fig.11 ). The IC50 of Ha15-10ac12vcMMAE was 0.9076, but the IC50 of M15-68(2)18 could not be calculated.

[0583] Example 7

[0584] Ha15-10ac12vcMMAE inhibits tumor growth in vivo

[0585] 158P1D7 is significantly expressed on the surface of tumor tissue cells and is restrictedly expressed in normal tissues, which makes 158P1D7 a good target for antibody therapy and similar therapy via ACD. Therefore, the therapeutic efficacy of Ha15-10ac12vcMMAE in human bladder cancer, lung cancer, breast cancer and glioblastoma xenograft mouse models was evaluated.

[0586] The efficacy of antibody drug conjugates on tumor growth and metastasis formation was studied in mouse cancer xenograft models (e.g., subcutaneous and orthotopic).

[0587] 5x 10 4 -10 6Cancer cells were mixed with Matrigel (Collaborative Research) at a 1:1 dilution and injected into the right axilla of male SCID mice to form subcutaneous (sc) tumors. To test the efficacy of ADC on tumor formation, ADC injections were started on the same day as the tumor cell injection. As controls, mice were injected with purified human IgG or PBS; or purified MAbs that recognize non-related antigens not expressed in human cells. In preliminary studies, no difference in the effect of control IgG or PBS on tumor growth was found. Tumor size was determined by caliper measurement and the width was measured as 2 Tumor volume was calculated as x length / 2, where width is the smallest dimension and length is the largest dimension. Mice were sacrificed when subcutaneous tumors were larger than 1.5 cm in diameter.

[0588] The advantage of xenograft cancer models is that new blood vessel formation and angiogenesis can be studied. Tumor growth depends in part on the development of new blood vessels. Although the capillary system and the developing vascular network are derived from the host, the initiation and structure of new blood vessel formation are regulated by xenograft tumors (Davidoff et al., Clin Cancer Res. (2001) 7: 2870; Solesvik et al., Eur J Cancer Clin Oncol. (1984) 20: 1295). The effects of antibodies and small molecules on new blood vessel formation are studied according to methods known in the art, such as IHC analysis of tumor tissue and its surrounding microenvironment.

[0589] 158P1D7 ADC:

[0590] Monoclonal antibodies against 158P1D7 were produced as described in the example entitled "Preparation of 158P1D7 Monoclonal Antibodies (MAbs)". MAbs were further conjugated to toxins to form Hal5-10ac12vcMMAE as described in the example entitled "Antibody Drug Conjugates of Hal5-10ac12 MAbs". Hal5-10ac12vcMMAE was characterized by FACS and its ability to bind to 158P1D7 was determined by other methods known in the art.

[0591] Cell lines and xenografts:

[0592] As known in the art, cells were maintained in DMEM supplemented with L-glutamine and 10% FBS. AG-B7, RT-4-XCL and NCI-H322M-XCL xenografts were maintained by continuous proliferation in SCID mice. SW780 and RT-4-XCL are cell-derived bladder cancer cell lines obtained from ATCC (Manassas, Virginia). AG-B7 and AG-B8 are patient-derived xenografts derived from human bladder cancer specimens. It will be appreciated by those skilled in the art that multiple experiments have been conducted on the ADC of the present invention in vivo. This is in part because, even in the same disease condition, each in vivo model will exhibit a certain level of unpredictability for those skilled in the art. Therefore, it will be appreciated by those skilled in the art that the use of several types of in vivo models enables those skilled in the art to better evaluate the ADC of the present invention.

[0593] Evaluation of Ha15-10ac12vcMMAE in the human bladder cancer AG-B7 xenograft model established subcutaneously in SCID mice

[0594] In this experiment, human bladder cancer AG-B7 cells (5 x 10 per mouse) were cultured. 6 cells) were injected into the flank of individual SCID mice and the tumors were allowed to grow without treatment until they reached 250 mm 3 The approximate volume of 100 mg / kg was obtained by ADC. At this point, animals were divided into groups based on the tumor volume at the start of treatment using Study Director Software (v.1.7; Studylog Systems, Inc., South San Francisco, California) to ensure that each group had a similar average tumor size and variance. All ADC-treated groups received a single dose of 10 mg / kg by intravenous push injection. Tumor growth in each group was monitored twice a week using caliper measurements until the study endpoint. Statistical analysis of tumor volume was performed at the final time point where data were available for all groups using nonparametric analysis of variance (ANOVA) for hierarchical data.

