antibody-drug conjugate comprising an anti-191p4d12 antibody conjugated to monomethyl auristatin and

专利摘要:
ANTIBODY-DRUG CONJUGATES (ADC) THAT BIND TO 191P4D12 PROTEINS. The present invention relates to antibody-drug conjugates (ADC's) that bind to 191P4D12 protein and variants thereof. 191P4D12 shows tissue-specific expression in normal adult tissue and is aberrantly expressed in the cancers mentioned in table I. Consequently, the ADC's of the invention provide a therapeutic composition for the treatment of cancer.
公开号:BR112013007309B1
申请号:R112013007309-8
申请日:2011-09-29
公开日:2021-07-06
发明作者:Daulet Satpaev；Robert Kendall Morrison；Karen Jane Meyrick Morrison；Jean Gudas；Aya Jakobovits；Michael Torgov；Zili An
申请人:Agensys, Inc.；Seattle Genetics, Inc.；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDERS
[001] This application is a non-provisional patent application claiming the priority benefit of US Provisional Patent Application No. 61/387,933, filed September 29, 2010. The contents of each application mentioned in this paragraph are fully incorporated. by reference in the present invention. REFERENCE TO THE SEQUENCE LISTING SUBMITTED THROUGH EFS-WEB
[002] The entire content of the following electronic submission of the string listing through the USPTO's EFS-WEB server, as authorized and presented in MPEP §1730 II.B.2(a)(C), is hereby incorporated by way of reference in its entirety for all purposes. The string listing is identified in the electronically deposited text file, as follows:
DECLARATION OF RIGHTS TO INVENTIONS MADE UNDER RESEARCH SPONSORED BY THE FEDERAL GOVERNMENT
[003] Not applicable. FIELD OF THE INVENTION
The invention described herein relates to antibodies, binding fragments, and their antibody-drug conjugates (ADCs), which bind to proteins called 191P4D12. The invention further relates to prognostic, prophylactic and therapeutic methods and compositions useful in the treatment of cancers expressing 191P4D12. BACKGROUND OF THE INVENTION
[005] Cancer is the second leading cause of human death after coronary heart disease. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer kills well over half a million people annually, with more than 1.2 million new cases diagnosed each year. While deaths from heart disease have dropped significantly, those resulting from cancer are generally on the rise. In the first part of the next century, cancer is predicted to become the leading cause of death.
[006] All over the world, several cancers stand out as the ones that kill the most. In particular, carcinomas of the lungs, prostate, breast, colon, pancreas, ovary, and bladder represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal element. With a few exceptions, metastatic disease from a carcinoma is fatal. Furthermore, even for cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties induced by the realization of the potential for recurrence or treatment failure. Many cancer patients suffer physical debilitation after treatment. Also, many cancer patients experience a recurrence.
[007] Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common cancer in men and is the second leading cause of cancer death in men. In the United States alone, well over 30,000 men die each year from this disease - second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiotherapy, hormone ablation therapy, surgical castration and chemotherapy remain the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.
[008] On the diagnostic front, the lack of a prostate tumor marker that can accurately detect early-stage localized tumors is still a significant limitation in the diagnosis and management of this disease. Although serum prostate specific antigen (PSA) testing has been a very useful tool, it is widely considered that its specificity and general utility lacks many important aspects.
[009] Progress in identifying additional specific markers for prostate cancer has been enhanced by the generation of prostate cancer xenografts that can recapitulate different stages of disease in mice. LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in mice with severe combined immune deficiency (SCID) and have shown the ability to mimic the transition from androgen dependence to androgen independence (Klein et al. ., 1997, Nat. Med. 3:402). The most recently identified markers 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 US A. 1999 Dec 7;96(25): 14523-8) and stem cell antigen. prostate (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).
[0010] Although previously identified markers such as PSA have facilitated efforts to diagnose and treat prostate cancer, there is a need to identify additional markers and therapeutic targets for prostate cancers and related cancers to further improve diagnosis and therapy. An estimated 130,200 cases of colorectal cancer occurred in 2000 in the United States, including 93,800 cases of colon cancer and 36,400 of rectal cancer.
[0011] Colorectal cancers are the third most common cancer in men and women. Incidence rates dropped significantly during the years 1992 to 1996 (-2.1% per year). Research suggests that these declines were caused by increased screening and removal of polyps, preventing the progression of polyps to invasive cancers. There were an estimated 56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer) in 2000, which corresponds to about 11% of all cancer deaths in the US.
[0012] At present, surgery is the most common form of therapy for colorectal cancer, and for cancers that have not spread, it is often curative. Chemotherapy, or chemotherapy plus radiation, is given before or after surgery for most patients whose cancer has already pierced deep into the intestinal wall or has spread to lymph nodes. A permanent colostomy (creating an abdominal opening to dispose of bodily waste) is occasionally needed for colon cancer and is rarely needed for rectal cancer. There is still a need for effective diagnostic and treatment modalities for colorectal cancer.
[0013] Of all new cancer cases in the United States, bladder cancer represents approximately 5 percent in men (the fifth most common cancer) and 3 percent in women (the eighth most common cancer). The incidence is slowly growing, concurrently with an increasingly older population. In 1998, there were an estimated 54,500 cases, including 39,500 in men and 15,000 in women. This age-adjusted incidence in the United States is 32 per 100,000 for men and eight per 100,000 for women. The historical male/female ratio of 3:1 may be declining relative to smoking patterns in women. 11,000 deaths from bladder cancer were estimated in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality increase dramatically with age and will be a growing problem as the population becomes older and older.
[0014] Most bladder cancers recur in the bladder. Bladder cancer is managed with a combination of transurethral bladder resection (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer indicates the limitations of TUR. Most muscle invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion are the most effective means of eliminating cancer, but they have an undeniable impact on urinary and sexual function. There is still a significant need for treatment modalities that are beneficial to bladder cancer patients.
[0015] There were an estimated 164,100 new cases of lung and bronchial cancer in 2000, which accounts for 14% of all cancer diagnoses in the US. The rate of incidence of lung and bronchial cancer is falling significantly in men, from high 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of increase among women began to decline. In 1996, the incidence rate in women was 42.3 per 100,000.
[0016] Lung and bronchial cancer caused an estimated 156,900 deaths in 2000, representing 28% of all cancer deaths. During 1992 to 1996, mortality from lung cancer dropped significantly among men (-1.7% per year) while rates for women were still increasing significantly (0.9% per year). Since 1987, more women have died each year from lung cancer than from breast cancer, which for more than 40 years was the leading cause of cancer death in women. The reduction in lung cancer incidence and mortality rates most likely resulted from the reduction in smoking rates over the previous 30 years; however, the reduction in smoking patterns among women lags behind that of men. Interestingly, although declines in adult tobacco use have slowed, tobacco use in young people is on the rise again.
[0017] Treatment options for lung and bronchial cancer are determined by the type and stage of cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is often the treatment of choice. Because the disease has usually spread by the time it is discovered, radiotherapy and chemotherapy are often needed in combination with surgery. Chemotherapy alone or combined with radiation is the treatment of choice for small cell lung cancer; on this regimen, a large percentage of patients experience remission, which in some cases is long-lasting. There is, however, a growing need for effective treatment and diagnostic approaches for lung and bronchial cancers.
[0018] An estimated 182,800 new invasive cases of breast cancer were expected to occur among women in the United States during the year 2000. Additionally, approximately 1,400 new cases of breast cancer were expected to be diagnosed in men in 2000. Afterwards. increasing by about 4% a year in the 1980s, breast cancer incidence rates in women balanced out in the 1990s to about 110.6 cases per 100,000.
[0019] In the US alone, there were 41,200 estimated deaths (40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer ranks second among cancer deaths in women. According to the most recent data, mortality rates decreased significantly during the years 1992 to 1996 with the greatest reductions in younger, white and black women. These reductions were likely the result of earlier detection and improved treatment.
[0020] Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer may involve lumpectomy (local removal of the tumor) and removal of lymph nodes under the arms; mastectomy (surgical removal of the breast) and removal of lymph nodes under the arms; radiotherapy; chemotherapy; or hormone therapy. Often two or more methods are used in combination. Several studies have shown that, for early-stage disease, long-term survival rates after lumpectomy plus radiotherapy are similar to survival rates after modified radical mastectomy. Significant advances in reconstructive techniques provide several options for breast reconstruction after mastectomy. Recently, such reconstruction has been done at the same time as the mastectomy.
Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast tissue can prevent local recurrence of DCIS. Radiation to the breast and/or tamoxifen may reduce the chance of DCIS occurring in remaining breast tissue. This is important because DCIS, if left untreated, can develop into invasive breast cancer. However, there are serious side effects or sequelae caused by these treatments. There is therefore a need for effective treatments for breast cancer.
[0022] There were an estimated 23,100 new cases of ovarian cancer in the United States in 2000. This represents 4% of all cancers among women and ranks second among gynecological cancers. During 1992 to 1996, ovarian cancer incidence rates were significantly declining. As a consequence of ovarian cancer, there were an estimated 14,000 deaths in 2000. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.
[0023] Surgery, radiotherapy, and chemotherapy are the treatment options for ovarian cancer. Surgery usually includes removing one or both of the ovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus (hysterectomy). In some very early tumors, only the involved ovary will be removed, especially in young women who want to become pregnant. In advanced disease, an attempt is made to remove all intra-abdominal disease to enhance the effect of chemotherapy. There is still an important need for effective treatment options for ovarian cancer.
[0024] There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past 20 years, rates of pancreatic cancer have fallen in men. Rates among women have remained roughly constant but may be starting to decline. Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the United States. Over the past 20 years, there has been a slight but significant reduction in mortality rates among men (about -0.9% per year) while rates have increased slightly among women.
[0025] Surgery, radiotherapy, and chemotherapy are the treatment options for pancreatic cancer. These treatment options can extend survival and/or alleviate symptoms in many patients, but are unlikely to produce a cure for most. There is a significant need for additional therapeutic and diagnostic options for cancers. These include the use of antibodies, vaccines, and small molecules as treatment modalities. Additionally, there is also a need to use these modalities as research tools to diagnose, detect, monitor, and advance the state of the art in all areas of cancer treatments and studies.
The therapeutic utility of monoclonal antibodies (mAbs) (G. Kohler and C. Milstein, Nature 256:495-497 (1975)) is being understood. Monoclonal antibodies have now been approved as therapies in transplantation, cancer, infectious disease, cardiovascular disease and inflammation. Different isotypes have different effector functions. Such differences in function are reflected in distinct three-dimensional structures for the various immunoglobulin isotypes (P.M. Alzari et al., Annual Rev. Immunol., 6:555-580 (1988)).
[0027] Because mice are convenient for immunization and recognize most human antigens as foreign, mAbs against human targets with therapeutic potential have typically been of murine origin. However, murine mAbs have inherent disadvantages as therapeutic agents for humans. They require more frequent dosing as mAbs have a shorter circulating half-life in humans than human antibodies. More critically, repeated administration of murine antibodies to the human immune system causes the human immune system to respond by recognizing the mouse protein as foreign and generating a human anti-mouse antibody (HAMA) response. Such a HAMA response can result in an allergic reaction and rapid clearance of the murine antibody from the system, thus rendering murine antibody treatment useless. To avoid these effects, attempts have been made to create human immune systems within mice.
Initial attempts had hoped to create transgenic mice capable of responding to antigens with antibodies with human sequences (see Bruggemann et al., Proc. Nat'l. Acad. Sci. USA 86:6709-6713 (1989)), but they were limited by the amount of DNA that could be stably maintained by available cloning vehicles. The use of yeast artificial chromosome (YAC) cloning vectors has shown the way to introduce large germline fragments of human Ig locus into transgenic mammals. Essentially, most of the human V, D, and J region genes arranged with the same spacing found in the human genome and the human constant regions were introduced into mice using YACs. One such transgenic mouse strain is known as XenoMouse® mice and is commercially available from Amgen Fremont, Inc. (Fremont CA). SUMMARY OF THE INVENTION
The invention provides antibodies, binding fragments, and their antibody-drug conjugates (ADCs) that bind to 191P4D12 proteins and to polypeptide fragments of 191P4D12 proteins. In some embodiments, the invention comprises fully human antibodies conjugated to a therapeutic agent. In certain embodiments, there is a proviso 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 certain embodiments, the entire nucleic acid sequence of Figure 3 is encoded and/or the entire amino acid sequence of Figure 2 is prepared, either in respective human unit dose forms.
[0030] The invention further provides various immunogenic or therapeutic compositions, e.g. antibody-drug conjugates, and strategies for treating cancers expressing 191P4D12, such as tissue cancers mentioned in table I. BRIEF DESCRIPTION OF THE FIGURES
[0031] Figure 1. The cDNA and amino acid sequence of 191P4D12 is shown in Figure 1. The start methionine is underlined. The open reading frame extends from nucleic acid 264 to 1796, including the stop codon.
[0032] Figures 2A-B. Nucleic acid and amino acid sequences of 191P4D12 antibodies. Figure 2A. The heavy chain cDNA and amino acid sequence of Ha22-2(2,4)6.1. The leader sequence is double underlined, the heavy chain variable region is underlined, and the human IgG1 constant region is underlined with a dashed line. Figure 2B. The cDNA and amino acid sequence of the light chain of Ha22-2(2,4)6.1. The leader sequence is double underlined, the light chain variable region is underlined, and the human kappa constant region is underlined with a dashed line.
