Antibody drug conjugates (ADC) that bind to 191p4d12 proteins

Abstract

This invention provides an antibody-drug conjugate (ADC) that binds to the 191P4D12 protein and its variants. 191P4D12 exhibits tissue-specific expression in normal adult tissues and shows aberrant expression in the cancers listed in Table I. Therefore, the ADC of this invention provides a therapeutic composition for treating cancer.

Description

Technical Field

[0001] The present invention described herein relates to antibodies that bind to a protein called 191P4D12, their binding fragments, and antibody-drug conjugates (ADCs). Furthermore, the present invention relates to prognostic, preventative, and therapeutic methods and compositions suitable for treating cancers exhibiting 191P4D12. Cross-citation of related applications

[0002] This application is a non-provisional patent application that claims priority to U.S. Provisional Patent Application No. 61 / 387,933, filed September 29, 2010. The contents of each application listed in this paragraph are incorporated herein by reference in their entirety. [Proof of this invention based on a federally funded research institute] []

[0003] not applicable. Prior Technology

[0004] Cancer is the second leading cause of death after coronary artery disease. Millions die from cancer each year worldwide. In the United States alone, the American Cancer Society reports well over half a million deaths annually, with more than 1.2 million new cases diagnosed each year. While deaths from heart disease have significantly decreased, deaths from cancer are generally rising. Cancer is projected to become the leading cause of death by the beginning of the next century.

[0005] Globally, several cancers have emerged as leading killers. Specifically, lung cancer, prostate cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, and bladder cancer represent the leading causes of cancer death. These, and almost all other cancers, share common lethal characteristics. With very few exceptions, metastatic cancer is fatal. Furthermore, even for those who initially survive their primary cancer, shared experiences have shown dramatic changes in their lifespan. Many cancer patients experience severe anxiety due to the knowledge of the possibility of recurrence or treatment failure. Many cancer patients become physically weak after treatment. In addition, many cancer patients experience recurrence.

[0006] Worldwide, prostate cancer is the fourth most common cancer among men. In North America and Northern Europe, it remains the most common cancer in men and the second leading cause of cancer death. In the United States alone, well over 30,000 men die from this disease each year, second only to lung cancer. Despite these large numbers, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone removal therapy, surgical castration, and chemotherapy remain the main treatment methods. Unfortunately, these treatments are ineffective for many men and often have adverse consequences.

[0007] At the forefront of diagnosis, the lack of accurate markers for detecting early localized prostate tumors remains a significant limitation in the diagnosis and control of this disease. Although serum prostate-specific antigen (PSA) testing has become a very useful tool, its specificity and general utility are generally considered to be lacking in several important aspects.

[0008] Advances in identifying other specific markers for prostate cancer have been made possible by the generation of prostate cancer xenografts that can reproduce different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenograft is a prostate cancer xenograft that has survived in severely combined immune deficient (SCID) mice and has demonstrated the ability to mimic the transition from androgen-dependent to androgen-independent behavior (Klein et al. 1997, Nat. Med. 3:402). In recent years, the markers identified for prostate cancer 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).

[0009] While previously identified markers (such as PSA) have been helpful in attempting to diagnose and treat prostate cancer, further identification of other markers and therapeutic targets for prostate and related cancers is needed to improve diagnosis and treatment. In 2000, an estimated 130,200 cases of colorectal cancer were reported in the United States, including 93,800 cases of colon cancer and 36,400 cases of rectal cancer.

[0010] Colorectal cancer is the third most common cancer among both men and women. Between 1992 and 1996, its incidence rate decreased significantly (by 2.1% annually). Studies indicate that this decrease is attributed to increased screening and polyp removal, thus preventing polyps from developing into invasive cancers. In 2000, an estimated 56,300 cancer deaths occurred (47,700 from colon cancer and 8,600 from rectal cancer), accounting for approximately 11% of all cancer deaths in the United States.

[0011] Currently, surgery is the most common form of treatment for colorectal cancer, and it is usually effective for cancers that have not yet spread. For most patients whose cancer has penetrated deep into the intestinal wall or spread to the lymph nodes, chemotherapy or chemotherapy plus radiation therapy is administered before or after surgery. Colorectal cancer sometimes requires a permanent colostomy (creating an opening in the abdomen to drain bodily waste), and rectal cancer occasionally does as well. Effective diagnostic and treatment methods for colorectal cancer are still needed.

[0012] Bladder cancer accounts for approximately 5% of all new cancer cases in the United States (the fifth most common cancer) in men and 3% in women (the eighth most common cancer). The incidence rate is slowly rising with the aging population. In 1998, an estimated 54,500 cases were reported, including 39,500 men and 15,000 women. The age-adjusted incidence rate in the United States was 32 per 100,000 men and 8 per 100,000 women. The historically low male-to-female ratio of 3:1 is likely declining, which is related to women's smoking habits. In 1998, bladder cancer was estimated to cause 11,000 deaths (7,800 men and 3,900 women). The incidence and mortality rates of bladder cancer are rising sharply with age and will become increasingly problematic due to population aging.

[0013] Most bladder cancers recur in the bladder. Transurethral resection (TUR) combined with intravesical chemotherapy or immunotherapy is used to control bladder cancer. The multifocal and recurrent nature of bladder cancer illustrates the limitations of TUR. Most invasive muscle cancers cannot be cured by TUR alone. Radical cystectomy and urinary diversion are the most effective methods for removing cancer, but they have undeniable impacts on urinary and sexual function. There is still a great need for treatment methods that are beneficial to bladder cancer patients.

[0014] In 2000, an estimated 164,100 new cases of lung and bronchial cancer were diagnosed, accounting for 14% of all cancer diagnoses in the United States. The incidence of lung and bronchial cancer has decreased significantly in men, from a high rate of 86.5 per 100,000 in 1984 to 70.0 per 100,000 in 1996. In the 1990s, the rate of increase in women began to slow. In 1996, the incidence rate in women was 42.3 per 100,000.

[0015] In 2000, lung and bronchial cancer was estimated to cause 156,900 deaths, accounting for 28% of all cancer deaths. Between 1992 and 1996, lung cancer mortality rates decreased significantly in men (1.7% annually), while mortality rates in women continued to rise significantly (0.9% annually). Since 1987, more women have died from lung cancer than from breast cancer each year, making it the leading cause of cancer death for women for over 40 years. The decline in lung cancer incidence and mortality rates is likely due to the reduction in smoking rates over the previous 30 years; however, the reduction in smoking behavior among women has lagged behind that of men. Worryingly, while the decline in adult tobacco use has slowed, tobacco use among adolescents is rising again.

[0016] Treatment options for lung and bronchial cancers are determined based on the type and stage of the cancer and include surgery, radiotherapy, and chemotherapy. For many localized cancers, surgery is generally the preferred treatment. Because the disease is often metastatic by the time it is discovered, radiotherapy and chemotherapy are often combined with surgery. Chemotherapy alone or in combination with radiotherapy is an option for treating small cell lung cancer; with this therapy, most patients experience remission, and in some cases, this remission is long-lasting. However, there is an ongoing need for effective treatments and diagnostic methods for lung and bronchial cancers.

[0017] An estimated 182,800 new cases of invasive breast cancer were expected among women in the United States during the 2000 period. Additionally, approximately 1,400 new cases of breast cancer were expected to be diagnosed among men in 2000. After increasing by about 4% annually in the 1980s, the incidence of breast cancer in women stabilized in the 1990s at approximately 110.6 cases per 100,000 women.

[0018] In the United States alone, an estimated 41,200 people (40,800 women and 400 men) died from breast cancer in 2000. Breast cancer is the second leading cause of cancer death among women. According to the latest data, the mortality rate decreased significantly between 1992 and 1996, with the largest decreases observed in young white and Black women. This reduction is largely attributed to earlier detection and improved treatment.

[0019] Considering the medical environment and patient preferences, breast cancer treatment may include mastectomy (local removal of the tumor) and axillary lymph node dissection; mastectomy (surgical removal of the breast) and axillary lymph node dissection; radiotherapy; chemotherapy; or hormone therapy. Two or more methods are often used in combination. Many studies have shown that for early-stage disease, the long-term survival rate after mastectomy plus radiotherapy is similar to that after modified radical mastectomy. Significant advances in reconstruction techniques have provided several options for breast reconstruction after mastectomy. Recently, this reconstruction has been performed simultaneously with mastectomy.

[0020] Local excision of ductal carcinoma in situ (DCIS) with sufficient surrounding normal breast tissue can prevent local recurrence of DCIS. Breast radiation and / or tamoxifen can reduce the incidence of DCIS in other breast tissues. This is important because, without treatment, DCIS can develop into invasive breast cancer. Nevertheless, these treatments still have serious side effects or sequelae. Therefore, effective breast cancer treatment is necessary.

[0021] An estimated 23,100 new cases of ovarian cancer were reported in the United States in 2000. It accounts for 4% of all cancers in women and ranks second among gynecological cancers. The incidence of ovarian cancer decreased significantly between 1992 and 1996. An estimated 14,000 deaths were attributed to ovarian cancer in 2000. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.

[0022] Surgery, radiation therapy, and chemotherapy are treatment options for ovarian cancer. Surgery generally involves the removal of one or both ovaries, fallopian tubes (salpingo-oophorectomy), and uterus (hysterectomy). In some very early-stage tumors, only the affected ovary is removed, especially in young women who wish to have children. In advanced disease, efforts are made to remove all intra-abdominal tissue to enhance the effectiveness of chemotherapy. Effective treatment options for ovarian cancer remain in high demand.

[0023] An estimated 28,300 new cases of pancreatic cancer were diagnosed in the United States in 2000. Over the past 20 years, the incidence of pancreatic cancer in men has decreased. The incidence in women has remained roughly constant and may be beginning to decline. An estimated 28,200 deaths from pancreatic cancer were reported in the United States in 2000. Over the past 20 years, there has been a small but significant decrease in male mortality rates (approximately 0.9% per year), while female mortality rates have increased slightly.

[0024] Surgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer. These options can prolong survival and / or alleviate symptoms in many patients, but are unlikely to be curative for most. There is an urgent need for alternative treatment and diagnostic options for cancer. These options include the use of antibodies, vaccines, and small molecules as treatment forms. Furthermore, these forms are needed in all areas of cancer treatment and research as diagnostic, detection, monitoring, and research tools to advance prior technologies.

[0025] The therapeutic efficacy of monoclonal antibodies (mAbs) (G. Kohler and C. Milstein, Nature 256:495-497 (1975)) is still being investigated. Monoclonal antibodies are currently approved as a therapy for transplantation, cancer, infectious diseases, cardiovascular diseases, and inflammation. Different isotypes have different effector functions. These functional differences are reflected in the different three-dimensional structures of various immunoglobulin isotypes (PM Alzari et al., Annual Rev. Immunol., 6:555-580 (1988)).

[0026] Because mice are easily immunized and recognize most human antigens as foreign antigens, mAbs targeting human targets with therapeutic potential are often derived from mice. However, mouse mAbs have inherent drawbacks as therapeutic agents for humans. Due to their shorter circulating half-life in humans compared to human antibodies, they require more frequent administration. More critically, repeated administration of mouse antibodies to the human immune system elicits a response in which the human immune system recognizes mouse proteins as foreign proteins and produces a human anti-mouse antibody (HAMA) response. This HAMA response can cause allergic reactions and rapid clearance of mouse antibodies from the system, rendering mouse antibody treatment ineffective. To avoid these effects, methods have been devised to induce a human immune system in mice.

[0027] Initial attempts aimed to generate transgenic mice responsive to antigens using antibodies with 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 available selection vectors. The use of yeast artificial chromosome (YAC) selection vectors has played a guiding role in introducing large germline fragments of human Ig loci into transgenic mammals. Most of the human V, D, and J region genes, which are arranged at equal intervals in the human genome and human constant regions, have been introduced into mice using YACs. One such transgenic mouse strain, called XenoMouse®, was purchased from Amgen Fremont, Inc. (Fremont CA). Summary of the Invention

[0028] This invention provides antibodies that bind to the 191P4D12 protein and its polypeptide fragment, binding fragments thereof, and antibody-drug conjugates (ADCs). In some embodiments, the invention comprises a whole-human antibody conjugated with a therapeutic agent. In some embodiments, the limitation is that the entire nucleic acid sequence of Figure 3 is not encoded and / or the entire amino acid sequence of Figure 2 is not prepared. In some embodiments, the entire nucleic acid sequence of Figure 3 is encoded and / or the entire amino acid sequence of Figure 2 is prepared, both using different human unit dosage forms.

[0029] The present invention further provides various immunogenic or therapeutic compositions, such as antibody-drug conjugates, and strategies for treating cancers exhibiting 191P4D12 (such as the tissue cancers listed in Table I). Simple Explanation of the Diagram

[0030] [picture] [1]. The cDNA (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of 191P4D12 are shown in Figure 1. The starting methionine is underlined. The open reading frame extends from nucleic acid 264 to nucleic acid 1796, including the stop codon.

[0031] [picture] [2]. Nucleic acid and amino acid sequences of the 191P4D12 antibody.

[0032] [picture] [2A] [。]Ha22-2(2,4)6.1 heavy chain cDNA (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4). Double underline the leader sequence, underline the variable region of the heavy chain, and dotted underline the human IgG1 constant region.

[0033] [picture] [2B] [。 ]Ha22-2(2,4)6.1 light chain cDNA (SEQ ID NO: 5) and amino acid sequence (SEQ ID NO: 6). Double underline the leader sequence, underline the variable region of the light chain, and add a dashed underline to the human κ constant region.

[0034] [picture] [3] [。] Amino acid sequence of antibody 191P4D12.

[0035] [picture] [3A] [。] The amino acid sequence of the heavy chain of Ha22-2(2,4)6.1 (SEQ ID NO: 7). Double underline the leader sequence, underline the variable region of the heavy chain, and add a dashed underline to the human IgG1 constant region.

[0036] [picture] [3B] [。 ]Ha22-2(2,4)6.1 amino acid sequence of the light chain (SEQ ID NO: 8). Double underline the leader sequence, underline the variable region of the light chain, and add a dashed underline to the human κ constant region.

[0037] [picture] [4]. Comparison of Ha22-2(2,4)6.1 antibody with human Ig germline.

[0038] [picture] [4A] Alignment of the heavy chain of Ha22-2(2,4)6.1 (a portion of SEQ ID NO: 3 and 4) with human Ig germline VH3-48 (SEQ ID NO: 9). []

[0039] [picture] [4B] Alignment of the light chain of Ha22-2(2,4)6.1 (part of SEQ ID NO: 5, 6) with human Ig germline L5 (SEQ ID NO: 10).

[0040] [picture] [5] [。 ]Ha22-2(2,4)6.1 MAb binding assay. [picture] [5A] [:] Flow cytometry was used to detect the binding of Ha22-2(2,4)6.1 MAb. RAT-control and RAT-191P4D12 cells were stained with Ha22-2(2,4)6.1 MAb derived from fusion tumors or CHO cells. Binding was detected by flow cytometry. Results showed that recombinant Ha22-2(2,4)6.1 MAb was secreted in CHO cells and specifically bound to 191P4D12 on the cell surface. [picture] [5B] [:] The binding of Ha22-2(2,4)6.1 Mab was detected using ELISA. The binding of Ha22-2(2,4)6.1 Mab from fusion tumors or CHO cells to the purified extracellular protein of recombinant 191P4D12 was tested by ELISA. The results showed that the binding to 191P4D12 protein from Ha22-2(2,4)6.1 derived from CHO and fusion tumors was consistent.

[0041] [picture] [6]. The affinity of Ha22-2(2,4)6.1vcMMAE was determined by FACS using PC3-human-191P4D12 cells. The affinity was 0.69 Kd.

[0042] [picture] [7]. The affinity of Ha22-2(2,4)6.1vcMMAE was determined by FACS using PC3-cynomolgus monkey-191P4D12 cells. The affinity was 0.34 Kd.

[0043] [picture] [8]. The affinity of Ha22-2(2,4)6.1vcMMAE was determined by FACS using PC3-rat-191P4D12 cells. The affinity was 1.6 Kd.

[0044] [picture] [9] [。] Cytotoxicity mediated by Ha22-2(2,4)6.1vcMMAE. [picture] [9A] PC3-Human-191P4D12: Killed in 4 days / 1500 cells per well. Cytotoxicity assays were performed using PC3-Human-191P4D12 cells. [picture] [9B] [:]PC3-cynomolgus monkey-191P4D12: 1500 cells per well killed in 4 days. Cytotoxicity was assessed using PC3-cynomolgus monkey-191P4D12 cells. [picture] [9C] PC3-rat-191P4D12: 1500 cells per well were killed after 4 days. Cytotoxicity was assessed using PC3-rat-191P4D12 cells. [picture] [9D] PC-3 Neo: 1500 cells per well killed in 4 days. Cytotoxicity was assessed using PC3-Neo cells. []

[0045] [picture]

[10] [。] The domain of Ha22-(2,4)6.1 MAb was located using FACS.

[0046] [picture]

[11] . The Ha22-2(2,4)6.1 MAb domain was located using Western ink dot analysis.

[0047] [picture]

[12] . Ha22-2(2,4)6.1 MAb was evaluated in a subcutaneous tumor formation model of human lung cancer xenograft AG-L4 in SCID mice. The results showed that 191P4D12 MAb did not significantly inhibit tumor growth in human lung cancer xenograft AG-L4 in SCID mice.

[0048] [picture]

[13] . Ha22-2(2,4)6.1 MAb was evaluated in a subcutaneous tumor formation model of human pancreatic cancer xenograft HPAC in SCID mice. The results showed that 191P4D12 MAb did not inhibit tumor growth in human pancreatic xenografts in SCID mice compared with the control antibody.

[0049] [picture]

[14] . Ha22-2(2,4)6.1 MAb was evaluated in a subcutaneous tumor formation model of human pancreatic cancer xenograft AG-Panc3 in SCID mice. The results showed that 191P4D12 MAb did not inhibit tumor growth in human pancreatic xenografts in SCID mice compared with the control antibody.

[0050] [picture]

[15] . Efficacy of Ha22-2(2,4)6.1-vcMMAE in subcutaneous human lung cancer xenografts AG-L4 from SCID mice. The results showed that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of subcutaneously implanted AG-L4 lung cancer xenografts in nude mice compared with the treated and untreated control groups.

[0051] [picture]

[16] . Efficacy of Ha22-2(2,4)6.1-vcMMAE in subcutaneously implanted human breast cancer xenografts BT-483 in SCID mice. The results showed that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of subcutaneously implanted BT-483 breast tumor xenografts in SCID mice compared with the treated and untreated control ADCs.

[0052] [picture]

[17] . Efficacy of Ha22-2(2,4)6.1-vcMMAE in human bladder cancer xenografts AG-B1 formed subcutaneously in SCID mice. The results showed that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of AG-B1 bladder cancer xenografts compared with the control ADC.

[0053] [picture]

[18] . Efficacy of Ha22-2(2,4)6.1-vcMMAE in human pancreatic cancer xenograft AG-Panc2 formed subcutaneously in SCID mice. The results showed that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of AG-Panc2 pancreatic cancer xenografts compared with the control ADC.

[0054] [picture]

[19] . Efficacy of Ha22-2(2,4)6.1-vcMMAE in human lung cancer xenografts AG-Panc4 formed subcutaneously in SCID mice. The results showed that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of AG-Panc4 pancreatic cancer xenografts compared with the control ADC.

[0055] [picture]

[20] . The efficacy of Ha22-2(2,4)6.1-vcMMAE at comparative doses in human bladder cancer xenografts AG-B8 formed subcutaneously in SCID mice was compared. The results showed that treatment with 10 mg / kg Ha22-2(2,4)6.1vcMMAE significantly inhibited the growth of AG-B8 bladder cancer xenografts compared with 5 mg / kg Ha22-2(2,4)6.1vcMMAE.

[0056] [picture]

[21] . Detection of 191P4D12 protein in cancer patient samples by immunohistochemistry (IHC). [picture] [21(A)] [and] [(B)] Displaying a bladder cancer sample. [picture] [21(C)] [and] [(D)] Displaying a breast cancer sample. [picture] [21(E)] [and] [(F)] Displaying a pancreatic cancer sample. [picture] [21(G)] [and] [(H)] Displays lung cancer sample. [picture] [21(I)] [and] [(J)] Displays ovarian cancer specimens. [picture] [21(K)] [and] [(L)] Displays an esophageal cancer sample. [picture] [21(M)] [and] [(N)] Displaying a sample of head and neck cancer. Implementation

[0057] [Chapter Outline] [] I.) Definition II.) 191P4D12 antibody III.) Overview of Antibody Drug Conjugates III(A). Maytansinoid III(B). Auristatin and dolostatin III(C). Calciamicin III(D). Other cytotoxic agents IV.) Antibody-drug conjugates that bind to 191P4D12 V.) Connecting Subunit VI.) Extended Subunit VII.) Amino acid units VIII.) Spacer Subunit IX.) Drug Unit X.) Drug load XI.) Methods for determining the cytotoxic effects of ADCs XII.) Treatment of cancer manifested by 191P4D12 XIII.) 191P4D12 as a target for antibody-based therapy XIV.) 191P4D12 ADC mixture XV.) Combination therapy XVI.) Sets / Products I.) Definition: Unless otherwise defined, all technical terms, annotations, and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art to which this invention pertains. In some cases, for clarity and / or for timely reference, terms having their commonly understood meanings are defined herein, and the inclusion of such definitions herein should not be construed as indicating a significant difference from definitions generally understood in this art. Many techniques and procedures described or referenced herein are well understood and are commonly used by those skilled in the art using familiar methods, such as Sambrook et al. The widely used molecular selection and colonization methods described in [Molecular Cloning: A Laboratory Manual] 2nd Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Unless otherwise stated, procedures involving the use of commercially available kits and reagents are generally performed according to the manufacturer's specified protocols and / or parameters, as appropriate.

[0058] Unless otherwise indicated herein, when a trade name is used herein, reference to a trade name also refers to a product formulation, a generic drug, or the active pharmaceutical ingredient of a product under that trade name.

[0059] The terms "advanced cancer," "locally advanced cancer," "advanced disease," and "locally advanced disease" refer to cancer that has penetrated the capsule of the relevant tissue, and are intended to include stage C disease according to the American Urological Association (AUA) system; stage C1-C2 disease according to the Whitmore-Jewett system; and stage T3-T4 and N+ disease according to the TNM (tumor, nodule, metastasis) system. Generally, surgery is not recommended for patients with locally advanced disease, and these patients have substantially worse outcomes compared to patients with clinically localized (organ-bound) cancer.

[0060] The abbreviation "AFP" refers to dimethylvaline-valine-dolaproline-phenylalanine-p-phenylenediamine (see formula XVI below).

[0061] The abbreviation "MMAE" refers to monomethyl auristatin E (see formula XI below).

[0062] The abbreviation "AEB" refers to the ester produced by reacting olistatin E with p-acetylbenzoic acid (see formula XX below).

[0063] The abbreviation "AEVB" refers to the ester produced by reacting ozogluconate E with benzoylvaleric acid (see formula XXI below).

[0064] The abbreviation "MMAF" refers to dovaline-valine-dolaisoleucine-dolaproline-phenylalanine (see formula XVIV below).

[0065] Unless otherwise stated, the term "alkyl" refers to a saturated straight-chain or branched hydrocarbon having about 1 to about 20 carbon atoms (and all combinations and sub-combinations of such carbon atoms within a range and specific number), preferably about 1 to about 8 carbon atoms. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, dibutyl, tributyl, 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.

[0066] The alkyl group, alone or as part of another group, may be substituted by one or more groups, preferably one to three groups (and any other substituents selected from halogens), including (but not limited to) -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), -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 each R' is independently selected from -H, -C1-C8 alkyl, -C 2-C8 alkenyl, -C2-C8 ynyl or -aryl, wherein the -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl and -C2-C8 ynyl may be further substituted by one or more groups, such groups including (but not limited to) -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH 2. -C(O)NHR'', -C(O)N(R'') 2, -NHC(O)R'', -SR'', -SO 3R'', -S(O) 2R'', -S(O)R'', -OH, -N 3, -NH 2, -NH(R''), -N(R'') 2 and -CN, wherein each R'' is independently selected from -H, -C 1-C 8 alkyl, -C 2-C 8 alkenyl, -C 2-C 8 alkynyl or -aryl.

[0067] Unless otherwise stated, the terms "alkenyl" and "alkynyl" refer to straight-chain and branched carbon chains having about 2 to about 20 carbon atoms (and all combinations and sub-combinations of such carbon atoms within a range and specific number), preferably about 2 to about 8 carbon atoms. The alkenyl chain has at least one double bond in the chain, and the alkynyl chain has at least one parabond 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, propynyl, ethynyl, propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1-butynyl.

[0068] Alkenyl and alkynyl groups, alone or as part of another group, may be substituted, as appropriate, with one or more groups, preferably one to three groups (and any other substituents selected from halogens), 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 each R' is independently selected from -H, -C1-C 8-alkyl, -C2-C8 alkenyl, -C2-C8 ynyl or -aryl, wherein the -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl and -C2-C8 ynyl may be further substituted with one or more substituents as appropriate, such substituents including (but not limited to) -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH 2. -C(O)NHR'', -C(O)N(R'') 2, -NHC(O)R'', -SR'', -SO 3R'', -S(O) 2R'', -S(O)R'', -OH, -N 3, -NH 2, -NH(R''), -N(R'') 2 and -CN, wherein each R'' is independently selected from -H, -C 1-C 8 alkyl, -C 2-C 8 alkenyl, -C 2-C 8 alkynyl or -aryl.

[0069] Unless otherwise stated, the term "alkylene" refers to a saturated branched or straight-chain hydrocarbon group having about 1 to about 20 carbon atoms (and all combinations and sub-combinations of such carbon atoms within a range and specific number), preferably about 1 to about 8 carbon atoms, and having two monovalent groups at the center obtained by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of the 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 similar groups. The alkyl group, alone or as part of another group, may be substituted by one or more groups, preferably one to three groups (and any other substituents selected from halogens), including (but not limited to) -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), -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 each R' is independently selected from -H, -C1-C8 alkyl, -C 2-C8 alkenyl, -C2-C8 ynyl or -aryl, wherein the -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl and -C2-C8 ynyl may be further substituted with one or more substituents as appropriate, such substituents including (but not limited to) -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH 2. -C(O)NHR'', -C(O)N(R'') 2, -NHC(O)R'', -SR'', -SO 3R'', -S(O) 2R'', -S(O)R'', -OH, -N 3, -NH 2, -NH(R''), -N(R'') 2 and -CN, wherein each R'' is independently selected from -H, -C 1-C 8 alkyl, -C 2-C 8 alkenyl, -C 2-C 8 alkynyl or -aryl.

[0070] Unless otherwise stated, the term "alkenyl" refers to an alkyl group, if applicable, containing at least one carbon-carbon double bond and which is substituted. Exemplary alkenyl groups include, for example, vinylene (-CH= CH-) and propenyl group (-CH=CHCH 2-).

[0071] Unless otherwise stated, the term "entynyl" refers to an alkyl group containing at least one carbon-carbon linkage, which may be substituted. Exemplary entynyl groups include, for example, acetylene (-C≡C-), propynyl (-CH 2C≡C-), and 4-pentynyl (-CH 2CH 2CH 2C≡CH-).

[0072] Unless otherwise stated, the term "aryl" refers to a monovalent aromatic hydrocarbon group having 6-20 carbon atoms (and all combinations and sub-combinations of such carbon atoms within a certain range and number) obtained by removing a hydrogen atom from a single carbon atom of a parent aromatic ring system. In illustrative structures, some aryl groups are indicated by "Ar". Typical aryl groups include (but are not limited to) groups derived from benzene, substituted benzene, phenyl, naphthalene, anthracene, biphenyl, and similar groups.

[0073] The aryl group, alone or as part of another group, may be substituted by one or more groups, preferably one to five or even one to two groups, as appropriate. These groups include (but are not limited to) -halogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), -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, -NH 2. -NH(R'), -N(R')2, and -CN, wherein each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl, or -aryl, and wherein such -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl, O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), and -aryl may be further substituted with one or more substituents as appropriate, such substituents including (but not limited to) -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), -H, -C2-C8 alkyl, -C2-C8 alkenyl, -H, -C2-C8 alkenyl ... -8-alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH 2, -C(O)NHR'', -C(O)N(R'') 2, -NHC(O)R'', -SR'', -SO 3R'', -S(O) 2R'', -S(O)R'', -OH, -N 3, -NH 2, -NH(R''), -N(R'') 2 and -CN, wherein each R'' is independently selected from -H, -C 1-C 8 alkyl, -C 2-C 8 alkenyl, -C 2-C 8 alkynyl or -aryl.

[0074] Unless otherwise stated, the term "extrinyl" refers to an aryl group that is, where appropriate, substituted, which is a divalent group (i.e., obtained by removing two hydrogen atoms from the same carbon atom or two different carbon atoms in the parent aromatic ring system) and can be configured in the ortho, meta, or para positions as illustrated by phenyl in the following structures. Typical "-(C1-C8 enylyl)aryl", "-(C2-C8 enylenyl)aryl" and "-(C2-C8 enylynyl)aryl" include (but are not limited to) benzyl, 2-phenylethyl-1-yl, 2-phenylvinyl-1-yl, naphthylmethyl, 2-naphthylethyl-1-yl, 2-naphthylvinyl-1-yl, naphthylbenzyl, 2-naphthylphenylethyl-1-yl and similar groups.

[0075] Unless otherwise stated, the term "heterocycle" refers to a monocyclic, bicyclic, or polycyclic system having 3 to 14 ring atoms (also called ring members), wherein at least one ring atom in at least one ring is a heteroatom selected from N, O, P, or S (and all combinations and sub-combinations of the range and specific number of carbon atoms and heteroatoms therein). A heterocycle may have 1 to 4 independently selected ring heteroatoms from N, O, P, or S. One or more N, C, or S atoms in the heterocycle may be oxidized. Monocyclic heterocycles preferably have 3 to 7 ring members (e.g., 2 to 6 carbon atoms and 1 to 3 independently selected heteroatoms from N, O, P, or S), and bicyclic heterocycles preferably have 5 to 10 ring members (e.g., 4 to 9 carbon atoms and 1 to 3 independently selected heteroatoms from N, O, P, or S). The ring including the heteroatom may be an aromatic or non-aromatic ring. Unless otherwise stated, heterocycles are attached to their side groups at any heteroatom or carbon atom that produces a stable structure.