[0595] The results showed that Ha15-10ac12vcMMAE showed a strong inhibitory effect compared with the untreated control (p<0.0001) ( Figure 5 ).

[0596] In SCID mice, subcutaneously implanted human bladder cancer RT-4-XCL

[0597] Evaluation of Ha15-10ac12vcMMAE

[0598] In another experiment, human bladder cancer xenograft AG-B7 primary tumors were aseptically harvested and minced into small pieces (approximately 1 mm 3 ). Six small pieces were implanted subcutaneously into the flank of a single SCID mouse. When the average tumor volume reached 100 mm3 When the predetermined size of the tumor was reached, the animals were randomly divided into ADC treatment groups and untreated control groups (see tumor volume figure) using the research supervisor software (v.1.7; Research Log Systems, South San Francisco, California), wherein each group had a similar average tumor size and variance. All ADC treatment groups (including two control ADCs) received a single dose of 5 mg / kg by intravenous bolus injection. Tumor growth in each group was monitored twice a week using caliper measurements until the end of the study. Statistical analysis of tumor volume was performed at the final time point where data were available for all groups using nonparametric analysis of variance (ANOVA) for hierarchical data.

[0599] The results showed that Ha15-10ac12vcMMAE showed a strong tumor inhibition effect compared with the untreated control or the corresponding ADC control H3-1.4.1.2vcMMAE (p<0.0001 in both cases) ( Figure 6 ).

[0600] Expression of Ha15-10ac12vcMMAE in subcutaneously established human lung cancer NCI-H322M-XCL implanted in SCID mice Evaluate

[0601] In another experiment, human lung cancer NCI-H322M-XCL cells (2.5 x 10 per mouse) were cultured in vitro. 6 cells) were injected into the flank of individual SCID mice and the tumors were allowed to grow without treatment until they reached 200 mm 3 The approximate volume of the tumor was 1.5 mg / kg. At this point, the animals were divided into groups based on the tumor volume at the start of treatment using the research supervisor software (v.1.7; Research Log Systems, South San Francisco, California) to ensure that each group had a similar average tumor size and variance. All ADC groups (including two control ADCs) were given a dose of 3 mg / kg twice a week by intravenous push injection, for a total of five doses. Tumor growth in each group was monitored twice a week using caliper measurements until the study endpoint. Statistical analysis of tumor volume was performed at the final time point where data were available for all groups using nonparametric analysis of variance (ANOVA) for hierarchical data.

[0602] The results showed that Ha15-10ac12vcMMAE showed a strong tumor inhibitory effect compared with the vehicle control (p<0.0001) or the corresponding ADC control Ha3-12bc1.1vcMMAE (p=0.0041). Figure 7 ).

[0603] Efficacy of Ha15-10ac12vcMMAE in the human bladder cancer AG-B7 xenograft model established subcutaneously in SCID mice

[0604] In another experiment, human bladder cancer xenograft AG-B7 primary tumors were aseptically harvested and minced into small pieces (approximately 1 mm 3Five small pieces were implanted subcutaneously in the flank of a single SCID mouse. Tumors were allowed to grow without treatment until they reached a volume of approximately 200 mm. 3 At this point, animals were divided into groups based on the tumor volume at the start of treatment using Research Director software (v.1.7; Research Log Systems, South San Francisco, California) to ensure similar mean tumor size and variance in each group. All ADC groups (including two control ADCs) were given a dose of 0.5 mg / kg twice a week by intravenous bolus injection for a total of seven doses. Tumor growth in each group was monitored twice a week using caliper measurements until the study endpoint. Statistical analysis of tumor volume was performed at the final time point where data were available for all groups using nonparametric analysis of variance (ANOVA) for hierarchical data.

[0605] The results showed that both Ha15-10ac12vcMMAE (Ha15-10ac12.1vcMMAE) produced in hybridomas and Ha15-10ac12vcMMAE (Ha15-10ac12vcMMAE) produced in CHO cells showed potent tumor inhibition compared with vehicle control (p<0.0001) or corresponding ADC control Ha3-12abc1vcMMAE (p<0.0001). Figure 8 ).