[0033] Figures 3A-B. Amino Acid Sequences of 191P4D12 Antibodies. Figure 3A. The heavy chain amino acid sequence of Ha22-2(2,4)6.1. The leader sequence is double underlined, the heavy chain variable region is underlined, and the human IgG1 constant region is underlined with a dashed line. Figure 3B. The light chain amino acid sequence of Ha22-2(2,4)6.1. The leader sequence is double underlined, the light chain variable region is underlined, and the human kappa constant region is underlined with a dashed line.
[0034] Figures 4A-B. Alignment of Ha22-2(2,4)6.1 antibodies to human Ig germline. Figure 4A. Alignment of Ha22-2(2,4)6.1 heavy chain to human Ig germline. Figure 4B. Alignment of Ha22-2(2,4)6.1 light chain to human Ig germline.
[0035] Figures 5A-B. Mab Ha22-2(2,4)6.1 binding assays. Figure 5A: RAT-control and RAT-191P4D12 cells were labeled with MAb Ha22-2(2,4)6.1 from hybridoma or CHO cells. Binding was detected by flow cytometry. The results show that recombinantly expressed MAb Ha22-2(2,4)6.1 in CHO cells is secreted and specifically binds to cell surface 191P4D12. Figure 5B: MAb Ha22-2(2,4)6.1 from hybridoma or CHO cells was tested for binding to recombinant purified extracellular 191P4D12 protein by ELISA. The results show that the binding of 191P4D12 protein to Ha22-2(2,4)6.1 derived from CHO and hybridoma was identical.
[0036] Figure 6 Affinity determination of Ha22-2(2,4)6.1vcMMAE by FACS using PC3-human-191P4D12 cells. Affinity is 0.69 Kd.
[0037] Figure 7 Affinity determination of Ha22-2(2,4)6.1vcMMAE by FACS using PC3-cynomolgus-191P4D12 cells. Affinity is 0.34 Kd.
[0038] Figure 8 Affinity determination of Ha22-2(2,4)6.1vcMMAE by FACS using PC3-mouse-191P4D12 cells. Affinity is 1.6 Kd.
[0039] Figures 9A-D. Ha22-2(2,4) 6.1vcMMAE-mediated cell cytotoxicity. Figure 9A: Cellular cytotoxicity assay using PC3-human-191P4D12 cells. Figure 9B: Cellular cytotoxicity assay using PC3-cynomolgus-191P4D12 cells. Figure 9C: Cellular cytotoxicity assay using PC3-mouse-191P4D12 cells. Figure 9D: Cell cytotoxicity assay using PC3-Neo cells.
[0040] Figure 10. Mab Ha22-(2,4)6.1 domain mapping by FACS.
[0041] Figure 11. Domain mapping of MAb Ha22-2(2,4)6.1 by Western Blot analysis.
[0042] Figure 12. Evaluation of MAb Ha22-2(2,4)6.1 in the AG-L4 human lung cancer xenograft subcutaneous tumor formation model in SCID mice. The results show that MAbs 191P4D12 did not significantly inhibit tumor growth in AG-L4 human lung cancer xenograft in SCID mice.
[0043] Figure 13 Evaluation of MAb Ha22-2(2,4)6.1 in the HPAC human pancreatic cancer xenograft subcutaneous tumor formation model in SCID mice. The results show that MAbs 191P4D12 did not inhibit tumor growth in a human pancreatic xenograft in SCID mice when compared to the control antibody.
[0044] Figure 14 Evaluation of MAb Ha22-2(2,4)6.1 in the AG-Panc3 human pancreatic cancer xenograft subcutaneous tumor formation model in SCID mice. The results show that MAbs 191P4D12 did not inhibit tumor growth in a human pancreatic xenograft in SCID mice when compared to the control antibody.
[0045] Figure 15 Efficacy of Ha22-2(2,4)6.1-vcMMAE in AG-L4 established subcutaneous human lung cancer xenograft in SCID mice. The results show 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 to treated and untreated controls.
[0046] Figure 16 Efficacy of Ha22-2(2,4)6.1-vcMMAE in established BT-483 subcutaneous human breast cancer xenograft in SCID mice. The results show that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of BT-483 breast tumor xenografts implanted subcutaneously in SCID mice compared to treated and untreated control ADCs.
[0047] Figure 17 Efficacy of Ha22-2(2,4)6.1-vcMMAE in AG-B1 established subcutaneous human bladder cancer xenograft in SCID mice. The results show that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of AG-B1 bladder cancer xenografts compared to control ADCs.
[0048] Figure 18 Efficacy of Ha22-2(2,4)6.1-vcMMAE in AG-Panc2 established subcutaneous human pancreatic cancer xenograft in SCID mice. The results show that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of AG-Panc2 pancreatic cancer xenografts compared to control ADCs.
[0049] Figure 19 Efficacy of Ha22-2(2,4)6.1-vcMMAE in AG-Panc4 established subcutaneous human lung cancer xenograft in SCID mice. The results show that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of AG-Panc4 pancreatic cancer xenografts compared to control ADCs.
[0050] Figure 20 Efficacy of Ha22-2(2,4)6.1-vcMMAE at comparative dosage in AG-B8 established subcutaneous human bladder cancer xenograft in SCID mice. The results show that treatment with Ha22-2(2,4)6.1vcMMAE at 10mg/kg significantly inhibited the growth of AG-B8 bladder cancer xenografts compared to Ha22-2(2,4)6.1vcMMAE at 5mg/ kg.
[0051] Figures 21A-N. Detection of 191P4D12 protein in cancer patient specimens by IHC. Figures 21A-B show bladder cancer specimens. Figures 21C-D show breast cancer specimens. Figures 21E-F show pancreatic cancer specimens. Figures 21G-H show lung cancer specimens. Figures 21I-J show ovarian cancer specimens. Figures 21K-L show esophageal cancer specimens. Figures 21M-N show esophageal cancer specimens.
[0052] Figures 22A-B. They show binding curves used to determine the affinity of MAb Ha22-2(2,4)6.1 and Ha22-2(2,4)6.1vcMMAE to purified recombinant 191P4D12 (ECD amino acids 1 to 348).
[0053] Figures 23A-D. They show the binding of Ha22-2(2,4)6.1 to PC3 cells expressing 191P4D12 (figure 23A) and cynomolgus monkey (figure 23B), rat (figure 23C) and mouse (figure 23D) orthologs.
[0054] Figures 24A-D. They show that the binding of Ha22-2(2,4)6.1 to the A76I double mutant, S91N is similar to the binding of the murine ortholog.
[0055] Figure 25 Shows a 191P4D12 V domain model based on published crystal structure data for 191P4D12 family members and Ig domain-containing proteins using PyMOL. The positions of Ala-76 (dotted) and Ser-91 (shaded with crossed lines) are shown.
[0056] Figures 26A-C. They show that Ha22-2(2,4)6.1 binds to cells expressing the V domain (Fig. 26A) as well as wild-type 191P4D12 (Fig. 26B), but not to cells expressing the C1C2 domain generated earlier (Fig. 26C). DETAILED DESCRIPTION OF THE INVENTION Section outline
[0057] I.) Definitions
[0058] II.) 191P4D12 Antibodies
[0059] III.) Antibody-drug conjugates in general
[0060] III(A). Maytansinoids
[0061] III(B). Auristatins and Dolastatins
[0062] III(C). calicheamicin
[0063] III(D). Other cytotoxic agents
[0064] 191P4D12 IV.) antibody-drug conjugates that bind to
[0065] V.) Binding units
[0066] VI.) The stretching unit
[0067] VII.) The amino acid unit
[0068] VIII.) The spacer unit
[0069] IX.) The drug unit
[0070] X.) Drug Loading
[0071] ADCs XI.) Methods for determining the cytotoxic effect of
[0072] 191P4D12 XII.) Treatment of cancer(s) that express(s)
[0073] antibody XIII.) 191P4D12 as a target for
[0074] XIV.) 191P4D12 ADC Cocktails
[0075] XV.) Combination Therapy
[0076] XVI.) Kits/articles of manufacture I.) Definitions:
[0077] Except where defined otherwise, all art terms, notations and other scientific terms or terminology used in the present invention are intended to have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions in the present invention should not necessarily be construed to represent a substantial difference from what is generally understood in the art. Many of the techniques and procedures described or mentioned herein are well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely used molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2a. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY As appropriate, procedures involving the use of commercially available kits and reagents are generally performed in accordance with the protocols and/or parameters defined by the manufacturer, except where specified in contrary.
[0078] When a trade name is used in the present invention, reference to the trade name also refers to the product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the name product commercial, except where the context indicates otherwise.
[0079] The terms "advanced cancer", "locally advanced cancer", "advanced disease" and "locally advanced disease" mean cancers that have extended through the relevant tissue capsule, 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, node) system , metastasis). In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients with clinically localized (organ-confined) cancer.
The abbreviation "AFP" refers to dimethylvaline-valine-dolaisoleucine-dolaproline-phenylalanine-p-phenylenediamine (see formula XVI below).
[0081] The abbreviation "MMAE" refers to monomethyl auristatin E (see formula XI below).
[0082] The abbreviation "AEB" refers to an ester produced by the reaction of auristatin E with para-acetyl benzoic acid (see formula XX below).
[0083] The abbreviation "AEVB" refers to an ester produced by the reaction of auristatin E with benzoyl valeric acid (see formula XXI below).
[0084] The abbreviation "MMAF" refers to dovaline-valine-dolaisoleucine-dolaproline-phenylalanine (see formula XVIV below).
[0085] Unless otherwise specified, the term "alkyl" refers to a straight or branched saturated hydrocarbon having from about 1 to about 20 carbon atoms (and all combinations and subcombinations of specific ranges and numbers of carbon atoms. contained therein), with from about 1 to about 8 carbon atoms being preferred. Examples of alkyl groups are methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2 -butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl , 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2 ,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl.
[0086] Alkyl groups, which alone or as part of another group, may optionally be substituted with one or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogen), including, but not limited to, limiting 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, where each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl, and wherein said groups -O-(C1-C8) alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl may be optionally further substituted with one or more groups including, but not limited to, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O- (C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR '', -C(O)N(R'')2, -NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R '', -OH, -N3, -NH2, -NH(R''), -N(R'')2 and -CN, where each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.
[0087] Unless otherwise specified, the terms "alkenyl" and "alkynyl" refer to straight or branched carbon chains having from about 2 to about 20 carbon atoms (and all combinations and subcombinations of specific ranges and numbers of carbon atoms contained therein), with from about 2 to about 8 carbon atoms being preferred. An alkenyl chain has at least one double bond in the chain and an alkynyl chain has at least one triple bond in the chain. Examples of alkenyl groups include, but are not limited to ethylene or vinyl, allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, - 2-methyl-2-butenyl, and -2,3-dimethyl-2-butenyl. Examples of alkynyl groups include, but are not limited to, acetylenic, propargyl, acetylenyl, propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1-butynyl.
[0088] Alkenyl and alkynyl groups, either alone or as part of another group, may optionally be substituted with one or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogen), including but not limited to -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC (O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', - SR', -SO3R', -S(O)2R', -S(O)R', -OH, =O, -N3, -NH2, -NH(R'), -N(R')2 and -CN, where each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl and wherein said groups -O-(C1-C8) alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl may be optionally further substituted with one or more substituents including, but not limited to, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C 8 alkenyl), -O-(C2C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2 , - C(O)NHR'', -C(O)N(R'')2, -NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', - S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2 and -CN, where each R'' is independently selected from -H , -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.
[0089] Unless otherwise specified, the term "alkylene" refers to a saturated straight or branched chain hydrocarbon radical having from about 1 to about 20 carbon atoms (and all combinations and subcombinations of specific ranges and numbers of carbon atoms contained therein), with from about 1 to about 8 carbon atoms being preferred and having two monovalent radical centers derived by removing two hydrogen atoms from the same carbon atom or from two different carbon atoms from an original alkane. Typical alkylenes include, but are not limited to methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, dekalene, 1,4-cyclohexylene, and the like. Alkylene groups, whether alone or as part of another group, may optionally be substituted with one or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogen), including, but not limited to - halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R' , -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SO3R ', -S(O)2 R', -S(O)R', -OH, =O, -N3, -NH2, -NH(R'), -N(R')2 and -CN, where each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl and wherein said -O-(C1-C8 alkyl), -O -(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl may be optionally further substituted with one or more substituents including, but not limited to, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), - O-(C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2 , -C(O)NHR'', - C(O)N(R'')2, - NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R'', - OH, -N3, -NH2, -NH(R''), -N(R'')2 and -CN, where each R'' is independently selected from -H, -C1-C8 alkyl, -C2 -C8 alkenyl, -C2-C8 alkynyl, or -aryl.
[0090] Unless otherwise specified, the term "alkenylene" refers to an optionally substituted alkylene group containing at least one carbon-carbon double bond. Exemplary alkenylene groups include, for example, ethenylene (-CH=CH-) and propenylene (-CH=CHCH2-).
[0091] Unless otherwise specified, the term "alkynylene" refers to an optionally substituted alkylene group containing at least one carbon-carbon triple bond. Exemplary alkynylene groups include, for example, acetylene (-C=C-), propargyl (-CH2C=C-), and 4-pentynyl (-CH2CH2CH2C=CH-).
[0092] Unless otherwise specified, the term "aryl" refers to a monovalent aromatic hydrocarbon radical of 6 to 20 carbon atoms (and all combinations and subcombinations of specific ranges and numbers of carbon atoms contained therein) derived by removing a hydrogen atom from a single carbon atom from an original aromatic ring system. Some aryl groups are represented by exemplifying structures such as "Ar". Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, phenyl, naphthalene, anthracene, biphenyl, and the like.
[0093] An aryl group, either alone or as part of another group, may optionally be substituted with one or more, preferably 1 to 5, or even 1 to 2 groups, including, but not limited to - halogen, - C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2 , -C(O)NHR', -C(O)N(R')2 , -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, -NO2, -N3, -NH2, -NH(R' ), -N(R')2 and -CN, where each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl and where said -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), and -aryl may be optionally additionally substituted with one or more substituents including, but not limited to -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl ), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl) , -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2 , -C(O)NHR'', -C( O) N(R'')2, -NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2 and -CN, where each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.
[0094] Unless otherwise specified, the term "arylene" refers to an optionally substituted aryl group that is divalent (ie, derived by removing two hydrogen atoms from the same carbon atom or from two different carbon atoms from an original aromatic ring system) and can be in ortho, meta, or para configurations, as shown in the following structures with phenyl as the exemplary aryl group.