[0076] Heterocyclic compounds are described in the following literature: Paquette, " "[Principles of Modern Heterocyclic Chemistry]" (WA Benjamin, New York, 1968), especially 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), especially Volumes 13, 14, 16, 19 and 28; and J. Am. Chem. Soc. 82:5566 (1960).

[0077] Examples of "heterocyclic" compounds include, but are not limited to, pyridinyl, dihydropyridinyl, tetrahydropyridinyl (piperidinyl), thiazolyl, pyrimidinyl, furanyl, thiopheneyl, pyrroloyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthyl, indoleyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidinoneyl, pyrrolodylinyl, 2-pyrrolodylinoneyl, pyrrololyl, tetrahydrofuranyl, bistetrahydrofuranyl, tetrahydropiperanyl, bistetrahydropiperanyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, and acrylonitrile. Triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl, thienyl, thienyl, piperanyl, isobenzofuranyl, Alkenyl, styryl, phenoxazyl, 2H-pyrroleyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indoleazinyl, isoydinolyl, 3H-indoleyl, 1H-indazoleyl, purineyl, 4H-quinazinyl, tarazinyl pyridyl, quinolinyl, quinazolinyl, Linolyl, pteridinyl, 4H-carbazolyl, carbazolyl, β-carbazolyl, phenidinyl, acridinel, pyrimidinyl, phenolinyl, phenazinyl, phenazinyl, furazinyl, phenoxazinyl, iso alkyl, Alkyl, imidazolidine, imidazolinyl, pyrazolidine, pyrazolidine, piperazinyl, indololinyl, isoindololinyl The preferred heterocyclic groups include (but are not limited to) benzofuranyl, benzothiopheneyl, indolyl, benzoxoxazolinyl, and isatinoyl.

[0078] The heterocyclic group, alone or as part of another group, may be substituted with one or more groups, preferably one or two groups, as appropriate. These groups include (but are not limited to) -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), -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 each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl or -aryl, and wherein such -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl and -aryl may be further substituted by one or more substituents as appropriate, such substituents including (but not limited to) -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), -C2-C8 ynyl, ... -8-alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH 2, -C(O)NHR'', -C(O)N(R'') 2, -NHC(O)R'', -SR'', -SO 3R'', -S(O) 2R'', -S(O)R'', -OH, -N 3, -NH 2, -NH(R''), -N(R'') 2 and -CN, wherein each R'' is independently selected from -H, -C 1-C 8 alkyl, -C 2-C 8 alkenyl, -C 2-C 8 alkynyl or aryl.

[0079] For example, and without limitation, the carbon-bonded heterocycle can be bonded at the following positions: pyridine at position 2, 3, 4, 5, or 6; pyridazine at position 3, 4, 5, or 6; pyrimidine at position 2, 4, 5, or 6; pyrazine at position 2, 3, 5, or 6; furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole at position 2, 3, 4, or 5; oxazole, imidazole, or thiazole at position 2, 4, or 5; isoxazole, pyrazole, or isothiazole at position 3, 4, or 5; aziridine at position 2 or 3; azacyclobutane at position 2, 3, or 4; quinoline at position 2, 3, 4, 5, 6, 7, or 8; or isoquinoline at position 1, 3, 4, 5, 6, 7, or 8. The heterocycles of the carbon-bonded structures more typically include 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.

[0080] For example, and without limitation, nitrogen-bonded heterocycles can be bonded at the following positions: aziridinium, aziridine, pyrrole, pyrrolidine, 2-pyrrololine, 3-pyrrololine, imidazole, imidazoline, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, or 1H-indazole at position 1; isoindole or isoindoline at position 2; morpholine at position 4; and carbazole or β-carbline at position 9. Nitrogen-bonded heterocycles more commonly include 1-aziridinyl, 1-aziridinebutyl (1-azetedyl), 1-pyrroleyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0081] Unless otherwise stated, the term "carbon ring" refers to a saturated or unsaturated non-aromatic monocyclic, bicyclic, or polycyclic system having 3 to 14 ring atoms (and all combinations and sub-combinations of the range and specific number of carbon atoms therein), wherein all ring atoms are carbon atoms. Monocyclic carbon rings preferably have 3 to 6 ring atoms, more preferably 5 or 6 ring atoms. Bicyclic carbon rings preferably have 7 to 12 ring atoms, for example, arranged in bicyclic [4,5], [5,5], [5,6], or [6,6] systems; or 9 or 10 ring atoms arranged in bicyclic [5,6] or [6,6] systems. The term "carbon ring" includes, for example, monocyclic carbon rings fused to aromatic rings (e.g., monocyclic carbon rings fused to benzene rings). Carbon rings preferably have 3 to 8 carbon ring atoms.

[0082] The carbocyclic group, alone or as part of another group, may be substituted, as appropriate, with one or more groups, preferably one or two groups (and any other substituents selected from halogens), including (but not limited to) -halogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), -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, -NH 2. -NH(R'), -N(R')2, and -CN, wherein each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl, or -aryl, and wherein such -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), and -aryl may be further substituted with one or more substituents as appropriate, such substituents including (but not limited to) -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 ynyl), -H, -C2-C8 alkyl, -C2-C8 alkenyl, -H, -C2-C8 alkenyl ... -8-alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH 2, -C(O)NHR'', -C(O)N(R'') 2, -NHC(O)R'', -SR'', -SO 3R'', -S(O) 2R'', -S(O)R'', -OH, -N 3, -NH 2, -NH(R''), -N(R'') 2 and -CN, wherein each R'' is independently selected from -H, -C 1-C 8 alkyl, -C 2-C 8 alkenyl, -C 2-C 8 alkynyl or -aryl.

[0083] 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-cycloheptadienyl, and -cyclooctadienyl.

[0084] The term "carbocyclic ring" used alone or as part of another group refers to a carbocyclic group as defined above, which is substituted as appropriate and is a divalent group (i.e. obtained by removing two hydrogen atoms from the same carbon atom or two different carbon atoms in the parent carbocyclic system).

[0085] Unless otherwise indicated herein, a hyphen (-) indicates a connection point with a lateral molecule. Therefore, the terms "-(C1-C8 alkyl)aryl" or "-C1-C8 alkyl(aryl)" refer to a C1-C8 alkyl group as defined herein, wherein the alkyl group is attached to a lateral molecule at any carbon atom of the alkyl group, and a hydrogen atom of the alkyl group carbon atom is replaced by an aryl group as defined herein.

[0086] When a particular group is "substituted", the group may have one or more substituents, preferably 1 to 5 substituents, more preferably 1 to 3 substituents, and most preferably 1 to 2 substituents, which are independently selected from the list of substituents. However, the group may generally have any number of substituents selected from halogens. Substituted groups are as indicated.

[0087] The definition of any substituent or variable at a specific position in a molecule is independent of its definition at other positions in that molecule. It should be understood that the substituents and substitution patterns on the compounds of this invention can be selected by those skilled in the art to provide chemically stable compounds that can be readily synthesized using techniques known in this art and the methods described herein.

[0088] As used herein, a protecting group refers to a group that temporarily or permanently and selectively blocks one reactive site in a multifunctional compound. The hydroxyl protecting groups suitable for this invention are pharmaceutically acceptable and may or may not require cleavage of the parent compound after administration to an individual to activate the compound. Cleavage is achieved through normal metabolic processes in the body. Hydroxyl protecting groups are well known in this art; see TW Greene and PGM Wuts. [Protective Groups in Organic Synthesis] (John Wiley & sons, 3rd edition) (incorporated hereto in its entirety for all purposes), and includes, for example, ethers (e.g., alkyl ethers and silyl ethers, including, for example, dialkylsilyl ethers, trialkylsilyl ethers, dialkylalkoxysilyl ethers), esters, carbonates, carbamates, sulfonates and phosphate protecting groups. Examples of hydroxyl protecting groups include (but are not limited to) methyl ethers; methoxymethyl ethers, methyl thiomethyl ethers, (phenyl dimethyl silyl)methoxymethyl ethers, benzoxymethyl ethers, p-methoxybenzoxymethyl ethers, p-nitrobenzoxymethyl ethers, o-nitrobenzoxymethyl ethers, (4-methoxyphenoxy)methyl ethers, guaiacol methyl ethers, tributoxymethyl ethers, 4-pentenoxymethyl ethers, silaneoxymethyl ethers, 2-methoxyethoxymethyl ethers, 2,2,2-trichloroethoxymethyl ethers, bis(2-chloroethoxy)methyl ethers, 2-(trimethyl silyl) Ethoxymethyl ether, methoxymethyl ether, tetrahydropiperanyl ether, 1-methoxycyclohexyl ether, 4-methoxytetrahydrothiopiperanyl ether, 4-methoxytetrahydrothiopiperanyl 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, tetrahydrothiofuranyl ether; substituted ethyl 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-benzoxyethyl ether, 1-methyl-1-benzoxy-2-fluoroethyl ether, 1-methyl-1-phenoxyethyl ether; 2-trimethylsilyl ether, tributyl 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, dimethyl isopropylsilyl ether, diethyl isopropylsilyl ether, dimethylhexylsilyl ether, tri... Butyl dimethyl silicate ether, diphenyl methyl silicate ether, benzoyl carbamate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenylacetate, benzoate, alkyl carbonate methyl ester, alkyl carbonate 9-pyromethyl ester, alkyl carbonate ethyl ester, alkyl carbonate 2,2,2,-trichloroethyl ester, 1,1,-dimethyl-2,2,2-trichloroethyl carbonate, alkyl sulfonate, methane sulfonate, benzyl methyl sulfonate, toluene sulfonate, methylene acetal, ethyl acetal, and tert-butylmethyl ketal.Preferred protecting groups are represented by the formulas -Ra, -Si(Ra)(Ra)(Ra), -C(O)Ra, -C(O)ORa, -C(O)NH(Ra), -S(O)2Ra, -S(O)2OH, P(O)(OH)2, and -P(O)(OH)ORa, wherein Ra is C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, -C1-C20 alkylene (carbocyclic), -C2-C20 alkenyl (carbocyclic), -C2-C20 alynyl (carbocyclic), -C6-C10 aryl, -C1-C20 alkylene (aryl), -C2-C20 alkenyl (aryl), -C2-C20 alynyl (aryl), -C1-C20 alkylene (aryl), -C2-C20 alynyl (aryl), -C1-C20 alkylene (aryl), -C2-C20 alkylene ... 20-Phenylalkyl (heterocyclic), -C 2-C 20-Phenylenyl (heterocyclic), or -C 2-C 20-Phenyneyl (heterocyclic), wherein such alkyl, alkenyl, ynylalkyl, phenylenyl, phenyneyl, aryl, carbocyclic, and heterocyclic groups, alone or as part of another group, are substituted as appropriate.

[0089] For the purposes of this article, "altering the natural glycosylation pattern" is intended to mean deleting one or more carbohydrate moieties present in the natural sequence 191P4D12 (by removing potential glycosylation sites or by deleting glycosylation via chemical and / or enzymatic methods), and / or adding one or more glycosylation sites not present in the natural sequence 191P4D12. Furthermore, this phrase includes qualitative changes in the glycosylation of natural proteins, involving changes in the properties and proportions of the various carbohydrate moieties present.

[0090] The term "analyte" refers to a molecule that is structurally similar to or shares similar or corresponding properties with another molecule (such as the 191P4D12-associated protein). For example, analogues of the 191P4D12 protein can be obtained by antibodies that specifically bind to 191P4D12 or by T cells that specifically bind to it.

[0091] Unless otherwise explicitly indicated, the term "antibody" is used in its broadest sense. Therefore, "antibody" can be a naturally occurring antibody or an artificial antibody, such as a monoclonal antibody prepared by a known fusion tumor technique. 191P4D12 antibodies include monoclonal and polyclonal antibodies and fragments containing an antigen-binding domain and / or one or more complementarity-determining regions of such antibodies. As used herein, the term "antibody" refers to any form of antibody or fragment thereof that specifically binds to 191P4D12 and / or exhibits the desired biological activity, and particularly encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they specifically bind to 191P4D12 and / or exhibit the desired biological activity. Any specific antibody may be used in the methods and compositions provided herein. Thus, in one embodiment, the term "antibody" encompasses a molecule comprising at least one variable region from an immunoglobulin light chain molecule and at least one variable region from a heavy chain molecule, the variable regions combining 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 suitable for use in the methods and compositions of this invention can be produced in cell cultures, bacteriophages, or various animals, including (but not limited to) cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. Therefore, in one embodiment, the antibody of this invention is a mammalian antibody. Phage technology can be used to isolate the initial antibody or to generate variants with altered specificity or affinity characteristics. These techniques are conventional and well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, recombinant antibodies can be produced by transfecting host cells with a vector containing a DNA sequence encoding the antibody. One or more vectors can be used to transfect a DNA sequence expressing at least one VL and at least one VH region into host cells. Illustrative descriptions of recombinant methods for antibody production and preparation 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 this invention can be modified by recombinant methods to enhance their efficacy in mediating desired functions.Therefore, modifying antibody systems by substitution using recombinant methods falls within the scope of this invention. Such substitutions are generally conservative. For example, at least one amino acid in the constant region of the antibody may be replaced by different residues. See, for example, U.S. Patent Nos. 5,624,821, 6,194,551, and WO 9958572; and Angal et al., Mol. Immunol. 30: 105-08 (1993). Amino acid modification includes the deletion, addition, and substitution of amino acids. In some cases, these changes are intended to reduce undesirable activities, such as complement-dependent cytotoxicity. Antibodies are typically labeled by covalent or non-covalent linkage to a substance that provides a detectable signal. Various labeling and conjugation techniques are known and widely reported in scientific and patent literature. Such antibodies can be screened for binding to normal or defective 191P4D12. See, for example. [Antibody Engineering: A Practical Approach](Oxford University Press, 1996). Suitable antibodies with the desired biological activity can be identified using the following in vitro assays, including (but not limited to): proliferation, migration, adhesion, soft agar growth, angiogenesis, intercellular communication, apoptosis, transport, signal transduction, and the following in vivo assays, such as inhibition of tumor growth. The antibodies described herein can also be used for diagnostic applications. As capture antibodies or non-neutralizing antibodies, they can be screened for their ability to bind to specific antigens without inhibiting receptor binding or biological activity of the antigen. As neutralizing antibodies, antibodies can be used for competitive binding assays. They can also be used to quantify 191P4D12 or its receptor.

[0092] As used herein, the term "antigen-binding portion" or "antibody fragment" (or simply "antibody portion") of an antibody refers to one or more fragments of the 191P4D12 antibody that retain the ability to specifically bind to antigens (e.g., 191P4D12 and its variants; Figure 1). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments covered by the term "antigen-binding portion" of an antibody include: (i) Fab fragments: monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments: bivalent fragments consisting of two Fab fragments connected by disulfide bridging bonds located in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of VH domains (Ward et al. (1989) Nature 341:544-546); and (vi) separated complementarity-determining regions (CDRs). Furthermore, although the two domains (VL and VH) of the Fv fragment are encoded by separate genes, they can be linked by a synthetic linker using recombination, which enables the preparation of a single protein chain (called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). These single-chain antibodies are also intended to be included within the term "antigen-binding portion" of the antibody. These antibody fragments were obtained using known techniques to those skilled in the art and were screened for efficacy in the same manner as intact antibodies.

[0093] As used herein, any form of "antigen" can be used to generate antibodies specific to 191P4D12. Therefore, the initiating antigen can be a single antigenic determinant, multiple antigenic determinants, or a single intact protein or a combination of an intact protein and one or more immunogenic enhancers known in this art. The initiating antigen can be an isolated full-length protein, a cell surface protein (e.g., immunizing cells transfected with at least a portion of the antigen), or a soluble protein (e.g., immunizing only the extracellular domain portion of the protein). The antigen can be produced in genetically modified cells. The DNA encoding the antigen can be genomic DNA or non-genomic DNA (e.g., cDNA) and encodes at least a portion of the extracellular domain. As used herein, the term "portion" refers to the minimum number of amino acids or nucleic acids that, where appropriate, constitute the immunogenic antigenic determinant of the antigen of interest. Any genetic vector suitable for transforming the cells of interest can be used, including (but not limited to) adenoviral vectors, plastids, and non-viral vectors such as cationic lipids. In one embodiment, the antibody in the methods and compositions herein specifically binds to at least a portion of the extracellular domain of 191P4D12 of interest.

[0094] The antibodies or antigen-binding fragments thereof provided herein may be conjugated with "bioactive agents". As used herein, the term "bioactive agent" refers to any synthetic or naturally occurring compound that binds to an antigen and / or enhances or mediates a desired biological effect to increase the cytotoxicity of the toxin. In one embodiment, the binding fragment suitable for the present invention is a bioactive fragment. As used herein, the term "bioactive" means that the antibody or antibody fragment is capable of binding to a desired antigenic determinant and exerting a biological effect directly or indirectly. Direct effects include (but are not limited to): regulating, stimulating, and / or inhibiting growth signaling; regulating, stimulating, and / or inhibiting anti-apoptotic signaling; regulating, stimulating, and / or inhibiting apoptosis or necrosis signaling; regulating, stimulating, and / or inhibiting ADCC cascade reactions; and regulating, stimulating, and / or inhibiting CDC cascade reactions.

[0095] "Bispecific" antibodies are also applicable to the methods and compositions of this invention. As used herein, the term "bispecific antibody" refers to an antibody that has binding specificity to at least two different antigenic determinants, typically a monoclonal antibody. In one embodiment, the antigenic determinants are derived from the same antigen. In another embodiment, the antigenic determinants are derived from two different antigens. Methods for preparing bispecific antibodies are known in the art. For example, bispecific antibodies can be recombinantly generated using the co-expression of two immunoglobulin heavy / light chain pairs. See, for example, Milstein et al., Nature 305:537-39 (1983). Alternatively, bispecific antibodies can be prepared using chemical bonding. See, for example, Brennan et al., Science 229:81 (1985). Bispecific antibodies comprise bispecific antibody fragments. See, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993), Gruber et al., J. Immunol. 152:5368 (1994).

[0096] The monoclonal antibodies described herein specifically include "chimeric" antibodies, wherein a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass; and fragments of such antibodies, provided that they specifically bind to the target antigen and / or exhibit the desired biological activity (US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)).

[0097] The term "chemotherapeutic agent" refers to all compounds that effectively inhibit tumor growth. Non-limiting examples of chemotherapeutic agents include alkylating agents, such as nitrogen mustard, imine compounds, and alkyl sulfonates; antimetabolites, such as folic acid, purine, or pyrimidine antagonists; mitotic inhibitors, such as anti-tubulin agents, such as vinca alkaloids, oliquistatin, and podophyllotoxin derivatives; cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication, such as DNA minor groove binders; and growth factor receptor antagonists. Additionally, chemotherapeutic agents include cytotoxic agents (as defined herein), antibodies, biomolecules, and small molecules.

[0098] The term "compound" means and encompasses the compound itself and the following (whether explicitly stated or not, and unless the context dictates otherwise): the amorphous and crystalline forms of the compound, including polycrystalline forms, wherein such forms may be part of a mixture or in a separated form; the free acid and free base forms of the compound, which are generally those shown in the structures provided herein; the isomers of the compound, which are optical isomers and tautomers, wherein optical isomers include enantiomers and diastereomers, pinecones and non-pinecones, and optical isomers include separated optical isomers and mixtures of optical isomers, including racemic and non-racemic mixtures; wherein the isomers may be in a separated form or in a mixture with one or more other isomers; and isotopes of the compound. This includes compounds containing deuterium and tritium, and compounds containing radioactive isotopes (including radioactive isotopes effective for treatment and diagnosis); polymeric forms of compounds, including dimers, trimers, etc.; salts of compounds, preferably pharmaceutically acceptable salts, including acid addition salts and base addition salts, including salts having organic and inorganic relative ions, and including zwitterionic forms, wherein if the compound associates with two or more relative ions, the two or more relative ions may be the same or different; and solvates of compounds, including hemisolvents, monosolvents, disolvents, etc., including organic solvates and inorganic solvates, such inorganic solvates including hydrates; wherein if the compound associates with two or more solvent molecules, the two or more solvent molecules may be the same or different. In some cases, references to compounds of the present invention herein will include explicit references to one or more of the above forms, such as salts and / or solvates; however, such references are for emphasis only and should not be construed as excluding other forms identified above.

[0099] As used herein, the term "conservative substitution" refers to an amino acid substitution known to those skilled in the art and generally permissible without altering the biological activity of the resulting molecule. Those skilled in the art will recognize that, in general, substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al., *MOLECULAR BIOLOGY OF THE GENE*, The Benjamin / Cummings Pub. Co., p. 224 (4th edition, 1987)). This illustrative substitution is preferably performed according to the substitutions shown in Tables II and III (ab). For example, such changes include replacing any one of these hydrophobic amino acids with any of isoleucine (I), valine (V), and leucine (L); replacing glutamic acid (E) with aspartic acid (D) and vice versa; replacing aspartic acid (N) with glutamic acid (Q) and vice versa; and replacing threonine (T) with serine (S) and vice versa. Other substitutions can also be considered conserved substitutions depending on the context of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) are generally interchangeable, just as alanine (A) and valine (V) are interchangeable. The relatively hydrophobic methionine (M) is generally interchangeable with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are typically interchangeable at positions where the charge of the amino acid residues is not significantly different and where the pK values ​​of these two amino acid residues are not significantly different. Other alterations may be considered "conservative" under specific circumstances (see, for example, Table III(a) in this paper; Biochemistry, 2nd ed., pp. 13-15, edited by Lubert Stryer (Stanford University); Henikoff et al., PNAS 1992, Vol. 89, pp. 10915-10919; Lei et al., J Biol Chem, May 19, 1995; 270(20):11882-11886). Other substitutions are also permissible and can be determined empirically or according to known conservative substitutions.

[0100] The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression of cellular activity, cellular function, and / or causes cellular damage. This term is intended to include radioactive isotopes, 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) auromycin (e.g., auromycin E, auromycin F, MMAE, and MMAF), auromycin, maytansine, ricin, ricin A chain, combrestatin, duocarmycin, sea hare toxin, doxorubicin, daunorubicin, taxol, cisplatin, CC1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin. Exotoxin (PE)A, PE40, absinthecin, absinthecin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curcin, crotin, kazimidic acid, Sapaonaria officinalis inhibitors, glucocorticoids and other chemotherapeutic agents, and radioactive isotopes such as At 211, I 131, I 125, Y 90, Re 186, Re 188, Sm 153, Bi 212 or 213, P 32 and Lu radioactive isotopes (including Lu 177). Antibodies can also be conjugated with anticancer prodrug activating enzymes capable of converting prodrugs to their active forms.

[0101] As used herein, the term "bifunctional antibody" refers to a small antibody fragment having two antigen-binding sites, which comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) linked together on the same polypeptide chain (VHVL). Because the linker used is too short to allow pairing between the two domains on the same chain, the domain is forced to pair with the complementary domain of the other chain, resulting in two antigen-binding sites. Bifunctional antibodies are described more fully in the following literature: e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993).

[0102] In the presence of 191P4D12 binders on 191P4D12-expressing cells, the term "vacuum" refers to a reduction or elimination of the number of 191P4D12-expressing cells.

[0103] The term "gene product" is used herein to refer to peptides / proteins or mRNA. For example, "gene product of the present invention" is sometimes referred to herein as "cancer amino acid sequence," "cancer protein," "cancer protein listed in Table I," "cancer mRNA," "mRNA of cancers listed in Table I," etc. In one embodiment, the cancer protein is encoded by the nucleic acid of Figure 1. The cancer protein may be a fragment or a full-length protein encoded by the nucleic acid of Figure 1. In one embodiment, sequence identity or similarity is determined using the cancer amino acid sequence. In another embodiment, the sequences are naturally occurring dual gene variants of the proteins encoded by the nucleic acids of Figure 1. In yet another embodiment, the sequences are sequence variants as further described herein.

[0104] "Heteroconjugated" antibodies are applicable to the methods and compositions of this invention. As used herein, the term "heteroconjugated antibody" refers to two antibodies covalently linked together. Such antibodies can be prepared using known methods in synthetic protein chemistry, including the use of cross-linking agents. See, for example, U.S. Patent No. 4,676,980.

[0105] The term "homology" refers to a molecule that exhibits homology with another molecule, for example, having the same or similar chemical residue sequences at corresponding positions.

[0106] In one embodiment, the antibody provided herein is a "human antibody." As used herein, the term "human antibody" refers to an antibody whose entire sequence of light and heavy chain sequences (including complementarity-determining regions (CDRs)) is substantially derived from human genes. In one embodiment, human monoclonal antibodies are prepared using techniques such as trioma technology, human B cell technology (see, for example, Kozbor et al. Immunol. Today 4: 72 (1983)), EBV conversion technology (see, for example, Cole et al. Monoclonal Antibodies And Cancer Therapy 77-96 (1985)), or phage presentation (see, for example, Marks et al. J. Mol. Biol. 222:581 (1991)). In a particular embodiment, human antibodies are generated in transgenic mice. Techniques for preparing such partial to complete human antibodies are known in this art and any of these techniques can be used. According to a particularly preferred embodiment, complete human antibody sequences are prepared in transgenic mice engineered to express human heavy and light chain antibody genes. An illustrative description of the preparation of transgenic mice capable of producing human antibodies can be found in application No. WO 02 / 43478 and U.S. Patent No. 6,657,103 (Abgenix) and subsequent applications. B cells from the transgenic mice producing the desired antibodies can then be fused to prepare fusion tumor cell lines for continuous antibody production. See, for example, U.S. Patent 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).

[0107] As used herein, the term "humanized antibody" refers to an antibody form containing sequences derived from both non-human (e.g., mouse) antibodies and human antibodies. These antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody will contain substantially all of at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the FR regions are FR regions of the human immunoglobulin sequence. The humanized antibody may also, where applicable, contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region. See, for example, U.S. Patent No. 4,816,567 to Cabilly; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; and [Antibody Engineering: A Practical Approach] (Oxford University Press 1996).

[0108] As used in this article, the term "suppression" or "suppression of..." means to reduce a measurable quantity or to prevent it completely.

[0109] The phrases "isolated" or "biologically pure" refer to a substance that is substantially or substantially free of the components that normally accompany it when it exists in its natural state. Therefore, the isolated peptides of this invention preferably do not contain substances that normally associate with such peptides in their original environment. For example, a polynucleotide is called an "isolated" polynucleotide when it is substantially isolated from contaminant polynucleotides that correspond to genes other than the 191P4D12 gene, or to polypeptides complementary to or encoding polypeptides other than the 191P4D12 gene product or fragments thereof. Those skilled in the art can easily obtain isolated 191P4D12 polynucleotides using nucleic acid isolation procedures. For example, a protein is called an "isolated" protein when 191P4D12 protein is removed from cellular components that normally associate with proteins using physical, mechanical, or chemical methods. Those skilled in the art can easily obtain isolated 191P4D12 protein using standard purification methods. Alternatively, isolated proteins can be prepared by chemical means.

[0110] Suitable "labels" include radionuclides, enzymes, receptors, cofactors, inhibitors, fluorescent portions, chemiluminescent portions, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Additionally, the antibodies provided herein can be used as antigen-binding components of fluorobodies. See, for example, Zeytun et al., Nat. Biotechnol. 21:1473-79 (2003).

[0111] 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.

[0112] 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 according to the AUA system and stage TxNxM+ disease according to the TNM system.

[0113] As used herein, the terms "modifier," "test compound," "drug candidate," or their grammatical equivalents describe any molecule intended to be tested for directly or indirectly altering the expression or function of a cancer sequence (e.g., nucleic acid or protein sequence) (e.g., signal transduction, gene expression, protein-protein interactions), such as proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, etc. In one embodiment, the modifier neutralizes the effect of the cancer protein of the present invention. "Neutralizes" means inhibiting or blocking protein activity and its consequent effects on cells. In another embodiment, the modifier neutralizes the effect of the gene and its corresponding protein of the present invention by normalizing the amount of the protein. In preferred embodiments, the modifier alters the phenotype, or the phenotype of the nucleic acid or protein provided herein, or downstream effector pathways. In one embodiment, the modifier suppresses the cancer phenotype to, for example, a normal tissue fingerprint. In another embodiment, the modifier induces the cancer phenotype. Parallel tests are typically performed on multiple test mixtures at different concentrations to obtain differential responses to various concentrations. One of these concentrations is usually used as a negative control, that is, zero concentration or below the detection level.

[0114] Modifiers, drug candidates, or test compounds encompass many chemical categories, but are generally organic molecules, preferably small organic compounds with a molecular weight greater than 100 Daltons and less than about 2,500 Daltons. Preferably, the small molecule is less than 2000 Da, or less than 1500 Da, or less than 1000 Da, or less than 500 Da. Drug candidates contain functional groups structurally necessary for protein interaction, especially hydrogen bonds, and generally include at least an amine, carbonyl, hydroxyl, or carboxyl group, preferably at least two of the functional groups. Drug candidates often contain cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Modifiers also include biomolecules such as peptides, sugars, fatty acids, steroids, purines, pyrimidines, their derivatives, structural analogs, or combinations thereof. Peptides are particularly preferred. One class of regulators are, for example, peptides having about 5 to about 35 amino acids, preferably about 5 to about 20 amino acids, and particularly preferably about 7 to about 15 amino acids. Preferably, the cancer regulatory protein is soluble, including a non-transmembrane region and / or having an N-terminal Cys that facilitates dissolution. In one embodiment, the C-terminus of the fragment remains in a free acid form and the N-terminus is a free amine that facilitates coupling (i.e., coupling to cysteine). In one embodiment, the cancer protein of the present invention is conjugated with an immunogenic agent as discussed herein. In one embodiment, the cancer protein is conjugated with BSA. The peptides of the present invention (e.g., having a preferred length) can be linked to each other or to other amino acids to form longer peptides / proteins. Regulatory peptides can be digests of naturally occurring proteins as outlined above, random peptides, or "biased" random peptides. In a preferred embodiment, the peptide / protein-based regulator is an antibody and its fragment as defined herein.