[0606] Efficacy of Ha15-10ac12vcMMAE in the human bladder cancer SW780 xenograft model established subcutaneously in SCID mice

[0607] In another experiment, human bladder cancer xenograft SW780 cells were implanted into the flanks of SCID mice and the tumors were allowed to grow until they reached approximately 200 mm 3 The volume of 100mg / kg was 100mg / kg. At this point, the animals were divided into several treatment groups according to the treatment volume at the start of treatment to ensure that each treatment group had a similar average tumor size and variance. With the help of the research supervisor software (v.1.7; Research Log Systems, South San Francisco, California), mice were divided into treatment groups to match the size of mice. At the beginning of the study, all ADC groups (including two control ADCs) were administered by intravenous push injection at a dosage of 1mg / kg. Tumor growth in each group was monitored twice a week using caliper measurements until the end of the study. Statistical analysis of tumor volume was performed at the final time point where data were available in all groups using nonparametric analysis of variance (ANOVA) for hierarchical data.

[0608] The results showed that the tumor inhibitory activity of Ha15-10ac12vcMMAE was superior to that of Ha15-10ac12 MAb. In addition, it can be inferred that Ha15-10ac12 MAb has no tumor inhibitory effect because the growth kinetics of tumors in this treatment group followed the isotype control antibody. In addition, after a single dose of 1 mg / kg, Ha15-10ac12vcMMAE showed statistically significant growth inhibition (p<0.001) compared with the isotype control ADC ( Fig.10 ).

[0609] Ha15- The efficacy of 10ac12vcMMAE

[0610] In another experiment, patient-derived human bladder cancer AG-B8 tumor pieces were implanted into the flanks of SCID mice and the tumors were allowed to grow until they reached approximately 200 mm 3 The volume of ADC was 500 mg / kg. At this point, animals were divided into several treatment groups according to the treatment volume at the start of treatment to ensure that each treatment group had a similar average tumor size and variance. With the help of the research supervisor software (v.1.7; Research Log Systems, South San Francisco, California), mice were divided into treatment groups to match the size of mice. At the beginning of the study (zero (0) day), all ADC groups (including two control ADCs) were administered by intravenous push injection at a dosage of 5 mg / kg. Tumor growth in each group was monitored twice a week using caliper measurements until the study endpoint. Statistical analysis of tumor volume was performed at the final time point where data were available in all groups using nonparametric analysis of variance (ANOVA) for hierarchical data.

[0611] The results showed that Ha15-10ac12vcMMAE had better tumor inhibitory activity than untreated controls (p<0.0001) or other corresponding ADCγ-2 controls (p<0.0001). In addition, Ha15ac12vcMMAE also showed a better statistically significant effect (p<0.0458) compared with Ha15-10ac12mcMMAF ( Fig.12 ).

[0612] in conclusion

[0613] In conclusion, Figure 5-812 show that the 158P1D7 ADC, designated as Ha15-10ac12vcMMAE, significantly inhibited the growth of tumor cells expressing 158P1D7 compared to the control ADC. Therefore, Ha15-10ac12vcMMAE can be used for therapeutic purposes to treat and control the cancers shown in Table I. Specifically, these results show that Ha15-10ac12vcMMAE has an inhibitory effect on multiple types of bladder cancer models, indicating that it is particularly therapeutically beneficial for treating bladder cancer.

[0614] Example 8

[0615] Human clinical trials using 158P1D7 ADC for the treatment and diagnosis of human cancer

[0616] 158P1D7 ADCs that specifically bind to 158P1D7 are used according to the present invention and are used to treat certain tumors, preferably those shown in Table I. For each of the cases shown, two clinical approaches were successfully pursued.

[0617] I.) Adjuvant therapy: In adjuvant therapy, 158P1D7 ADC is used to treat patients in combination with chemotherapy or anti-tumor agents and / or radiotherapy or a combination thereof. Primary cancer targets (such as those listed in Table I) are treated with standard regimens by adding 158P1D7 ADC to standard first-line and second-line therapies. The regimen design addresses effectiveness issues based on the following example evaluations, including but not limited to: reduction in primary tumor mass or metastatic lesions, increase in progression-free survival, overall survival, improvement in patient health, disease stabilization, and the ability to reduce standard chemotherapy and other biological agents standard doses. These dose reductions can allow additional and / or extended treatment by reducing the dose-related toxicity of chemotherapy or biological agents. 158P1D7 ADC is used in combination with chemotherapy or anti-tumor agents in several adjuvant clinical trials.