[0095] Typical "-(C1-C8 alkylene)aryl," "-(C2-C8 alkenylene)aryl", "and -(C2-C8 alkynylene)aryl" groups include, but are not limited to benzyl, 2- phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethane-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthylethane-1-yl and the like.
[0096] Unless otherwise specified, the term "heterocycle," refers to a monocyclic, bicyclic, or polycyclic ring system having from 3 to 14 ring atoms (also called ring members) in which at least one atom A ring in at least one ring is a heteroatom selected from N, O, P, or S (and all combinations and subcombinations of specific ranges and numbers of carbon atoms and heteroatoms contained therein). The heterocycle can have from 1 to 4 ring heteroatoms independently selected from N, O, P, or S. One or more N, C, or S atoms in a heterocycle can be oxidized. A monocyclic heterocycle preferably has 3 to 7 ring members (for example, 2 to 6 carbon atoms and 1 to 3 heteroatoms independently selected from N, O, P, or S) and a bicyclic heterocycle has, preferably, 5 to 10 ring members (for example, 4 to 9 carbon atoms and 1 to 3 heteroatoms independently selected from N, O, P, or S). The ring including the heteroatom can be aromatic or non-aromatic. Unless otherwise specified, the heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure.
[0097] Heterocycles are described in Paquette, "Principles of Modern Heterocyclic Chemistry" (W.A. Benjamin, New York, 1968), particularly in chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to the present), in particular, Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. 82:5566 (1960).
[0098] Examples of "heterocycle" groups include, by way of example and not limitation, pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl hydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H - 1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl, isobenzofuranyl, cromenyl, xanthenyl, fenoxatinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, cinoxalinyl, inyl, 4H-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, fenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, morpholinyl, indolylinyl oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, and isatinoyl. Preferred "heterocycle" groups include, but are not limited to, benzofuranyl, benzothiophenyl, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyrazinyl , isothiazolyl, isoxazolyl and tetrazolyl.
[0099] A heterocycle group, either alone or as part of another group, may optionally be substituted with one or more groups, preferably 1 to 2 groups, including, but not limited to, -C1-C8 alkyl, -C2 -C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O )R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC( O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, -N3, -NH2, -NH(R'), -N(R' )2 and -CN, where each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl and wherein said groups -O-( C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, and -aryl may optionally be further substituted with one or more substituents including, but not limited to -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2 -C8 alkenyl), -O-(C2-C8 alkynyl), -ar ila, -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, where each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or aryl.
By way of example, and not limitation, carbon-linked heterocycles may be attached at the following positions: position 2, 3, 4, 5, or 6 of a pyridine; position 3, 4, 5, or 6 of a pyridazine; position 2, 4, 5, or 6 of a pyrimidine; position 2, 3, 5, or 6 of a pyrazine; 2, 3, 4, or 5 position of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole; 2-, 4-, or 5-position of an oxazol, imidazole or thiazole; 3-, 4-, or 5-position of an isoxazole, pyrazole, or isothiazole; 2- or 3-position of an aziridine; position 2, 3, or 4 of an azetidine; position 2, 3, 4, 5, 6, 7, or 8 of a quinoline; or position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline. More typically still, carbon-linked heterocycles 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.
By way of example, and not limitation, nitrogen-linked heterocycles may be linked at the 1-position of an aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazolidine, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, or 1H-indazole; position 2 of an isoindole, or isoindoline; position 4 of a morpholine; and position 9 of a carbazole, or β-carboline. More typically still, nitrogen-linked heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.
[00102] Unless otherwise specified, the term "carbocycle," refers to a non-aromatic saturated or unsaturated monocyclic, bicyclic, or polycyclic ring system having from 3 to 14 ring atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms contained therein) wherein all ring atoms are carbon atoms. Monocyclic carbocycles preferably have 3 to 6 ring atoms, more preferably 5 or 6 ring atoms. Bicyclic carbocycles preferably have 7 to 12 ring atoms, for example arranged as a [4.5], [5.5], [5.6] or [6.6], or 9 or 10 ring atoms arranged as a [5,6] or [6,6] bicyclic system. The term "carbocycle" includes, for example, a monocyclic carbocycle ring fused to an aryl ring (for example, a monocyclic carbocycle ring fused to a benzene ring). Carbocycles preferably have 3 to 8 carbon ring atoms.
[00103] Carbocycle groups, which alone or as part of another group, may optionally be substituted with, for example, one or more groups, preferably 1 or 2 groups (and any additional substituents selected from halogen), including , but not limited to -halogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O- (C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2 , -C(O)NHR', - C(O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, =O, - N3, -NH2, -NH(R'), -N(R')2 and -CN, where each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2 -C8 alkynyl, or -aryl and wherein said groups -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl) , -O-(C2-C8 alkynyl), and -aryl may be optionally further substituted with one or more substituents including, but not limited to -C1-C8 alkyl, -C2-C8 alkenyl, - C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2 , -C(O)NHR'', -C(O)N(R'')2, -NHC( O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R'') , -N(R'')2 and -CN, where each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.
[00104] Examples of monocyclic carbocyclic substituents include -cyclopropyl, -cyclobutyl, -cyclopentyl, -1-cyclopent-1-enyl, -1-cyclopent-2-enyl, -1-cyclopent-3-enyl, cyclohexyl, - 1-cyclohex-1-enyl, -1-cyclohex-2-enyl, -1-cyclohex-3-enyl, -cycloheptyl, -cyclooctyl. - 1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -1,3-cycloheptadienyl, - 1,3,5-cycloheptatrienyl, and -cyclooctadienyl.
[00105] A "carbocycle," whether used alone or as part of another group, refers to an optionally substituted carbocycle group, as defined above, which is divalent (ie, derived by removing two hydrogen atoms from the same atom of carbon or two carbon atoms different from an original carbocyclic ring system).
[00106] Except where the context indicates otherwise, a hyphen (-) designates the point of attachment to the pendant molecule. Accordingly, the term "-(C1-C8 alkylene)aryl" or "-C1-C8 alkylene(aryl)" refers to a C1-C8 alkylene radical, as defined herein, wherein the alkylene radical is attached to the pendant molecule. on any of the carbon atoms of the alkylene radical and one of the hydrogen atoms bonded to a carbon atom of the alkylene radical is replaced by an aryl radical, as defined herein.
[00107] When a particular group is "substituted", that group may have one or more substituents, preferably from one to five substituents, more preferably from one to three substituents, most preferably from one to two substituents, selected independently from the list of substituents. The group can, however, generally have any number of substituents selected from halogen. The groups that are replaced are indicated as such.
[00108] The definition of any substituent or variable at a specific location in a molecule is intended to be independent of its definitions anywhere in this molecule. It is understood that the substituents and substitution patterns in the compounds of this invention can be selected by one of skill in the art so as to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as the methods herein. described.
[00109] Protecting groups for use in the present invention refer to groups that selectively, temporarily or permanently block a reactive site on a multifunctional compound. Suitable hydroxy protecting groups for use in the present invention are pharmaceutically acceptable and may or may not need to be cleaved from the parent compound after administration to an individual for the compound to be active. Cleavage is through normal metabolic processes within the body. Hydroxy protecting groups are well known in the art, see Protective Groups in Organic Synthesis by TW Greene and PGM Wuts (John Wiley & Sons, 3rd edition) incorporated herein in its entirety by reference and for all purposes and include, for example , ether protecting groups (for example, alkyl ethers and silyl ethers including, for example, dialkylsilyl ether, trialkylsilyl ether, dialkylalkoxysilyl ether), ester, carbonate, carbamates, sulfonate, and phosphate. Examples of hydroxy protecting groups include, but are not limited to, methyl ether; methoxy methyl ether, methylthiomethyl ether, (phenyldimethylsilyl)methoxymethyl ether, benzyloxymethyl ether, p-methoxybenzyloxymethyl ether, p-nitrobenzyloxymethyl ether, o-nitrobenzyloxymethyl ether, (4-methoxyphenoxy)methyl ether, guaiacolmethyl ether, t-butoxymethyl ether pentenyloxymethyl, siloxymethyl ether, 2-methoxyethoxymethyl ether, 2,2,2-trichloroethoxymethyl ether, bis(2-chloroethoxy)methyl ether, 2-(trimethylsilyl)ethoxymethyl ether, menthoxymethyl ether, tetrahydropyranyl ether, 1-methoxycyclohexyl ether , 4-methoxytetrahydrothiopyranyl ether, 4-methoxytetrahydrothiopyranyl ether, S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl ether, 1-(2 -fluorophenyl)-4-methoxypiperidin-4-yl, 1,4-dioxan-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-benzyloxyethyl ether, 1-methyl-1-benzyloxy-2-fluoroethyl ether, 1-methyl-1-phenoxyethyl ether, 2-trimethylsilyl ether, t-butyl ether, allyl ether, propargyl ethers, p-chlorophenyl ether, p-methoxyphenyl ether, benzyl ether , p-methoxybenzyl ether, 3,4-dimethoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, tripropylsilyl ether, dimethylisopropylsilyl ether, diethylisopropylsilyl ether, dimethylhexylsilyl ether, t-butyldimethylsilyl ether, diphenylmethylsilyl ether, benzoyl ester acetate ester, ether chloroacetate, dichloroacetate ester, trichloroacetate ester, trifluoroacetate ester, methoxyacetate ester, triphenylmethoxyacetate ester, phenylacetate ester, benzoate ester, alkyl methyl carbonate, alkyl 9-fluorenylmethyl carbonate, alkyl ether carbonate ila, 2,2,2-alkyl-trichloroethyl carbonate, 1,1,-dimethyl-2,2,2-trichloroethyl carbonate, alkylsulfonate, methanesulfonate, benzylsulfonate, tosylate, methylene acetal, ethylidene acetal, and t-butylmethylidene 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, where Ra is C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, - C1-C20 alkylene(carbocycle), -C2-C20 alkenylene(carbocycle), -C2-C20 alkynylene(carbocycle), -C6-C10 aryl, -C1-C20 alkylene(aryl), -C2-C20 alkenylene(aryl), -C2-C20 alkynylene(aryl), -C1-C20 alkylene(heterocycle), -C2-C20 alkenylene(heterocycle), or -C2-C20 alkynylene(heterocycle) wherein said alkyl, alkenyl, alkynyl, alkylene, alkenylene radicals , alkynylene, aryl, carbocycle, and heterocycle, either alone or as part of another group are optionally substituted.
[00110] "Changing the native glycosylation pattern" is intended, for the purposes of the present invention, to mean deleting one or more carbohydrate moieties found in the native sequence of 191P4D12 (by removing the underlying glycosylation site or by deleting glycosylation by means chemical and/or enzymatic), and/or addition of one or more glycosylation sites that are not present in the native 191P4D12 sequence. Furthermore, the expression includes qualitative changes in the glycosylation of native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
[00111] The term "analog" refers to a molecule that is structurally similar or shares similar or corresponding attributes with another molecule (eg, a 191P4D12-related protein). For example, an analog of a 191P4D12 protein can be specifically bound by an antibody or T cell that specifically binds to 191P4D12.
[00112] The term "antibody" is used in its broadest sense unless clearly indicated otherwise. Therefore, an "antibody" can be naturally or artificially occurring, such as, for example, monoclonal antibodies produced by conventional hybridoma technology. 191P4D12 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen binding domain and/or one or more complementarity determining regions of these antibodies. As used herein, the term "antibody" refers to any form of antibody or fragment thereof that specifically binds to 191P4D12 and/or has the desired biological activity and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments as long as they specifically bind 191P4D12 and/or have the desired biological activity. Any specific antibody can be used in the methods and compositions provided herein. Thus, in one embodiment, the term "antibody" encompasses a molecule that comprises at least one variable region of a light chain immunoglobulin molecule and at least one variable region of a heavy chain molecule that, in combination, form a site specific binding to the target antigen. In one embodiment, the antibody is an IgG antibody. For example, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. Antibodies useful in the present methods and compositions can be generated in cell culture, on phages, or in 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, an antibody of the present invention is a mammalian antibody. Phage techniques can be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics. Such techniques are routine and well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, a recombinant antibody can be produced by transfecting a host cell with a vector that comprises a DNA sequence that encodes the antibody. One or more vectors can be used to transfect DNA sequence expressing at least one VL region and one VH region into the host cell. Exemplary descriptions of recombinant means for antibody generation and production include Delves, ANTIBODY PRODUCTION: ESSENTIAL TECHNIQUES (Wiley, 1997); Shephard, et al., MONOCLONAL ANTIBODIES (Oxford University Press, 2000); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (Academic Press, 1993); and CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons, most recent edition). An antibody of the present invention can be modified by recombinant means to increase the antibody's effectiveness in mediating the desired function. Thus, it is within the scope of the invention that antibodies can be modified by substitutions using recombinant means. Typically, substitutions will be conservative substitutions. For example, at least one amino acid in the antibody constant region can be replaced by a different residue. See, for example, US Patent 5,624,821, US Patent 6,194,551, Application No. WO 9958572; and Angal, et al., Mol. Immunol. 30: 105-08 (1993). Amino acid modification includes amino acid deletions, additions, and substitutions. In some cases, such changes are made to reduce unwanted activities, eg complement dependent cytotoxicity. Antibodies are often labeled by binding, covalently or non-covalently, a substance that provides a detectable signal. A wide variety of labeling and conjugation techniques are known and are reported extensively in the scientific and patent literature. These antibodies can be selected for binding to the normal or defective 191P4D12 molecule. See, for example, Antibody Engineering: A Practical Approach (Oxford University Press, 1996). Suitable antibodies with the desired biological activities can be identified using the following in vitro assays, including, but not limited to: proliferation, migration, adhesion, growth on soft agar, angiogenesis, cell-cell communication, apoptosis, transport, transduction of signal, and the following in vivo assays, such as tumor growth inhibition. The antibodies provided herein may also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can be selected for their ability to bind specific antigen without inhibiting receptor binding or biological activity of the antigen. As neutralizing antibodies, the antibodies can be useful in competitive binding assays. They can also be used to quantify 191P4D12 or its receiver.
The term "antigen-binding portion" or "antibody fragment" of an antibody (or simply "antibody portion"), for use herein, refers to one or more fragments of a 191P4D12 antibody that guard the ability to specifically bind an antigen (eg, 191P4D12 and 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 encompassed by the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain ; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that allows them to be produced as a single protein chain in the which VL and VH regions pair to form monovalent molecules (known as 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 encompassed by the term "antigen binding portion" of an antibody. These antibody fragments are obtained using standard techniques known to those of skill in the art, and fragments are selected for their usefulness in the same way as intact antibodies.
[00114] For use in the present invention, any form of the "antigen" can be used to generate an antibody that is specific for 191P4D12. Thus, the generating antigen can be a single epitope, multiple epitopes, or the entire protein alone or in combination with one or more immunogenicity enhancing agents known in the art. The generating antigen can be an isolated full-length protein, a cell surface protein (eg immunization with cells transfected with at least a portion of the antigen), or a soluble protein (eg immunization with only the extracellular domain portion of the protein). The antigen can be produced in a genetically modified cell. The DNA encoding the antigen can be genomic or non-genomic (eg, 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, as appropriate, to constitute an immunogenic epitope of the antigen of interest. Any suitable genetic vectors for transforming the cells of interest can be employed, including but not limited to adenoviral vectors, plasmids, and non-viral vectors such as cationic lipids. In one embodiment, the antibody of the methods and compositions of the present invention specifically binds to at least a portion of the 191P4D12 extracellular domain of interest.
The antibodies or antigen-binding fragments thereof provided herein may be conjugated to a "bioactive agent." As used herein, the term "bioactive agent" refers to any synthetic or naturally occurring compound that binds to antigen and/or enhances or mediates a desired biological effect to enhance cell-killing toxins. In one embodiment, the binding fragments useful in the present invention are biologically active fragments. As used herein, the term "biologically active" refers to an antibody or antibody fragment that is capable of binding to the desired antigenic epitope and directly or indirectly exerting a biological effect. Direct effects include, but are not limited to, modulation, stimulation, and/or inhibition of a growth signal, modulation, stimulation, and/or inhibition of an anti-apoptotic signal, modulation, stimulation, and/or inhibition of an apoptotic or necrotic signal, modulating, stimulating, and/or inhibiting the ADCC cascade, and modulating, stimulating, and/or inhibiting the CDC cascade.
"Bispecific" antibodies are also useful in the present methods and compositions. As used herein, the term "bispecific antibody" refers to an antibody, typically a monoclonal antibody, that has binding specificities for at least two different antigenic epitopes. In one embodiment, the epitopes are from the same antigen. In another modality, the epitopes are from two different antigens. Methods for making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using the co-expression of two immunoglobulin heavy chain/light chain pairs. See, for example, Milstein et al., Nature 305:537-39 (1983). Alternatively, bispecific antibodies can be prepared using chemical linkage. See, for example, Brennan, et al., Science 229:81 (1985). Bispecific antibodies include bispecific antibody fragments. See, for example, Hollinger, et al., Proc. Natl. Academic Sci. U.S.A. 90:6444-48 (1993), Gruber, et al., J. Immunol. 152:5368 (1994).
The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a specific class or subclass of antibody , while the remainder of the chain(s) is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibody, as well as fragments of these antibodies, as long as they specifically bind to the antigen target and/or have the desired biological activity (US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