[0115] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical except for the possible presence of small amounts of naturally occurring mutations. Monoclonal antibodies exhibit high specificity against a single antigenic determinant. In contrast, conventional (multiclonal) antibody formulations typically comprise multiple antibodies targeting (or specific to) different antigenic determinants. In one embodiment, a multiclonal antibody comprises a plurality of monoclonal antibodies that, within a single antigen containing multiple antigenic determinants, exhibit different antigenic determinant specificities, affinities, or affinity. The modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, a monoclonal antibody intended for use according to the invention may be prepared by the fusion-tumor method originally described by Kohler et al., Nature 256:495 (1975), or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques described, for example, those described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991). Generally, these monoclonal antibodies are measured by ELISA and are typically bound to a concentration of at least about 1 μM, more generally at least about 300 nM, usually at least about 30 nM, preferably at least about 10 nM, more preferably at least about 3 nM, or better.

[0116] "Pharmaceutical excipients" include substances such as adjuvants, carriers, pH adjusters and buffers, tension modifiers, humectants, preservatives and similar pharmaceutical agents.

[0117] "Medically acceptable" means a composition that is non-toxic, inert, and / or physiologically compatible with humans or other mammals.

[0118] The term "polynucleotide" refers to a polymer of nucleotides (ribonucleotides, deoxynucleotides, or modified forms of any type of nucleotide) with a length of at least 10 base pairs, and is intended to include both single-stranded and double-stranded forms of DNA and / or RNA. In this art, this term is often used interchangeably with "oligonucleotide." Polynucleotides may contain the nucleotide sequences disclosed herein, where, for example, thymidine (T) shown in Figure 1 may also be uracil (U); this definition relates to the differences in the chemical structures of DNA and RNA, particularly to the observation that one of the four major bases in RNA is uracil (U) rather than thymidine (T).

[0119] The term "peptide" refers to a polymer having at least about 4, 5, 6, 7, or 8 amino acids. Throughout this specification, the standard three-letter or one-letter names for amino acids are used. In this art, this term is often used interchangeably with "peptide" or "protein."

[0120] "Recombinant" DNA or RNA molecules are DNA or RNA molecules that have been manipulated in vitro.

[0121] As used herein, the terms "single-chain Fv" or "scFv" or "single-chain" antibody system refer to antibody fragments containing both the VH and VL domains of the antibody, wherein these domains are contained within a single polypeptide chain. Fv polypeptides generally further include a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun. [The Pharmacology Of Monoclonal Antibodies] [,] Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0122] As used herein, the terms "specificity" and "specific binding" refer to the selective binding of an antibody to a target antigen determinant. The binding specificity of an antibody is tested by comparing its binding to an appropriate antigen with its binding to an irrelevant antigen or a mixture of antigens under a specific set of conditions. An antibody is considered specific if its binding to an appropriate antigen is at least 2, 5, 7, and preferably 10 times greater than its binding to an irrelevant antigen or a mixture of antigens. In one embodiment, a specific antibody is an antibody that binds only to the 191P4D12 antigen and not to irrelevant antigens. In another embodiment, a specific antibody is an antibody that binds to the human 191P4D12 antigen but not to non-human 191P4D12 antigen, and has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% amino acid homology to the 191P4D12 antigen. In another embodiment, the specific antibody is an antibody that binds to both human and mouse 191P4D12 antigens, but with a higher degree of binding to the human antigen. In another embodiment, the specific antibody is an antibody that binds to both human and primate 191P4D12 antigens, but with a higher degree of binding to the human antigen. In another embodiment, the specific antibody binds to both human 191P4D12 antigen and any non-human 191P4D12 antigen, but with a higher degree of binding to the human antigen or any combination thereof.

[0123] As used herein, “treatment” or “therapeutic” and related grammatical terms refer to any improvement in the outcome of any disease, such as increased survival time, reduced morbidity, and / or reduced side effects as a byproduct of alternative treatments; as is readily understood in this technique, complete cure of the disease is not required for the treatment, but is preferred.

[0124] The term "variant" refers to a molecule that exhibits a variation from the described type or normal type, such as a protein having one or more different amino acid residues at the corresponding position in a specifically described protein (e.g., the 191P4D12 protein shown in Figure 1). Analogs are one example of variant proteins. Splice isoforms and single nucleotide polymorphisms (SNPs) are other examples of variants.

[0125] The "191P4D12 protein" and / or "191P4D12-related proteins" of this invention include proteins specifically identified herein (see Figure 1), as well as paired gene variants, conserved substitution variants, analogs, and homologs that can be isolated / produced and characterized without excessive experimentation using methods readily available herein or in this art. It also includes fusion proteins combining different 191P4D12 proteins or portions thereof, and fusion proteins of 191P4D12 protein and heterologous polypeptides. These 191P4D12 proteins are collectively referred to as 191P4D12-related proteins, the proteins of this invention, or 191P4D12. The term "191P4D12-related protein" refers to a polypeptide fragment or 191P4D12 protein sequence having the following numbers of amino acids: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more; or at least 30, 35, 40, 45, 50, 55, 60, 65, 7 0, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 330, 335, 339 or more amino acids. II.) 191P4D12 antibody

[0126] Another embodiment of the present invention provides an antibody that binds to a 191P4D12-related protein (see Figure 1). In one embodiment, the antibody binding to the 191P4D12-related protein is an antibody that specifically binds to the 191P4D12 protein containing the amino acid sequence of SEQ ID NO.: 2. Antibodies that specifically bind to the 191P4D12 protein containing the amino acid sequence of SEQ ID NO.: 2 include antibodies that can bind to other 191P4D12-related proteins. For example, an antibody binding to the 191P4D12 protein containing the amino acid sequence of SEQ ID NO.: 2 can bind to 191P4D12-related proteins, such as 191P4D12 variants and their homologs or analogs.

[0127] The 191P4D12 antibody of this invention is particularly suitable for prognostic, imaging, and therapeutic methods for cancer (see, for example, Table I). Similarly, these antibodies are suitable for the treatment and / or prognosis of colon cancer and other cancers to the extent that 191P4D12 is also expressed or overexpressed in these other cancers. Furthermore, intracellular expression antibodies (e.g., single-chain antibodies) are therapeutically suitable for treating cancers involving 191P4D12 expression, such as advanced or metastatic colon cancer or other advanced or metastatic cancers.

[0128] Various methods for preparing antibodies, specifically monoclonal antibodies, are well known in this art. For example, antibodies can be prepared by immunizing a suitable mammalian host with 191P4D12-related proteins, peptides, or fragments in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). Alternatively, fusion proteins of 191P4D12, such as the 191P4D12 GST fusion protein, can also be used. In one particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence shown in Figure 1 is prepared and then used as an immunogen to generate appropriate antibodies. In another embodiment, the 191P4D12-related protein is synthesized and used as an immunogen.

[0129] In addition, the naked DNA immunization technique known in this technology (with or without purified 191P4D12-associated protein or 191P4D12-expressing cells) was used to generate an immune response against the encoded immunogen (for comments, see Donnelly et al. 1997, Ann. Rev. Immunol. 15: 617-648).

[0130] The amino acid sequence of the 191P4D12 protein, as shown in Figure 1, can be analyzed to select specific regions of the 191P4D12 protein for antibody production. For example, hydrophobicity and hydrophilicity analysis of the 191P4D12 amino acid sequence can be used to identify hydrophilic regions within the 191P4D12 structure. Regions of the 191P4D12 protein exhibiting immunogenic structures, as well as other regions and domains, can be readily identified using a variety of other methods known in this technique, such as the Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz, or Jameson-Wolf assays. Hydrophilicity profiles can be generated using the methods described in Hopp, TP, and Woods, KR, 1981, Proc. Natl. Acad. Sci. USA 78:3824-3828. Hydrophobicity profiles can be generated using the methods described in Kyte, J. and Doolittle, RF, 1982, J. Mol. Biol. 157:105-132. Approachable residue percentage (%) profiles can be generated using the methods described in Janin J., 1979, Nature 277:491-492. Average flexibility profiles can be generated using the methods described in Bhaskaran R., Ponnuswamy PK, 1988, Int. J. Pept. Protein Res. 32:242-255. β-turn profiles can be generated using the methods described in Deleage, G., Roux B., 1987, Protein Engineering 1:289-294. Therefore, each region identified by any of these procedures or methods is within the scope of this invention. Preferred methods for generating 191P4D12 antibodies are further illustrated by the examples provided herein. Methods for preparing proteins or peptides for use as immunogens are well known in this art. Methods for preparing immunogenic conjugates of proteins and carriers (such as BSA, KLH, or other carrier proteins) are also well known in this art. In some cases, direct conjugation is used, employing, for example, a carbodiimide reagent; in others, a ligation reagent (such as those supplied by Pierce Chemical Co. (Rockford, IL)) is effective. The 191P4D12 immunogen is typically administered by injection over a suitable period of time using a suitable adjuvant, as understood in this art. During the immunization schedule, antibody titers can be obtained to determine the adequacy of antibody formation.

[0131] 191P4D12 monoclonal antibodies can be produced using various methods well-known in this technology. For example, immortalized cell lines secreting the desired monoclonal antibody are prepared using Kohler and Milstein's standard fusion tumor technique, or a modification thereof, to immortalize antibody-producing B cells, as is generally known. Immortalized cell lines secreting the desired antibody are screened by immunoassay of the antigen being a 191P4D12-related protein. Once a suitable immortalized cell culture is identified, the cells can be propagated and produce antibodies from live in vitro cultures or ascites fluid.

[0132] The antibodies or fragments of this invention can also be generated through recombinant methods. Regions that specifically bind to the desired region of the 191P4D12 protein can also be generated using chimeric antibodies or complementary determinant region (CDR) transplanted antibodies derived from multiple species. Humanized or human 191P4D12 antibodies can also be generated, and these are preferred for therapeutic applications. Methods for humanizing murine and other non-human antibodies by replacing the corresponding human antibody sequence with one or more non-human antibody CDRs are well known (see, for example, 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.

[0133] In a preferred embodiment, the antibody of the present invention comprises a fully human 191P4D12 antibody (191P4D12 MAb). Various methods in this art provide ways to generate a fully human 191P4D12 MAb. For example, a preferred embodiment provides a technique for generating antibody-inactivated transgenic mice engineered with a human heavy and light chain locus called Xenomouse (Amgen Fremont, Inc.). An illustrative description of the preparation of transgenic mice that generate human antibodies can be found in US 6,657,103. See also U.S. Patents 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).

[0134] In addition, the human antibody of the present invention can be produced using HuMAb mice (Medarex, Inc.), which contain small loci of human immunoglobulin genes encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, as well as targeted mutations of inactive endogenous μ and κ chain loci (see, for example, Lonberg et al. (1994) Nature 368(6474): 856-859).

[0135] In another embodiment, the all-human antibody of the present invention can be bred using mice carrying human immunoglobulin sequences on the transgene and transchomosome (such as mice carrying human heavy chain transgene and human light chain transchomosome). These mice are referred to herein as "KM mice," and are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727 and Tomizuka et al. PCT Publication WO 02 / 43478.

[0136] The human monoclonal antibodies of this invention can also be prepared using phage presentation methods for screening human immunoglobulin gene libraries. These phage presentation methods for isolating human antibodies are established in this technique. See, for example: U.S. Patents 5,223,409, 5,403,484, and 5,571,698 to Ladner et al.; U.S. Patents 5,427,908 and 5,580,717 to Dower et al.; U.S. Patents 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Patents 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081 to Griffiths et al.

[0137] The human monoclonal antibody of the present invention can also be prepared using SCID mice in which human immune cells have been reconstructed to produce a human antibody response after immunization. Such mice are described, for example, in U.S. Patents 5,476,996 and 5,698,767 to Wilson et al.

[0138] In a preferred embodiment, the 191P4D12 MAb of the present invention comprises the heavy and light chain variable regions of an antibody called Ha22-2(2,4)6.1 generated from a fusion tumor registered with the American Type Culture Collection (ATCC) under the registration number PTA-11267 (see Figure 3), or the heavy and light chain variable regions of an amino acid sequence homologous to the amino acid sequence of the heavy and light chain variable regions of Ha22-2(2,4)6.1, wherein such antibodies retain the desired functional properties of the 191P4D12 MAb of the present invention. The heavy chain variable region of Ha22-2(2,4)6.1 consists of an amino acid sequence ranging from residue E at position 20 to residue S at position 136 of SEQ ID NO:7, and the light chain variable region of Ha22-2(2,4)6.1 consists of an amino acid sequence ranging from residue D at position 23 to residue R at position 130 of SEQ ID NO:8. Any subclass of the constant region can be selected as the constant region of the antibody of the present invention. In one embodiment, the human IgG1 constant region can be used as the heavy chain constant region and the human Igκ constant region can be used as the light chain constant region.

[0139] For example, the present invention provides an isolated monoclonal antibody or its antigen-binding portion comprising a heavy chain variable region and a light chain variable region, wherein: (a) The heavy chain variable region contains an amino acid sequence that is at least 80% homologous to the amino acid sequence of the heavy chain variable region shown in Figure 3; and (b) The light chain variable region contains an amino acid sequence that is at least 80% homologous to the amino acid sequence of the light chain variable region shown in Figure 3.

[0140] In other embodiments, the VH and / or VL amino acid sequences may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the VH and VL sequences shown in Figure 3.

[0141] In another embodiment, the present invention provides an isolated monoclonal antibody or its antigen-binding portion, comprising a humanized heavy chain variable region and a humanized light chain variable region, wherein: (a) The heavy chain variable region contains a complementarity-determining region (CDR) having the amino acid sequence of the heavy chain variable region CDR shown in Figure 3; (b) The light chain variable region contains a CDR having the amino acid sequence of the light chain variable region shown in Figure 3.

[0142] The engineered antibodies of this invention include antibodies whose framework residues within the VH and / or VL have been modified (e.g., to improve antibody properties). Such framework modifications are typically performed to reduce the immunogenicity of the antibody. For example, one method involves "backmutating" one or more framework residues into their corresponding germline sequences. More specifically, antibodies that have undergone recipient cell mutations may contain framework residues different from the germline sequence from which the antibody was obtained. These residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody was obtained. To restore the framework region sequence to its germline configuration, somatic mutations can be "backmutated" into germline sequences (e.g., "backmutated" from leucine to methionine) by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be covered by this invention.

[0143] Another type of skeletal modification involves mutating one or more residues within a skeletal region or even one or more CDR regions to remove T-cell antigenic determinants, thereby reducing the potential immunogenicity of the antibody. This method is also known as "deimmunization" and is further described in detail in U.S. Patent Publication No. 2003 / 0153043 to Carr et al.

[0144] In addition to modifications made within the framework region or CDR region, or alternatively, the antibodies of the present invention can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity. Furthermore, the 191P4D12 MAb of the present invention can be chemically modified (e.g., one or more chemical moieties can be linked to the antibody) or modified to alter its glycosylation, again altering one or more functional properties of the MAb. Each of these embodiments is further described in detail below.

[0145] In one embodiment, the hinge region of CH1 is modified to alter the number of cysteine ​​residues in that hinge region, for example, by increasing or decreasing it. This method is further described in U.S. Patent No. 5,677,425 to Bodmer et al. The alteration of the number of cysteine ​​residues in the hinge region of CH1 can, for example, facilitate the assembly of light and heavy chains or enhance or reduce the stability of 191P4D12 MAb.

[0146] In another embodiment, the Fc hinge region of the antibody is mutated to shorten the biological half-life of 191P4D12 MAb. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment to weaken the binding of the antibody to Staphylococcyl protein A (SpA) relative to the binding of SpA to the native Fc hinge domain. This method is further described in detail in U.S. Patent No. 6,165,745 to Ward et al.

[0147] In another embodiment, 191P4D12 MAb is modified to extend its biological half-life. Several methods are feasible. For example, a mutation may be introduced as described in Ward's U.S. Patent No. 6,277,375. Alternatively, to extend the biological half-life, the antibody may be altered in the CH1 or CL region to contain a rescue receptor-binding antigenic determinant with two loops derived from the CH2 domain of the Fc region of IgG, as described in Presta et al.'s U.S. Patents Nos. 5,869,046 and 6,121,022.

[0148] In other embodiments, the effector function of 191P4D12 MAb is altered by replacing at least one amino acid residue with a different amino acid residue in the Fc region. For example, one or more amino acids selected from amino acid-specific residues can be replaced with different amino acid residues to alter the antibody's affinity for the effector ligand while retaining the antigen-binding ability of the parent antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This method is further described in detail in U.S. Patents 5,624,821 and 5,648,260 to Winter et al.

[0149] The reactivity of 191P4D12 antibodies with 191P4D12-associated proteins can be determined using various well-known methods, including Western blot, immunoprecipitation, ELISA, and FACS analysis, depending on the use of 191P4D12-associated proteins, 191P4D12-expressing cells, or their extracts. 191P4D12 antibodies or fragments thereof can be labeled with detectable markers or conjugated with a second molecule. Suitable detectable markers include (but are not limited to) radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators, or enzymes. Furthermore, bispecific antibody systems specific to two or more 191P4D12 antigenic determinants can be generated using methods generally known in this technique. Hypodimeric antibodies can also be generated using cross-linking techniques known in this technique (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

[0150] In another preferred embodiment, the 191P4D12 MAb of the present invention is an antibody comprising a heavy chain and a light chain of an antibody called Ha22-2(2,4)6.1. The heavy chain of Ha22-2(2,4)6.1 consists of an amino acid sequence ranging from residue E at position 20 to residue K at position 466 of SEQ ID NO:7, and the light chain of Ha22-2(2,4)6.1 consists of an amino acid sequence ranging from residue D at position 23 to residue C at position 236 of the sequence of SEQ ID NO:8. These sequences are shown in Figures 2 and 3. In a preferred embodiment, Ha22-2(2,4)6.1 is conjugated with a cytotoxic agent.

[0151] The generation is called Fusion tumors of the antibody [Ha22-2(2,4)6.1] were found in...

[2010] [Year] [8] [moon]

[18] [Japan] (via Federal Express) sent to the American Type Culture Collection Center (ATCC), P.O. Box 1549, Manassas, VA 20108, and given a deposit number. [PTA-11267]. III.) Overview of Antibody-Drug Conjugates

[0152] In another embodiment, the present invention provides an antibody-drug conjugate (ADC) comprising an antibody conjugated with a cytotoxic agent, such as a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope (i.e., a radioconjugate). In another embodiment, the present invention further provides a method of using the ADC. In one embodiment, the ADC comprises any of the 191P4D12 MAb herein covalently linked to a cytotoxic agent or a detectable drug.

[0153] The use of antibody-drug conjugates for local delivery of cytotoxic agents or cell growth inhibitors (i.e., drugs that kill or inhibit tumor cells in the treatment of cancer) (Syrigos and Epinetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151-172; US Patent 4,975,278) allows for the partial targeted delivery of drugs to tumors and their intracellular accumulation within the tumor. Systemic administration of such unconjugated drug agents can produce unacceptable levels of toxicity to normal cells and to tumor cells that are being eliminated (Baldwin et al. (1986) Lancet pp. (March 15, 1986):603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," Monoclonal Antibodies '84: Biological And Clinical Applications, A). Pinchera et al. (eds., pp. 475-506). Therefore, efforts should be made to minimize toxicity while maximizing efficacy. Both polyclonal and monoclonal antibodies have been reported to be suitable for such 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) ibid.).The toxins used in antibody-toxin conjugates include bacterial toxins, such as diphtheria toxin; plant toxins, such as ricin; small molecule toxins, such as 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), maytansine (EP 1391213;Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and cazithromycin (Lode et al. (1998) Cancer Res. 58:2928;Hinman et al. (1993) Cancer Res. 58:2928). (Res. 53:3336-3342). These toxins exert their cytotoxic and inhibitory effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to become inactive or weakly active when conjugated with large antibody or protein receptor ligands.

[0154] An example of an antibody drug conjugate is ZEVALIN® (ibritumomab tiuxetan, Biogen / Idec), which is an antibody-radioisotope conjugate consisting of a mouse IgG1κ monoclonal antibody against the CD20 antigen present on the surface of normal and malignant B lymphocytes and a 111In or 90Y radioisotope bound by a thiourea linker-chelator (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).

[0155] In addition, MYLOTARG™ (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody-drug conjugate consisting of a huCD33 antibody linked to chazim, was approved in 2000 for the injection treatment of acute myeloid leukemia (Drugs of the Future (2000) 25 (7):686; US Patent Nos. 4970198, 5079233, 5585089, 5606040, 5693762, 5739116, 5767285, and 5773001).

[0156] In addition, Cantuzumab mertansine (Immunogen, Inc.), an antibody-drug conjugate consisting of a huC242 antibody linked to the maytansine-like drug moiety DM1 via a disulfide linker SPP, is entering a Phase II trial for the treatment of CanAg-expressing cancers (such as colon cancer, pancreatic cancer, gastric cancer, and other cancers).

[0157] In addition, MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), an antibody-drug conjugate consisting of a monoclonal antibody against prostate-specific membrane antigen (PSMA) linked to the maytansine-like drug moiety DM1, is being developed as a potential treatment for prostate cancer.

[0158] Finally, olistatin peptide, olistatin E (AE), and monomethyl olistatin (MMAE), synthetic analogs of sea hare toxin, are conjugated with chimeric monoclonal antibodies cBR96 (specific to Lewis Y on cancer) and cAC10 (specific to CD30 on hematologic malignancies) (Doronina et al. (2003) Nature Biotechnology 21 (7):778-784) and are under therapeutic development.

[0159] Furthermore, this document describes chemotherapeutic agents suitable for generating ADCs. Enzymatically active toxins and their fragments that can be used include diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-ascorbic acid, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, jatropha toxin, croton toxin, soapwort inhibitor, leucopicrin, mitogen, localized ascorbic acid, phenolmycin, enoxacin, and mycotoxins. See, for example, WO 93 / 21232, published October 28, 1993. Various radionuclides can be used to generate radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re. Antibody-cytotoxic agent conjugates can be prepared using a variety of bifunctional protein conjugates, such as N-succinimino-3-(2-pyridyldithiol)propionate (SPDP), iminothiacyclopentane (IT), bifunctional derivatives of succinimino esters (such as dimethyl diimino adipate hydrochloride), active esters (such as disuccinimino octanoate), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), dinitrogen salt derivatives (such as bis(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and difluorinated compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described by Vitetta et al. (1987) Science, 238:1098. 1-Isothiocyanophenylmethyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) labeled with carbon-14 is an exemplary chelating agent for conjugating radioactive nucleotides with antibodies (WO94 / 11026).

[0160] This article also covers conjugates of antibodies with one or more small molecule toxins, such as chachiin, maytansin, saliton, oligostatin, trichothecene, and CC1065, as well as derivatives of these toxins with toxic activity. [III(A).] [Like-Liketansin] []

[0161] Maytansine compounds suitable for use as maytansine-like drug fractions are well known in this technology and can be isolated from natural sources by known methods, produced by genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973), or synthesized by known methods to prepare maytansinol and maytansinol analogues.

[0162] Exemplary maytansine-like drug portions include maytansine-like drug portions having modified aromatic rings, such modifications being: C-19-dechlorination (US 4256746) (prepared by reduction with lithium aluminum hydride of ansamytocin P2); C-20-hydroxy (or C-20-demethyl) + / -C-19-dechlorination (US Patents 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or by dechlorination using LAH); and C-20-demethoxy, C-20-acetoxy (-OCOR), + / -dechlorination (US Patent 4,294,757) (prepared by acetylation); and maytansine-like drug portions having modifications at other positions.

[0163] Exemplary maytansine-like drug portions also include maytansine-like drug portions having modifications such as: C-9-SH (US 4,424,219) (prepared by reacting maytanol with H₂S or P₂S₅); C-14-alkoxymethyl (demethoxy / CH₂OR) (US 4,331,598); C-14-hydroxymethyl or acetoxymethyl (CH₂OH or CH₂OAc) (US 4,450,254) (prepared by Nocardia); C-15-hydroxy / acetoxy (US 4,364,866) (prepared by converting maytanol with Streptomyces); C-15-methoxy (US Patent Nos. 4,313,946 and 4,315,929) (from Trewia). (nudlflora) separation); C-18-N-demethyl (US Patent Nos. 4,362,663 and 4,322,348) (prepared by demethylation of maytanol by Streptomyces); and 4,5-deoxy (US 4,371,533) (prepared by reduction of maytanol by titanium trichloride / LAH).

[0164] ADCs containing maytansine-like compounds, methods for their preparation, and their therapeutic uses are disclosed, for example, in U.S. Patents 5,208,020, 5,416,064, and 6,441,163 and European Patent EP 0 425 235 B1, the disclosures of which are expressly incorporated herein by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996), describe an ADC comprising maytansine-like compounds (referred to as DM1) linked to a monoclonal antibody C242 against human colorectal cancer. This conjugate has been found to be highly cytotoxic to cultured colon cancer cells and exhibits antitumor activity in in vivo tumor growth assays. Chari et al. (Cancer Research 52:127-131, 1992) described an ADC in which maytansine-like antibodies are conjugated via a disulfide linker to a murine antibody A7 that binds to an antigen on a human colon cancer cell line, or to another murine monoclonal antibody TA.1 that binds to the HER-2 / neu oncogene. In vitro testing of the TA.1-maytansine-like conjugate showed cytotoxicity against the human breast cancer cell line SK-BR-3, which exhibits 3 × 10⁵ HER-2 surface antigens per cell. The drug conjugate achieved a level of cytotoxicity similar to that of free maytansine-like antibodies, which could be increased by increasing the number of maytansine-like compound molecules per antibody molecule. The A7-maytansine-like conjugate showed low systemic cytotoxicity in mice. [III(B).] [Oristatin and sea hare toxin] []

[0165] In some embodiments, the ADC comprises the antibody of the present invention conjugated with salipodin or salipodin peptide analogs and derivatives, and olistatin (US Patent Nos. 5,635,483 and 5,780,588). Salipodin and olistatin 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 activity (US 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). The salipodin or olistatin drug moiety may be linked to the antibody via the N-terminus (amino group) or C-terminus (carboxyl group) of the peptide drug moiety (WO 02 / 088172).

[0166] Exemplary examples of olistatin include the N-terminal linked monomethyl olistatin drug portions DE and DF disclosed in Senter et al., Proceedings of the American Association for Cancer Research, Vol. 45, Abstract No. 623, filed 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 entirety.

[0167] An example of an indicative oprestatin embodiment is MMAE (where the wavy lines indicate linkers (L) covalently linked to the antibody-drug conjugate). MMAE

[0168] Another example of an indicative oprestatin embodiment is MMAF, wherein the wavy line indicates the linker (L) covalently linked to the antibody-drug conjugate (US 2005 / 0238649): MMAF.

[0169] Other exemplary embodiments comprising MMAE or MMAF and various linker components (further described herein) have the following structures and abbreviations (where Ab means antibody, S is the sulfur of the antibody, and p is 1 to about 8): Ab-MC-vc-PAB-MMAF Ab-MC-vc-PAB-MMAE Ab-MC-MMAF.

[0170] Peptide drug moieties can typically be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. These peptide bonds can be prepared, for example, by liquid-phase synthesis methods well known in the field of peptide chemistry (see E. Schröder and K. Lübke, "The Peptides", Vol. 1, pp. 76-136, 1965, Academic Press). The olistatin / sea haretoxin drug fraction 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. 1 5:859-863; and Doronina (2003) Nat Biotechnol 21(7):778-784. [III(C).] [Kazimycin] []

[0171] In other embodiments, the ADC comprises the antibody of the present invention conjugated with one or more chachiomycin molecules. The chachiomycin family of antibiotics can produce double-stranded DNA fragments at subpimolar concentrations. For the preparation of chachiomycin 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 analogues of chachiin that can be used include (but are not limited to) γ1I, α2I, α3I, N-acetylg-γ1I, PSAG, and θI1 (Hinman et al., Cancer Research 53:3336-3342 (1993); Lode et al., Cancer Research 58:2925-2928 (1998); and the aforementioned U.S. patents belonging to American Cyanamid). Another antitumor drug that can be conjugated with antibodies is QFA, an antifolate agent. Both chachiin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these drugs via antibody-mediated internalization can significantly enhance their cytotoxic effects. [III(D).] [Other cytotoxic agents] []

[0172] Other antitumor agents that can be conjugated with the antibodies of this invention include BCNU, streptozoticin, vincristine, and 5-fluorouracil, the family of agents collectively referred to as the LL-E33288 complex as described in U.S. Patent Nos. 5,053,394 and 5,770,710, and esperamicin (U.S. Patent No. 5,877,296).

[0173] Usable enzymatically active toxins and their fragments include diphtheria A chain, non-conjugated active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), pectin A chain, absinthecin A chain, modizol A chain, α-ascorbic acid, tung oil protein, caryophyllin protein, pokeweed protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, jatropha toxin, croton toxin, soapwort inhibitor, leucopicrin, mitogen, localized ascorbic acid, phenolmycin, enoxacin, and mycotoxins. See, for example, WO 93 / 21232, published on October 28, 1993.

[0174] The present invention further covers ADCs formed between antibodies and compounds with nuclear degradation activity (e.g., ribonucleases or DNA endonucleases, such as deoxyribonucleases; DNases).

[0175] To selectively destroy tumors, antibodies can contain highly radioactive atoms. A variety of radioactive isotopes can be used to generate radioconjugated antibodies. Examples include radioisotopes such as At-211, I-131, I-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, Pb-212, and Lu. When using conjugates for detection, they can contain radioactive atoms used in scintillation studies, such as tc-99m or I-123; or spin labels used in nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, thorium, manganese, or iron.

[0176] Radiolabeled or other labels can be incorporated into conjugates in known ways. For example, peptides can be biosynthesized or synthesized by chemical amino acid synthesis using suitable amino acid precursors, such as those involving the substitution of hydrogen with fluorine-19. Labels such as TC-99M or I-123, Re-186, Re-188, and In-111 can be linked via cysteine ​​residues in the peptide. Yttrium-90 can be linked via lysine residues. The IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123. Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989). IV.) Antibody-drug conjugates that bind to 191P4D12

[0177] This invention provides antibody-drug conjugate compounds for targeted drug delivery. The inventors have discovered that antibody-drug conjugate compounds possess effective cytotoxic and / or cell growth inhibitory activity against cells expressing 191P4D12. The antibody-drug conjugate compounds comprise antibody units covalently linked to at least one drug unit. These drug units may be covalently linked directly or via a linker unit (LU).