[0618] II.) Monotherapy: Regarding the use of 158P1D7 ADC in tumor monotherapy, 158P1D7 ADC is administered to patients without chemotherapeutic agents or anti-tumor agents. In one embodiment, clinical monotherapy is carried out in terminal cancer patients with extensive metastatic disease. The design of the program addresses effectiveness issues based on the following example evaluations, including but not limited to: reduction of primary tumor mass or metastatic lesions, increase in progression-free survival, overall survival, improvement of patient health status, disease stabilization, and the ability to reduce the commonly used doses of standard chemotherapy and other biological agents.

[0619] dose

[0620] The dosage regimen can be adjusted to provide the best desired response. For example, a single bolus dose can be given, divided into several doses over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the treatment situation. Particularly advantageous are parenteral compositions formulated in unit dosage form for ease of administration and dosage uniformity. The dosage unit form used herein refers to physically discrete units used as a single dose for a mammalian subject to be treated, each unit containing a predetermined amount of active compound that is calculated to produce the desired therapeutic effect associated with the desired pharmaceutical carrier. The specifications of the dosage unit form of the present invention are determined by and directly depend on (a) the unique characteristics of the antibody and / or ADC and the specific therapeutic or preventive effects that can be achieved, and (b) the limitations inherent in the technology of compounding the active compound with respect to the sensitivity of the individual to be treated.

[0621] According to the present invention, a non-limiting range example of a therapeutically effective amount of the combined administration of 158P1D7 ADC is about 0.5 to about 10 mg / kg, about 1 to about 5 mg / kg, at least 1 mg / kg, at least 2 mg / kg, at least 3 mg / kg or at least 4 mg / kg. Other exemplary non-limiting ranges are, for example, about 0.5 to about 5 mg / kg, or, for example, about 0.8 to about 5 mg / kg, or, for example, about 1 to about 7.5 mg / kg. High-dose embodiments of the present invention involve doses exceeding 10 mg / kg. It should be noted that the dosage value may vary depending on the type and severity of the condition to be alleviated, and may include a single dose or multiple doses. It is further understood that for any specific object, the specific dosing regimen should be adjusted over time according to the needs of the individual and the personal professional judgment of administering or supervising the administration of the composition, and the dosage ranges listed herein are merely exemplary and are not intended to limit the scope or embodiment of the claimed composition.

[0622] Clinical Development Plan (CDP)

[0623] CDP follows and develops 158P1D7 ADC treatment for adjuvant or monotherapy. Each trial initially confirms the safety of repeated doses and then determines its effectiveness. Each trial is an open label that compares standard chemotherapy with standard therapy plus 158P1D7 ADC. It should be understood that a non-limiting criterion that can be used in conjunction with recruiting patients is the 158P1D7 expression level in the patient's tumor measured by biopsy.

[0624] As with any protein or antibody infusion-based therapy, safety concerns primarily concern (i) cytokine release syndrome, i.e., hypertension, fever, shivering, chills; (ii) development of an immune response to the material (i.e., development of human antibodies by the patient in response to the antibody treatment, or generation of a HAMA response); and (iii) toxicity to normal cells expressing 158P1D7. Standard testing and follow-up are used to monitor a variety of these safety issues. The 158P1D7 ADC was found to be safe for human administration.