[00118] The term "chemotherapeutic agent" refers to all chemical compounds that are effective in inhibiting tumor growth. Some non-limiting examples of chemotherapeutic agents include alkylating agents; for example nitrogen mustards, ethyleneimine compounds and alkyl sulfonates; antimetabolites, for example folic acid, purine or pyrimidine antagonists; mitotic inhibitors, for example anti-tubulin agents like vinca alkaloids, auristatins and podophyllotoxin derivatives; cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication, for example, DNA minor groove ligands; and growth factor receptor antagonists. In addition, chemotherapeutic agents include cytotoxic agents (as defined herein), antibodies, biological molecules, and small molecules.
[00119] The term "compound" refers to and encompasses the chemical compound itself as well as, whether explicitly stated or not, and unless the context makes it clear that the following are to be excluded: amorphous and crystalline forms of the compound, including forms polymorphic, where these forms can be part of a mixture or can be in isolation; free acid and free base forms of the compound, which are typically the forms shown in the structures provided herein; compound isomers, which refers to optical isomers, and tautomeric isomers, where optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and optical isomers include isolated optical isomers as well as mixtures of non-optical isomers and mixtures of optical isomers including racemic; where an isomer can be in isolated form or in admixture with one or more other isomers; isotopes of the compound, including compounds containing deuterium and tritium, and including compounds containing radioisotopes, including therapeutically and diagnostically effective radioisotopes; multimeric forms of the compound, including dimeric, trimeric, etc. forms; salts of the compound, preferably pharmaceutically acceptable salts, including acid addition salts and base addition salts, including salts having organic counterions and inorganic counterions, and including zwitterionic forms, where if a compound is associated with two or more counterions , the two or more counterions can be the same different; and solvates of the compound, including hemisolvates, monosolvates, dissovates, etc., including organic solvates and inorganic solvates, said inorganic solvates including hydrates; where if a compound is associated with two or more solvent molecules, the two or more solvent molecules can be the same or different. In some cases, reference made herein to a compound of the invention will include an explicit reference to one of the above forms, for example, salts and/or solvates; however, this reference is for emphasis only and should not be construed as exclusive to other of the above forms as identified above.
[00120] As used herein, the term "conservative substitution" refers to amino acid substitutions that are known to those of skill in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson, et al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin/Cummings Pub. Co., p. 224 (4th edition, 1987)). Such exemplary substitutions are preferably made in accordance with those shown in Table II and Table(s) III(a-b). For example, such changes include replacing any one of isoleucine (I), valine (V), and leucine (L) with any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions may also be considered conservative, depending on the particular amino acid's environment and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can often be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can often be exchanged for leucine and isoleucine, and sometimes for valine. Lysine (K) and arginine (R) are often interchangeable at sites where the significant characteristic of the amino acid residue is its charge and the different pK's of these two amino acid residues are not significant. Still other alterations may be considered "conservative" in particular environments (see, for example, Table III(a) of the present invention; pages 13 to 15 "Biochemistry" 2nd ED. Lubert Stryer ed (Stanford University); Henikoff et al. ., PNAS 1992 Vol 89 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.
[00121] The term "cytotoxic agent" refers to a substance that inhibits or prevents cell expression activity, cell function and/or causes cell destruction. The 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, auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), auromycins, maytansinoids, ricin, ricin A chain, combrestatin, duocarmycins, dolastatins, doxorubicin, daunoisrubicin, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, That of modecin, alpha-sarcin, gelonin, mitogelin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, and glucocorticoids and other chemotherapeutic agents, as well as radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32 and radioactive isotopes of Lu including Lu177. Antibodies can also be conjugated to an anticancer prodrug activating enzyme capable of converting the prodrug into its active form.
[00122] As used herein, the term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are more fully described, for example, in EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Academic Sci. USA 90:6444-48 (1993).
[00123] The term "deplete," in the context of the effect of a 191P4D12 binding agent on cells expressing 191P4D12, refers to a reduction in the number or elimination of cells expressing 191P4D12.
[00124] The term "gene product" is used in the present invention to indicate a peptide/protein or mRNA. For example, a "gene product of the invention" is sometimes referred to here as "cancer amino acid sequence", "cancer protein", "protein from a cancer mentioned in table I", a "cancer mRNA", "mRNA of a cancer mentioned in table I", etc. In one embodiment, the cancer protein is encoded by a nucleic acid of Figure 1. The cancer protein can be a fragment, or alternatively, it can be the full length protein encoded by the nucleic acids of Figure 1. In one embodiment, a cancer amino acid sequence is used to determine sequence identity or similarity. In another embodiment, the sequences are naturally occurring allelic variants of a protein encoded by a nucleic acid of Figure 1. In another embodiment, the sequences are sequence variants, as further described herein.
"Heteroconjugate" antibodies are useful in the present methods and compositions. As used herein, the term "heteroconjugate antibody" refers to two covalently joined antibodies. Such antibodies can be prepared using known methods in synthetic protein chemistry, including using crosslinking agents. See, for example, US patent 4,676,980.
[00126] The term "homologous" refers to a molecule that has homology to another molecule, for example, by having sequences of chemical residues that are the same or similar in corresponding positions.
[00127] In one embodiment, the antibody provided herein is a "human antibody." As used herein, the term "human antibody" refers to an antibody in which essentially the entire sequences of the light chain and heavy chain sequences, including the complementarity determining regions (CDRs), are from human genes. In one embodiment, human monoclonal antibodies are prepared by the trioma technique, the human B cell technique (see, for example, Kozbor, et al., Immunol. Today 4: 72 (1983), EBV transformation technique (see, , for example, Cole et al. Monoclonal Antibodies And Cancer Therapy 77-96 (1985)), or using phage display (see, for example, Marks et al., J. Mol. Biol. 222:581 (1991)) In a specific embodiment, the human antibody is generated in a transgenic mouse. Procedures for making such antibodies partially to fully human are known in the art and any such procedures can be used. Fully human antibodies are produced in a transgenic mouse engineered to express human heavy and light chain antibody genes. An exemplary description of the preparation of transgenic mice that produce human antibodies is found in p ed. No. WO 02/43478 and in US patent 6,657,103 (Abgenix) and its progeny. B cells from transgenic mice that produce the desired antibody can then be fused to make hybridoma cell lines for continuous antibody production. See, for example, US 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).
[00128] As used herein, the term "humanized antibody" refers to those forms of antibodies which contain sequences from non-human (eg murine) antibodies as well as human antibodies. Such antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are of a sequence of human immunoglobulin. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, for example, Cabilly US Patent 4,816,567; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; and Antibody Engineering: A Practical Approach (Oxford University Press 1996).
[00129] The terms "inhibit" or "inhibition of" for use in the present invention mean to reduce by a measurable amount, or even to completely prevent.
[00130] The phrases "isolated" or "biologically pure" refer to material that is substantially or essentially free of components that normally accompany the material as it is found in its native state. Thus, the isolated peptides according to the invention preferably do not contain materials normally associated with the peptides in their in situ environment. For example, a polynucleotide is said to be "isolated" when it is substantially separated from contaminating polynucleotides that correspond or are complementary to genes other than the 191P4D12 genes or that encode polypeptides other than the 191P4D12 gene product or fragments thereof. One skilled in the art can readily employ nucleic acid isolation procedures to obtain an isolated 191P4D12 polynucleotide. A protein is called "isolated," for example, when physical, mechanical, or chemical methods are employed to remove 191P4D12 proteins from cellular constituents that are normally associated with the protein. One skilled in the art can easily employ standard purification methods to obtain an isolated 191P4D12 protein. Alternatively, an isolated protein can be prepared by chemical means.
Suitable "markers" include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such markers include US Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. In addition, the antibodies provided herein may be useful as the antigen-binding component of fluorobodies. See for example, Zeytun et al., Nat. Biotechnol. 21:1473-79 (2003).
[00132] 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.
[00133] The terms "metastatic cancer" and "metastatic disease" mean cancers that have spread to regional lymph nodes or to distant locations, and are intended to include stage D disease according to the AUA system and stage TxNxM+ according to the system TNM.
[00134] The term "modulator" or "test compound" or "drug candidate" or grammatical equivalents, for use in the present invention, describes any molecule, for example, protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc. ., to be tested for the ability to directly or indirectly alter the cancer phenotype or the expression of a cancer sequence, for example, nucleic acid or protein sequences, or effects of cancer sequences (for example, signaling, gene expression , protein interaction, etc.) In one aspect, a modulator will neutralize the effect of a cancer protein of the invention. By "neutralizing" it is meant that an activity of a protein is inhibited or blocked, along with the consequent effect on the cell. In another aspect, a modulator will neutralize the effect of a gene, and its corresponding protein, of the invention by normalizing the levels of said protein. In preferred embodiments, the modulators alter expression profiles, or expression profiles nucleic acids or proteins provided herein, or downstream effector pathways. In one embodiment, the modulator suppresses a cancer phenotype, for example, for a normal tissue fingerprint. In another modality, a modulator induced a cancer phenotype. In general, a plurality of test mixtures are run in parallel with different concentrations of agent to obtain a differential response at the various concentrations. Typically, one of these concentrations serves as a negative control, that is, at zero concentration or below the detection level.
[00135] Modulators, drug candidates, or test compounds span numerous chemical classes, although typically they are organic molecules, preferably small organic compounds that have a molecular weight of more than 100 and less than about 2,500 Daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least one group amine, carbonyl, hydroxyl or carboxyl, preferably at least two of the functional chemical groups. Candidate agents often comprise cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Modulators also comprise biomolecules such as peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogues or combinations thereof. Peptides are particularly preferred. One class of modulators are peptides, for example, from about five to about 35 amino acids, with from about five to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. Preferably, the cancer modulating protein is soluble, includes a non-transmembrane region, and/or has an N-terminal Cys to aid solubility. In one embodiment, the C-terminus of the fragment is maintained as a free acid and the N-terminus is a free amine to aid in coupling, ie, to cysteine. In one embodiment, a cancer protein of the invention is conjugated to an immunogenic agent as discussed herein. In one embodiment, the cancer protein is conjugated to BSA. Peptides of the invention, for example, of preferred lengths, can be linked together or to other amino acids to create a longer peptide/protein. Modulator peptides can be naturally occurring protein digestion products as described above, random peptides, or "induced" random peptides. In a preferred embodiment, the peptide/protein-based modulators are antibodies, and fragments thereof, as defined herein.
[00136] The term "monoclonal antibody", for use in the present invention, refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies that make up the population are identical with the exception of possible mutations naturally occurring that may be present in smaller amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a variety of antibodies directed against (or specific for) different epitopes. In one embodiment, the polyclonal antibody contains a plurality of monoclonal antibodies with different epitope specificities, affinities, or avidity within a single antigen that contains multiple antigenic epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be produced by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or can be produced by recombinant DNA methods (see , for example, US Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991), for example. These monoclonal antibodies will generally bind with at least a Kd of about 1 µM, more generally at least about 300 nM, typically at least about 30 nM, preferably at least about 10 nM, more preferably at least about of 3 nM or better, usually determined by ELISA.
[00137] A "pharmaceutical excipient" comprises a material, such as an adjuvant, a vehicle, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like.
[00138] "Pharmaceutically acceptable" refers to a non-toxic, inert composition that is physiologically compatible with humans or other mammals.
[00139] The term "polynucleotide" means a polymeric form of nucleotides of at least 10 bases or base pairs in length, ribonucleotides or deoxynucleotides or a modified form of any type of nucleotide, and is intended to include single and double stranded forms of DNA and/or RNA. In the art, this term is often used interchangeably with "oligonucleotide". A polynucleotide may comprise a nucleotide sequence shown herein, wherein the thymidine (T), as shown, for example, in Figure 1 may also be uracil (U); this definition refers to the differences between the chemical structures of DNA and RNA, in particular the observation that one of the four main bases in RNA is uracil (U) rather than thymidine (T).
[00140] The term "polypeptide" means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the descriptive report, standard three-letter or one-letter designations for amino acids are used. In the art, this term is often used interchangeably with "peptide" or "protein".
[00141] A "recombinant" DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.
As used herein, the term "single-chain Fv" or "scFv" or "single-chain" antibody refers to antibody fragments comprising the VH and VL domains of the antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide additionally comprises a polypeptide linker between VH and VL domains that allows the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology Of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
As used herein, the terms "specific", "specifically binds" and "specifically binds" refer to the selective binding of the antibody to the target antigen epitope. Antibodies can be tested for binding specificity by comparing binding to an appropriate antigen with binding to an irrelevant antigen or mixture of antigens under a given set of conditions. If the antibody binds to the appropriate antigen at least 2, 5, 7, and preferably 10 times more than the irrelevant antigen or mixture of antigens, then it is considered specific. In one embodiment, a specific antibody is one that binds only to the 191P4D12 antigen but does not bind the irrelevant antigen. In another embodiment, a specific antibody is one that binds to the human 191P4D12 antigen but does not bind to the non-human 191P4D12 antigen with 70%, 75%, 80%, 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid homology to the 191P4D12 antigen. In another embodiment, a specific antibody is one that binds to the human 191P4D12 antigen and binds to the murine 191P4D12 antigen, but with a higher degree of binding to the human antigen. In another embodiment, a specific antibody is one that binds to the human 191P4D12 antigen and binds to the primate 191P4D12 antigen, but with a higher degree of binding to the human antigen. In another embodiment, the specific antibody binds to the human 191P4D12 antigen and any non-human 191P4D12 antigen, but with a greater degree of binding to the human antigen or any combination thereof.
[00144] For use in the present invention "treat" or "therapeutic" and grammatically related terms refer to any amelioration of any consequence of a disease, such as prolonged survival, less morbidity, and/or a reduction in the side effects that are the by-products of an alternative therapeutic modality; as is readily understood in the art, complete eradication of the disease is preferred, but not a requirement for an act of treatment.
[00145] The term "variant" refers to a molecule that exhibits a variation of a described type or norm, such as a protein that has one or more different amino acid residues at the corresponding position(s) of a specifically described protein (for example, the 191P4D12 protein shown in Figure 1.) An analog is an example of a variant protein. Splice isoforms or single nucleotide polymorphisms (SNPs) are additional examples of variants.
The "191P4D12 proteins" and/or "191P4D12-related proteins" of the invention include those specifically identified in the present invention (see, Figure 1), as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated / generated and characterized without undue experimentation according to the methods described herein or readily available in the art. Fusion proteins that combine parts of different 191P4D12 proteins or fragments thereof, as well as fusion proteins of a 191P4D12 protein and a heterologous polypeptide are also included. Such 191P4D12 proteins are collectively called 191P4D12-related proteins, the proteins of the invention, or 191P4D12. The term "191P4D12-related protein" refers to a polypeptide fragment or a 191P4D12 protein sequence having 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; or at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 330, 335, 339 or more amino acids. 191P4D12 Antibodies
Another aspect of the invention provides antibodies that bind to 191P4D12-related proteins (see Figure 1). In one embodiment, the antibody that binds to 191P4D12-related proteins is an antibody that specifically binds to the 191P4D12 protein comprising the amino acid sequence of SEQ ID NO.: 2. The antibody that specifically binds to the 191P4D12 protein comprising the sequence of amino acids of SEQ ID NO.: 2 include antibodies that can bind to other 191P4D12-related proteins. For example, antibodies that bind to 191P4D12 protein that comprise the amino acid sequence of SEQ ID NO.:2 can bind to 191P4D12-related proteins, such as 191P4D12 variants and their homologues or analogs.
The 191P4D12 antibodies of the invention are particularly useful in cancer (see, for example, table I) prognostic assays, imaging, and therapeutic methodologies. Similarly, such antibodies are useful in the treatment, and/or prognosis of colon cancer and other cancers, as 191P4D12 is also expressed or overexpressed in these other cancers. In addition, intracellularly expressed antibodies (eg, single-chain antibodies) are therapeutically useful in treating cancers in which 191P4D12 expression is involved, such as advanced or metastatic colon cancers or other advanced or metastatic cancers.