[0178] In some embodiments, the antibody drug conjugate compound has the following formula: L-(LU-D) p [(I)] Or a pharmaceutically acceptable salt or solvate thereof, wherein L represents an antibody unit, such as the 191P4D12 MAb of this invention, and (LU-D) represents the connecting subunit-drug unit portion, where: LU- is the connecting subunit, and -D represents a drug unit possessing cell growth inhibitory or cytotoxic activity against target cells; and p is an integer from 1 to 20.

[0179] In some embodiments, p is in the range of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is in the range of 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is 1, 2, 3, 4, 5, or 6. In some embodiments, p is 2 or 4.

[0180] In some embodiments, the antibody drug conjugate compound has the following formula: L-(A aW wY yD) p [(II)] Or a pharmaceutically acceptable salt or solvate thereof, wherein: L represents an antibody unit, such as 191P4D12 MAb; and -A aW wY y- represents the connection subunit (LU), where: -A- represents the extended subunit. a is 0 or 1, Each -W- is an independent amino acid unit. w is an integer in the range of 0 to 12. -Y- represents a self-decomposing interlayer subunit. y is 0, 1, or 2; -D represents a drug unit possessing cell growth inhibitory or cytotoxic activity against target cells; and p is an integer from 1 to 20.

[0181] 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 is in the range of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is in the range of 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is 1, 2, 3, 4, 5, or 6. In some embodiments, p is 2 or 4. In some embodiments, y is 1 or 2 when w is not 0. In some embodiments, y is 1 or 2 when w is 1 to 12. In some embodiments, w is 2 to 12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0.

[0182] For compositions containing multiple antibodies, drug loading is represented by the average number p of drug molecules per antibody. Drug loading can range from 1 to 20 drug molecules (D) per antibody. The average number of drugs per antibody during preconjugation can be characterized using conventional methods such as mass spectrometry, ELISA, and HPLC. The quantitative distribution of the antibody-drug conjugate can also be determined based on p. In some cases, homogeneous antibody-drug conjugates can be separated, purified, and characterized by methods such as reverse-phase HPLC or electrophoresis, where p is a value for the antibody-drug conjugate with other drug loadings. In exemplary examples, p is 2 to 8.

[0183] Antibody-drug conjugate compounds can be produced using any technique known to those skilled in the art. In short, an antibody-drug conjugate compound comprises 191P4D12 MAb as an antibody unit, a drug, and, where applicable, a linker connecting the drug and a conjugate. In a preferred embodiment, the antibody is 191P4D12 MAb comprising the heavy and light chain variable regions of an antibody referred to as Ha22-2(2,4)6.1. In a more preferred embodiment, the antibody is 191P4D12 MAb comprising the heavy and light chains of an antibody referred to as Ha22-2(2,4)6.1. Various reactions can be used to covalently link the drug and / or linker to a conjugate. This is typically accomplished by reacting amino acid residues of the conjugate (e.g., an antibody molecule), including the amino group of lysine, the free carboxyl group of glutamic acid and aspartic acid, the hydrogen sulfide group of cysteine, and various portions of aromatic amino acids. One of the most common non-specific methods for covalent linkage is the carbodiimide reaction, which links the carboxyl (or amino) group of a compound to the amino (or carboxyl) group of an antibody. Alternatively, bifunctional agents (such as dialdehydes or amide esters) have been used to link the amino group of a compound to the amino group of an antibody molecule. The Schiff base reaction can also be used to link drugs to conjugates. This method involves the oxidation of periodate containing a diol or hydroxyl group of a drug to form an aldehyde, which is then reacted with a conjugate. Linkage is achieved by forming a Schiff base with the amino group of the conjugate. Isothiocyanates can also be used as coupling agents for covalently linking drugs to conjugates. Other techniques are known to those skilled in the art and are within the scope of this invention.

[0184] In some embodiments, an intermediate serving as a linker precursor is reacted with the drug under appropriate conditions. In some embodiments, reactive groups on the drug and / or intermediate are used. Subsequently, the reaction product or derivative of the drug and intermediate is reacted with 191P4D12 MAb under appropriate conditions.

[0185] This document describes in more detail the specific units of the antibody-drug conjugate compound. The synthesis and structure of illustrative linker units, extension units, amino acid units, self-degrading spacer units, and drug units are also described in U.S. Patent Application Publications Nos. 2003-0083263, 2005-0238649, and 2005-0009751, each of which is incorporated herein by reference in its entirety for all purposes. V.) Connecting Subunit

[0186] Typically, antibody-drug conjugate compounds include a linker unit between the drug unit and the antibody unit. In some embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker in the intracellular environment releases the drug unit from the antibody. In other embodiments, the linker unit is not cleavable and the drug is released, for example, by antibody degradation.

[0187] In some embodiments, the linker may be cleaved by a lysing agent present in the intracellular environment (e.g., within a lysosome, endosome, or caveole). The linker may be, for example, a peptide linker, which may be cleaved by intracellular peptidases or proteases (including, but not limited to, lysosomal or endosomal proteases). In some embodiments, the peptide linker is at least two amino acids long or at least three amino acids long. The lysing agent may include cathepsins B and D and fibrinolysins, all known to hydrolyze dipeptide drug derivatives, thereby releasing the active drug within the target cell (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). More typically, a peptide linker may be cleaved by an enzyme present in the cell, represented by 191P4D12. For example, peptide linkers that can be cleaved by cathepsin B, a thiol-dependent protease highly expressed in cancerous tissues (e.g., the Phe-Leu or Gly-Phe-Leu-Gly linker (SEQ ID NO: 9)) can be used. Other examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, which is incorporated herein by reference in its entirety for all purposes. In a particular embodiment, the peptide linker that can be cleaved by an intracellular protease is the Val-Cit linker or the Phe-Lys linker (see, for example, U.S. Patent 6,214,345, which describes the synthesis of doxorubicin having the Val-Cit linker). One advantage of intracellular proteolytic release agents is that the agent is generally attenuated when conjugated and the serum stability of the conjugate is generally high.

[0188] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, pH-sensitive linkers can be hydrolyzed under acidic conditions. For example, acid-instable linkers that can be hydrolyzed in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, aconitine, orthoesters, acetals, ketals, or the like) can be used. (See, for example, 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.) These linkers are relatively stable under neutral pH conditions (such as the pH conditions in blood), but unstable below pH 5.5 or 5.0 (approximate to the pH of lysosomes). In some embodiments, the hydrolyzable linker is a thioether linker (such as a thioether linked to a therapeutic agent via a acetylated hydrazone bond (see, for example, U.S. Patent No. 5,622,929)). []

[0189] In other embodiments, the linker may be cleaved under reducing conditions (e.g., disulfide linkers). Various disulfide linkers are known in this art, including, for example, disulfide linkers formed using: SATA (N-succinimidyl-S-acetylsuloacetate), 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, for example, Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel, ed., Oxford U. Press, 1987. See also U.S. Patent No. 4,880,935.)

[0190] In other specific embodiments, the linker is a malonate linker (Johnson et al. 1995, Anticancer Res. 15:1387-93), a maleic anisodiminobenzoyl linker (Lau et al. 1995, Bioorg-Med-Chem. 3(10):1299-1304) or a 3'-N-acetylamine analog (Lau et al. 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0191] In other embodiments, the linker unit is non-cleavable and the drug is released by antibody degradation. (See U.S. Publication No. 2005 / 0238649, which is incorporated herein by reference in its entirety for all purposes.)

[0192] Typically, linkers are not highly sensitive to the extracellular environment. As used herein, in the case of linkers, "not highly sensitive to the extracellular environment" means that when the antibody-drug conjugate compound is presented in an extracellular environment (e.g., in plasma), no more than about 20%, usually no more than about 15%, more usually no more than about 10%, and even more usually no more than about 5%, no more than about 3%, or no more than about 1% of the linker in the sample of the antibody-drug conjugate compound is lysed. Whether a linker is not highly sensitive to the extracellular environment can be determined, for example, by incubating the antibody-drug conjugate compound with plasma for a predetermined time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free drug present in the plasma.

[0193] In other non-mutually exclusive embodiments, the linker promotes cell internalization. In some embodiments, it promotes cell internalization when the linker is conjugated with a therapeutic agent (i.e., in the context of the linker-therapeutic agent portion of an antibody-drug conjugate compound as described herein). In other embodiments, it promotes cell internalization when the linker is conjugated with an olistatin compound and 191P4D12 MAb.

[0194] Various exemplary connectors that can be used in the compositions and methods of the present invention are described in WO 2004-010957, U.S. Publication No. 2006 / 0074008, U.S. Publication No. 20050238649 and U.S. Publication No. 2006 / 0024317 (each of which is incorporated herein by reference in its entirety for all purposes).

[0195] A "linker unit" (LU) is a bifunctional compound that can be used to link a drug unit and an antibody unit to form an antibody-drug conjugate compound. In some embodiments, the linker unit has the following formula: -A aW wY y- Where: -A- represents the extended subunit, a is 0 or 1, Each -W- is an independent amino acid unit. w is an integer in the range of 0 to 12. -Y- represents a self-decomposing interlayer unit, and y is 0, 1 or 2.

[0196] 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, when w is 1 to 12, y is 1 or 2. In some embodiments, w is 2 to 12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0. VI.) Extended Subunit

[0197] The extension subunit (A) (if present) enables the antibody unit to be linked to an amino acid unit (-W-) (if present), to a spacer subunit (-Y-) (if present), or to a drug unit (-D). Suitable functional groups, either natural or chemically treated, that may be present on 191P4D12 MAb (e.g., Ha22-2(2,4)6.1) include (but are not limited to) sulfhydryl groups, amino groups, hydroxyl groups, mutarotatory isomers of carbohydrates, and carboxyl groups. Suitable functional groups are sulfhydryl and amino groups. In one example, a sulfhydryl group can be generated by reducing an intramolecular disulfide bond in 191P4D12 MAb. In another embodiment, a sulfhydryl group can be generated by reacting the amino group of the lecithic acid moiety of 191P4D12 MAb with 2-iminothiocyclopentane (Traut's reagent) or other sulfhydryl group generating agents. In some embodiments, 191P4D12 MAb is a recombinant antibody and engineered to carry one or more lysines. In some other embodiments, recombinant 191P4D12 MAb is engineered to carry other hydrogen sulfide groups, such as other cysteines.

[0198] In one embodiment, the extended subunit forms a bond with the sulfur atom of the antibody unit. This sulfur atom may be derived from the hydrogen sulfide group of the antibody. Representative extended subunits of this embodiment are depicted in square brackets of formulas IIIa and IIIb, wherein L-, -W-, -Y-, -D, w, and y are as defined above, and R 17 is selected from -C1-C10 alkyl-, -C1-C10 alkenyl-, -C1-C10 alkyne-, carbocyclic-, -O-(C1-C8 alkyl)-, O-(C1-C8 alkenyl)-, -O-(C1-C8 alkyne)-, -alkenyl-, -C1-C10 alkyl-alkenyl-, -C2-C10 alkenyl-alkenyl, -C2-C10 alkyne-alkenyl, -alkenyl-C1-C10 alkyl-, -alkenyl-C2-C10 alkenyl-, -alkenyl-C 2-C 10-Phenyneyl-, -C 1-C 10-Phenylalkyl-(carbocyclic)-, -C 2-C 10-Phenenyl-(carbocyclic)-, -C 2-C 10-Phenyneyl-(carbocyclic)-, -(carbocyclic)-C 1-C 10-Phenylalkyl-, -(carbocyclic)-C 2-C 10-Phenenyl-, -(carbocyclic)-C 2-C 10-Phenyneyl-, -heterocyclic-, -C 1-C 10-Phenylalkyl-(heterocyclic)-, -C 2-C 10-Phenenyl-(heterocyclic)-, -(heterocyclic)-C 1-C 10-Phenylalkyl-, -(heterocyclic)-C 2-C 10-Phenylalkyl-, -(heterocyclic)-C 1-C 10. The radical is argynyl-, -(CH₂CH₂O)r-, or -(CH₂CH₂O)r-CH₂-, where r is an integer in the range of 1-10, wherein the alkyl, alkenyl, alkynyl, alkyl, alkenyl, alkynyl, aryl, carbocyclic, carbocyclic, heterocyclic, and aryl groups, individually or as part of another group, are substituted as appropriate. In some embodiments, the alkyl, alkenyl, alkynyl, alkyl, alkenyl, alkynyl, aryl, carbocyclic, carbocyclic, heterocyclic, and aryl groups, individually or as part of another group, are unsubstituted. In some embodiments, R 17 is selected from -C 1-C 10 alkyl-, -carbocyclic-, -O-(C 1-C 8 alkyl)-, -aryl-, -C 1-C 10 alkyl-aryl-, -aryl-C 1-C 10 alkyl-, -C 1-C 10 alkyl-(carbocyclic)-, -(carbocyclic)-C 1-C 10 alkyl-, -C 3-C 8 heterocyclic-, -C 1-C 10 alkyl-(heterocyclic)-, -(heterocyclic)-C 1-C 10 alkyl-, -(CH 2CH 2O)r- and -(CH 2CH 2O)r-CH 2-; and r is an integer in the range of 1-10, wherein the alkyl groups are unsubstituted and the remaining portions of the groups are substituted as appropriate.

[0199] As will be understood from all exemplary embodiments, unless otherwise specified, 1 to 20 pharmaceutical moieties may be attached to the antibody (p=1-20). IIIa IIIb

[0200] The illustrative extended subunit is the extended subunit of equation IIIa, where R 17 is -(CH 2) 5-: .

[0201] Another illustrative extension subunit is the extension subunit of formula IIIa, where R 17 is -(CH 2CH 2O) r-CH 2-; and r is 2: .

[0202] The illustrative extended subunit is an extended subunit of formula IIIa, wherein R 17 is an aryl- or an aryl-C1-C10 aryl-. In some embodiments, the aryl group is an unsubstituted phenyl group.

[0203] Another illustrative extension subunit is the extension subunit of formula IIIb, where R 17 is -(CH 2) 5-: .

[0204] In some embodiments, the extension subunit is connected to the antibody unit via a disulfide bond between the sulfur atom of the antibody unit and the sulfur atom of the extension subunit. A representative extension subunit of this embodiment is depicted in square brackets of Formula IV, wherein R17, L-, -W-, -Y-, -D, w, and y are as defined above. [IV] []

[0205] It should be noted that in this application, unless otherwise indicated herein, the S part in the following formula refers to the sulfur atom of the antibody unit.

[0206] In other embodiments, the elongator contains a reactive site that can form a one-bond with a primary or secondary amino group of the antibody. Examples of such reactive sites include (but are not limited to) activated esters, such as dibutylimide, 4-nitrophenyl, pentafluorophenyl, tetrafluorophenyl, acid anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. Representative elongator units of this embodiment are depicted in square brackets of formulas Va and Vb, wherein -R17-, L-, -W-, -Y-, -D, w, and y are as defined above; [Va] [] Vb.

[0207] In some embodiments, the elongator contains a reactive site that can react with the (-CHO) group of a modified carbohydrate that may be present on an antibody. For example, the carbohydrate may be moderately oxidized using a reagent such as sodium periodate, and the resulting oxidized carbohydrate (-CHO) unit may condense with an elongator containing a functional group such as acehydrazine, oxime, primary or secondary amine, hydrazine, thioheptacarbazone, carboxylic acid hydrazine, and arylacehydrazine, such as the elongator described in Kaneko et al. 1991, Bioconjugate Chem. 2:133-41. Representative elongator units of this embodiment are depicted in square brackets of formulas VIa, VIb, and VIc, wherein -R17-, L-, -W-, -Y-, -D, w, and y are as defined above. [Via] [] [VIb] [] Vic VII.) Amino acid units

[0208] If the spacer unit is present, the amino acid unit (-W-) (if present) links the extension unit to the spacer unit; if the spacer unit is not present, the extension unit links to the drug unit; and if neither the extension unit nor the spacer unit is present, the antibody unit links to the drug unit.

[0209] Ww- can be, for example, a monopeptide, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide unit. Each Ww- unit independently has the formula shown in the square brackets below, where w is an integer in the range of 0 to 12: or Where R 19 represents hydrogen, methyl, isopropyl, isobutyl, dibutyl, benzyl, p-hydroxybenzyl, -CH 2OH, -CH(OH)CH 3, -CH 2CH 2SCH 3, -CH 2CONH 2, -CH 2COOH, -CH 2CH 2CONH 2, -CH 2CH 2COOH, -(CH 2) 3NHC(=NH)NH 2 , -(CH 2) 3NH 2, -(CH 2) 3NHCOCH 3, -(CH 2) 3NHCHO, -(CH 2) 4NHC(=NH)NH 2 , -(CH 2) 4NH 2, -(CH 2) 4NHCOCH 3, -(CH 2) 4NHCHO, -(CH 2) 3NHCONH 2 -(CH 2) 4NHCONH 2, -CH 2CH 2CH(OH)CH 2NH 2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl

[0210] In some embodiments, the amino acid unit may be cleaved by one or more enzymes (including cancer or tumor-associated proteases) using enzymatic methods to release the drug unit (-D), which, in one embodiment, is protonated in vivo after release to deliver the drug (D).

[0211] In some 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. The illustrative Ww unit is represented by formulas (VII)-(IX): ( [VII]) R20 and R21 are as follows: R 20 R 21 benzyl (CH₂)₄NH₂; methyl (CH₂)₄NH₂; Isopropyl (CH₂)₄NH₂; Isopropyl (CH₂)₃NHCONH₂; benzyl (CH₂)₃NHCONH₂; Isobutyl (CH₂)₃NHCONH₂; Second butyl (CH₂)₃NHCONH₂; (CH₂)₃NHCONH₂; benzyl methyl; benzyl (CH₂)₃NHC(=NH)NH₂; ( [VIII]) R20, R21, and R22 are as follows: R 20 R 21 R 22 benzyl benzyl (CH₂)₄NH₂; Isopropyl benzyl (CH₂)₄NH₂; and H benzyl (CH₂)₄NH₂; ( [IX]) R20, R21, R22 and R23 are as follows: R 20 R 21 R 22 R 23 H benzyl Isobutyl H; and methyl Isobutyl methyl Isobutyl.

[0212] Exemplary amino acid units include (but are not limited to) units of formula VII, wherein: R20 is benzyl and R21 is -(CH2)4NH2; R20 is isopropyl and R21 is -(CH2)4NH2; or R20 is isopropyl and R21 is -(CH2)3NHCONH2. Another exemplary amino acid unit is a unit of formula VIII, wherein R20 is benzyl, R21 is benzyl, and R22 is -(CH2)4NH2.

[0213] The applicable -W w- unit can be designed and optimized for its selectivity in enzymatic cleavage by a specific enzyme (e.g., tumor-associated protease). In one embodiment, the -W w- unit is the unit for cleavage catalyzed by cathepsins B, C, and D or plasminase protease.

[0214] In one embodiment, -W w- is a dipeptide, tripeptide, tetrapeptide, or pentapeptide. When R19, R20, R21, R22, or R23 is not hydrogen, the carbon atom bonded to R19, R20, R21, R22, or R23 is an antagonistic carbon atom.

[0215] Each carbon atom connected to R19, R20, R21, R22 or R23 is independently in an (S) or (R) configuration.

[0216] In one state of the amino acid unit, the amino acid unit is valine-citrulline (VC or Val-Cit). In another state, the amino acid unit is phenylalanine-lysine (i.e., FK). In yet another state, the amino acid unit is N-methylvaline-citrulline. In yet another state, the amino acid unit is 5-aminovaleric acid, homophenylalanine-lysine, tetraisoquinoline carboxylic acid-lysine, cyclohexylalanine-lysine, isoperidine carboxylic acid-lysine, β-alanine-lysine, glycine, serine, valine, glutamine, and isoperidine carboxylic acid. VIII.) Spacer Subunit

[0217] When an amino acid unit is present, the spacer subunit (-Y-) (if present) links the amino acid unit to the drug unit. Alternatively, when an amino acid unit is absent, the spacer subunit links the extension subunit to the drug unit. When neither an amino acid unit nor the extension subunit is present, the spacer subunit also links the drug unit to the antibody unit.

[0218] Spacer units generally fall into two types: non-self-degrading and self-degrading. Non-self-degrading spacer units are those in which part or all of the amino acid unit remains bound to the drug moiety after cleavage (especially enzymatic cleavage) of the antibody-drug conjugate. Examples of non-self-degrading spacer units include (but are not limited to) glycine-glycine spacer units and glycine spacer units (both depicted in process 1) (hereinafter). When a conjugate containing a glycine-glycine spacer unit or a glycine spacer unit undergoes enzymatic cleavage via an enzyme (e.g., tumor cell-associated protease, cancer cell-associated protease, or lymphocyte-associated protease), the glycine-glycine-drug moiety or glycine-drug moiety cleaves from L-Aa-Ww. In one embodiment, a separate hydrolysis reaction occurs within the target cell, thereby cleaving the glycine-drug moiety bond and releasing the drug.

[0219] Process 1

[0220] In some embodiments, the non-self-decomposing spacer subunit (-Y-) is -Gly-. In some embodiments, the non-self-decomposing spacer subunit (-Y-) is -Gly-Gly-.

[0221] In one embodiment, a drug-linker conjugate or a pharmaceutically acceptable salt or solvate thereof without spacer subunits (-Y y-, where y=0) is provided.

[0222] Alternatively, conjugates containing self-decomposing spacer units can release -D. As used herein, the term "self-decomposing spacer" refers to a bifunctional chemical part that can covalently link the chemical parts of two spacers together to form a stable triplet molecule. If its bond with the first part breaks, it will spontaneously separate from the second chemical part.

[0223] In some embodiments, -Y y- is a p-aminobenzyl alcohol (PAB) unit (see processes 2 and 3), the extended phenyl portion of which is substituted with Q m, wherein Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 ynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 ynyl), -halogen, -nitro, or -cyano; and m is an integer in the range of 0-4. Alkyl, alkenyl, and ynyl groups, alone or as part of another group, may be substituted as appropriate.

[0224] In some embodiments, -Y- is a PAB group that is attached to -W w- via an amino nitrogen atom of a PAB group and directly attached to -D via a carbonate, carbamate, or ether group. Without being bound by any particular theory or mechanism, Procedure 2 describes a possible mechanism of drug release via a PAB group directly attached to -D via a carbamate or carbonate group, as described by Toki et al. 2002, J. Org. Chem. 67:1866-1872. [process] [2] []

[0225] In process 2, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 ynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 ynyl), -halogen, -nitro, or -cyano; m is an integer in the range of 0-4; and p is in the range of 1 to about 20. Alkyl, alkenyl, and ynyl groups, alone or as part of another group, may be substituted as appropriate.

[0226] Without being bound by any particular theory or mechanism, process 3 describes a possible mechanism for drug release via a PAB group directly attached to -D via an ether or amine bond, where D includes an oxygen or nitrogen group that is part of the drug unit. Process 3 []

[0227] In process 3, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 ynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 ynyl), -halogen, -nitro, or -cyano; m is an integer in the range of 0-4; and p is in the range of 1 to about 20. Alkyl, alkenyl, and ynyl groups, alone or as part of another group, may be substituted as appropriate.

[0228] Other examples of self-decomposing spacers include (but are not limited to) aromatic compounds electronically similar to PAB groups, such as 2-aminoimidazolium-5-methanol derivatives (Hay et al. 1999, Bioorg. Med. Chem. Lett. 9:2237) and o-aminobenzyl acetal or p-aminobenzyl acetal. Spacers that undergo cyclization after hydrolysis of the amide bond can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al. 1995, Chemistry Biology 2:223); appropriately substituted bicyclic [2.2.1] and bicyclic [2.2.2] ring systems (Storm et al. 1972, J. Amer. Chem. Soc. 94:5815); and 2-aminophenylpropionate amides (Amsberry et al. 1990, J. Org. Chem. 55:5867). Amine-containing drugs with α-substitution of glycine (Kingsbury et al. 1984, J. Med. Chem. 27:1447) are also examples of self-disintegrating spacers.

[0229] In one embodiment, the spacer subunit is a branched bis(hydroxymethyl)-styrene (BHMS) unit as described in process 4, which can be used to incorporate multiple drugs and release multiple drugs. [process] [4] []

[0230] In process 4, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 ynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 ynyl), -halogen, -nitro, or -cyano; m is an integer in the range of 0-4; n is 0 or 1; and p is in the range of 1 to about 20. Alkyl, alkenyl, and ynyl groups, alone or as part of another group, may be substituted as appropriate.

[0231] In some embodiments, the -D portion is the same. In another embodiment, the -D portion is different.

[0232] In a single-state sample, the spacer subunit (-Y y-) is given by equation ( [X])-( [XII]) indicates: [X] [] Where Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 ynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 ynyl), -halogen, -nitro, or -cyano; and m is an integer in the range of 0-4. Alkyl, alkenyl, and ynyl groups, alone or as part of another group, may be substituted as appropriate. [XI] [] and [XII].

[0233] Examples comprising formulas I and II of antibody-drug conjugate compounds may include: Where w and y are 0, 1, or 2 respectively, and Where w and y are both 0. and . IX.) Drug Unit

[0234] The pharmaceutical moiety (D) can be any cytotoxic, cell growth inhibitory, or immunomodulatory (e.g., immunosuppressive) drug. D is a pharmaceutical unit (mole) having atoms that can form a bond with a spacer subunit, an amino acid subunit, an extension subunit, or an antibody unit. In some embodiments, the pharmaceutical unit D has a nitrogen atom that can form a bond with a spacer subunit. As used herein, the terms "pharmaceutical unit" and "pharmaceutical moiety" are synonyms and are used interchangeably.

[0235] Applicable types of cytotoxic agents, cell growth inhibitors, or immunomodulators include, for example, anti-microtubule agents, DNA minor groove binders, DNA replication inhibitors, and alkylating agents.

[0236] In some embodiments, the drug is olistatin, such as olistatin E (also referred to in this art as a derivative of salitonin-10) or a derivative thereof. Olistatin may be, for example, an ester formed between olistatin E and a keto acid. For example, olistatin E may be reacted with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEB, respectively. Other typical olistatins include AFP, MMAF, and MMAE. The synthesis and structure of illustrative olistatins are described in the following patents: U.S. Patent Application Publication No. 2003-0083263; International Patent Publication No. WO 04 / 010957, International Patent Publication No. WO US Patent Nos. 02 / 088172 and 7,498,298, 6,884,869, 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; and 5,530 No. 097; No. 5,521,284; No. 5,504,191; No. 5,410,024; No. 5,138,036; No. 5,076,973; No. 4,986,988; No. 4,978,744; No. 4,879,278; No. 4,816,444; and No. 4,486,414, each of which is incorporated herein by reference in its entirety and for all purposes.

[0237] Orestatin has been shown to interfere with microtubule dynamics, cell nucleus and cell division, and possess anticancer activity. Orestatin binds to tubulin and can exert cytotoxic or growth-inhibiting effects on 191P4D12 expression cells. Several different assays known in this technique exist that can be used to determine whether orestatin or the resulting antibody-drug conjugate exerts growth-inhibiting or cytotoxic effects on desired cell lines.

[0238] Methods for determining whether a compound binds to tubulin are known in this 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 this invention, the relative affinity of a compound for tubulin can be determined. Some preferred olistatin of this invention has a tubulin-binding affinity ranging from 1 / 10 of the affinity of MMAE for tubulin (weak affinity) to 10, 20, or even 100 times the affinity of MMAE for tubulin (higher affinity). []

[0239] In some embodiments, -D is the formula [D, E , ]or [D, F Orestatin: [D, E , ] [, , ] [, , ] [D, F , ] [, , ] [, , ] Or in a medically acceptable salt or solvate form; Independently in each location: The waveform line indicates one-click operation; [R, 2 , ] represents -C1-C20 alkyl, -C2-C20 alkenyl, or -C2-C20 ynyl; [R, 3 , ] represents -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 ynyl, -carbocyclic, -C1-C20 alkyl (carbocyclic), -C2-C20 alkenyl (carbocyclic), -C2-C20 alynyl (carbocyclic), -aryl, -C1-C20 alkyl (aryl), -C2-C20 alkenyl (aryl), -C2-C20 alynyl (aryl), heterocyclic, -C1-C20 alkyl (heterocyclic), -C2-C20 alkenyl (heterocyclic), or -C2-C20 alynyl (heterocyclic); [R, 4 , ] represents -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 ynyl, carbocyclic, -C1-C20 alkyl (carbocyclic), -C2-C20 alkenyl (carbocyclic), -C2-C20 alynyl (carbocyclic), -aryl, -C1-C20 alkyl (aryl), -C2-C20 alkenyl (aryl), -C2-C20 alynyl (aryl), -heterocyclic, -C1-C20 alkyl (heterocyclic), -C2-C20 alkenyl (heterocyclic), or -C2-C20 alynyl (heterocyclic); [R, 5 , ] represents -H or -C1-C8 alkyl; or [R, 4 , ] [and] [R, 5 , ] together form a carbocyclic ring and have the formula -(CR aR b) s-, wherein Ra and R b are independently -H, -C 1-C 20 alkyl, -C 2-C 20 alkenyl, -C 2-C 20 ynyl or -carbocyclic, and s is 2, 3, 4, 5 or 6, [R, 6 , ] represents -H, -C1-C20 alkyl, -C2-C20 alkenyl, or -C2-C20 ynyl; [R, 7, ] represents -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 ynyl, carbocyclic, -C1-C20 alkyl (carbocyclic), -C2-C20 alkenyl (carbocyclic), -C2-C20 alynyl (carbocyclic), -aryl, -C1-C20 alkyl (aryl), -C2-C20 alkenyl (aryl), -C2-C20 alynyl (aryl), heterocyclic, -C1-C20 alkyl (heterocyclic), -C2-C20 alkenyl (heterocyclic), or -C2-C20 alynyl (heterocyclic); each [R, 8 , ] independently can be -H, -OH, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 ynyl, -O-(C1-C20 alkyl), -O-(C2-C20 alkenyl), -O-(C1-C20 ynyl) or -carbocyclic; [R, 9 , ] represents -H, -C1-C20 alkyl, -C2-C20 alkenyl, or -C2-C20 ynyl; [R, 24 , ] represents -aryl, -heterocyclic, or -carbocyclic; [R, 25 , ] represents -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 ynyl, -carbocyclic, -O-(C1-C20 alkyl), -O-(C2-C20 alkenyl), -O-(C2-C20 ynyl) or O [R, 18 , ],in [R, 18 , ] represents -H, hydroxyl protecting group, or when O [R, 18 , ] indicates that when =0, it is a direct key; [R, 26 , ] represents -H, -C1-C20 alkyl, -C2-C20 alkenyl or -C2-C20 alkynyl, -aryl, -heterocyclic or -carbocyclic; [R, 10 , ] represents -aryl or -heterocyclic; [Z] is -O, -S, -NH, or -NR 12, where [R, 12 , ] represents -C1-C20 alkyl, -C2-C20 alkenyl, or -C2-C20 ynyl; [R, 11 , ] represents -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl, -aryl, -heterocyclic, -(R13O)mR14 or -(R13O)m-CH(R15)2; [m] is an integer in the range of 1-1000 or m=0-1000; [R, 13 , ] represents -C 2-C 20 alkyl, -C 2-C 20 alkenyl, or -C 2-C 20 ynyl; [R, 14 , ] represents -H, -C1-C20 alkyl, -C2-C20 alkenyl, or -C2-C20 ynyl; [R, 15 Each time it appears, it is independently -H, -COOH, -(CH2)nN(R16)2, -(CH2)n-SO3H, -(CH2)n-SO3-C1-C20alkyl, -(CH2)n-SO3-C2-C20alkenyl, or -(CH2)n-SO3-C2-C20ynyl. [R, 16 Each time it appears, it is independently -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 ynyl, or -(CH2)n-COOH; and [n] is an integer in the range of 0 to 6; The alkyl, alkenyl, ynyl, alkylene, alkenyl, alkenyl, aryl, carbocyclic and heterocyclic groups, either alone or as part of another group, may be substituted as appropriate.