[0625] Example 9

[0626] Detection of 158P1D7 protein in cancer patient samples by IHC

[0627] Patient tumor specimens from patients with (i) bladder cancer, (ii) breast cancer, (iii) lung cancer, and (iv) glioblastoma were tested for expression of 158P1D7 protein by immunohistochemistry. Briefly, formalin-fixed, paraffin-embedded tissues were cut into four (4) micron sections and mounted on slides. The sections were dewaxed, rehydrated, and treated with Citra antigen retrieval solution (Biogenex, San Ramon, CA) at 95°C for 15 minutes in an EZ-Retriever microwave (Biogenex, San Ramon, CA). The sections were then treated with a 3% hydrogen peroxide solution to inactivate endogenous peroxidase activity. Serum-free protein block (Dako, Carpentria, CA) was used to inhibit nonspecific binding and then incubated with monoclonal mouse anti-158P1D7 antibody or isotype control. Next, the sections were stained with Super Sensitive Immunofluorescence ... TM The sections were processed with a polymer-horseradish peroxidase (HRP) detection system consisting of the following steps: TM The sections were then incubated in 1% paraformaldehyde (PAGE) and 1% paraformaldehyde (PAGE) reagents and then incubated with polymer-HRP secondary antibody conjugate (Biogene, San Ramon, California). The sections were then developed using a DAB kit (Biogene, San Ramon, California). Nuclear staining was performed using hematoxylin and analyzed by bright field microscopy. Specific staining indicated by brown staining in patient samples was detected using 158P1D7 immunoreactive antibody. See Figures 9 (A), 9 (C), 9 (E) and 9 (G). In contrast, the control antibody did not stain any patient samples. See Figures 9 (B), 9 (D), 9 (F) and 9 (H).

[0628] The results showed the expression of 158P1D7 in tumor cells of patient bladder cancer, breast cancer, lung cancer and glioblastoma. These results show that 158P1D7 is expressed in human cancers and that antibodies against this antigen and an antibody drug conjugate called Ha15-10ac12vcMMAE can be used for diagnostic and therapeutic purposes. (Figure 9).

[0629] Various website data contents, various publications, patent applications and patents are cited throughout this application. (Websites can be cited by their uniform resource locators or URLs, addresses on the World Wide Web.) The disclosures of each of these references are incorporated herein by reference in their entirety.

[0630] The present invention is not limited to the scope of the embodiments described herein, which are intended only to illustrate individual aspects of the present invention, and any functional equivalents are also within the scope of the present invention. Various modifications to the inventive models and methods other than those described herein will be apparent to those skilled in the art through the above description and teachings, and the modifications are also intended to fall within the scope of the present invention. Such modifications or other embodiments may be implemented without departing from the true scope and spirit of the present invention.

[0631] sheet

[0632] Table I: Tissues expressing 158P1D7 during malignant transformation Malignant glioma

[0633] lung

[0634] bladder

[0635] Mammary Table II: Amino Acid Abbreviations

[0636] Single letter Three letters Full name F Phe Phenylalanine L Leu Leucine S Ser Serine Y Tyr Tyrosine C Cys Cysteine W Trp Tryptophan P Pro Proline H His Histidine Q Gln Glutamine R Arg Arginine I Ile Isoleucine M Met Methionine T Thr Threonine N Asn Asparagine K Lys Leucine V Val Valine A Ala Alanine D Asp Aspartic acid E Glu Glutamate G Gly Glycine

[0637] Table III: Amino Acid Substitution Matrix

[0638] Modified from GCG software 9.0 BLOSUM62 amino acid substitution matrix (module substitution matrix). The higher the value, the greater the probability of finding the substitution in the related natural protein.

[0639]

[0640] Table IV. General method for synthesizing vcMMAE

[0641] in: AA1 = amino acid 1

[0642] AA2 = amino acid 2

[0643] AA5 = amino acid 5

[0644] DIL = Aplysia isoleucine DAP = Aplysia proline Linker = Val-Cit (vc)

[0645]

[0646] Table V Positions of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 identified by Kabat, Chothia and contact schemes, respectively. For CDR-H1, the residue numbers are listed using the Kabat and Chothia numbering schemes.

[0647]

[0648] Table VI. Mean fluorescence intensity (MFI) value in FACS assay

[0649] nM Ha15-10ac12vcMMAE 6.667 155.0 2.222 149.6 0.741 140.9 0.247 133.8 0.082 124.0 0.027 107.6 0.0091 87.0 0.0030 60.8 0.0010 25.7 0.0003 14.9 0.0001 4.3

[0650] Table VII. Ha15-10ac12vcMMAE FACS MFA values ​​for binding assay

[0651] UMUC-AGS15 SW780 CHP-212 MFI 115 159 142 .