[00149] Various methods for preparing antibodies, specifically, monoclonal antibodies, are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host using a 191P4D12-related protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane ( 1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, 191P4D12 fusion proteins can also be used, such as a 191P4D12 GST fusion protein. In a particular embodiment, a GST fusion protein comprising all or nearly all of the amino acid sequence of Figure 1 is produced and then used as an immunogen to generate the appropriate antibodies. In another embodiment, a 191P4D12-related protein is synthesized and used as an immunogen.
In addition, naked DNA immunization methods known in the art are used (with or without purified 191P4D12-related protein or cells expressing 191P4D12) to generate an immune response to the encoded immunogen (for a review, see Donnelly et al. ., 1997, Ann. Rev. Immunol. 15: 617-648).
[00151] The amino acid sequence of a 191P4D12 protein as shown in figure 1 can be analyzed to select specific regions of the 191P4D12 protein to generate antibodies. For example, hydrophobic capacity and hydrophilic capacity analyzes of a 191P4D12 amino acid sequence are used to identify the hydrophilic regions in the 191P4D12 structure. Regions of a 191P4D12 protein that show immunogenic structure, as well as other regions and domains, can be easily identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz analysis or Jameson-Wolf. Hydrophilic capacity profiles can be generated using the method of Hopp, T.P. and Woods, K.R., 1981, Proc. Natl. Academic Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated using the method of Kyte, J. and Doolittle, R.F., 1982, J. Mol. Biol. 157:105-132. Percent (%) accessible waste profiles can be generated using the method of Janin J., 1979, Nature 277:491-492. Average flexibility profiles can be generated using the method of Bhaskaran R., Ponnuswamy P.K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-loop profiles can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289294. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Preferred methods for generating 191P4D12 antibodies are further illustrated by means of the examples provided herein. Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art. Methods for making immunogenic conjugates of a protein with a carrier such as BSA, KLH or other carrier protein are also well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents is used; in other cases, binding reagents such as those supplied by Pierce Chemical Co., Rockford, IL, are effective. Administration of a 191P4D12 immunogen is often conducted by injection over a suitable period of time and with the use of a suitable adjuvant, as is understood in the art. During the immunization schedule, antibody titrations can be taken to determine the adequacy of antibody formation.
191P4D12 monoclonal antibodies can be produced by various means well known in the art. For example, immortalized cell lines that secrete a desired monoclonal antibody are prepared using standard hybridoma technology of Kohler and Milstein or modifications that immortalize antibody-producing B cells, as is well known. Immortalized cell lines that secrete the desired antibodies are selected by immunoassay, in which the antigen is a 191P4D12-related protein. When the appropriate immortalized cell culture is identified, cells can be expanded and antibodies produced from in vitro cultures or from ascites fluid.
Antibodies or fragments of the invention can also be produced by recombinant means. Regions that specifically bind to the desired regions of a 191P4D12 protein can also be produced in the context of chimeric antibodies or grafted with a complementarity determining region (CDR) of multiple species origin. Humanized or human 191P4D12 antibodies can also be produced, and are preferred for use in therapeutic contexts. Methods for humanizing murine antibodies and other non-human antibodies by substituting one or more of the non-human antibody CDRs with corresponding human antibody sequences 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. Academic Sci. USA 89:4285 and Sims et al., 1993, J. Immunol. 151: 2296.
In a preferred embodiment, the antibodies of the present invention comprise fully human 191P4D12 antibodies (MAbs 191P4D12). Various methods in the art provide a means to produce fully human 191P4D12 MAbs. For example, a preferred modality provides techniques that use inactivated transgenic mice to produce antibodies, engineered with human heavy and light chain loci called the Xenomouse (Amgen Fremont, Inc.). An exemplary description of the preparation of transgenic mice that produce human antibodies can be found in U.S. 6.657,103. See also US Patent Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; and Mendez. et. al. Nature Genetics, 15: 146-156 (1998); Kellerman, S.A. & Green, L.L., Curr. Opinion Biotechnol 13, 593-597 (2002).
[00155] In addition, human antibodies of the invention can be generated using mouse HuMAb (Medarex, Inc.) that contain human immunoglobulin gene miniloci encoding unrearranged human heavy chain (mu and gamma) and chain immunoglobulin sequences kappa light, along with targeted mutations that inactivate endogenous mu and kappa chain loci (see, for example, Lonberg, et al. (1994) Nature 368(6474): 856-859).
In another embodiment, the fully human antibodies of the invention can be created using a mouse that carries human immunoglobulin sequences on transgenes and transchromosomes, such as a mouse that carries a human heavy chain transgene and a chain transchromosome light human. Such mice, called in the present invention as "mouse KM", are described Tomizuka et al. (2000) Proc. Natl. Academic Sci. USA 97:722-727 and PCT publication WO 02/43478 to Tomizuka, et al.
The human monoclonal antibodies of the invention can also be prepared using phage display methods for selecting human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are established in the art. See for example: US Patent Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; US Patent Nos. 5,427,908 and 5,580,717 to Dower et al.; US Patent Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and US Patent Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 for Griffiths et al.
The human monoclonal antibodies of the invention can also be prepared using SCID mice in which human immune cells have been reconstituted so that a human antibody response can be generated upon immunization. Such mice are described, for example, in US Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.
In a preferred embodiment, the MAbs 191P4D12 of the invention comprise heavy and light chain variable regions of an antibody designated Ha22-2(2,4)6.1 produced by a hybridoma deposited under accession number of the American Type Culture Collection ( ATCC): PTA-11267 (see, Figure 3), or heavy and light variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the heavy and light chain variable regions of Ha22-2(2,4)6.1, and wherein the antibodies retain the desired functional properties of the 191P4D12 MAbs of the invention. The heavy chain variable region of Ha22-2(2,4)6.1 consists of the amino acid sequence from the 20th E residue to the 136th S residue of SEQ ID NO.7, and the light chain variable region of Ha22-2(2,4)6.1 consists of the amino acid sequence from the 23rd residue of D to the 130th residue of R of SEQ ID NO:8. As the constant region of the antibody of the invention, any subclass of the constant region can be chosen. In one embodiment, one can use the human IgG1 constant region as the heavy chain constant region and the human Ig kappa constant region as the light chain constant region.
[00160] For example, the invention features an isolated monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein:
The heavy chain variable region immunogen comprises an amino acid sequence that is at least 80% homologous to the heavy chain variable region amino acid sequence shown in Figure 3; and
(a) the light chain variable region comprises an amino acid sequence that is at least 80% homologous to the light chain variable region amino acid sequence shown in Figure 3.
[00163] 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.
[00164] In another embodiment, the invention features an isolated monoclonal antibody, or antigen-binding portion thereof, comprising a humanized heavy chain variable region and a humanized light chain variable region, wherein:
(a) the heavy chain variable region comprises complementarity determining regions (CDRs) having the amino acid sequences of the heavy chain variable region CDRs shown in Figure 3;
[00166] (b) the light chain variable region comprises CDRs having the amino acid sequences of the light chain variable region CDRs shown in Figure 3.
Engineered antibodies of the invention include those in which modifications have been made to framework residues within the VH and/or VL (e.g., to optimize the properties of the antibody). Typically, these structural modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, somatic mutations can be "backmutated" to the germline sequence, eg, by site-directed mutagenesis or PCR-mediated mutagenesis (eg, "backmutated" from leucine to methionine). Such "remutated" antibodies are also encompassed by the invention.
Another type of structural modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also called "deimmunization" and is described in more detail in U.S. Patent Publication No. 2003/0153043 by Carr et al.
In addition or alternatively to modifications produced within framework regions or CDRs, antibodies of the 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, binding complement, Fc receptor binding and/or antigen-dependent cellular cytotoxicity. Furthermore, a MAb 191P4D12 of the invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or can be modified to alter its glycosylation, again to alter one or more functional properties of the MAb. Each of these modalities is described in more detail below.
[00170] In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is changed, e.g., increased or decreased. This approach is further described in US patent 5,677,425 by Bodmer et al. The number of cysteine residues in the CH1 hinge region is altered, for example, to facilitate heavy and light chain assembly or to increase or decrease the stability of MAb 191P4D12.
In another embodiment, the Fc hinge region of an antibody is mutated to reduce the biological half-life of MAb 191P4D12. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment so that the antibody has binding to Staphylococcal protein A (SpA) defective with respect to binding to SpA of the Fc domain -native hinge. This approach is described in more detail in US patent 6,165,745 by Ward et al.
[00172] In another embodiment, MAb 191P4D12 is modified to increase its biological half-life. Several approaches are possible. For example, mutations can be introduced, as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to increase the biological half-life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in US patents 5,869,046 and 6,121,022 by Presta et al.
In still other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of MAb 191P4D12. For example, one or more amino acids selected from specific amino acid residues can be replaced with a different amino acid residue so that the antibody has an altered affinity for an effector ligand, but retains the antigen-binding capacity of the source antibody. The effector ligand for which the affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in US Patent Nos. 5,624,821 and 5,648,260, both to Winter et al.
The reactivity of 191P4D12 antibodies with a 191P4D12-related protein can be established by a number of well-known means, including Western blot, immunoprecipitation, ELISA, and FACS analysis using, as appropriate, 191P4D12-related proteins, 191P4D12 expressing cells or your extracts. A 191P4D12 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable labels include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelating agent or an enzyme. Additionally, bispecific antibodies specific for two or more epitopes of 191P4D12 are generated using methods well known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (for example, Wolff et al., Cancer Res. 53: 2560-2565).
In yet another preferred embodiment, the MAb 191P4D12 of the invention is an antibody comprising the heavy and light chain of an antibody designated Ha22-2(2,4)6.1. The heavy chain of Ha22-2(2,4)6.1 consists of the amino acid sequence from the 20th residue of E to the 466th residue of K of SEQ ID NO:7 and the light chain of Ha22-2(2, 4)6.1 is the amino acid sequence from the 23rd D residue to the 236th C residue of the sequence of SEQ ID NO:8. The sequence of which is shown in Figure 2 and Figure 3. In a preferred embodiment, Ha22-2(2,4)6.1 is conjugated to a cytotoxic agent.
The hybridoma producing the antibody designated Ha22-2(2,4)6.1 was shipped (via Federal Express) to the American Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108 on August 18, 2010 and the access number PTA-11267 was assigned.
[00177] Antibody-drug conjugates in general
[00178] In another aspect, the invention provides antibody-drug conjugates (ADCs), comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (ie, a radioconjugate). In another aspect, the invention further provides methods of using ADCs. In one aspect, an ADC comprises any of the 191P4D12 MAbs of the present invention covalently linked to a cytotoxic agent or a detectable agent.
[00179] The use of antibody-drug conjugates for the local application of cytotoxic or cytostatic agents, that is, drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu -Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151-172; US patent 4,975,278) allows targeted application of the drug portion to tumors, and intracellular accumulation there, where systemic administration of these pharmaceutical agents unconjugated can result in unacceptable levels of toxicity to normal cells as well as tumor cells that one seeks to eliminate (Baldwin et al., (1986) Lancet pp. (Mar. 15, 1986):603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (ed.s), pp. 475506). Maximum efficacy with minimal toxicity is thus sought. Both polyclonal antibodies and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra). Toxins used in antibody-toxin conjugates include bacterial toxins 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), maytansinoides (EP 1391213; Liu et al., (1996) ) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:33363342). Toxins can express their cytotoxic and cytostatic effects by mechanisms that include tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.
[00180] Examples of antibody-drug conjugates are, ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) which is an antibody-radioisotope conjugate composed of a murine IgG1 kappa monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes and the 111In or 90Y radioisotope bound by a thiourea ligand 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).
[00181] Additionally, MYLOTARGTM (gemtuzumab ozogamycin, Wyeth Pharmaceuticals), an antibody-drug antibody conjugate composed of a hu CD33 antibody linked to calicheamicin, was approved in 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future ( 2000) 25(7):686; US Patent Nos. 4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001).
In addition, Cantuzumab mertansine (Immunogen, Inc.), an antibody-drug conjugate composed of the antibody huC242 linked through the SPP disulfide linker to the maytansinoid drug moiety, DM1, is progressing to phase II trials for the treatment of cancers that express CanAg, such as colon, pancreatic, gastric, and others.
[00183] Additionally, MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), an antibody-drug conjugate composed of the monoclonal antibody anti-prostate-specific membrane antigen (PSMA) linked to the maytansinoid drug moiety, DM1, is under development for the potential treatment of prostate tumors.
[00184] Finally, the peptides of auristatin, auristatin E (AE) and monomethylauristatin (MMAE), synthetic analogues of dolastatin, were conjugated to the chimeric monoclonal antibodies cBR96 (specific for Lewis Y in carcinomas) and cAC10 (specific for CD30 in hematologic malignancies ) (Doronina et al (2003) Nature Biotechnology 21(7):778-784) and are under therapeutic development.
[00185] Additionally, chemotherapeutic agents useful in the generation of ADCs are described in the present invention. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modecin A chain , alpha-sarcin, Aleurites fordii proteins, diantin proteins, American Phytolaca proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcina, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin , enomicin, and trichothecenes. See, for example, WO 93/21232 published October 28, 1993. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re. Antibody and cytotoxic agent conjugates are produced using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazonium-benzoyl) )-ethylenediamine), diisocyanates (such as 2,6-toluene diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al (1987) Science, 238:1098. Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to antibody (WO94/11026).
[00186] Conjugates of an antibody and one or more small molecule toxins, such as a calicheamicin, maytansinoids, dolastatins, auristatins, a trichothecene, and CC1065, and derivatives of these toxins that have toxin activity, are also contemplated in the present invention . III(A). Maytansinoids
Maytansine compounds suitable for use as maytansinoid drug moieties are well known in the art, and can be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al (2002) PNAS 99:7968-7973), or maytansinol and maytansinol analogues prepared synthetically according to known methods.
Exemplary maytansinoid drug moieties include those having a modified aromatic ring, such as: C-19-dechlorine (US 4256746) (prepared by lithium aluminum hydride reduction of ansamitocin P2); C-20-hydroxy (or C-20-demethyl) +/- C-19-dechloro (US Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (-OCOR), +/-dechlor (US Patent No. 4,294,757) (prepared by acylation using acyl chlorides) and those having modifications at other positions.
[00189] Exemplary maytansinoid drug moieties also include those having modifications such as: C-9-SH (US 4,424,219) (prepared by reacting maytansinol with H2S or P2S5); C-14-alkoxymethyl (demethoxy/CH2 OR)(US 4331598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (US 4450254) (prepared from Nocardia); C-15-hydroxy/acyloxy (US 4,364,866) (prepared by conversion of maytansinol by Streptomyces); C-15-methoxy (US Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudlflora); C-18-N-demethyl (US Pat. Nos. 4,362,663 and 4,322,348) (prepared by the demethylation of maytansinol by Streptomyces); and 4,5-deoxy (US 4,371,533) (prepared by titanium trichloride/LAH reduction of maytansinol).
[00190] ADCs containing maytansinoids, methods for manufacturing them, and their therapeutic use are disclosed, for example, in US patents No. 5,208,020; 5,416,064; 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. Academic Sci. USA 93:8618-8623 (1996) described ADCs comprising a maytansinoid designated DM1 linked to monoclonal antibody C242 directed against human colorectal cancer. The conjugate was observed to be highly cytotoxic to cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay. Chari et al., Cancer Research 52:127-131 (1992) describe ADCs in which a maytansinoid has been conjugated via a disulfide linker to the murine antibody A7 that binds to an antigen on human colon cancer cell lines, or to another murine monoclonal antibody TA.1 that binds to the HER-2/neu oncogene. The cytotoxicity of TA.1-maytansinoid conjugate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3 x 105 HER-2 surface antigens per cell. The drug conjugate achieved a similar degree of cytotoxicity as the free maytansinoid drug, which can be increased by increasing the number of maytansinoid molecules per antibody molecule. The A7-maytansinoid conjugate showed low systemic cytotoxicity in mice. III(B). Auristatins and Dolastatins
In some embodiments, the ADC comprises an antibody of the invention conjugated to dolastatins or peptide analogues of dolastatin and derivatives, auristatins (US Patent Nos. 5,635,483; 5,780,588). Dolastatins and auristatins 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 fungicide (Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). The drug portion of dolastatin or auristatin can be attached to the antibody via the N-terminus (amino) or the C-terminus (carboxyl) of the peptide drug portion (WO 02/088172).
Exemplary auristatin modalities include the DE and DF drug moieties of monomethylauristatin linked to the N-terminus, disclosed in "Senter et al, Proceedings of the American Association for Cancer Research, Volume 45, abstract number 623, filed 28 of March 2004 and described in US Patent Publication No. 2005/0238649, the description of which is expressly incorporated in its entirety by reference.
[00193] An exemplary modality of auristatin is MMAE (where the wavy line indicates covalent binding to a linker (L) of an antibody-drug conjugate).