[0240] Mode [D, E Orestatin includes unsubstituted alkyl, alkenyl, ynyl, alkylene, alkenyl, ynylene, aryl, carbocyclic and heterocyclic olates.

[0241] Mode [D, E Oristatin includes osristatin in which the R2, R3, R4, R5, R6, R7, R8 and R9 groups are unsubstituted and the R19, R20 and R21 groups are substituted as described herein, where appropriate.

[0242] Mode [D, E The olaristatin group includes the following olaristatins, among which... [R, 2 , ] represents C1-C8 alkyl groups; [R, 3 , ]、 [R, 4 , ]and [R, 7 The radical , is independently selected from -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 ynyl, monocyclic C3-C6 carbon ring, -C1-C20 alkylene (monocyclic C3-C6 carbon ring), -C2-C20 alkenyl (monocyclic C3-C6 carbon ring), -C2-C20 ynylene (monocyclic C3-C6 carbon ring), C6-C10 aryl, -C1-C20 alkylene (C6-C10 aryl), -C2-C20 alkenyl (C6-C10 aryl), -C2-C20 ynylene (C6-C10 aryl), heterocycle, -C1-C20 alkylene (heterocycle), -C2-C 20-Alkenyl (heterocyclic) or -C 2-C 20-Alynyl (heterocyclic); wherein the alkyl, alkenyl, ynyl, alkyl, alkenyl, ynynyl, carbocyclic, aryl, and heterocyclic groups are substituted as appropriate; [R, 5 , ] represents -H; [R, 6 , ] represents -C1-C8 alkyl; each [R, 8 The , ] is independently selected from -OH, -O-(C1-C20 alkyl), -O-(C2-C20 alkenyl) or -O-(C2-C20 alkynyl), wherein such alkyl, alkenyl and alkynyl groups are substituted as appropriate; [R, 9, ] represents -H or -C1-C8 alkyl; [R, 24 , ] represents a substituted -phenyl group, depending on the situation; [R, 25 , ] is -O [R, 18 , ];in [R, 18 , ] represents H, hydroxyl protecting group, or when O [R, 18 , ] indicates that when =0, it is a direct key; [R, 26 ] is selected from -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl or -carbocyclic; wherein such alkyl, alkenyl, alkynyl and carbocyclic groups are substituted as appropriate; or in the form of a pharmaceutically acceptable salt or solvate thereof.

[0243] Mode [D, E The olaristatin group includes the following olaristatins, among which... [R, 2 , ] represents methyl; [R, 3 , ] is -H, -C1-C8 alkyl, -C2-C8 alkenyl or C2-C8 ynyl, wherein such alkyl, alkenyl and ynyl groups are substituted as appropriate; [R, 4 , ] represents -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 ynyl, monocyclic C3-C6 carbon ring, -C6-C10 aryl, -C1-C8 alkylene (C6-C10 aryl), -C2-C8 alkenyl (C6-C10 aryl), -C2-C8 alynyl (C6-C10 aryl), -C1-C8 alkylene (monocyclic C3-C6 carbon ring), -C2-C8 alkenyl (monocyclic C3-C6 carbon ring), -C2-C8 alynyl (monocyclic C3-C6 carbon ring); wherein the alkyl, alkenyl, ynylene, alkylene, alkenylene, alynyl, aryl, and carbocyclic groups, alone or as part of another group, are substituted as appropriate; [R, 5, ] represents H; [R, 6 , ] represents methyl; [R, 7 , ] represents -C1-C8 alkyl, -C2-C8 alkenyl, or -C2-C8 ynyl; each [R, 8 , ] represents a methoxy group; [R, 9 , ] represents -H or -C1-C8 alkyl; [R, 24 , ] represents -phenyl; [R, 25 , ] is -O [R, 18 , ];in [R, 18 , ] represents H, hydroxyl protecting group, or when O [R, 18 , ] indicates that when =0, it is a direct key; [R, 26 , ] represents methyl; Or its medically acceptable salt form.

[0244] Mode [D, E The olaristatin group includes the following olaristatins, among which... [R, 2 , ] represents methyl; [R, 3 , ] represents -H or -C1-C3 alkyl; [R, 4 , ] represents a -C1-C5 alkyl group; [R, 5 , ] represents -H; [R, 6 , ] represents methyl; [R, 7 , ] represents isopropyl or second butyl; [R, 8 , ] represents a methoxy group; [R, 9 , ] represents -H or -C1-C8 alkyl; [R, 24 , ] represents phenyl; [R, 25 , ] is -O [R, 18 , ];in [R, 18 , ] represents a -H, a hydroxyl protecting group, or a direct bond when OR 18 represents =O; and [R, 26 , ] is methyl; or a pharmaceutically acceptable salt or solvate thereof.

[0245] Mode [D, E The olaristatin group includes the following olaristatins, among which... [R, 2 , ] represents methyl or C1-C3 alkyl; [R, 3 , ] represents -H or -C1-C3 alkyl; [] [R, 4 , ] represents a -C1-C5 alkyl group; [R, 5 , ] represents H; [R, 6 , ] represents C1-C3 alkyl groups; [R, 7 , ] represents a -C1-C5 alkyl group; [R] 8 represents a -C1-C3 alkoxy group; [R] 9 is -H or -C1-C8 alkyl; [R, 24 , ] represents phenyl; [R, 25 , ] is -O [R, 18 , ];in [R,18 , ] represents -H, hydroxyl protecting group, or when O [R, 18 , ] indicates that when =0, it is a direct key; and [R, 26 , ] represents a -C1-C3 alkyl group; Or its medically acceptable salt form.

[0246] Mode [D, F The olaristatin group includes the following olaristatins, among which... [R, 2 , ] represents methyl; [R, 3 , ]、 [R, 4 , ]and [R, 7 The radical , is independently selected from -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 ynyl, monocyclic C3-C6 carbon ring, -C1-C20 alkylene (monocyclic C3-C6 carbon ring), -C2-C20 alkenyl (monocyclic C3-C6 carbon ring), -C2-C20 ynylene (monocyclic C3-C6 carbon ring), -C6-C10 aryl, -C1-C20 alkylene (C6-C10 aryl), -C2-C20 alkenyl (C6-C10 aryl), -C2-C20 ynylene (C6-C10 aryl), heterocycle, -C1-C20 alkylene (heterocycle), -C2-C 20-Phenylene (heterocyclic) or -C 2-C 20-Phenyne (heterocyclic); wherein such alkyl, alkenyl, ynylene, alkylene, alkenylene, alkenylene, carbocyclic, aryl, and heterocyclic groups, alone or as part of another group, are substituted as appropriate; [R, 5 , ] represents -H; [R, 6 , ] represents methyl; each [R, 8 , ] represents a methoxy group; [R, 9, ] represents -H, -C1-C20 alkyl, -C2-C20 alkenyl or -C2-C20 ynyl; wherein such alkyl, alkenyl and ynyl groups are substituted as appropriate; [R, 10 , ] represents a substituted aryl group or a substituted heterocyclic group, depending on the situation; [Z] is -O-, -S-, -NH-, or -NR 12-, where [R, 12 , ] represents -C1-C20 alkyl, -C2-C20 alkenyl, or -C2-C20 ynyl, each substituted as appropriate; [R, 11 , ] represents -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl, -aryl, -heterocyclic, -(R13O)mR14 or -(R13O)m-CH(R15)2, wherein such alkyl, alkenyl, alkynyl and heterocyclic groups are substituted as appropriate; [m] is an integer in the range of 1-1000 or m=0; [R, 13 , ] is a -C 2-C 20 alkyl, -C 2-C 20 alkenyl, or -C 2-C 20 ynyl, each substituted as appropriate; [R, 14 , ] is -H, -C1-C20 alkyl, -C2-C20 alkenyl or -C2-C20 ynyl, wherein such alkyl, alkenyl and ynyl are substituted as appropriate; [R, 15 Each time it appears, it is independently -H, -COOH, -(CH2)nN(R16)2, -(CH2)n-SO3H, -(CH2)n-SO3-C1-C20alkyl, -(CH2)n-SO3-C2-C20alkenyl or -(CH2)n-SO3-C2-C20ynyl, wherein such alkyl, alkenyl and ynyl groups are substituted as appropriate; [R, 16, ] is independently -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 ynyl or -(CH2)n-COOH each time it appears, wherein such alkyl, alkenyl and ynyl are substituted as appropriate; [n] is an integer in the range of 0 to 6; Or a medically acceptable salt.

[0247] In some of these embodiments, [R, 10 , ] represents a phenyl group that has been substituted, depending on the situation.

[0248] Mode [D, F Oristatin includes osristatin in which the R2, R3, R4, R5, R6, R7, R8 and R9 groups are unsubstituted and the R10 and R11 groups are as described herein.

[0249] Mode [D, F Orestatin includes unsubstituted alkyl, alkenyl, ynyl, alkylene, alkenyl, ynylene, aryl, carbocyclic and heterocyclic olates.

[0250] Mode [D, F The olaristatin group includes the following olaristatins, among which... [R, 2 , ] represents a -C1-C3 alkyl group; [R, 3 , ] represents -H or -C1-C3 alkyl; [R, 4 , ] represents a -C1-C5 alkyl group; [R, 5 , ] represents -H; [R, 6 , ] represents a -C1-C3 alkyl group; [R, 7 , ] represents a -C1-C5 alkyl group; [R, 8 , ] represents a -C1-C3 alkoxy group; [R, 9 , ] represents -H or -C1-C8 alkyl; [R, 10 , ] represents a substituted phenyl group, depending on the context; [Z] is -O-, -S-, or -NH-; [R, 11 , ] as defined herein; or a medically acceptable salt thereof.

[0251] Mode [D, F The olaristatin group includes the following olaristatins, among which... [R, 2 , ] represents methyl; [R, 3 , ] represents -H or -C1-C3 alkyl; [R, 4 , ] represents a -C1-C5 alkyl group; [R, 5 , ] represents -H; [R, 6 , ] represents methyl; [R, 7 , ] represents isopropyl or second butyl; [R, 8 , ] represents a methoxy group; [R, 9 , ] represents -H or -C1-C8 alkyl; [R, 10 , ] represents a substituted phenyl group, depending on the context; [Z] is -O-, -S-, or -NH-; and R [, 11 , ] as defined herein; or a medically acceptable salt thereof.

[0252] Mode [D, F The olaristatin group includes the following olaristatins, among which... [R, 2 , ] represents methyl; [R, 3 , ] represents -H or -C1-C3 alkyl; [R, 4 , ] represents a -C1-C5 alkyl group; [R, 5 , ] represents -H; [R, 6 , ] represents methyl; [R, 7 , ] represents isopropyl or second butyl; [R, 8 , ] represents a methoxy group; [R, 9 , ] represents -H or C1-C8 alkyl; [R, 10 , ] is phenyl; and Z is -O- or -NH-, and R [, 11 As defined herein, it is preferably hydrogen; or a pharmaceutically acceptable salt thereof.

[0253] Mode [D, F The olaristatin group includes the following olaristatins, among which... [R, 2 , ] represents a -C1-C3 alkyl group; [R, 3 , ] represents -H or -C1-C3 alkyl; [R, 4 , ] represents a -C1-C5 alkyl group; [R, 5 , ] represents -H; [R, 6 , ] represents a -C1-C3 alkyl group; [R, 7 , ] represents a -C1-C5 alkyl group; [R, 8 , ] represents a -C1-C3 alkoxy group; [R, 9 , ] represents -H or -C1-C8 alkyl; [R, 10 , ] represents phenyl; and [Z] is -O- or -NH-, and R [, 11 As defined herein, it is preferably hydrogen; or a pharmaceutically acceptable salt thereof.

[0254] Mode [D, E , ]or [D, FOristatin comprising R3, R4, and R7 being independently isopropyl or second-butyl and R5 being -H. In one exemplary embodiment, R3 and R4 are each isopropyl, R5 is H, and R7 is second-butyl. The remaining substituents are as defined herein.

[0255] Mode [D, E , ]or [D, F Orestatin of the , ] type includes olrestatin in which R2 and R6 are both methyl and R9 is H. Other substituents are as defined herein.

[0256] Mode [D, E , ]or [D, F Orestatin includes ozogatidine with R8 as -OCH3 in each occurrence. Other substituents are as defined herein.

[0257] Mode [D, E , ]or [D, F The osrestatin , ] includes the following osrestatin, wherein R3 and R4 are each isopropyl, R2 and R6 are each methyl, R5 is H, R7 is dibutyl, R8 is -OCH3 each time it appears, and R9 is H. The remaining substituents are as defined herein.

[0258] Mode [D, F Orestatin of the , ] category includes olarestatin in which Z is -O- or -NH-. Other substituents are as defined herein.

[0259] Mode [D, F Orestatin of the , ] class includes olarestatin with R 10 being aryl. Other substituents are as defined herein.

[0260] Mode [D, F Orestatin of the , ] class includes olarestatin with R 10 being -phenyl. Other substituents are as defined herein.

[0261] Mode [D,F Orestatins of the denominator , ] include those with Z being -O- and R 11 being H, methyl, or tert-butyl. Other substituents are as defined herein.

[0262] Mode [D, F The osrestatin , ] comprises the following osrestatin, wherein when Z is -NH-, R11 is -(R13O)m-CH(R15)2, where R15 is -(CH2)nN(R16)2, and R16 is -C1-C8 alkyl or -(CH2)n-COOH. Other substituents are as defined herein.

[0263] Mode [D, F The oseristatin , ] includes the following oseristatin, wherein when Z is -NH-, R11 is -(R13O)m-CH(R15)2, and R15 is -(CH2)n-SO3H. The remaining substituents are as defined herein.

[0264] In a preferred embodiment, when D is the equation [D, E When using osmaristatin, w is an integer in the range of 1 to 12, preferably 2 to 12, y is 1 or 2, and a is preferably 1.

[0265] In some embodiments, when D is the equation [D, F When , ] is ozogatidine, a is 1 and w and y are 0.

[0266] The illustrative pharmaceutical unit (-D) includes a pharmaceutical unit having the following structure: , , , , , , , , , , , and Or its pharmaceutically acceptable salts or solvates.

[0267] In one state, hydrophilic groups such as (but not limited to) triethylene glycol ester (TEG) can be attached to R11 of the drug unit. Without being bound by theory, the hydrophilic groups help the drug unit internalize and prevent aggregation.

[0268] In some embodiments, the pharmaceutical unit is not TZT-1027. In some embodiments, the pharmaceutical unit is not olistatin E, saliton toxin 10, or olistatin PE.

[0269] An exemplary antibody-drug conjugate compound has the following structure, wherein "L" or "mAb-s" represents the 191P4D12 MAb described herein as Ha22-2(2,4)6.1: [L-MC-vc-PAB-MMAF] [] [] or [L-MC-vc-PAB-MMAE.] [] or [] [L-MC-MMAF] [] [] Or a medically acceptable salt.

[0270] In some embodiments, the pharmaceutical unit is chalcogenide, camptothecin, maytansine, or anthracycline. In some embodiments, the pharmaceutical unit is taxane, topoisomerase inhibitor, vinca alkaloid, or an analogue thereof.

[0271] 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. Patent No. 6,130,237), pyroxine, taxanes (e.g., paclitaxel and docetaxel), puromycin, and vinca alkaloids. Other cytotoxic agents include, for example, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorolino-doxorubicin, echinomycin, cobustatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, leutherobin, discodermolide, eleutherobin, and mitoxantrone.

[0272] In some embodiments, the drug is an anti-tubulin agent. Examples of anti-tubulin agents include oliquistatin, taxanes (e.g., Taxol®, Taxotere®), T67 (Tularik), and vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine, and vinorelbine). Other anti-tubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epokine A and B), nocodazole, colchicine and colcimid, estradiol, novocyclophosphamide, simadalide, maytansine, cobustatin, disoproxil fumarate, and ilexol.

[0273] In some embodiments, the cytotoxic agent is a maytansine-like agent, another group of anti-microtubule agents. For example, in a particular embodiment, the maytansine-like agent is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari et al. 1992, Cancer Res. 52:127-131).

[0274] In some embodiments, the cytotoxic agent or cell growth inhibitor is salipodoxomil. In some embodiments, the cytotoxic agent or cell growth inhibitor is an olistatin. Therefore, in a particular embodiment, the cytotoxic agent or cell growth inhibitor is MMAE (formula omitted). [XI]). In another specific embodiment, the cytotoxic agent or cell growth inhibitor is AFP (formula...). [XVI]). [(XI)] [] [] In some embodiments, the cytotoxic agent or cell growth inhibitor is of the formula... [XII-XXI] compounds or their pharmaceutically acceptable salts: [(XII)] [] [(XIII)] [] [(XIV)] [] [] [(XV)] [] [] [(XVI)] [] [] [] [(XVII)] [] [] ( [XVIII]) [(XVIV)] [(XX)] [] [(XXI)] [] X.) Drug load

[0275] Drug load is represented by p and is the average number of drug moieties in each antibody in the molecule. Drug load can range from 1 to 20 drug moieties (D) per antibody. The ADC of the present invention comprises a collection of antibodies conjugated with drug moieties ranging from 1 to 20. The average number of drug moieties in each antibody in the preparation of the ADC by the conjugation reaction can be characterized by conventional means such as mass spectrometry and ELISA assay. The quantitative distribution of p in the ADC can also be determined. In some cases, homogeneous ADCs can be separated, purified, and characterized by means such as electrophoresis, where p is a value of an ADC with other drug loads.

[0276] For some antibody-drug conjugates, p may be limited by the number of linker sites on the antibody. For example, if the linker is a cysteine ​​sulfhydryl group as illustrated in the above examples, the antibody may have only one or more cysteine ​​sulfhydryl groups, or it may have only one or more sufficiently reactive sulfhydryl groups, through which the linker can be linked. In some embodiments, a higher drug loading (e.g., p > 5) may cause some antibody-drug conjugates to agglutinate, become insoluble, become toxic, or lose cell permeability. In some embodiments, the drug loading of the ADC of the present invention is in the following ranges: 1 to 8; about 2 to 6; about 3 to 5; about 3 to 4; about 3.1 to 3.9; about 3.2 to 3.8; about 3.2 to 3.7; about 3.2 to 3.6; about 3.3 to 3.8; or about 3.3 to 3.7. In fact, it has been shown that for some ADCs, the optimal ratio of the drug portion per antibody may be less than 8, and may be about 2 to about 5. See U.S. Patent No. 7,498,298 (which is incorporated herein by reference in its entirety).

[0277] In some embodiments, less than the theoretical maximum amount of the drug moiety is conjugated with the antibody during the conjugation reaction. The antibody may contain, for example, lysine residues that do not react with drug-linker intermediates or linker reagents, as discussed below. Generally, antibodies do not contain many free and reactive cysteine ​​thiols that can be linked to the drug moiety; in fact, most cysteine ​​thiols in antibodies exist as disulfide bridges. In some embodiments, the antibody may be reduced under partial or complete reducing conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to produce reactive cysteine ​​thiols. In some embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine.

[0278] The loading (drug / antibody ratio) of an ADC can be controlled in various ways, such as by: (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody; (ii) limiting the conjugation reaction time or temperature; (iii) partially limiting or restricting the reduction conditions of cysteine ​​thiol modification; (iv) engineering the amino acid sequence of the antibody using recombinant technology to adjust the number and position of cysteine ​​residues to control the number and / or position of linker-drug linkages (such as thioMab or thioFab prepared as disclosed herein and in WO 2006 / 034488 (incorporated herein by reference in its entirety).

[0279] It should be understood that when one or more nucleophilic groups react with a drug-linker intermediate or linker reagent, followed by a drug moiety reagent, the resulting product is a mixture of ADC compounds having one or more drug moieties linked to antibodies. The average number of drugs per antibody can be calculated from this mixture using a dual ELISA antibody assay that is specific for both antibodies and drugs. Individual ADC molecules can be identified in the mixture by mass spectrometry and separated by HPLC (e.g., hydrophobic interaction chromatography) (see, for example, 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, Proceedings of the AACR, 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, Proceedings of the AACR, Vol. 45, March 2004). In some embodiments, homogeneous ADCs with a single loading value can be separated from conjugated mixtures by electrophoresis or chromatography. XI.) Methods for determining the cytotoxic effects of ADCs

[0280] Methods for determining whether a drug or antibody-drug conjugate exerts cell growth inhibition and / or cytotoxic effects on cells are known. Generally, the cytotoxic or cell growth-inhibiting activity of an antibody-drug conjugate can be measured by: exposing mammalian cells expressing the target protein of the antibody-drug conjugate to cell culture medium; culturing these cells for a period of approximately 6 hours to approximately 5 days; and measuring cell viability. Cell-based in vitro assays can be used to quantitatively measure the activity (proliferation), cytotoxicity, and apoptosis induction (caspase activation) of antibody-drug conjugates.

[0281] To determine whether an antibody drug conjugate exerts an inhibitory effect on cell growth, a thymidine incorporation assay can be used. For example, cancer cells representing the target antigen can be cultured in 96-well plates at a density of 5,000 cells per well for 72 hours, and exposed to 0.5 μCi of 3H-thymidine during the last 8 hours of the 72-hour period. The incorporation of 3H-thymidine into the cells of the culture is measured in both the presence and absence of the antibody drug conjugate.

[0282] To determine cytotoxicity, necrosis or apoptosis (progressive cell death) can be measured. Necrosis is usually accompanied by increased plasma membrane permeability, cell swelling, and plasma membrane rupture. Apoptosis is typically characterized by membrane bubbling, cytoplasmic condensation, and activation of endogenous nucleases. Measuring either of these effects on cancer cells indicates that the antibody-drug conjugate is suitable for cancer treatment.

[0283] Cell viability can be measured by determining the uptake of dyes (such as neutral red, trypan blue, or ALAMAR™ blue) in cells (see, for example, Page et al. 1993, Intl. J. Oncology 3:473-476). In this assay, cells are cultured in a dye-containing medium, washed, and the remaining dye is measured spectrophotometrically, reflecting the cells' uptake of the dye. Cytotoxicity can also be measured using the protein-binding dye sulforhodamine B (SRB) (Skehan et al. 1990, J. Natl. Cancer Inst. 82:1107-12).

[0284] Alternatively, a tetrazolium salt such as MTT can be used in quantitative colorimetric assays for determining mammalian cell viability and proliferation by detecting live rather than dead cells (see, for example, Mosmann, 1983, J. Immunol. Methods 65:55-63).

[0285] Apoptosis can be quantified by measurements such as DNA fragmentation. Commercially available spectrophotometric methods for the in vitro quantification of DNA fragmentation can be used. Examples of such assays, including those based on TUNEL (which detects the incorporation of labeled nucleotides into DNA fragments) and ELISA, are described in Biochemica, 1999, Vol. 2, pp. 34-37 (Roche Molecular Biochemicals).

[0286] Apoptosis can also be determined by measuring morphological changes in cells. For example, similar to necrosis, loss of plasma membrane integrity can be determined by measuring the uptake of certain dyes (e.g., fluorescent dyes such as acridine orange or ethidium bromide). Methods for measuring the number of apoptotic cells have been described by Duke and Cohen, 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 chromatin condensation and erosion along the inner nuclear membrane can be observed. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane bubbling, and cell contraction.

[0287] The presence of apoptotic cells can be measured in both the attached and "floating" portions of the culture. For example, the supernatant can be removed from both collected portions, the attached cells can be trypsinized, the preparations can be combined after a centrifugation and washing step (e.g., at 2000 rpm for 10 minutes), and apoptosis can be detected (e.g., by measuring DNA breaks). (See, for example, Piazza et al. 1995, Cancer Research 55:3110-16).

[0288] The effects of 191P4D12 therapeutic compositions can be evaluated in vivo in suitable animal models. For example, allogeneic cancer models can be used, in which cancer explants or subsequent xenograft tissues are introduced into immunocompromised animals, such as nude mice or SCID mice (Klein et al. 1997, Nature Medicine 3: 402-408). For instance, PCT patent application WO 98 / 16628 and US patent 6,107,540 describe various xenograft models of human prostate cancer that can recreate the development of the primary tumor, micrometastases, and the formation of osteoblastic metastases characteristic of advanced disease. Predictive efficacy can be assessed using measures of tumorigenesis inhibition, tumor regression or metastasis, and similar processes.

[0289] In vivo assays to assess accelerated apoptosis are suitable for evaluating therapeutic compositions. In one embodiment, the presence of apoptotic centers in xenografts from mice with tumors treated with the therapeutic composition can be examined and compared with untreated control mice with xenografts. The degree of presence of apoptotic foci in the tumors of the treated mice indicates the therapeutic efficacy of the composition.

[0290] Therapeutic compositions used to carry out the above methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any substance that, when combined with a therapeutic composition, retains the antitumor function of the therapeutic composition and is normally unresponsive to the patient's immune system. Examples include (but are not limited to) any of a variety of standard pharmaceutical carriers, such as sterile phosphate-buffered saline solutions, antibacterial water, and the like (see generally Remington's Pharmaceutical Sciences, 16th edition, ed. A. Osal, 1980).

[0291] Therapeutic formulations are soluble and can be administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include (but are not limited to) intravenous, non-enteral, intraperitoneal, intramuscular, intratumoral, intradermal, intra-organ, orthotopic, and similar routes. Preferred formulations for intravenous injection comprise the therapeutic composition in a solution of preservative-treated sterile water, unpreservative-treated sterile water, and / or diluted in a polyvinyl chloride or polyethylene bag containing 0.9% sterile sodium chloride for injection (USP). Therapeutic protein formulations may be lyophilized and can be stored in sterile powder form, preferably under vacuum, followed by reconstitution with sterile water (containing, for example, a preservative such as benzyl alcohol) or sterile water before injection.

[0292] The dosage and administration regimen for treating cancer using the above methods will vary depending on the method and the target cancer, and will generally depend on a number of other factors understood in this technology. XII.) Treatment of cancer manifested by 191P4D12

[0293] Identifying 191P4D12 as a protein that functions normally in a limited group of tissues but also in cancers (such as those listed in Table I) opens up a variety of treatment options for these cancers.

[0294] It is worth noting that targeted anti-tumor therapy is useful even when the target protein is present in normal tissues, or even in normal living organs. Living organs are those essential for maintaining life, such as the heart or colon. Non-living organs are those that allow the individual to survive after removal. Examples of non-living organs include the ovary, breast, and prostate.

[0295] The expression of a target protein in normal tissues, or even normal living tissues, does not preclude the efficacy of the protein's target agent as a therapeutic agent for certain tumors where the protein is also overexpressed. For example, expression in living organs is not inherently harmful. Furthermore, organs considered unnecessary (such as the prostate and ovaries) can be removed without affecting lifespan. Finally, some living organs are unaffected by expression in normal organs due to immune pardon. Immune pardoned organs are those protected by the blood-organ barrier and isolated from the blood, thus inaccessible to immunotherapy. Examples of immune pardoned organs include the brain and testes.

[0296] Therefore, treatments that inhibit 191P4D12 protein activity are suitable for patients with cancers exhibiting 191P4D12 activity. These treatments are generally classified into three categories. The first category modulates 191P4D12 function because it is associated with tumor cell growth, thereby inhibiting or delaying tumor cell growth or inducing its killing. The second category includes various methods that inhibit the binding or association of the 191P4D12 protein with its binding complexes or with other proteins. The third category includes various methods that inhibit the transcription of the 191P4D12 gene or the translation of 191P4D12 mRNA.

[0297] Therefore, to assess the presence and level of 191P4D12 expression in cancer patients, immunohistochemical evaluation of tumor tissue, quantitative 191P4D12 imaging, or other techniques that reliably indicate the presence and extent of 191P4D12 expression are preferred. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissue are well known in this technique. XIII.) 191P4D12 as a target for antibody-based therapy

[0298] 191P4D12 is an attractive target for antibody-based therapeutic strategies. Various antibody strategies targeting extracellular and intracellular molecules are known in this technology (see, for example, complement and ADCC-mediated killing and the use of intracellular antibodies). Because 191P4D12 is expressed by various lineages of cancer cells relative to their corresponding normal cells, systemic administration of 191P4D12 immunoreactive compositions is prepared to exhibit excellent sensitivity to non-target organs and tissues without toxicity, non-specificity, and / or non-targeting effects. Antibodies that specifically react with the domains of 191P4D12 are suitable for systemic treatment of cancers expressing 191P4D12, preferably in the form of antibody-drug conjugates (i.e., ADCs) containing a toxin or therapeutic agent.

[0299] Those skilled in this art will understand that antibodies can be used to specifically target and bind to immunogenic molecules, such as the immunogenic region of the 191P4D12 sequence shown in Figure 1. Furthermore, those skilled in this art will understand that conjugation of antibodies with cytotoxic agents is common practice (see, for example, 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 conjugation with an antibody that is specific to the molecules expressed by those cells (e.g., 191P4D12)), the cytotoxic agents exert known biological effects (i.e., cytotoxicity) on those cells.