Claims

1. An antibody-drug conjugate comprising: An antibody or antigen-binding fragment thereof that binds to a 158P1D7 protein, wherein the antibody or antigen-binding fragment thereof is coupled to 1-20 units of monomethyl auristatin E (MMAE) via a linker, and wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a CDR-H1 consisting of an amino acid sequence in the range of 31-35 of SEQ ID NO:7, a CDR-H2 consisting of an amino acid sequence in the range of 50-66 of SEQ ID NO:7, and a CDR-H3 consisting of an amino acid sequence in the range of 99-109 of SEQ ID NO:7, and the light chain variable region comprising a CDR-L1 consisting of an amino acid sequence in the range of 24-39 of SEQ ID NO:8, a CDR-L2 consisting of an amino acid sequence in the range of 55-61 of SEQ ID NO:8, and a CDR-L3 consisting of an amino acid sequence in the range of 94-102 of SEQ ID NO:

8.

2. The antibody-drug conjugate of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region consisting of an amino acid sequence in the range of positions 1 to 120 of SEQ ID NO: 7 and a light chain variable region consisting of an amino acid sequence in the range of positions 1 to 113 of SEQ ID NO:

8.

3. The antibody drug conjugate of claim 1, wherein the antibody comprises a heavy chain consisting of an amino acid sequence in the range of positions 1 to 446 of SEQ ID NO: 7 and a light chain consisting of an amino acid sequence in the range of positions 1 to 219 of SEQ ID NO:

8.

4. An antibody-drug conjugate, comprising an antibody or an antigen-binding fragment thereof that binds to a 158P1D7 protein, wherein the antibody or the antigen-binding fragment thereof is coupled to 1-20 units of monomethyl auristatin E (MMAE) via a linker, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR consisting of an amino acid sequence according to Kabat numbering of a complementarity determining region (CDR) in a heavy chain variable region of an antibody produced by Chinese hamster ovary (CHO) and deposited with American Type Culture Collection (ATCC) Accession No. PTA-13102, and the light chain variable region comprises a CDR consisting of an amino acid sequence according to Kabat numbering of a CDR in a light chain variable region of an antibody produced by Chinese hamster ovary (CHO) and deposited with American Type Culture Collection (ATCC) Accession No. PTA-13102.

5. The antibody-drug conjugate of claim 4, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is composed of the amino acid sequence of the heavy chain variable region of an antibody produced by Chinese hamster ovary (CHO) deposited with American Type Culture Collection (ATCC) Accession No. PTA-13102, and the light chain variable region is composed of the amino acid sequence of the light chain variable region of an antibody produced by Chinese hamster ovary (CHO) deposited with American Type Culture Collection (ATCC) Accession No. PTA-13102.

6. The antibody drug conjugate of claim 4, wherein the antibody comprises a heavy chain and a light chain, the heavy chain consisting of the amino acid sequence of the heavy chain of an antibody produced by Chinese hamster ovary (CHO) deposited with American Type Culture Collection (ATCC) Accession No. PTA-13102, and the light chain consisting of the amino acid sequence of the light chain of an antibody produced by Chinese hamster ovary (CHO) deposited with American Type Culture Collection (ATCC) Accession No. PTA-13102.

7. The antibody drug conjugate of any one of claims 1, 2, 4 or 5, wherein the antigen binding fragment is Fab, F(ab')2, Fv or scFv.

8. The antibody drug conjugate of any one of claims 1 to 6, wherein the antibody is a fully human antibody.

9. The antibody drug conjugate of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is recombinantly produced.

10. The antibody drug conjugate of any one of claims 1 to 6, wherein the antibody drug conjugate comprises 1 to 8 units of MMAE per antibody or antigen-binding fragment thereof.

11. The antibody drug conjugate of any one of claims 1 to 6, wherein the antibody drug conjugate comprises 3 to 5 units of MMAE per antibody or antigen-binding fragment thereof.

12. The antibody drug conjugate of any one of claims 1-6, wherein the linker comprises valine-citrulline (Val-Cit).

13. The antibody drug conjugate of any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof is connected to MMAE via a linker unit of the formula -Aa-Ww-Yy-; wherein -A- is a stretching unit, a is 0 or 1; -W- is an amino acid unit, w is an integer of 0-12; and -Y- is a self-immolative spacer unit, y is 0, 1 or 2.

14. The antibody drug conjugate of claim 13, wherein the stretching unit has a structure of Formula I; the amino acid unit is Val-Cit; and the spacer unit is a PAB group having a structure of Formula II:

15. The antibody drug conjugate of claim 14, wherein the stretcher forms a bond with a sulfur atom of the antibody or antigen-binding fragment thereof; and wherein the spacer is linked to MMAE via a carbamate group.

16. The antibody drug conjugate according to any one of claims 1 to 6, wherein the antibody drug conjugate has the following structure: Wherein L- represents an anti-158P1D7 antibody or an antigen-binding fragment thereof, and the range of p is 1-8. The antibody drug conjugate of claim 16 , wherein p is in the range of 3-4.

18. The antibody drug conjugate of any one of claims 1-6, wherein the antibody is a fully human monoclonal antibody, wherein the antibody comprises a human heavy chain IgG2 constant region and a human light chain Ig kappa constant region, and wherein the antibody is coupled to 3-4 units of MMAE.

19. A pharmaceutical composition comprising a therapeutically effective amount of the antibody drug conjugate according to any one of claims 1 to 6 and a pharmaceutically acceptable excipient.

20. The pharmaceutical composition of claim 19, wherein the antibody or antigen-binding fragment thereof is conjugated to 3.5-3.7 units of MMAE.

21. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition is used to treat glioblastoma, lung cancer, bladder cancer or breast cancer.

22. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition is administered in combination with a radiotherapeutic or chemotherapeutic agent.

23. The pharmaceutical composition of claim 19, further comprising a chemotherapeutic agent.

24. Use of the antibody drug conjugate of any one of claims 1 to 6 in the preparation of a medicament for treating cancer in a subject, wherein the cancer is selected from glioblastoma, lung cancer, bladder cancer and breast cancer.

25. Use of the combination of an antibody drug conjugate according to any one of claims 1 to 6 and a chemotherapeutic agent in the preparation of a medicament for treating a cancer in a subject, the cancer being selected from the group consisting of glioblastoma, lung cancer, bladder cancer and breast cancer.

26. Use of the antibody drug conjugate of any one of claims 1 to 6 in the preparation of a medicament for treating cancer in a subject, the cancer being selected from glioblastoma, lung cancer, bladder cancer and breast cancer, wherein the subject is also receiving radiotherapy.

27. The use of claim 24, wherein the subject is a human subject.

28. The use of claim 25, wherein the subject is a human subject.

29. The use of claim 26, wherein the subject is a human subject.

30. The use of claim 27, wherein the cancer is glioblastoma.

31. The use of claim 28, wherein the cancer is glioblastoma.

32. The use of claim 29, wherein the cancer is glioblastoma.

33. The use of claim 27, wherein the cancer is lung cancer.

34. The use of claim 28, wherein the cancer is lung cancer.

35. The use of claim 29, wherein the cancer is lung cancer.

36. The use of claim 27, wherein the cancer is bladder cancer.

37. The use of claim 28, wherein the cancer is bladder cancer.

38. The use of claim 29, wherein the cancer is bladder cancer.

39. The use of claim 27, wherein the cancer is breast cancer.

40. The use of claim 28, wherein the cancer is breast cancer.

41. The use of claim 29, wherein the cancer is breast cancer.

42. The use of claim 24, wherein the antibody drug conjugate is formulated for administration to a subject in an amount of 1, 2, 3, 4, 5 or 6 mg / kg body weight.

43. The use of claim 25, wherein the antibody drug conjugate is formulated for administration to a subject in an amount of 1, 2, 3, 4, 5 or 6 mg / kg body weight.

44. The use of claim 26, wherein the antibody drug conjugate is formulated for administration to a subject in an amount of 1, 2, 3, 4, 5 or 6 mg / kg body weight.

45. The use of claim 24, wherein the antibody drug conjugate is formulated for administration to a subject in an amount of 1 to 5 mg / kg body weight.

46. ​​The use of claim 25, wherein the antibody drug conjugate is formulated for administration to a subject in an amount of 1 to 5 mg / kg body weight.

47. The use of claim 26, wherein the antibody drug conjugate is formulated for administration to a subject in an amount of 1 to 5 mg / kg body weight.

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