[0300] Various compositions and methods for killing cells using antibody-cytotoxic agent conjugates are known in this art. In the case of cancer, a typical method requires administering a bioeffective amount of a conjugate to a mammal with a tumor, the conjugate comprising a selected cytotoxic agent and / or therapeutic agent linked to a target agent (e.g., 191P4D12 MAb, preferably Ha22-2(2,4)6.1) that binds to an antigen (e.g., 191P4D12) expressed, readily bound to, or localized on the cell surface. A typical embodiment is a method of delivering a cytotoxic agent and / or therapeutic agent to cells expressing 191P4D12, comprising conjugating the cytotoxic agent to an antibody that specifically binds to the 191P4D12 antigenic determinant and exposing the cells to an antibody-drug conjugate (ADC). Another illustrative embodiment is a method of treating an individual suspected of having metastatic cancer, comprising administering a pharmaceutical composition, non-enteral, to the individual comprising a therapeutically effective amount of an antibody and a conjugate of a cytotoxic agent and / or therapeutic agent.

[0301] Cancer immunotherapy using the 191P4D12 antibody can be performed using 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 (Ozaki 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 (Zhong et al. 1996, Leuk. Res. 20:581-589), and 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 involve conjugating naked antibodies with toxins or radioisotopes, such as conjugating Y91 or I131 with anti-CD20 antibodies (e.g., Zevalin™ (IDEC Pharmaceuticals Corp.) or Bexxar™ (Coulter Pharmaceuticals)), while other methods involve co-administering antibodies with other therapeutic agents, such as co-administering Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc). In a preferred embodiment, the antibody is conjugated with a cytotoxic agent (see above), preferably a piracetam derivative called MMAE (Seattle Genetics, Inc).

[0302] Although 191P4D12 antibody therapy is applicable to all stages of cancer, it is particularly suitable for advanced or metastatic cancer. Treatment with the antibody therapy of this invention is suitable for patients who have received one or more rounds of chemotherapy. Alternatively, the antibody therapy of this invention can be combined with chemotherapy or radiotherapy for patients who have not yet received chemotherapy. In addition, antibody therapy can be used with low doses of concomitant chemotherapy, especially for patients who cannot tolerate the toxicity of chemotherapy agents very 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 use of various antibodies in conjunction with chemotherapy agents.

[0303] 191P4D12 monoclonal antibodies for treating the cancers shown in Table I include antibodies that elicit an effective immune response against the tumor or antibodies that are directly cytotoxic. In this regard, 191P4D12 monoclonal antibodies (MAbs) can induce tumor cell lysis via complement-mediated or antibody-dependent cytotoxicity (ADCC) mechanisms, both of which require the interaction of the intact Fc portion of the immunoglobulin molecule with the effector cell Fc receptor site on complement proteins. Additionally, 191P4D12 MAbs that exert direct biological effects on tumor growth are suitable for treating cancers exhibiting 191P4D12. The mechanisms by which cytotoxic MAbs act directly include: inhibiting cell growth, regulating cell differentiation, modulating tumor angiogenesis profiles, and inducing apoptosis. As is generally known in this art, various in vitro assays are used to assess the mechanisms by which specific 191P4D12 MAbs exert their antitumor effects, such as ADCC and complement-mediated cell death.

[0304] Therefore, the preferred monoclonal antibody used in the treatment method of the present invention is a fully human monoclonal antibody and a monoclonal antibody that specifically binds to the target 191P4D12 antigen with high affinity. [XIV.) 191P4D12 ADC] [mixture] []

[0305] The treatment methods of this invention encompass the administration of a single 191P4D12 ADC and combinations or mixtures of different MAbs (i.e., 191P4D12 MAb or a Mab binding to another protein). Such MAb mixtures may have certain advantages due to containing MAbs targeting different antigenic determinants, utilizing different effector mechanisms, or combining direct cytotoxic MAbs with MAbs that depend on immune effector functions. These combined forms of MAbs can exhibit synergistic therapeutic effects. Furthermore, 191P4D12 MAb can be administered in conjunction with other therapeutic modalities, including (but not limited to) various chemotherapeutic agents and biologics, androgen blockers, immunomodulators (e.g., IL-2, GM-CSF), surgery, or radiation. In a preferred embodiment, 191P4D12 MAb is administered in a conjugated form.

[0306] 191P4D12 ADC formulations are administered via any route capable of delivering antibodies to tumor cells. Routes of administration include (but are not limited to) intravenous, intraperitoneal, intramuscular, intratumoral, intradermal, and similar routes. Treatment typically involves repeated administration of 191P4D12 MAb via an acceptable route of administration, such as intravenous (IV) injection, at doses typically including (but 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 per kilogram of body weight. Generally, a dose range of 10–1,000 mg MAb per week is effective and well-tolerated.

[0307] Based on clinical experience with Herceptin® (trastuzumab) in the treatment of metastatic breast cancer, an initial intravenous rapid-acting dose of approximately 4 mg per kilogram of patient body weight (MAb), followed by approximately 2 mg per kilogram of patient body weight intravenously weekly, represents an acceptable dosing regimen. The initial rapid-acting dose is preferably administered via infusion over 90 minutes or longer. Regular maintenance doses are administered via infusion over 30 minutes or longer, subject to the condition that the initial dose is well tolerated. As those skilled in the art will understand, several factors can influence the ideal dosing regimen in specific circumstances. These factors include, for example, the binding affinity and half-life of the MAb used; the degree of 191P4D12 expression in the patient; the extent of circulatory excretion of the 191P4D12 antigen; the required steady-state antibody concentration level; the frequency of treatment; and the effect of combining chemotherapy agents or other agents with the treatment methods of this invention, as well as the specific patient's health condition.

[0308] As appropriate, the patient's 191P4D12 levels (e.g., circulating 191P4D12 antigen and / or 191P4D12-expressing cell levels) should be assessed in specific samples to help determine the most effective dosing regimen. These assessments are also used to monitor treatment objectives and are suitable for evaluating treatment outcomes in conjunction with assessments of other parameters (e.g., urinary cytology and / or ImmunoCyt levels in bladder cancer therapy, or similarly, serum PSA levels in prostate cancer therapy).

[0309] The object of this invention is to provide a 191P4D12 ADC that inhibits or delays the growth of tumor cells expressing 191P4D12. Another object of this invention is to provide a method for using such 191P4D12 ADCs, and especially for using such 191P4D12 ADCs in combination with other drugs or immunologically effective treatments, to inhibit angiogenesis and other biological functions, thereby reducing tumor growth in mammals (preferably humans). [XV.)] [Combination therapy] []

[0310] In one embodiment, a synergistic effect exists when 191P4D12 ADC is combined with chemotherapy or radiation, or a combination thereof, to treat tumors (including human tumors). In other words, when combined with chemotherapy or radiation, or a combination thereof, the inhibition of tumor growth by 191P4D12 ADC is enhanced beyond expectation. The synergistic effect can be demonstrated, for example, by the combination therapy inhibiting tumor growth beyond the effect expected by treatment using 191P4D12 ADC alone, or by the additive effect of using 191P4D12 ADC with chemotherapy or radiation. Preferably, the synergistic effect is illustrated by cancer remission, wherein treatment using 191P4D12 ADC or treatment using a cumulative combination of 191P4D12 ADC with chemotherapy or radiation did not result in expected remission.

[0311] Methods of using 191P4D12 ADCs in conjunction with chemotherapy or radiation, or a combination of both, to inhibit tumor cell growth include administering 191P4D12 ADCs before, during, or after the initiation of chemotherapy or radiation therapy, and any combination thereof (i.e., before and during, before and after, during and after, or before, during, and after). For example, 191P4D12 ADCs are typically administered 1 to 60 days, preferably 3 to 40 days, and even more preferably 5 to 12 days before the initiation of radiation therapy and / or chemotherapy. However, depending on the treatment regimen and the specific needs of the patient, this method may provide the most effective treatment and ultimately prolong the patient's life.

[0312] Chemotherapy agents can be administered in various ways, including systemic administration via non-enteric and enteral routes. In one embodiment, the 191P4D12 ADC and the chemotherapeutic agent are administered as separate molecules. Specific examples of chemotherapeutic agents or chemotherapy include cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), etc. Mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), adriamycin, daunomycin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vincristine, bleomycin, paclitaxel, taxotere, aldesleukin, aspartame, busulfan, carboplatin, cladribine. Carbazine, fluxuridine, fludarabine, hydroxyurea, ifosfamide, interferon-alpha, leuprolide, megestrol, melphalan, mecaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipebroman, streptozomycin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, gemcitabine, chlorambucil, paclitaxel, and combinations thereof.

[0313] The radiation source used in conjunction with the 191P4D12 ADC can be external or internal to the patient being treated. When the radiation source is external to the patient, this therapy is called external beam radiation therapy (EBRT). When the radiation source is internal to the patient, this therapy is called brachytherapy (BT).

[0314] The above treatment regimens can be further combined with other cancer therapeutics and / or regimens, such as other chemotherapy, cancer vaccines, signal transduction inhibitors, agents suitable for treating abnormal cell growth or cancer, antibodies (e.g., the anti-CTLA-4 antibody described in WO / 2005 / 092380 (Pfizer)) or other ligands that inhibit tumor growth by binding to IGF-1R and cytokines.

[0315] The aforementioned chemotherapeutic agents can be used when mammals are undergoing other chemotherapy treatments. Additionally, growth factor inhibitors, biological response modifiers, anti-hormonal therapies, selective estrogen receptor modulators (SERMs), angiogenesis inhibitors, and anti-androgens can be used. For example, anti-hormonal agents such as anti-estrogens, such as Nolvadex (tamoxifen), or anti-androgens, such as Casodex (4'-cyano-3-(4-fluorobenzenesulfonylurea)-2-hydroxy-2-methyl-3'-(trifluoromethyl)propionic acid), can be used.

[0316] The above-described treatment methods can be combined with any of a variety of surgical, chemotherapy, or radiotherapy regimens. The treatment methods of this invention allow for the use of low-dose chemotherapy (or other therapies) and / or infrequent administration, which is an advantage for all patients, especially those who cannot well tolerate the toxicity of chemotherapy agents. XVI.) Sets / Products

[0317] Kits used for the laboratory, prognostic, preventive, diagnostic, and therapeutic applications described herein are within the scope of this invention. Such kits may comprise carriers, packages, or containers divided into compartments to accommodate one or more containers (such as vials, test tubes, and the like), each container containing one of the respective elements to be used in the method, along with a label or insert containing instructions for use (such as those described herein). For example, a container may contain a labeled detectable or an antibody that can label a detectable. Kits may comprise containers containing pharmaceutical units. Kits may include all or part of the amino acid sequences shown in Figure 2 or 3, or analogues thereof, or nucleic acid molecules encoding such amino acid sequences.

[0318] The kit of the present invention will typically include the aforementioned container and one or more other related containers containing substances required from a commercial and user standpoint, including buffer solutions, diluents, filters, needles, syringes; carriers, packaging, containers, vials and / or test tube labels listing the contents and / or instructions for use; and packaging inserts with instructions for use.

[0319] Labels may be present on or with the container to indicate that the composition is intended for a specific therapeutic or non-therapeutic application, such as prognostic, preventative, diagnostic, or laboratory use, and may also indicate instructions for in vivo or in vitro use, such as those described herein. Instructions and / or other information may also be included on inserts or labels incorporated into or on the kit. Labels may be located on or with the container. When the letters, numbers, or other characters forming the label are molded or etched into the container itself, the label may be located on the container; when the label is present within a reservoir or carrier that also contains the container, it may accompany the container, such as a packaging insert. Labels may indicate that the composition is intended for the diagnosis, treatment, prevention, or prognosis of conditions, such as tissue cancers as shown in Table I.

[0320] The terms "set" and "product" can be used as synonyms.

[0321] In another embodiment of the invention, an article is provided comprising a composition, such as an antibody or antibody-drug conjugate (ADC), for example, a substance suitable for the diagnosis, prognosis, prevention, and / or treatment of tissue cancers (such as those shown in Table I). The article typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers may be formed from a variety of materials, such as glass, metal, or plastic. Containers may contain amino acid sequences, small molecules, nucleic acid sequences, cell populations, and / or antibodies. In another embodiment, the container comprises an antibody, its binding fragment, or specifically binding protein for assessing the protein expression of 191P4D12 in cells and tissues or for related laboratory, prognostic, diagnostic, preventative, and therapeutic purposes; instructions and / or guidance regarding such uses may be included on or with the container, and reagents and other compositions or tools for such purposes may also be included on or with the container.

[0322] The container may contain a composition for the effective treatment, diagnosis, prognosis, or prevention of a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous injection needle). The active agent in the composition may be an antibody that specifically binds to 191P4D12 or an antibody-drug conjugate that specifically binds to 191P4D12.

[0323] The product may further include a second container containing a pharmaceutically acceptable buffer solution, such as phosphate-buffered saline, Ringer's solution, and / or dextran solution. It may further include other substances required from a commercial and user standpoint, including other buffer solutions, diluents, filters, stirrers, needles, syringes, and / or packaging inserts with usage instructions and / or guidance. [Example:] []

[0324] The following examples further describe and illustrate various aspects of the present invention, but it is not intended that these examples limit the scope of the present invention. [Example] [1] [191P4D12] [antigen] []

[0325] The 191P4D12 gene sequence was visualized using suppression subtractive hybridization (SSH), a technique known in this field. The 223 bp 191P4D12 SSH sequence was identified by subtracting cDNA from a mixture of nine normal tissues from bladder tumor tissue using standard methods. A full-length cDNA line of 191P4D12 was isolated from a bladder cancer cDNA library. The cDNA was 3464 bp in length and encoded a 510-amino acid ORF (see Figure 1). The 191P4D12 gene showed homology with the Nectin-4 gene. For further references, see US 2004 / 0083497 (Agensys, Inc., Santa Monica, CA) and PCT Publication WO 2004 / 016799 (Agensys, Inc., Santa Monica, CA). An illustrative example of the 191P4D12 antigen is shown in Figure 1. [Example] [2] [produce] [191P4D12] [Single-strain antibody] [(MAb)]

[0326] In one embodiment, therapeutic monoclonal antibodies (“MAbs”) of 191P4D12 and 191P4D12 variants comprise monoclonal antibodies that react with antigenic determinants specific to each protein, or monoclonal antibodies specific to sequences common to the variants. These MAbs bind to 191P4D12 or the 191P4D12 variant, internalizing it, disrupting or modulating its biological function, such as by disrupting interactions with ligands, receptors, and binding complexes. Immunogens generating these MAbs include those designed to encode or contain an extracellular domain or the entire 191P4D12 protein sequence, regions expected to contain functional motifs, and regions of antigenic 191P4D12 protein variants predicted by computer analysis of amino acid sequences. Immunogens include peptides and recombinant proteins, such as tag5-191P4D12, a protein derived from purified mammalian cells that is a marker of His. Additionally, mice were immunized using cells engineered to express high levels of 191P4D12 (such as RAT1-191P4D12 or 300.19-191P4D12).

[0327] The 191P4D12 MAb was generated using XenoMouse technology® (Amgem Fremont), in which mouse heavy chain and κ light chain loci were deactivated and inserted into most human heavy chain and κ light chain immunoglobulin loci. This is called... [Ha22-2(2,4)6.1] MAb was produced by autoimmunization in xenomice (XenoMice) with pTag5 / mychis-191P4D12 (amino acid 23-351) producing human γ1.

[0328] 191P4D12 MAb [Ha22-2(2,4)6.1] was found by ELISA to specifically bind to the pTag5 / mychis-191P4D12 protein and to cells expressing recombinant 191P4D12 and various cancer cell lines expressing 191P4D12.

[0329] The generation is called Fusion tumors of the antibody [Ha22-2(2,4)6.1] were found in...

[2010] [Year] [8] [moon]

[18] [Japan] (via Federal Express) sent to the American Type Culture Collection Center (ATCC), P.O. Box 1549, Manassas, VA 20108, and given a deposit number. [PTA-11267].

[0330] mRNA encoded by 191P4D12 was isolated from individual fusion tumor cells using Trizol reagent (Life Technologies, Gibco BRL). DNA with the sequence [Ha22-2(2,4)6.1].

[0331] The following protocol was used to sequence the heavy and light chain variable nucleic acid sequences of autofusion tumor cells against 191P4D12 Ha22-2(2,4)6.1. Ha22-2(2,4)6.1 autofusion tumor cells secreting Ha22-2(2,4)6.1 were lysed using Trizol reagent (Life Technologies, Gibco BRL). Total RNA was purified and quantified. First-strand cDNA was generated from total RNA using the Gibco-BRL Superscript Preamplification system activated with oligo(dT)12-18. The first-strand cDNA was amplified using human immunoglobulin variable heavy chain and human immunoglobulin variable light chain primers. The PCR products were sequenced, and the variable heavy and light chain regions were determined.

[0332] The nucleic acid and amino acid sequences of the variable heavy and light chain regions are shown in Figures 2 and 3. The comparison between Ha22-2(2,4)6.1 MAb and human Ig germline is shown in Figures 4A-4B. [Example] [3] [Use Recombination] [DNA] [Method Performance] [Ha22-2(2,4)6.1]

[0333] To recombinantly express Ha22-2(2,4)6.1 MAb in transfected cells, the variable heavy and light chain sequences of Ha22-2(2,4)6.1 MAb were selected upstream of the human heavy chain IgG1 and human light chain Igκ constant regions, respectively. The intact Ha22-2(2,4)6.1 MAb human heavy and light chain cartridges were selected downstream of the CMV promoter / enhancer in the selection vector. A polyadenylation site is located downstream of the MAb coding sequence. The constructs expressing recombinant Ha22-2(2,4)6.1 MAb were transfected into CHO cells. The binding of Ha22-2(2,4)6.1 MAb secreted by the recombinant cells to 191P4D12 on the cell surface was assessed by flow cytometry (Figure 5A). RAT-control and RAT-191P4D12 cells were stained with Ha22-2(2,4)6.1 MAb from fusion tumors or CHO cells transfected with Ha22-2(2,4)6.1 heavy and light chain vector constructs. Binding was detected by flow cytometry.

[0334] The results showed that, similar to the purified Ha22-2(2,4)6.1 from the fusion tumor, recombinant Ha22-2(2,4)6.1 bound to 191P4D12 in CHO cells. The binding of Ha22-2(2,4)6.1 MAb secreted by the recombinant cells to the recombinant 191P4D12 protein was also assessed by ELISA. As shown in Figure 5B, the binding of Ha22-2(2,4)6.1 to 191P4D12 protein was consistent between MAb substances derived from CHO and fusion tumor cells. [Example] [4] [Ha22-2(2,4)6.1 MAb] [Antibody-drug conjugation] []

[0335] Using the following protocol, Ha22-2(2,4)6.1 MAb (Figure 2) is conjugated with an olritamine derivative called MMAE (Formula XI) using the vc(Val-Cit) linker described herein to form the antibody-drug conjugate (ADC) of the present invention, called Ha22-2(2,4)6.1vcMMAE. The vc(Val-Cit) linker is conjugated with MMAE (Seattle Genetics, Inc., Seattle, WA) using the general method shown in Table IV to form the cytotoxic vcMMAE (see U.S. Patent No. 7,659,241).

[0336] The antibody-drug conjugate (ADC) of the present invention, known as Ha22-2(2,4)6.1vcMMAE, was then prepared using the following protocol.

[0337] In short, a solution of 15 mg / mL Ha22-2(2,4)6.1MAb in 10 mM acetate (pH 5.0), 1% sorbitol, and 3% L-arginine was added to adjust the pH of the solution to 7.5, along with 20% (v / v) 0.1 M TrisCl (pH 8.4), 25 mM EDTA, and 750 mM NaCl. The MAb was then partially reduced by adding 2.3 molar equivalents of TCEP (moles relative to the MAb), followed by stirring at 37°C for 2 hours. The partially reduced MAb solution was then cooled to 5°C, and 4.4 molar equivalents of vcMMAE (moles relative to the antibody) were added in 6% (v / v) DMSO solution. The mixture was stirred at 5°C for 60 minutes, followed by the addition of 1 molar equivalent of N-acetylcysteine ​​(relative to vcMMAE) and stirring for another 15 minutes. Excess quenched vcMMAE and other reaction components were removed by ultrafiltration / permeation of the antibody-drug conjugate (ADC) with 10 volumes of 20 mM histamine (pH 6.0).

[0338] The resulting antibody-drug conjugate (ADC) is named Ha22-2(2,4)6.1vcMMAE and has the following formula: The MAb is Ha22-2(2,4)6.1 (Figures 2 and 3) and p ranges from 1 to 8. The p-value of the antibody-drug conjugate shown in this example is approximately 3.8. [Example] [5] [Ha22-2(2,4)6.1vcMMAE] [The characteristics of] []

[0339] The procedure shown in the example titled "Antibody-Drug Conjugation of Ha22-2(2,4)6.1 MAb" was used to generate antibody-drug conjugates that bind to 191P4D12, and the known assay combinations in this technique were used for screening, identification and characterization. [A.] [By means] [FACS] [Affinity determination] []

[0340] The binding affinity of Ha22-2(2,4)6.1vcMMAE to 191P4D12 expressed on the surface of PC3-human-191P4D12, PC3-cynomolgus monkey-191P4D12, and PC3-rat-191P4D12 cells was tested. In short, 11 dilutions of Ha22-2(2,4)6.1vcMMAE were incubated overnight at 4°C with each cell type (50,000 cells per well) to final concentrations ranging from 160 nM to 0.011 nM. At the end of the incubation, the cells were washed and incubated at 4°C for 45 minutes with anti-hIgG-PE detection antibody. After washing away unbound detection antibody, the cells were analyzed by FACS. The mean fluorescence intensity (MFI) values ​​are shown in Figures 6-8. The MFI values ​​were input into Graphpad Prisim software and analyzed using the unit point binding (hyperbolic) equation Y=Bmax*X / (Kd+X) to obtain the saturation curves of Ha22-2(2,4)6.1vcMMAE, which are shown in Figures 6-8. Bmax is the maximum binding MFI value between Ha22-2(2,4)6.1vcMMAE and 191P4D12; Kd is the Ha22-2(2,4)6.1vcMMAE concentration required to achieve half-maximal binding.

[0341] The calculated affinity (Kd) of Ha22-2(2,4)6.1vcMMAE for 191P4D12 expressed on the surface of PC3-human-191P4D12, PC3-cynomolgus monkey-191P4D12 and PC3-rat-191P4D12 cells was 0.69 nM (Fig. 6), 0.34 nM (Fig. 7) and 1.6 nM (Fig. 8), respectively. [B.] [By means] [SPR] [Affinity determination] []

[0342] The affinity of Ha22-2(2,4)6.1 MAb and Ha22-2(2,4)6.1vcMMAE for purified recombinant 191P4D12 (ECD amino acid 1-348) was determined by surface plasma resonance (SPR) (BIAcore). In short, goat-anti-human Fcγ polyclonal Abs (Jackson Immuno Research Labs, Inc.) were covalently immobilized on the surface of a CM5 sensing chip (Biacore). Purified Ha22-2(2,4)6.1 MAb or Ha22-2(2,4)6.1vcMMAE was then captured onto the chip surface. An average of approximately 300 RU of test Ha22-2(2,4)6.1 MAb or Ha22-2(2,4)6.1vcMMAE was captured per cycle. Subsequently, a series of 5 to 6 dilutions of recombinant 191P4D12 (ECD amino acid 1-348) in the range of 1 nM to 100 nM were injected onto the surface to generate binding curves (sensor maps). These curves were processed using BIAevaluation 3.2 and CLAMP software (Myszka and Morton, 1998) and fitted to a 1:1 interaction model (Table V). Table VI summarizes the association and dissociation rate constants and the affinity of Ha22-2(2,4)6.1MAb and Ha22-2(2,4)6.1vcMMAE for recombinant 191P4D12 (ECD amino acid 1-348). [C. Ha22-2(2,4)6.1 MAb] [Domain Location] []

[0343] To locate the binding sites of the Ha22-2(2,4)6.1 MAb to specific domains of the 191P4D12 protein, several rat 1(E) recombinant cell lines expressing these domains (or combinations thereof) were generated (Table VII). The binding of Ha22-2(2,4)6.1 to the cell surface was assessed using standard FACS protocols. As shown in Figure 10, Ha22-2(2,4)6.1 MAb bound to cells expressing the VC1 domain and wild-type 191P4D12, but not to cells expressing the C1C2 domain. Additionally, another 191P4D12 MAb, designated Ha22-8e6.1, recognized the C1C2 domain of 191P4D12 on the cell surface, but not the VC1 domain. This indicates that the binding site of Ha22-2(2,4)6.1 MAb is located in the 1-147 aa domain of 191P4D12, rather than each MAb that binds to 191P4D12 recognizing this domain.

[0344] To further confirm the results shown in Figure 10, Western blotting analysis was performed. In short, the entire extracellular portion of the full-length 191P4D12 and its specific domains, as shown in Table VII, appeared as a purified mouse Fc fusion protein in 293T cells. Goat anti-mouse HRP was used as a control. As shown in Figure 11, when analyzed on SDS-PAGE (non-reducing) using Ha22-2(2,4)6.1-biotin, followed by streptavidin-HRP as probes, bands corresponding to the full-length 191P4D12 (lane 1), V (lane 2), and VC1 (lane 3) fusion constructs, but not the C1C2 fusion construct (lane 4), were identified. This further indicates that the binding antigenic determinant of Ha22-2(2,4)6.1 MAb is located in the 1-147 aa domain of 191P4D12. [Example] [6] [Depend on] [Ha22-2(2,4)6.1vcMMAE] [Mediated Cytotoxicity] []

[0345] The ability of Ha22-2(2,4)6.1vcMMAE to mediate 191P4D12-dependent cytotoxicity was evaluated in PC3 cells engineered to represent human 191P4D12, cynomolgus monkey 191P4D12, and rat 191P4D12. In short, on day 1, PC3-Neo, PC3-human-191P4D12, PC3-cynomolgus monkey-191P4D12, or PC3-rat-191P4D12 cells (1500 cells per well) were seeded into 96-well plates. On day 2, an equal volume of medium containing a specified concentration of Ha22-2(2,4)6.1vcMMAE or a control MAb conjugate with vcMMAE (i.e., control-vcMMAE) was added to each well. Cells were cultured at 37°C for 4 days. At the end of the incubation period, Alamar Blue was added to each well, and incubation continued for another 4 hours. The fluorescence was detected using a Biotek culture plate reader at an excitation wavelength of 620 nM and an emission wavelength of 540 nM.

[0346] The results in Figures 9A-9D show that Ha22-2(2,4)6.1vcMMAE mediated cytotoxicity in PC3-human-191P4D12 (Figure 9A), PC3-cynomolgus monkey-191P4D12 (Figure 9B), and PC3-rat-191P4D12 cells (Figure 9C), while the control human IgG conjugated with vcMMAE was ineffective. The specificity of Ha22-2(2,4)6.1vcMMAE was further demonstrated by the lack of cytotoxicity in PC3-Neo cells that do not express 191P4D12 (Figure 9D). Therefore, these results indicate that Ha22-2(2,4)6.1vcMMAE can selectively deliver cytotoxic drugs to 191P4D12 expressing cells, thereby killing them. [Example] [7] [Ha22-2(2,4)6.1vcMMAE] [In vivo inhibition of tumor growth] []

[0347] The significant expression of 191P4D12 on the cell surface of tumor tissues and its limited expression in normal tissues make 191P4D12 a good target for antibody therapy and similar therapies via ADCs. Therefore, we evaluated the therapeutic efficacy of Ha22-2(2,4)6.1vcMMAE in mouse models of human bladder cancer, lung cancer, breast cancer, and pancreatic cancer xenografts.

[0348] Investigate the efficacy of antibody drug conjugates against tumor growth and metastasis in mouse cancer xenograft models (e.g., subcutaneous and orthotopic).

[0349] Subcutaneous (sc) tumors were induced by injecting 5 × 10⁴–10⁶ cancer cells mixed with Matrigel (Collaborative Research) at a 1:1 dilution into the right ventricular region of male SCID mice. To test the efficacy of the ADC (antibody-adjuvant drug) in tumorigenesis, ADC injections were initiated on the same day as the tumor cell injection. As a control group, mice were injected with purified human IgG or PBS; or purified MAb (antibody-associated antigen) that recognizes irrelevant antigens not expressed in human cells. In the preliminary study, no difference was found between the control IgG and PBS in the effect on tumor growth. Tumor size was determined by diameter measurement, and tumor volume was calculated as width × length / 2, where width was the minimum dimension and length was the maximum dimension. Mice with subcutaneous tumors larger than 1.5 cm in diameter were euthanized.

[0350] Ovarian tumors typically metastasize and grow within the peritoneal cavity. Therefore, intraperitoneal growth of ovarian tumors in mice was induced by direct intraperitoneal injection of 2 million cells into the peritoneum of female mice. The general health, physical activity, and appearance of the mice were monitored until they were near death. At sacrifice, the peritoneal cavity was examined to determine tumor burden, and the lungs were collected to assess metastasis to distal sites. Alternatively, death could be used as the endpoint. The mice were then divided into groups receiving intraperitoneal injection of 191P4D12 or appropriate treatment with control MAb.

[0351] The advantage of xenograft cancer models lies in their ability to study angiogenesis and the capacity for blood vessel formation. Tumor growth is partly dependent on new blood vessel development. Although the capillary system and developing blood network originate from the host, the initiation and structure of new blood vessels are regulated by the xenograft tumor (Davidoff et al., Clin Cancer Res. (2001) 7:2870; Solesvik et al., Eur J Cancer Clin Oncol. (1984) 20:1295). Procedures known in this technique, such as IHC analysis of tumor tissue and its surrounding microenvironment, can be used to study the effects of antibodies and small molecules on angiogenesis.

[0352] Ha22-2(2,4)6.1ADC inhibits xenograft formation in lung, bladder, breast, and pancreatic cancers. These results indicate the efficacy of Ha22-2(2,4)6.1ADC in the treatment of locally and advanced cancers, preferably those shown in Table I. [191P4D12 ADC] [:] []

[0353] As described in the example titled "Generation of 191P4D12 Monoclonal Antibody (MAb)," a monoclonal antibody against 191P4D12 was generated. Furthermore, as described in the example titled "Antibody-Drug Conjugation of Ha22-2(2,4)6.1 MAb," the MAb was conjugated with a toxin to form Ha22-2(2,4)6.1vcMMAE. Ha22-2(2,4)6.1vcMMAE was characterized using FACS and other methods known in this technique to determine its ability to bind to 191P4D12. [Cell lines and xenografts:] []

[0354] As is known in this technique, BT-483 and HPAC cells were maintained in DMEM supplemented with L-glutamic acid and 10% FBS. AG-B8, AG-Panc4, AG-Panc2, AG-B1, AG-L4, and AG-Panc3 xenografts were maintained in SCID mice through continuous propagation. [exist] [SCID] [Mouse xenografts for human lung cancer] [AG-L4] [Evaluated in a subcutaneous tumor formation model] [Ha22-2(2,4)6.1vcMMAE MAb]

[0355] In this experiment, AG-L4 xenografts derived from patients were maintained in SCID mice through continuous subculture. Tumor material was aseptically collected and enzymatically digested into a single-cell suspension. Two million cells were implanted into the flank of individual SCID mice. The animals were then randomly assigned to seven groups: six 191P4D12 antibody treatment groups and one control antibody H3-1.10.1.2 group (n=10). All antibodies were administered intraperitoneally at 750 μg per animal twice weekly until the end of the study. Tumor growth was monitored every 3 to 4 days using a diameter. Tumor volume was calculated as width × length / 2, where width was the minimum dimension and length was the maximum dimension.

[0356] The results showed that 191P4D12 MAb did not significantly inhibit tumor growth in AG-L4 xenografts of human lung cancer from SCID mice. Additionally, other 191P4D12 MAbs were used in this study. Results are not shown (Figure 12). [exist] [SCID] [Mouse xenografts for human pancreatic cancer] [HPAC] [Evaluated in a subcutaneous tumor formation model] [Ha22-2(2,4)6.1 MAb]

[0357] In another experiment, human pancreatic cancer HPAC cells (two million per mouse) were injected into the flank of individual SCID mice. The animals were then randomly assigned to eight groups: seven groups treated with the 191P4D12 antibody and one control antibody group treated with H3-1.4.1.2 (n=10). All antibodies were administered intraperitoneally at 500 μg per animal twice weekly until the end of the study. Tumor growth was monitored every 3 to 4 days using a diaphragm. Tumor volume was calculated as width × length / 2, where width was the minimum dimension and length was the maximum dimension.

[0358] The results showed that, compared with the control antibody, 191P4D12 MAb did not inhibit tumor growth in human pancreatic xenografts from SCID mice. Additionally, other 191P4D12 MAbs were used in this study. Results are not shown (Figure 13).

[0359] Evaluation of Ha22-2(2,4)6.1 MAb in a subcutaneous tumor formation model of human pancreatic cancer xenograft AG-Panc3 in SCID mice.

[0360] In another experiment, a patient-derived pancreatic cancer xenograft, AG-Panc3, was maintained in SCID mice via continuous subculture. Tumor material was aseptically collected and chopped into 1 mm³ pieces. Six pieces were implanted into the flank of individual SCID mice. The animals were then randomly assigned to the following groups (n=10): two 191P4D12 MAb treatment groups and one control antibody H3-1.4.1.2 group. All antibodies were administered intraperitoneally at 500 μg per animal twice weekly until the end of the study. Tumor growth was monitored every 3 to 4 days using a diameter. Tumor volume was calculated as width × length / 2, where width was the minimum dimension and length was the maximum dimension.

[0361] The results showed that, compared with the control antibody, 191P4D12 MAb did not inhibit tumor growth in human pancreatic xenografts from SCID mice. Additionally, other 191P4D12 MAbs were used in this study. Results are not shown (Figure 14). [Ha22-2(2,4)6.1-vcMMAE] [exist] [SCID] [Human lung cancer xenografts formed subcutaneously in mice] [AG-L4] [Efficacy of Traditional Chinese Medicine] []

[0362] In another experiment, a patient-derived lung cancer xenograft, AG-L13, was maintained in SCID mice via successive subcultures. Tumor material was aseptically collected and minced into 1 mm³ pieces. Six pieces were implanted into the flank of individual SCID mice. Tumors were allowed to grow untreated until they reached a volume of approximately 200 mm³. Every seven days, Ha22-2(2,4)6.1vcMMAE and a control ADC were administered twice via rapid intravenous injection. The ADC dosage was based on the individual animal's body weight obtained prior to administration. Tumor growth was monitored every 3 to 4 days using a diaphragm. Tumor volume was calculated as width × length / 2, where width was the minimum dimension and length was the maximum dimension.

[0363] The results showed that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of AG-L4 lung cancer xenografts subcutaneously implanted in nude mice compared with the control ADC. Additionally, other 191P4D12 MAb was used in this study. Results are not shown (Figure 15). [Ha22-2(2,4)6.1-vcMMAE] [exist] [SCID] [Human breast cancer xenografts formed subcutaneously in mice] [BT-483] [Efficacy of Traditional Chinese Medicine] []

[0364] In this experiment, xenograft material was generated using human breast cancer BT-483 cells, which were maintained in SCID mice through continuous subculture. Tumor material was aseptically collected and minced into 1 mm³ pieces. Six pieces were implanted into the flank of individual SCID mice. Tumors were allowed to grow untreated until they reached a volume of approximately 100 mm³. Ha22-2(2,4)6.1vcMMAE and a control ADC were administered intravenously four times every four days via rapid intravenous injection. The ADC dosage was based on the individual body weight of each animal obtained prior to administration. Tumor growth was monitored every 3 to 4 days using a diaphragm. Tumor volume was calculated as width × length / 2, where width was the minimum dimension and length was the maximum dimension.

[0365] The results showed that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of BT-483 mammary tumor xenografts subcutaneously implanted in SCID mice compared with the control ADC. Additionally, other 191P4D12 MAb was used in this study. Results are not shown (Figure 16). [Ha22-2(2,4)6.1-vcMMAE] [exist] [SCID] [Human bladder cancer xenografts formed subcutaneously in mice] [AG-B1] [Efficacy of Traditional Chinese Medicine] []

[0366] In another experiment, a patient-derived bladder cancer xenograft, AG-B1, was maintained in SCID mice via successive subcultures. Tumor material was aseptically collected and minced into 1 mm³ pieces. Six pieces were implanted into the flank of individual SCID mice. Tumors were allowed to grow untreated until they reached a volume of approximately 230 mm³. Ha22-2(2,4)6.1vcMMAE and a control ADC were administered intravenously via rapid injection. The ADC dosage was based on the individual animal's body weight obtained prior to administration. Tumor growth was monitored every 3 to 4 days using a diaphragm. Tumor volume was calculated as width × length / 2, where width was the minimum dimension and length was the maximum dimension.

[0367] The results showed that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of AG-B1 bladder cancer xenografts compared to the control ADC. Additionally, other 191P4D12 MAb was used in this study. Results are not shown (Figure 17). [Ha22-2(2,4)6.1-vcMMAE] [exist] [SCID] [Human pancreatic cancer xenograft formed subcutaneously in mice] [AG-Panc2] [Efficacy of Traditional Chinese Medicine] []

[0368] In another experiment, a patient-derived pancreatic cancer xenograft, AG-Panc2, was maintained in SCID mice via continuous subculture. Tumor material was aseptically collected and minced into 1 mm³ pieces. Five pieces were implanted into the flank of individual SCID mice. Tumors were allowed to grow untreated until they reached a volume of approximately 100 mm³. Ha22-2(2,4)6.1vcMMAE and a control ADC were administered intravenously four times every four days via rapid intravenous injection. The ADC dosage was based on the individual animal's body weight obtained prior to administration. Tumor growth was monitored every 3 to 4 days using a diaphragm. Tumor volume was calculated as width × length / 2, where width was the minimum dimension and length was the maximum dimension.

[0369] The results showed that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of AG-Panc2 pancreatic cancer xenografts compared with the control ADC. Additionally, other 191P4D12 MAb was used in this study. Results are not shown (Figure 18). [Ha22-2(2,4)6.1-vcMMAE] [exist] [SCID] [Human pancreatic cancer xenograft formed subcutaneously in mice] [AG-Panc4] [Efficacy of Traditional Chinese Medicine] []

[0370] In another experiment, a patient-derived pancreatic cancer xenograft, AG-Panc4, was maintained in SCID mice via continuous subculture. Tumor material was aseptically collected and minced into 1 mm³ pieces. Six pieces were implanted into the flank of individual SCID mice. Ha22-2(2,4)6.1vcMMAE and a control ADC were administered three times daily via rapid intravenous injection every seven days. The ADC dosage was based on the individual animal's body weight obtained prior to administration. Tumor growth was monitored every 3 to 4 days using a diaphragm. Tumor volume was calculated as width × length / 2, where width was the minimum dimension and length was the maximum dimension.

[0371] The results showed that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of AG-Panc4 pancreatic cancer xenografts compared with the control ADC. Additionally, other 191P4D12 MAb was used in this study. Results are not shown (Figure 19). [Ha22-2(2,4)6.1-vcMMAE] [At comparative doses] [SCID] [Human bladder cancer xenografts formed subcutaneously in mice] [AG-B8] [Efficacy of Traditional Chinese Medicine] []

[0372] In this experiment, a patient-derived bladder cancer xenograft, AG-B8, was maintained in SCID mice via continuous subculture. Tumor material was aseptically collected and minced into 1 mm³ pieces. Six pieces were implanted into the flank of individual SCID mice. Tumors were allowed to grow untreated until they reached a volume of approximately 200 mm³. The animals were then randomly assigned to three groups (n=6): two Ha22-2(2,4)6.1-vcMMAE treatment groups and one control ADC VCD37-5ce5p-vcMMAE group. Ha22-2(2,4)6.1-vcMMAE was administered at 5 mg / kg or 10 mg / kg, along with a control ADC at 5 mg / kg. All ADCs were administered once via rapid intravenous injection. ADC dosage was based on individual animal body weight obtained prior to administration. Tumor growth was monitored every 3 to 4 days using a diaphragm. The tumor volume is calculated as width 2 × length / 2, where width is the minimum dimension and length is the maximum dimension.

[0373] The results showed that treatment with 10 mg / kg Ha22-2(2,4)6.1vcMMAE inhibited the growth of AG-B8 bladder cancer xenografts compared with 5 mg / kg Ha22-2(2,4)6.1vcMMAE (Figure 20). [in conclusion] []

[0374] In summary, Figures 12-20 demonstrate that the 191P4D12 ADC, named Ha22-2(2,4)6.1vcMMAE, significantly inhibited the growth of tumor cells expressing 191P4D12 compared to the control ADC. Therefore, Ha22-2(2,4)6.1vcMMAE can be used for therapeutic purposes to treat and control the cancers shown in Table I. [Example] [8] [Through use] [191P4D12 ADC] [Human clinical trials for the treatment and diagnosis of human cancers] []

[0375] According to the present invention, a 191P4D12 ADC that specifically binds to 191P4D12 is used, and it is used to treat certain tumors, preferably those listed in Table I. Two clinical approaches have been successfully performed for each of these indications.

[0376] I.) Adjuvant Therapy: In adjuvant therapy, patients are treated with 191P4D12 ADC in combination with chemotherapy or anti-hypertrophic agents and / or radiotherapy, or a combination thereof. Primary cancer targets such as those listed in Table I are treated by adding 191P4D12 ADC to standard first- and second-line therapies, according to standard protocols. Protocol designs are aimed at efficacy as assessed by examples including (but not limited to) reduction in tumor mass of primary or metastatic lesions, increased progression-free survival, increased overall survival, improved patient health, disease stabilization, and the ability to reduce the commonly used doses of standard chemotherapy and other biologics. These dose reductions enable additional and / or prolonged treatment due to reduced dose-related toxicities of chemotherapy or biologics. The combination of 191P4D12 ADC with chemotherapy or anti-hypertrophic agents has been used in several adjuvant clinical trials.

[0377] II.) Monotherapy: Regarding the use of 191P4D12 ADC in cancer monotherapy, 191P4D12 ADC is administered to patients not receiving chemotherapy or anti-metastatic agents. In one embodiment, monotherapy is clinically administered to patients with advanced cancer and extensive metastatic disease. The protocol design aims to achieve efficacy as assessed by examples including (but not limited to) reduction in tumor mass of primary or metastatic lesions, increased progression-free survival, increased overall survival, improved patient health, disease stabilization, and the ability to reduce the commonly used doses of standard chemotherapy and other biologics. [dose] []

[0378] The dosing regimen can be adjusted to provide the optimal desired response. For example, a single dose can be administered; several fractionated doses can be administered over time; or the dose can be proportionally reduced or increased as needed for the treatment situation. For ease of administration and dosage uniformity, it is particularly advantageous to formulate non-enteral compositions into unit dosage forms. As used herein, a unit dosage form refers to a physical individual unit suitable for use as a single dose in the treatment of a mammalian individual; each unit contains a predetermined amount of the active compound, calculated to produce the desired therapeutic effect, bound to the desired pharmaceutical carrier. The description of the unit dosage forms of this invention is determined and directly depends on: (a) the unique characteristics of the antibody and / or ADC and the specific therapeutic or prophylactic effect to be achieved; and (b) the inherent limitations of this technology in mixing the active compound for treating individual sensitivities.

[0379] The illustrative, non-limiting range of therapeutically effective doses of the 191P4D12 ADC administered according to the present invention 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 illustrative, 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 relate to doses greater than 10 mg / kg. It should be noted that dose values ​​may vary depending on the type and severity of the symptoms to be alleviated, and may include single or multiple doses. It should be further understood that, for any particular individual, a specific dosing regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition, and the dose ranges described herein are merely illustrative and not intended to limit the scope or practice of the proposed compositions. [Clinical Development Plan] [(CDP)]

[0380] Regarding adjuvant or monotherapy, CDP was subsequently conducted, and the treatment with the developed 191P4D12 ADC was performed. The trial initially demonstrated safety and subsequently confirmed efficacy with repeated doses. The trial was an open-label trial comparing standard chemotherapy with standard chemotherapy plus the 191P4D12 ADC. As should be understood, a non-restrictive criterion for patient recruitment was the level of 191P4D12 expression in the tumor, as determined by biopsy.

[0381] As with any protein- or antibody-based infusion therapy, safety considerations primarily concern: (i) cytokine release syndrome, i.e., hypotension, fever, shivering, and chills; (ii) immunogenicity to the substance (i.e., the patient develops human antibodies against the antibody therapy, or a HAHA response); and (iii) toxicity to normal cells expressing 191P4D12. Each of these safety considerations was monitored using standard tests and follow-up. The 191P4D12 ADC was found to be safe after administration to humans. [Example] [9] [By means] [IHC] [Detected in samples from cancer patients] [191P4D12] [protein] []

[0382] Immunohistochemistry was used to test the expression of 191P4D12 protein in tumor samples from patients with (i) bladder cancer, (ii) breast cancer, (iii) pancreatic cancer, (iv) lung cancer, (v) ovarian cancer, (vi) esophageal cancer, and (vii) head and neck cancer. Briefly, formalin-fixed, paraffin-embedded tissue was sectioned into 4-micrometer sections and mounted on glass slides. The paraffin was removed from these sections, which were then rehydrated and treated at 95°C in an EZ-Retriever microwave (Biogenex, San Ramon, CA) with EDTA antigen retrieval solution (Biogenex, San Ramon, CA) for 30 minutes. The sections were then treated with 3% hydrogen peroxide solution to deactivate endogenous peroxidase activity. Non-specific binding was inhibited using a serum-free protein blocker (Dako, Carpenteria, CA), followed by incubation with monoclonal mouse anti-191P4D12 antibody or isotype control groups. Subsequently, sections were treated with the Super Sensitive™ polymer-horseradish peroxidase (HRP) detection system, consisting of incubation in Super Enhancer™ reagent followed by incubation with a polymer-HRP secondary antibody conjugate (BioGenex, San Ramon, CA). Sections were then developed using a DAB kit (BioGenex, San Ramon, CA). Cell nuclei were stained with hematoxylin and analyzed by bright-field microscopy. Specific staining was detected in patient samples using a 191P4D12 immunoreactive antibody, as indicated by brown staining (see Figures 21(A), 21(C), 21(E), 21(G), 21(I), 21(K), and 21(M)). In contrast, the control antibody did not stain any patient samples (see Figures 21(B), 21(D), 21(F), 21(H), 21(J), 21(L), and 21(N)).

[0383] The results showed that 191P4D12 was expressed in tumor cells of bladder cancer, breast cancer, pancreatic cancer, lung cancer, ovarian cancer, esophageal cancer, and head and neck cancer tissues from patients. These results indicate that 191P4D12 is expressed in human cancers and that antibodies against this antigen and antibody-drug conjugates called Ha22-2(2,4)6.1vcMMAE are suitable for diagnostic and therapeutic purposes (Figure 21). [Example]

[10] [Measurement] [Ha22-2(2,4)6.1 MAb] [Binding antigenic determinant] []

[0384] Recombinant overexpression of 191P4D12 protein from humans, cynomolgus monkeys, rats, and mice in PC3 cell lines was performed to determine the cross-reactivity of Ha22-2(2,4)6.1 with these orthologs. High cross-reactivity of Ha22-2(2,4)6.1 with 191P4D12 orthologs from cynomolgus monkeys and rats is shown (Table VIII). EC50 binding values ​​are presented in... [surface] In [IX], the binding of Ha22-2(2,4)6.1 to its murine ortholog showed a significantly reduced EC50 value, indicating that the substitution of important amino acids in the V domain (compared to human and rat sequences) affected the affinity of Ha22-2(2,4)6.1 for 191P4D12.

[0385] [surface] [X] Shows the sequence alignment of protein aa 1-180 containing the V domain of the 191P4D12 ortholog. The rat ortholog sequence contains only two amino acids, Thr-75 and Ser-90, which replace Ile and Asn in the mouse ortholog sequence, respectively. [Marked in red]). Note that the corresponding amino acids in the human sequence are Ala-76 and Ser-91. To determine whether these amino acids contain the binding antigenic determinant Ha22-2(2,4)6.1, several mutant constructs of 191P4D12 and its murine orthologs were generated and expressed in PC3 cells. [surface] [XI]). The substitution mutation of "mouse" amino acid for standard alanine was induced and introduced into human sequences and also into mouse sequences.

[0386] The Ser-91 mutation to Asn in 191P4D12 severely weakens the binding of Ha22-2(2,4)6.1, thus confirming that the amino acid Ser-91 is essential for binding and must contain an antigenic determinant recognized by Ha22-2(2,4)6.1 MAb. Other mutations of Ala at position 76 (A76I and S91N double mutants) were also introduced into 191P4D12. The binding of Ha22-2(2,4)6.1 to the double mutants A76I and S91N was shown to be highly similar to the binding of murine orthologs. [surface] [XII]). Conversely, the Asn-90 mutation in the murine sequence significantly modifies the binding of Ha22-2(2,4)6.1 to its murine mutant ortholog, further confirming the importance of this amino acid for Ha22-2(2,4)6.1 binding. The binding of Ha22-2(2,4)6.1 to the murine ortholog dual mutants A90S and I75A appears to be very similar to that of the human ortholog of 191P4D12.

[0387] In summary, these data demonstrate that Ser-91 and Ala-76 play a crucial role in the binding of Ha22-2(2,4)6.1 to the 191P4D12 protein on the cell surface and constitute part of the antigenic determinants on the 191P4D12 surface recognized by Ha22-2(2,4)6.1.

[0388] To observe this concept, a computer model of the V domain of 191P4D12 was generated using PyMOL, based on publicly available crystal structure data of 191P4D12 and other protein family members containing the Ig domain. [surface] [XIII]). Show the positions of Ala-76 (green) and Ser-91 (red).

[0389] In addition, to further modify the Ha22-2(2,4)6.1 binding site on the 191P4D12 molecule, a fragment of 191P4D12 corresponding to the V domain on the surface of rat (1)E cells was designed and represented. The following constructs were generated in the retroviral vector: 191P4D12(aa1-150, 347-510).

[0390] The binding of Ha22-2(2,4)6.1 MAb was evaluated using FACS. [surface] As shown in [XIV], Ha22-2(2,4)6.1 binds to cells expressing the V domain (A) and wild-type 191P4D12 (B), but not to cells expressing the earlier C1C2 domain (C). This confirms that the binding site of this antibody is located in the first 150 amino acids of the V domain of 191P4D12.

[0391] The results showed that Ha22-2(2,4)6.1 Mab binds to the v domain of the 191P4D12 protein from position aa 1-150, and further showed that specific antigenic determinants including aa Ser-91 and aa Ala-76 are key to binding Ha22-2(2,4)6.1 MAb.

[0392] Throughout this application, references are made to numerous websites, publications, patent applications, and patents. (Websites are referenced based on their Uniform Resource Locator (URL) or URL on the World Wide Web.) The disclosures of each of these references are incorporated herein by reference in their entirety.

[0393] The scope of this invention is not intended to be limited to the embodiments disclosed herein, which are intended to illustrate individual aspects of the invention, and any functionally equivalent embodiments are within the scope of this invention. Various modifications to the model and methods of this invention, in addition to those described herein, will be readily apparent to those skilled in the art from the foregoing description and teachings, and are also intended to be within the scope of this invention. Such modifications or other embodiments may be implemented without departing from the true scope and spirit of this invention. [sheet] [] [surface] [I] [Manifestations when it is a malignant disease] [191P4D12] [The organization.] [] colon pancreas ovaries breast lung bladder [surface] [II] [Amino acid abbreviation] [] [Single letter] [Three letters] [Full Name] F Phe Phenylan L Leu Leucine S Ser serine Y Tyr Tyrosine C Cys Cysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamic acid R Arg Arginine I Ile Isoleucine M Met Methionine T Thr threonine N Asn Aspartic acid K Lys lysine V Val Valine A Ala alanine D Asp Aspartic acid E Glu glutamic acid G Gly Glycine [surface] [III] [Amino acid substitution matrix] [] This is adapted from the BLOSUM62 amino acid substitution matrix (block substitution matrix) of GCG software 9.0. The higher the value, the more likely the substitution is to exist in the relevant natural protein. [surface] [IV]. General Methods for Synthesizing vcMMAE [Where]: AA1 = Amino acid 1 AA2 = Amino acid 2 AA5 = Amino Acid 5 DIL = Dolaisolexine DAP = Dopamine Connector = Val-Cit(vc) [surface] [V] [。] Binding curves (sensor maps) used to determine affinity via SPR. [surface] [VI] [。] Calculation results of Bayker association and dissociation rates and the resulting affinity kon, M-1s-1 koff, s-1 KD, M [Ha22-2(2,4)6.1] 3.8E+05 5.8E-03 1.6E-08 [Ha22-2(2,4)6.1vcMMAE] 4.5E+05 5.2E-03 1.1E-08 [surface] [VII] [。] Used for structural domain location verification of structure 191P4D12 [Construction] [name] 191P4D12(aa 1-242、347-510) VC1, rat 1(E) phenotype 191P4D12(aa 1-31、147-510) C1C2, rat 1(E) strain 191P4D12(aa 1-242) mFc-VC1, fusion protein 191P4D12(aa 1-31、147-346) mFc-C1C2, fusion protein 191P4D12(aa 1-141) mFc-V, fusion protein [surface] [VIII] Ha22-2(2,4)6.1 binds to PC3 cells expressing 191P4D12(A) and orthologs from cynomolgus monkeys (B), rats (C), and mice (D). AB CD [surface] [IX] [] [PC3-191P4D12] [Direct homolog of the crab-eating macaque] [Rat ortholog] [Rodent direct homologs] [Bmax(MFI)] 816 1146 679 325 [EC, 50 , (nM) ] 0.28 0.30 0.44 70.3 [surface] [X] [, , ] [] [surface] [XI] Wild-type structures Mutant constructs Dual mutant construct 191P4D12, Wild Type S91N S91N, A76I 191P4D12 is a rodent direct homolog, wild type. N90S N90S, I75A [surface] [XII] [surface] [XIII] [, , ] [] [surface] [XIV] [ABC]

[0394] Food Industry Development Institute; December 8, 2011; BCRC 960435 United States; American Type Culture Collection (ATCC); August 19, 2010; PTA-11267

[0395] <![CDATA[ <110> AGENSYS, INC. (USA) Seattle Genetics, Inc. (USA) <![CDATA[ <120> Antibody-drug conjugates (ADCs) that bind to the 191P4D12 protein <![CDATA[ <130> TW 109135992]]> TW 107143990 TW 107103146 TW 104140581 TW 100135340 TW 111117461 <![CDATA[ <150> US 61 / 387,933 <![CDATA[<151> 2010-09-29 ]]> <![CDATA[<160> 13]]> <![CDATA[<170> FastSEQ for Windows Version 4.0]]> <![CDATA[<210> 1]]> <![CDATA[<211> 3464]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<220> ]]> <![CDATA[<221> CDS ]]> <![CDATA[<222> (264)...(1796)]]> <![CDATA[<220> ]]> <![CDATA[<221> misc_feature ]]> <![CDATA[<222> (1)...(3464)]]> <![CDATA[<223> 191P4D12]]> <![CDATA[<400> 1]]> ggccgtcgtt gttggccaca gcgtgggaag cagctctggg ggagctcgga gctcccgatc 60 acggcttctt gggggtagct acggctgggt gtgtagaacg gggccggggc tggggctggg 120 tcccctagtg gagacccaag tgcgagaggc aagaactctg cagcttcctg ccttctgggt 180 cagttcctta ttcaagtctg cagccggctc ccagggagat ctcggtggaa cttcagaaac 240 gctgggcagt ctgcctttca acc atg ccc ctg tcc ctg gga gcc gag atg tgg 293 Met Pro Leu Ser Leu Gly Ala Glu Met Trp 1 5 10 ggg cct gag gcc tgg ctg ctg ctg ctg cta ctg ctg gca tca ttt aca 341 Gly Pro Glu Ala Trp Leu Leu Leu Leu Leu Leu Leu Ala Ser Phe Thr 15 20 25 ggc cgg tgc ccc gcg ggt gag ctg gag acc tca gac gtg gta act gtg 389 Gly Arg Cys Pro Ala Gly Glu Leu Glu Thr Ser Asp Val Val Thr Val 30 35 40 gtg ctg ggc cag gac gca aaa ctg ccc tgc ttc tac cga ggg gac tcc 437 Val Leu Gly Gln Asp Ala Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser 45 50 55 ggc gag caa gtg ggg caa gtg gca tgg gct cgg gtg gac gcg ggc gaa 485 Gly Glu Gln Val Gly Gln Val Ala Trp Ala Arg Val Asp Ala Gly Glu 60 65 70 ggc gcc cag gaa cta gcg cta ctg cac tcc aaa tac ggg ctt cat gtg 533 Gly Ala Gln Glu Leu Ala Leu Leu His Ser Lys Tyr Gly Leu His Val 75 80 85 90 agc ccg gct tac gag ggc cgc gtg gag cag ccg ccg ccc cca cgc aac 581 Ser Pro Ala Tyr Glu Gly Arg Val Glu Gln Pro Pro Pro Pro Arg Asn 95 100 105 ccc ctg gac ggc tca gtg ctc ctg cgc aac gca gtg cag gcg gat gag 629 Pro Leu Asp Gly Ser Val Leu Leu Arg Asn Ala Val Gln Ala Asp Glu 110 115 120 ggc gag tac gag tgc cgg gtc agc acc ttc ccc gcc ggc agc ttc cag 677 Gly Glu Tyr Glu Cys Arg Val Ser Thr Phe Pro Ala Gly Ser Phe Gln 125 130 135 gcg cgg ctg cgg ctc cga gtg ctg gtg cct ccc ctg ccc tca ctg aat 725 Ala Arg Leu Arg Leu Arg Val Leu Val Pro Pro Leu Pro Ser Leu Asn 140 145 150 cct ggt cca gca cta gaa gag ggc cag ggc ctg acc ctg gca gcc tcc 773 Pro Gly Pro Ala Leu Glu Glu Gly Gln Gly Leu Thr Leu Ala Ala Ser 155 160 165 170 tgc aca gct gag ggc agc cca gcc ccc agc gtg acc tgg gac acg gag 821 Cys Thr Ala Glu Gly Ser Pro Ala Pro Ser Val Thr Trp Asp Thr Glu 175 180 185 gtc aaa ggc aca acg tcc agc cgt tcc ttc aag cac tcc cgc tct gct 869 Val Lys Gly Thr Thr Ser Ser Arg Ser Phe Lys His Ser Arg Ser Ala 190 195 200 gcc gtc acc tca gag ttc cac ttg gtg cct agc cgc agc atg aat ggg 917 Ala Val Thr Ser Glu Phe His Leu Val Pro Ser Arg Ser Met Asn Gly 205 210 215 cag cca ctg act tgt gtg gtg tcc cat cct ggc ctg ctc cag gac caa 965 Gln Pro Leu Thr Cys Val Val Ser His Pro Gly Leu Leu Gln Asp Gln 220 225 230 agg atc acc cac atc ctc cac gtg tcc ttc ctt gct gag gcc tct gtg 1013 Arg Ile Thr His Ile Leu His Val Ser Phe Leu Ala Glu Ala Ser Val 235 240 245 250 agg ggc ctt gaa gac caa aat ctg tgg cac att ggc aga gaa gga gct 1061 Arg Gly Leu Glu Asp Gln Asn Leu Trp His Ile Gly Arg Glu Gly Ala 255 260 265 atg ctc aag tgc ctg agt gaa ggg cag ccc cct ccc tca tac aac tgg 1109 Met Leu Lys Cys Leu Ser Glu Gly Gln Pro Pro Pro Ser Tyr Asn Trp 270 275 280 aca cgg ctg gat ggg cct ctg ccc agt ggg gta cga gtg gat ggg gac 1157 Thr Arg Leu Asp Gly Pro Leu Pro Ser Gly Val Arg Val Asp Gly Asp 285 290 295 act ttg ggc ttt ccc cca ctg acc act gag cac agc ggc atc tac gtc 1205 Thr Leu Gly Phe Pro Pro Leu Thr Thr Glu His Ser Gly Ile Tyr Val 300 305 310 tgc cat gtc agc aat gag ttc tcc tca agg gat tct cag gtc act gtg 1253 Cys His Val Ser Asn Glu Phe Ser Ser Arg Asp Ser Gln Val Thr Val 315 320 325 330 gat gtt ctt gac ccc cag gaa gac tct ggg aag cag gtg gac cta gtg 1301 Asp Val Leu Asp Pro Gln Glu Asp Ser Gly Lys Gln Val Asp Leu Val 335 340 345 tca gcc tcg gtg gtg gtg gtg ggt gtg atc gcc gca ctc ttg ttc tgc 1349 Ser Ala Ser Val Val Val Val Gly Val Ile Ala Ala Leu Leu Phe Cys 350 355 360 ctt ctg gtg gtg gtg gtg gtg ctc atg tcc cga tac cat cgg cgc aag 1397 Leu Leu Val Val Val Val Val Leu Met Ser Arg Tyr His Arg Arg Lys 365 370 375 gcc cag cag atg acc cag aaa tat gag gag gag ctg acc ctg acc agg 1445 Ala Gln Gln Met Thr Gln Lys Tyr Glu Glu Glu Leu Thr Leu Thr Arg 380 385 390 gag aac tcc atc cgg agg ctg cat tcc cat cac acg gac ccc agg agc 1493 Glu Asn Ser Ile Arg Arg Leu His Ser His His Thr Asp Pro Arg Ser 395 400 405 410 cag ccg gag gag agt gta ggg ctg aga gcc gag ggc cac cct gat agt 1541 Gln Pro Glu Glu Ser Val Gly Leu Arg Ala Glu Gly His Pro Asp Ser 415 420 425 ctc aag gac aac agt agc tgc tct gtg atg agt gaa gag ccc gag ggc 1589 Leu Lys Asp Asn Ser Ser Cys Ser Val Met Ser Glu Glu Pro Glu Gly 430 435 440 cgc agt tac tcc acg ctg acc acg gtg agg gag ata gaa aca cag act 1637 Arg Ser Tyr Ser Thr Leu Thr Thr Val Arg Glu Ile Glu Thr Gln Thr 445 450 455 gaa ctg ctg tct cca ggc tct ggg cgg gcc gag gag gag gaa gat cag 1685 Glu Leu Leu Ser Pro Gly Ser Gly Arg Ala Glu Glu Glu Glu Asp Gln 460 465 470 gat gaa ggc atc aaa cag gcc atg aac cat ttt gtt cag gag aat ggg 1733 Asp Glu Gly Ile Lys Gln Ala Met Asn His Phe Val Gln Glu Asn Gly 475 480 485 490 acc cta cgg gcc aag ccc acg ggc aat ggc atc tac atc aat ggg cgg 1781 Thr Leu Arg Ala Lys Pro Thr Gly Asn Gly Ile Tyr Ile Asn Gly Arg 495 500 505 gga cac ctg gtc tga cccaggcctg cctcccttcc ctaggcctgg ctccttctgt 1836 Gly His Leu Val * 510 tgacatggga gattttagct catcttgggg gcctccttaa acacccccat ttcttgcgga 1896 agatgctccc catcccactg actgcttgac ctttacctcc aacccttctg ttcatcggga 1956 gggctccacc aattgagtct ctcccaccat gcatgcaggt cactgtgtgt gtgcatgtgt 2016 gcctgtgtga gtgttgactg actgtgtgtg tgtggagggg tgactgtccg tggaggggtg 2076 actgtgtccg tggtgtgtat tatgctgtca tatcagagtc aagtgaactg tggtgtatgt 2136 gccacgggat ttgagtggtt gcgtgggcaa cactgtcagg gtttggcgtg tgtgtcatgt 2196 ggctgtgtgt gacctctgcc tgaaaaagca ggtattttct cagaccccag agcagtatta 2256 atgatgcaga ggttggagga gagaggtgga gactgtggct cagacccagg tgtgcgggca 2316 tagctggagc tggaatctgc ctccggtgtg agggaacctg tctcctacca cttcggagcc 2376 atgggggcaa gtgtgaagca gccagtccct gggtcagcca gaggcttgaa ctgttacaga 2436 agccctctgc cctctggtgg cctctgggcc tgctgcatgt acatattttc tgtaaatata 2496 catgcgccgg gagcttcttg caggaatact gctccgaatc acttttaatt tttttctttt 2556 ttttttcttg ccctttccat tagttgtatt ttttatttat ttttattttt atttttttttt 2616 agagatggag tctcactatg ttgctcaggc tggccttgaa ctcctgggct caagcaatcc 2676 tcctgcctca gcctccctag tagctgggac tttaagtgta caccactgtg cctgctttga 2736 atcctttacg aagagaaaaa aaaaattaaa gaaagccttt agatttatcc aatgtttact 2796 actgggattg cttaaagtga ggcccctcca acaccagggg gttaattcct gtgattgtga 2856 aaggggctac ttccaaggca tcttcatgca ggcagcccct tgggagggca cctgagagct 2916 ggtagagtct gaaattaggg atgtgagcct cgtggttact gagtaaggta aaattgcatc 2976 caccattgtt tgtgatacct tagggaattg cttggacctg gtgacaaggg ctcctgttca 3036 atagtggtgt tggggagaga gagagcagtg attatagacc gagagagtag gagttgaggt 3096 gaggtgaagg aggtgctggg ggtgagaatg tcgcctttcc ccctgggttt tggatcacta 3156 attcaaggct cttctggatg tttctctggg ttggggctgg agttcaatga ggtttatttt 3216 tagctggccc acccagatac actcagccag aatacctaga tttagtaccc aaactcttct 3276 tagtctgaaa tctgctggat ttctggccta agggagaggc tcccatcctt cgttccccag 3336 ccagcctagg acttcgaatg tggagcctga agatctaaga tcctaacatg tacattttat 3396 gtaaatatgt gcatatttgt acataaaatg atattctgtt tttaaataaa cagacaaaac 3456 ttgaaaaa 3464 <![CDATA[<210> 2]]> <![CDATA[<211> 510]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<220> ]]> <![CDATA[<221> misc_feature ]]><0​​​​​​​​​​​​Met Pro Leu Ser Leu Gly Ala Glu Met Trp Gly Pro Glu Ala Trp Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu Ala Ser Phe Thr Gly Arg Cys Pro Ala Gly 20 25 30 Glu Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly Gln Asp Ala 35 40 45 Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser Gly Glu Gln Val Gly Gln 50 55 60 Val Ala Trp Ala Arg Val Asp Ala Gly Glu Gly Ala Gln Glu Leu Ala 65 70 75 80 Leu Leu His Ser Lys Tyr Gly Leu His Val Ser Pro Ala Tyr Glu Gly 85 90 95 Arg Val Glu Gln Pro Pro Pro Pro Arg Asn Pro Leu Asp Gly Ser Val 100 105 110 Leu Leu Arg Asn Ala Val Gln Ala Asp Glu Gly Glu Tyr Glu Cys Arg 115 120 125 Val Ser Thr Phe Pro Ala Gly Ser Phe Gln Ala Arg Leu Arg Leu Arg 130 135 140 Val Leu Val Pro Pro Leu Pro Ser Leu Asn Pro Gly Pro Ala Leu Glu 145 150 155 160 Glu Gly Gln Gly Leu Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly Ser 165 170 175 Pro Ala Pro Ser Val Thr Trp Asp Thr Glu Val Lys Gly Thr Thr Ser 180 185 190 Ser Arg Ser Phe Lys His Ser Arg Ser Ala Ala Val Thr Ser Glu Phe 195 200 205 His Leu Val Pro Ser Arg Ser Met Asn Gly Gln Pro Leu Thr Cys Val 210 215 220 Val Ser His Pro Gly Leu Leu Gln Asp Gln Arg Ile Thr His Ile Leu 225 230 235 240 His Val Ser Phe Leu Ala Glu Ala Ser Val Arg Gly Leu Glu Asp Gln 245 250 255 Asn Leu Trp His Ile Gly Arg Glu Gly Ala Met Leu Lys Cys Leu Ser 260 265 270 Glu Gly Gln Pro Pro Pro Ser Tyr Asn Trp Thr Arg Leu Asp Gly Pro 275 280 285 Leu Pro Ser Gly Val Arg Val Asp Gly Asp Thr Leu Gly Phe Pro Pro 290 295 300 Leu Thr Thr Glu His Ser Gly Ile Tyr Val Cys His Val Ser Asn Glu 305 310 315 320 Phe Ser Ser Arg Asp Ser Gln Val Thr Val Asp Val Leu Asp Pro Gln 325 330 335 Glu Asp Ser Gly Lys Gln Val Asp Leu Val Ser Ala Ser Val Val Val 340 345 350 Val Gly Val Ile Ala Ala Leu Leu Phe Cys Leu Leu Val Val Val Val 355 360 365 Val Leu Met Ser Arg Tyr His Arg Arg Lys Ala Gln Gln Met Thr Gln 370 375 380 Lys Tyr Glu Glu Glu Leu Thr Leu Thr Arg Glu Asn Ser Ile Arg Arg 385 390 395 400 Leu His Ser His His Thr Asp Pro Arg Ser Gln Pro Glu Glu Ser Val 405 410 415 Gly Leu Arg Ala Glu Gly His Pro Asp Ser Leu Lys Asp Asn Ser Ser 420 425 430 Cys Ser Val Met Ser Glu Glu Pro Glu Gly Arg Ser Tyr Ser Thr Leu 435 440 445 Thr Thr Val Arg Glu Ile Glu Thr Gln Thr Glu Leu Leu Ser Pro Gly 450 455 460 Ser Gly Arg Ala Glu Glu Glu Glu Asp Gln Asp Glu Gly Ile Lys Gln 465 470 475 480 Ala Met Asn His Phe Val Gln Glu Asn Gly Thr Leu Arg Ala Lys Pro 485 490 495 Thr Gly Asn Gly Ile Tyr Ile Asn Gly Arg Gly His Leu Val 500 505 510 <![CDATA[<210> 3]]><00032­03> <![CDATA[<211> 1432]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<220> ]]> [[ID=­33]]<![CDATA[<221> CDS ]]> <![CDATA[<222> (32)...(1432)]]> <![CDATA[<220> ]]> <00­03222><![CDATA[<221> misc_feature ]]> <![CDATA[<222> (1)...(1432)]]> <![CDATA[<223> Ha22-2(2,4)6.1 heavy chain]]> <![CDATA[<400> 3]]> ggtgatcagc actgaacaca gaggactcac c atg gag ttg ggg ctg tgc tgg 52 Met Glu Leu Gly Leu Cys Trp 1 5 gtt ttc ctt gtt gct att tta gaa ggt gtc cag tgt gag gtg cag ctg 100 Val Phe Leu Val Ala Ile Leu Glu Gly Val Gln Cys Glu Val Gln Leu 10 15 20 gtg gag tct ggg gga ggc ttg gta cag cct ggg ggg tcc ctg aga ctc 148 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 25 30 35 tcc tgt gca gcc tct gga ttc acc ttc agt agc tat aac atg aac tgg 196 Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Asn Met Asn Trp 40 45 50 55 gtc cgc cag gct cca ggg aag ggg ctg gag tgg gtt tca tac att agt 244 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser 60 65 70 agt agt agt agt acc ata tac tac gca gac tct gtg aag ggc cga ttc 292 Ser Ser Ser Ser Thr Ile Tyr Ala Asp Ser Val Lys Gly Arg Phe 75 80 85 acc atc tcc aga gac aat gcc aag aac tca ctg tct ctg caa atg aac 340 Thr Ile Serves Arg Asp Asn Only Lys Asn Serves Leu Ser Leu Gln Met Asn 90 95 100 agc ctg aga gac gag gac acg gct gtg tat tac tgt gcg aga gca tac 388 Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Cys Ala Arg Ala Tyr 105 110 115 tac tac ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tcc 436 Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 120 125 130 135 tca gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc 484 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 140 145 150 aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac 532 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 155 160 165 tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc 580 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 170 175 180 agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac 628 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 185 190 195 tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acc cag 676 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 200 205 210 215 acc tac atc tgc aac gtg aat cac aag ccc agc aac acc aag gtg gac 724 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 220 225 230 aag aga gtt gag ccc aaa tct tgt gac aaa act cac aca tgc cca ccg 772 Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 235 240 245 tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc 820 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 250 255 260 cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca 868 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 265 270 275 tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac 916 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 280 285 290 295 tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg 964 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 300 305 310 gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc 1012 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 315 320 325 ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc 1060 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 330 335 340 aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa 1108 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 345 350 355 ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg gag 1156 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 360 365 370 375 gag atg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc 1204 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 380 385 390 tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag 1252 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 395 400 405 aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc 1300 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 410 415 420 ttc ctc tat agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg 1348 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 425 430 435 aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac 1396 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 440 445 450 455 acg cag aag agc ctc tcc ctg tcc ccg ggt aaa tga 1432 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys * 460 465 <![CDATA[<210> 4]]> <![CDATA[<211> 466]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<220> ]]> <![CDATA[<221> misc_feature ]]> <![CDATA[<222> (1)...(466)]]> <![CDATA[<223> Ha22-2(2,4)6.1 heavy chain]]> <![CDATA[ <400> 4]]> Met Glu Leu Gly Leu Cys Trp Val Phe Leu Val Ala Ile Leu Glu Gly 1 5 10 15 Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Tyr Ile Ser Ser Ser Ser Thr Ile Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Ser Leu Ser Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln 115 120 125 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460 Gly Lys 465 <![CDATA[ <210> 5]]> <![CDATA[ <211> 735]]> <![CDATA[ <212> DNA <![CDATA[ <213> Homo sapiens <![CDATA[ <220> ]]> <![CDATA[ <221> CDS <![CDATA[ <222> (25)...(735)]]> <![CDATA[ <220> ]]> <![CDATA[ <221> misc_feature <![CDATA[ <222> (1)...(735)]]> <![CDATA[ <223> Ha22-2(2,4)6.1 light chain]]> <![CDATA[ <400> 5]]> agtcagaccc agtcaggaca cagc atg gac atg agg gtc ccc gct cag ctc 51 Met Asp Met Arg Val Pro Ala Gln Leu 1 5 ctg ggg ctc ctg ctg ctc tgg ttc cca ggt tcc aga tgc gac atc cag 99 Leu Gly Leu Leu Leu Leu Trp Phe Pro Gly Ser Arg Cys Asp Ile Gln 10 15 20 25 atg acc cag tct cca tct tcc gtg tct gca tct gtt gga gac aga gtc 147 Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val 30 35 40 acc atc act tgt cgg gcg agt cag ggt att agc ggc tgg tta gcc tgg 195 Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Gly Trp Leu Ala Trp 45 50 55 tat cag cag aaa cca ggg aaa gcc cct aag ttc ctg atc tat gct gca 243 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile Tyr Ala Ala 60 65 70 tcc act ttg caa agt ggg gtc cca tca agg ttc agc ggc agt gga tct 291 Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 75 80 85 ggg aca gat ttc act ctc acc atc agc agc ctg cag cct gaa gat ttt 339 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 90 95 100 105 gca act tac tat tgt caa cag gct aac agt ttc cct ccc act ttc ggc 387 Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro Thr Phe Gly 110 115 120 gga ggg acc aag gtg gag atc aaa cga act gtg gct gca cca tct gtc 435 Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val 125 130 135 ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga act gcc tct 483 Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 140 145 150 gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag 531 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 155 160 165 tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc 579 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 170 175 180 185 aca gag cag gac agc aag gac agc acc tac agc ctc agc agc acc ctg 627 Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 190 195 200 acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa 675 Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 205 210 215 gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg 723 Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 220 225 230 gga gag tgt tag 735 Gly Glu Cys * 235 <![CDATA[<210> 6]]> <![CDATA[<211> 236]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<220> ]]> <![CDATA[<221> misc_feature ]]> <![CDATA[ <222> (1)...(236)]]> <![CDATA[ <223> Ha22-2(2,4)6.1]]> <![CDATA[ <400> 6]]> Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Ser Gly Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Phe Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Ala Asn Ser Phe Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <![CDATA[<210> 7]]> <![CDATA[<211> 466]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<220> ]]> <![CDATA[<221> misc_feature ]]> <![CDATA[<222> (1)...(466)]]> <![CDATA[<223> Ha22-2(2,4)6.1 heavy chain]]> <![CDATA[<400> 7]]> Met Glu Leu Gly Leu Cys Trp Val Phe Leu Val Ala Ile Leu Glu Gly 1 5 10 15 Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Tyr Ile Ser Ser Ser Ser Thr Ile Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Ser Leu Ser Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln 115 120 125 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460 Gly Lys 465 <![CDATA[<210> 8]]> <![CDATA[<211> 236]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<220> ]]> <![CDATA[<221> misc_feature ]]> <![CDATA[ <222> (1)...(236)]]> <![CDATA[ <223> Ha22-2(2,4)6.1]]> <![CDATA[ <400> 8]]> Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Ser Gly Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Phe Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Ala Asn Ser Phe Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <![CDATA[<210> 9]]> <![CDATA[<211> 98]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<220> ]]> <![CDATA[<221> misc_feature ]]> <![CDATA[<222> (1)...(98)]]> <![CDATA[<223> Human Ig germline VH3-48]]> <![CDATA[<400> 9]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <![CDATA[<210> 10]]> <![CDATA[<211> 96]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<220> ]]> <![CDATA[<221> misc_feature ]]> <![CDATA[<222> (1)...(96)]]> <![CDATA[<223> Human Ig germline L5]]> <![CDATA[<400> 10]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro 85 90 95 <![CDATA[<210> 11]]> <![CDATA[ <211> 179]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Little house mouse]]> <![CDATA[ <220> ]]> <![CDATA[ <221> misc_feature <![CDATA[ <222> (1)...(179)]]> <![CDATA[ <223> Contains 191P4D12 orthologs of the V domain. <![CDATA[ <400> 11]]> Met Pro Leu Ser Leu Gly Ala Glu Met Trp Gly Pro Glu Ala Trp Leu 1 5 10 15 Arg Leu Leu Phe Leu Ala Ser Phe Thr Gly Gln Tyr Ser Ala Gly Glu 20 25 30 Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly Gln Asp Ala Lys 35 40 45 Leu Pro Cys Phe Tyr Arg Gly Asp Pro Asp Glu Gln Val Gly Gln Val 50 55 60 Ala Trp Ala Arg Val Asp Pro Asn Glu Gly Ile Arg Glu Leu Ala Leu 65 70 75 80 Leu His Ser Lys Tyr Gly Leu His Val Asn Pro Ala Tyr Glu Asp Arg 85 90 95 Val Glu Gln Pro Pro Pro Pro Arg Asp Pro Leu Asp Gly Ser Val Leu 100 105 110 Leu Arg Asn Ala Val Gln Ala Asp Glu Gly Glu Tyr Glu Cys Arg Val 115 120 125 Ser Thr Phe Pro Ala Gly Ser Phe Gln Ala Arg Met Arg Leu Arg Val 130 135 140 Leu Val Pro Pro Leu Pro Ser Leu Asn Pro Gly Pro Pro Leu Glu Glu 145 150 155 160 Gly Gln Gly Leu Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly Ser Pro 165 170 175 Ala Pro Serum <![CDATA[ <210> 12]]> <![CDATA[ <211> 179]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Brown rat <![CDATA[ <220> ]]> <![CDATA[ <221> misc_feature <![CDATA[ <222> (1)...(179)]]> <![CDATA[ <223> Contains 191P4D12 orthologs of the V domain. <![CDATA[ <400> 12]]> Met Pro Leu Ser Leu Gly Ala Glu Met Trp Gly Pro Glu Ala Trp Leu 1 5 10 15 Leu Leu Leu Phe Leu Ala Ser Phe Thr Gly Arg Tyr Ser Ala Gly Glu 20 25 30 Leu Glu Thr Ser Asp Leu Val Thr Val Val Leu Gly Gln Asp Ala Lys 35 40 45 Leu Pro Cys Phe Tyr Arg Gly Asp Pro Asp Glu Gln Val Gly Gln Val 50 55 60 Ala Trp Ala Arg Val Asp Pro Asn Glu Gly Thr Arg Glu Leu Ala Leu 65 70 75 80 Leu His Ser Lys Tyr Gly Leu His Val Ser Pro Ala Tyr Glu Asp Arg 85 90 95 Val Glu Gln Pro Pro Pro Arg Asp Pro Leu Asp Gly Ser Ile Leu 100 105 110 Leu Arg Asn Ala Val Gln Ala Asp Glu Gly Glu Tyr Glu Cys Arg Val 115 120 125 Ser Thr Phe Pro Ala Gly Ser Phe Gln Ala Arg Met Arg Leu Arg Val 130 135 140 Leu Val Pro Pro Leu Pro Ser Leu Asn Pro Gly Pro Pro Leu Glu Glu 145 150 155 160 Gly Gln Gly Leu Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly Ser Pro 165 170 175 Ala Pro Serum <![CDATA[ <210> 13]]> <![CDATA[ <211> 180]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Homo sapiens <![CDATA[ <220> ]]> <![CDATA[ <221> misc_feature <![CDATA[ <222> (1)...(180)]]> <![CDATA[ <223> Contains 191P4D12 orthologs of the V domain. <![CDATA[ <400> 13]]> Met Pro Leu Ser Leu Gly Ala Glu Met Trp Gly Pro Glu Ala Trp Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu Ala Ser Phe Thr Gly Arg Cys Pro Ala Gly 20 25 30 Glu Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly Gln Asp Ala 35 40 45 Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser Gly Glu Gln Val Gly Gln 50 55 60 Val Ala Trp Ala Arg Val Asp Ala Gly Glu Gly Ala Gln Glu Leu Ala 65 70 75 80 Leu Leu His Ser Lys Tyr Gly Leu His Val Ser Pro Ala Tyr Glu Gly 85 90 95 Arg Val Glu Gln Pro Pro Pro Pro Arg Asn Pro Leu Asp Gly Ser Val 100 105 110 Leu Leu Arg Asn Ala Val Gln Ala Asp Glu Gly Glu Tyr Glu Cys Arg 115 120 125 Val Ser Thr Phe Pro Ala Gly Ser Phe Gln Ala Arg Leu Arg Leu Arg 130 135 140 Val Leu Val Pro Pro Leu Pro Ser Leu Asn Pro Gly Pro Ala Leu Glu 145 150 155 160 Glu Gly Gln Gly Leu Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly Ser 165 170 175 For Ala For Ser 180

Claims

1. An antibody-drug conjugate comprising an anti-191P4D12 antibody or an antigen-binding fragment thereof, wherein: a) The anti-191P4D12 antibody or its antigen-binding fragment binds to the epitope in the V domain of 191P4D12, wherein the V domain contains amino acids 1 to 150 of SEQ ID NO: 2, and wherein the antibody or its antigen-binding fragment binds at least A76 and S91 of SEQ ID NO: 2; or b) The antibody or its antigen-binding fragment binds to the extracellular domain of 191P4D12 at 1.6 nM or less Kd, as determined by fluorescence activated cell sorting (FACS), wherein the extracellular domain of 191P4D12 contains amino acids 1 to 348 of SEQ ID NO:

2.

2. The antibody-drug conjugate of claim 1, wherein the antibody or its antigen-binding fragment binds to the extracellular domain of 191P4D12 at a concentration of 1.6 nM or less Kd, as determined by fluorescence activated cell sorting (FACS), wherein the extracellular domain of 191P4D12 contains amino acids 1 to 348 of SEQ ID NO: 2, and wherein the antibody or its antigen-binding fragment binds at least A76 and S91 of SEQ ID NO:

2.

3. The antibody-drug conjugate of claim 1, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence of the light chain variable region shown in SEQ ID NO:

8.

4. The antibody-drug conjugate of claim 1, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, or 90% homologous to the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:7, and wherein the light chain variable region comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, or 90% homologous to the amino acid sequence of the light chain variable region shown in SEQ ID NO:

8.

5. The antibody drug conjugate as requested in item 1, wherein the fragment is a Fab, F(ab')2, Fv, or scFv fragment.

6. The antibody-drug conjugate of claim 1, wherein the antibody is a human antibody.

7. The antibody-drug conjugate of claim 1, wherein the antibody is generated through recombination.

8. The antibody-drug conjugate of claim 1, wherein the antibody or its antigen-binding fragment is conjugated via a linker MMAE.

9. The antibody-drug conjugate of claim 7, wherein the linker comprises valine-citrulline.

10. The antibody-drug conjugate of claim 7, wherein the linker is an enzyme-cleavable linker, wherein the linker unit forms a bond with the sulfur atom of the antibody or its antigen-binding fragment.

11. The antibody-drug conjugate of claim 7, wherein the linker unit has the following formula: -Aa-Ww-Yy-; wherein -A- is an extension unit, a is 0 or 1; -W- is an amino acid unit, w is an integer in the range of 0 to 12; and -Y- is a spacer unit, y is 0, 1 or 2; wherein the extension unit has the structure of the following formula (1); the amino acid unit is valine or citrulline; and the spacer unit is a PAB group having the structure of the following formula (2); Formula (1) Formula (2); and wherein the extension unit forms a bond with the sulfur atom of the antibody or its antigen-binding fragment; and wherein the spacer unit is linked to MMAE through a carbamate group.

12. The antibody-drug conjugate of claim 1, comprising 1 to 10 units of MMAE in each antibody or its antigen-binding fragment.

13. The antibody-drug conjugate of claim 1, comprising 2 to 5 units of MMAE in each antibody or its antigen-binding fragment.

14. The antibody-drug conjugate of claim 1, comprising 3 to 5 units of MMAE in each antibody or its antigen-binding fragment.

15. The antibody-drug conjugate of claim 1, wherein the antibody-drug conjugate has the following structure: wherein L- is the anti-191P4D12 antibody or its antigen-binding fragment, and p is in the range of 1 to 10.

16. The antibody drug conjugate as requested in item 14, wherein p is in the range of 3 to 5.

17. The antibody drug conjugate as requested in item 14, wherein p is 3.

8.

18. A composition comprising an antibody drug conjugate of any one of claims 15 to 17, wherein the average p-value of the antibody drug conjugate in the composition is about 3.

8.

19. A pharmaceutical composition comprising a therapeutically effective amount of an antibody drug conjugate of any one of claims 1 to 17 and a pharmaceutically acceptable excipient.

20. A pharmaceutical composition comprising a therapeutically effective amount of an antibody drug conjugate of any one of claims 15 to 17 and a pharmaceutically acceptable excipient, wherein the average p-value of the antibody drug conjugate in the pharmaceutical composition is about 3.

8.

21. An anti-191P4D12 antibody or its antigen-binding fragment, wherein: a) The anti-191P4D12 antibody or its antigen-binding fragment binds to the antigen-binding site in the V domain of 191P4D12, wherein the V domain contains amino acids 1 to 150 of SEQ ID NO: 2, and wherein the antibody or its antigen-binding fragment binds at least A76 and S91 of SEQ ID NO: 2; or b) The antibody or its antigen-binding fragment binds to the extracellular domain of 191P4D12 at 1.6 nM or less Kd, as determined by fluorescence activated cell sorting (FACS), wherein the extracellular domain of 191P4D12 contains amino acids 1 to 348 of SEQ ID NO:

2.

22. The antibody or antigen-binding fragment thereof of claim 21, wherein the anti-191P4D12 antibody or antigen-binding fragment thereof binds to the extracellular domain of 191P4D12 at a concentration of 1.6 nM or less Kd, as determined by fluorescence activated cell sorting (FACS), wherein the extracellular domain of 191P4D12 contains amino acids 1 to 348 of SEQ ID NO: 2, and wherein the antibody or antigen-binding fragment thereof binds at least A76 and S91 of SEQ ID NO:

2.

23. The antibody or antigen-binding fragment thereof as claimed in claim 21, 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 an amino acid sequence that is at least 80% homologous to the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:7, and the light chain variable region comprising an amino acid sequence that is at least 80% homologous to the amino acid sequence of the light chain variable region shown in SEQ ID NO:

8.

24. The antibody or antigen-binding fragment thereof as claimed in claim 23, 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 an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, or 90% homologous to the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:7, and the light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, or 90% homologous to the amino acid sequence of the light chain variable region shown in SEQ ID NO:

8.

25. The antibody or antigen-binding fragment thereof as claimed in claim 21, wherein the antigen-binding fragment is a Fab, F(ab')2, Fv or scFv fragment.

26. The antibody or antigen-binding fragment thereof as claimed in claim 21, wherein the antibody is a human antibody.

27. The antibody or antigen-binding fragment thereof as claimed in claim 21, wherein the antibody or antigen-binding fragment thereof is generated by recombination.

28. Use of an antibody-drug conjugate as claimed in any one of claims 1 to 17, a composition as claimed in claim 18, or a pharmaceutical composition as claimed in claim 19 or 20, for the preparation of a medicament for the prevention or treatment of cancer in an individual.

29. As used in claim 28, wherein the individual is a human individual.

30. As claimed in claim 28, wherein the cancer comprises tumor cells expressing 191P4D12.

31. As claimed in claim 28, wherein the cancer is colon cancer, ovarian cancer, esophageal cancer, head and neck cancer, pancreatic cancer, lung cancer, bladder cancer, or breast cancer.

32. As used in claim 28, wherein the cancer is bladder cancer.

33. As used in claim 28, wherein the cancer is breast cancer.

34. As used in claim 28, wherein the cancer is esophageal cancer.

35. As used in claim 28, wherein the cancer is head and neck cancer.

36. As requested in claim 32, wherein the bladder cancer is advanced bladder cancer.

37. As used in claim 32, wherein the bladder cancer is metastatic bladder cancer.

38. Use of an antibody or antigen-binding fragment thereof as claimed in any one of claims 21 to 27, for the preparation of a medicament for the prevention or treatment of cancer in an individual.

39. As used in claim 38, wherein the individual is a human individual.

40. As claimed in claim 38, wherein the cancer comprises tumor cells expressing 191P4D12.

41. As claimed in claim 38, wherein the cancer is colon cancer, ovarian cancer, esophageal cancer, head and neck cancer, pancreatic cancer, lung cancer, bladder cancer, or breast cancer.

42. As used in claim 41, wherein the cancer is bladder cancer.

43. As used in claim 41, wherein the cancer is breast cancer.

44. As used in claim 41, wherein the cancer is esophageal cancer.

45. As used in claim 41, wherein the cancer is head and neck cancer.

46. ​​As requested in claim 42, wherein the bladder cancer is advanced bladder cancer.

47. As used in claim 42, wherein the bladder cancer is metastatic bladder cancer.

Images