[00194] Another exemplary modality of auristatin is MMAF, in which the wavy line indicates covalent binding to a linker (L) of an antibody-drug conjugate (US 2005/0238649):

[00195] Additional exemplary embodiments comprising MMAE or MMAF and various linker components (described further in the present invention) have the following structures and abbreviations (where Ab means antibody, S is an antibody sulfur, and p is 1 to about 8) :

Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schroder and K. Lübke, "The Peptides", volume 1, pp 76-136, 1965, Academic Press) which is well known in the field of peptide chemistry. Auristatin/dolastatin drug moieties can be prepared according to the methods of: 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, G.R., et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol 21(7):778-784. III(C). calicheamicin
In other embodiments, the ADC comprises an antibody of the invention conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics is capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, 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. 5,877,296 (all to American Cyanamid Company). Structural analogues of calicheamicin that can be used include, but are not limited to YI1,αZ α3I, N-acetyl-YiI, PSAG and θIi (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al. , Cancer Research 58:2925-2928 (1998) and the aforementioned US patents for American Cyanamid). Another anti-tumor drug to which the antibody can be conjugated is QFA which is an antifolate. Both calicheamicin and FFQ have intracellular sites of action and do not easily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody-mediated internalization greatly enhances their cytotoxic effects. III(D). Other cytotoxic agents
Other antitumor agents that can be conjugated to the antibodies of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents collectively known as the LL-E33288 complex described in US patents 5,053,394, 5,770,710 as well as the esperamycins (US patent 5,877,296).
Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active diphtheria toxin fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, chain That of modecin, alpha-sarcin, Aleurites fordii proteins, diantin proteins, American Phytolaca proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcina, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and the trichothecenes. See, for example, WO 93/21232 published October 28, 1993.
The present invention further contemplates an ADC formed between an antibody and a compound with nucleolytic activity (for example, a ribonuclease or a DNA endonuclease, such as a deoxyribonuclease; DNase).
[00201] For selective tumor destruction, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the conjugate is used for detection, it can comprise a radioactive atom for scintigraphic studies, eg tc99m or I123, or a spin tag for nuclear magnetic resonance (NMR) imaging (also known as resonance imaging magnetic, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
Radiolabels or other labels can be incorporated into the conjugate in known ways. For example, the peptide can be biosynthesized or can be synthesized by chemical amino acid synthesis using suitable amino acid precursors, involving, for example, fluorine-19 in place of hydrogen. Tags such as tc99m or I123, .Re186, Re188 and In111 can be linked through a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80:49-57 can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail .
[00203] Antibody-drug conjugated compounds that bind to 191P4D12
[00204] The present invention presents, among others, conjugated antibody-drug compounds for targeted application of drugs. The inventors made the discovery that the antibody-drug conjugated compounds have potent cytotoxic and/or cytostatic activity against cells expressing 191P4D12. Antibody-drug conjugate compounds comprise an antibody moiety covalently linked to at least one drug moiety. The drug units can be covalently linked directly or through a linker unit (LU).
[00205] In some embodiments, the antibody-drug conjugate compound has the following formula:

[00206] or a pharmaceutically acceptable salt or solvate thereof; on what:
[00207] L is the antibody moiety, e.g. MAb 191P4D12 of the present invention, and
[00208] (LU-D) is a drug unit-binding unit portion, where:
[00209] LU- is a binding unit, and
[00210] -D is a drug unit that has cytostatic or cytotoxic activity against a target cell; and
[00211] p is an integer from 1 to 20.
[00212] In some modalities, p ranges from 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 modalities , p ranges from 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 modalities, p is 1, 2, 3, 4, 5 or 6. In some modes, p is 2 or 4.
[00213] In some embodiments, the antibody-drug conjugate compound has the following formula:

[00214] or a pharmaceutically acceptable salt or solvate thereof, wherein:
[00215] L is the antibody moiety, for example, MAb 191P4D12; and
[00216] -Aa-Ww-Yy- is a binding unit (LU), in which:
[00217] -A- is a stretching unit,
[00218] a is 0 or 1,
[00219] each -W- is independently an amino acid unit,
[00220] w is an integer in the range 0 to 12,
[00221] -Y- is a self-immolative spacer unit,
[00222] y is 0, 1 or 2;
[00223] -D is a drug unit that has cytostatic or cytotoxic activity against the target cell; and
[00224] p is an integer from 1 to 20.
[00225] 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 In some modalities, p ranges from 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 modalities, p ranges from 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3. In other modes, p is 1, 2, 3, 4, 5 or 6. In some modes, p is 2 or 4. In some modes, when w is non-zero, y is 1 or 2. In some modes, when w is 1 to 12, y is 1 or 2. In some modes, w is 2 to 12 and y is 1 or 2. In some modes, a is 1 and w and y are 0.
For compositions comprising a plurality of antibodies, the drug loading is represented by p, the average number of drug molecules per antibody. Drug loading can range from 1 to 20 drugs (D) per antibody. The average number of drugs per antibody in preparing conjugation reactions can be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC. The quantitative distribution of antibody-drug conjugates in terms of p can also be determined. In some cases, the separation, purification, and characterization of homogeneous antibody-drug conjugates, where p is a value determined from antibody-drug conjugates with other drug loadings can be achieved by means such as reverse-phase HPLC or electrophoresis . In exemplary modalities, p is from 2 to 8.
[00227] The generation of conjugated antibody-drug compounds can be done by any technique known to those skilled in the art. Briefly, antibody-drug conjugated compounds comprise MAb 191P4D12 as the antibody moiety, a drug, and optionally, a linker that joins the drug and the binding agent. In a preferred embodiment, the antibody is MAb 191P4D12 which comprises heavy and light chain variable regions of an antibody designated Ha22-2(2,4)6.1 described above. In a more preferred embodiment, the antibody is MAb 191P4D12 which comprises the heavy and light chain of an antibody designated Ha22-2(2,4)6.1 described above. Several different reactions are available for the covalent attachment of drugs and/or ligands to binding agents. This is often done by reacting the amino acid residues of the binding agent, eg antibody molecule, including the amino groups of lysine, the free carboxylic acid groups of glutamic and aspartic acid, the sulfhydryl groups of cysteine and the various moieties of aromatic amino acids. One of the most commonly used nonspecific methods of covalent bonding is the carbodiimide reaction to link a carboxy (or amino) group of a compound to the amino (or carboxy) groups of the antibody. Additionally, bifunctional agents such as dialdehydes or imidoesters have been used to link the amino group of a compound to the amino groups of an antibody molecule. Schiff's base reaction is also available for drug binding to binding agents. This method involves periodate oxidation of a drug containing glycol or hydroxy groups, thereby forming an aldehyde which is then reacted with the coupling agent. Fixation takes place through the formation of a Schiff base with amino groups of the binding agent. Isothiocyanates can also be used as coupling agents to covalently link drugs to linkers. Other techniques are known to the person skilled in the art and are within the scope of the present invention.
[00228] In certain embodiments, an intermediate, which is the precursor of the ligand, is reacted with the drug under the appropriate conditions. In certain embodiments, reactive groups are used in the drug and/or intermediate. The product of the reaction between the drug and the intermediate, or the derivatized drug, is subsequently reacted with MAb 191P4D12 under suitable conditions.
[00229] Each of the particular units of the antibody-drug conjugate compounds is described in more detail in the present invention. The synthesis and structure of exemplary linker units, stretcher units, amino acid units, self-immolative spacer unit, and drug units are also described in US patent application publications Nos. 2003-0083263, 2005-0238649 and 2005-0009751, each is incorporated herein in its entirety, by reference and for all purposes. binding units
[00230] Typically, antibody-drug conjugate compounds comprise a linker moiety between the drug moiety and the antibody moiety. In some embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the drug moiety of the antibody into the intracellular environment. In still other embodiments, the linker moiety is not cleavable and the drug is released, for example, by antibody degradation.
[00231] In some embodiments, the ligand is cleavable by a cleavage agent that is present in the intracellular environment (eg, within a lysosome or endosome or caveola). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or an endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleavage agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of the active drug within target cells (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most typical are peptidyl linkers that are cleavable by enzymes that are present in cells expressing 191P4D12. For example, a peptidyl linker that is cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancer tissue, can be used (eg, a Phe-Leu linker or a Gly-Phe-Leu-Gly linker ( SEQ ID NO:9)). Other examples of such binders are described, for example, in US patent 6,214,345, incorporated herein in its entirety, by way of reference and for all purposes. In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, for example, US patent 6,214,345, which describes the synthesis of doxorubicin with the Val- linker. Cit). An advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.
[00232] In other embodiments, the cleavable linker is pH sensitive, that is, sensitive to hydrolysis at certain pH values. Typically, the pH sensitive binder is hydrolysable under acidic conditions. For example, an acid-labile binder that is hydrolysable in the lysosome (for example, a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used. (See, for example, US 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. 264:1465314661.) Such binders are relatively stable under neutral pH conditions such as blood, but are unstable at pH below 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, for example, a thioether linked to the therapeutic agent via an acylhydrazone linkage (see, for example, US patent 5,622,929).
[00233] In still other embodiments, the linker is cleavable under reducing conditions (eg, a disulfide linker). Various disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N -succinimidyl-3-(2-pyridyldithio) butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio) toluene), SPDB and SMPT. (See, for example, Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimaging and Therapy of Cancer (CW Vogel ed., Oxford U. Press, 1987) See also US Patent 4,880,935).
In yet other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:138793), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).
[00235] In still other embodiments, the linker moiety is not cleavable and the drug is released by antibody degradation. (See US Publication No. 2005/0238649 incorporated herein by reference in its entirety and for all purposes).
[00236] Typically, the ligand is not substantially sensitive to the extracellular environment. For use in the present invention, "not substantially sensitive to the extracellular environment," in the context of a binder, means at most about 20%, typically at most about 15%, more typically at most about 10%, and even more typically at most about 5%, at most about 3%, or at most about 1% of the ligands, in an antibody-drug conjugate compound sample, are cleaved when the antibody-drug conjugate compound presents itself in an extracellular environment (eg in plasma). It can be determined whether a binder will not be substantially sensitive to the extracellular environment, for example, by incubating the antibody-drug conjugated compound with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free drug present in the plasma.
[00237] In other non-mutually exclusive modalities, the ligand promotes cell internalization. In certain embodiments, the linker promotes cellular internalization when conjugated to the therapeutic agent (i.e., in the vicinity of the linker-therapeutic agent portion of the antibody-drug conjugated compound, as described herein). In still other embodiments, the ligand promotes cell internalization when conjugated to the auristatin compound and MAb 191P4D12.
[00238] Various exemplary binders that can be used with the present compositions and methods are described in WO 2004010957, US publication no. 2006/0074008, US publication no. 20050238649, and US publication no. herein incorporated by reference into the present invention in its entirety and for all purposes).
[00239] A "linker moiety" (LU) is a bifunctional compound that can be used to link a drug moiety and an antibody moiety to form an antibody-drug conjugated compound. In some modalities, the binding unit has the formula:

[00240] where: -A- is a stretching unit,
[00241] a is 0 or 1,
[00242] each -W- is independently an amino acid unit,
[00243] w is an integer in the range 0 to 12,
[00244] -Y- is a self-immolative spacer unit, and
[00245] y is 0, 1 or 2.
[00246] In some modes, a is 0 or 1, w is 0 or 1, and y is 0, 1 or 2. In some modes, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In In some modes, when w is 1 to 12, y is 1 or 2. In some modes, w is 2 to 12, and y is 1 or 2. In some modes, a is 1 and w and y are 0.
[00247] The stretching unit
[00248] The stretcher unit ( A ), when present, is capable of linking an antibody unit to an amino acid unit (-W-), if present, to a spacer unit (-Y-), if present; or to a drug unit (-D). Useful functional groups that may be present in a MAb 191P4D12 (eg, Ha22-2(2,4)6.1), naturally or through chemical manipulation include, but are not limited to, sulfhydryl, amino, hydroxyl, the hydroxyl group anomeric of a carbohydrate, and a carboxyl. Suitable functional groups are sulfhydryl and amino. In one example, sulfhydryl groups can be generated by reducing the intramolecular disulfide bonds of a MAb 191P4D12. In another embodiment, sulfhydryl groups can be generated by reacting an amino group of a lysine moiety of a MAb 191P4D12 with 2-iminothiolane (Traut's reagent) or other sulfhydryl-generating reagents. In certain embodiments, MAb 191P4D12 is a recombinant antibody and is engineered to carry one or more lysines. In certain other embodiments, the recombinant MAb 191P4D12 is engineered to carry additional sulfhydryl groups, e.g., additional cysteines.
[00249] In one embodiment, the stretcher unit forms a bond with a sulfur atom of the antibody unit. The sulfur atom can be derived from the sulfhydryl group of an antibody. Representative stretching units of this embodiment are shown within brackets of formulas IIIa and IIIb, wherein L-, -W-, -Y-, -D, w and y are as defined above, and R17 is selected from -C1-C10 alkylene -, -C1-C10 alkenylene-, -C1-C10 alkynylene-, carbocycle-, -O-(C1-C8 alkylene)-, O-(C1-C8 alkenylene)-, -O-(C1-C8 alkynylene)- , -arylene-, -C1-C10 alkylene-arylene-, -C2-C10 alkenylene-arylene, -C2-C10 alkynylene-arylene, -arylene-C1-C10 alkylene-, -arylene-C2-C10 alkenylene-, -arylene -C2-C10 alkynylene-, -C1-C10 alkylene-(carbocycle)-, -C2-C10 alkenylene-(carbocycle)-, -C2-C10 alkynylene- (carbocycle)-, -(carbocycle)-C1-C10 alkylene- , -(carbocycle)-C2-C10 alkenylene-, -(carbocycle)-C2-C10 alkynylene, -heterocycle-, -C1-C10 alkylene-(heterocycle)-, -C2-C10 alkenylene-(heterocycle)-, -C2 -C10 alkynylene-(heterocycle)-, -(heterocycle)-C1-C10 alkylene-, -(heterocycle)-C2-C10 alkenylene-, -(heterocycle)-C1-C10 alkynylene-, -(CH2CH2O)r-, or- (CH2CH2O)r-CH2-, er is an integer in the range of 1 to 10, said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, carbocycle, heterocycle, and arylene radicals, either alone or as part of another group, are optionally substituted. In some embodiments, said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocyclic, carbocycle, heterocycle, and arylene radicals, either alone or as part of another group, are unsubstituted. In some embodiments, R17 is selected from -C1-C10 alkylene-, -carbocycle-, -O-(C1-C8 alkylene)-, -arylene-, -C1-C10 alkylene-arylene-, -arylene-C1-C10 alkylene-, -C1-C10 alkylene-(carbocycle)-, -(carbocycle)-C1-C10 alkylene-, -C3-C8 heterocycle-, -C1-C10 alkylene-(heterocycle)-, -(heterocycle)-C1- C10 alkylene-, -(CH2CH2O)r-, and -(CH2CH2O)r-CH2-; and r is an integer in the range of 1 to 10, said alkylene groups being unsubstituted and the remainder of the groups being optionally substituted.
It should be understood from all exemplary embodiments that even when not expressly denoted, 1 to 20 drug moieties can be linked to an antibody (p = 1-20).


[00251] An illustrative stretching unit is that of formula IIIa where R17 is -(CH2)5-:

[00252] Another illustrative stretching unit is that of formula IIIa wherein R17 is -(CH2CH2O)r-CH2-; er is 2:

[00253] An illustrative stretching unit is that of formula IIIa, wherein R17 is arylene- or arylene-C1-C10 alkylene-. In some embodiments, the aryl group is an unsubstituted phenyl group.
[00254] Yet another illustrative stretching unit is that of formula IIIb, where R17 is -(CH2)5-:

In certain embodiments, the stretcher unit is linked to the antibody moiety through a disulfide bond between a sulfur atom of the antibody moiety and a sulfur atom of the stretcher moiety. A representative stretching unit of this modality is shown within brackets of Formula IV, where R17, L-, -W-, -Y-, -D, w and y are as defined above.

[00256] It should be noted that throughout this application, the S portion in the formula below refers to a sulfur atom of the antibody moiety, except where otherwise indicated by the context.
L-if-
[00257] In still other embodiments, the stretcher contains a reactive site that can form a bond with a primary or secondary amino group of an antibody. Examples of such reactive sites include, but are not limited to activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. Representative stretching units of this embodiment are shown within the brackets of formulas Va and Vb, where -R17-, L-, -W-, -Y-, -D, w and y are as defined above;

[00258] In some embodiments, the stretcher contains a reactive site that is reactive to a modified carbohydrate group (-CHO) that may be present on an antibody. For example, a carbohydrate can be moderately oxidized using a reagent such as sodium periodate and the resulting moiety (-CHO) of the oxidized carbohydrate can be condensed with a stretcher that contains a functionality such as a hydrazide, an oxime, a primary amine or secondary, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, and an arylhydrazide such as those described by Kaneko et al., 1991, Bioconjugate Chem. 2:133-41. Representative stretching units of this embodiment are shown within the brackets of formulas VIa, VIb, and VIc, where -R17-, L-, -W-, -Y-, -D, w and y are as defined above.

[00259] The amino acid unit
[00260] The amino acid unit (-W-), when present, connects the stretcher unit to the spacer unit, if the spacer unit is present, connects the stretcher unit to the drug portion if the spacer unit is absent, and turns on the spacer unit of antibody to the drug unit if the stretcher unit and the spacer unit are absent.
[00261] Ww- can be, for example, a monopeptide, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit. Each unit -W- independently has the formula denoted below in square brackets, and w is an integer in the range 0 to 12:

[00262] wherein R19is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2 COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3 NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2 )4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH2, 2-pyridyl methyl-, 3-pyridyl methyl-, 4-pyridyl methyl-, phenyl, cyclohexyl,

[00263] In some embodiments, the amino acid unit can be enzymatically cleaved by one or more enzymes, including a cancer-associated protease or tumor to release the drug unit (-D) which, in one embodiment, is protonated in vivo by release to deliver a drug (D).
In certain embodiments, the amino acid unit can comprise natural amino acids. In other embodiments, the amino acid unit can comprise unnatural amino acids. The illustrative Ww units are represented by formulas (VII)-(IX):
where R20 and R21 are as follows:
where R20, R21 and R22 are as follows:

where R20, R21, R22 and R23 are as follows:

Exemplary amino acid moieties include, but are not limited to Formula VII moieties where: R20 is benzyl and R21 is -(CH2)4 NH2; R20 is isopropyl and R21 is -(CH2)4NH2; or R20 is isopropyl and R21 is -(CH2)3 NHCONH2. Another exemplary amino acid moiety is a Formula VIII moiety where R20 is benzyl, R21 is benzyl, and R22 is -(CH2)4NH2.
[00266] Useful -Ww- units can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzyme, for example, a tumor associated protease. In one embodiment, a -Ww - unit is one whose cleavage is catalyzed by cathepsin B, C, and D, or a plasmin protease.
[00267] In one embodiment, -Ww- is a dipeptide, tripeptide, tetrapeptide or pentapeptide. When R19, R20, R21, R22 or R23 is other than hydrogen, the carbon atom to which R19, R20, R21, R22 or R23 is attached is chiral.
[00268] Each carbon atom to which R19, R20, R21, R22 or R23 is attached is independently in the (S) or (R) configuration.
[00269] In one aspect of the amino acid unit, the amino acid unit is valine-citrulline (vc or Val-Cit). In another aspect, the amino acid moiety is phenylalanine-lysine (i.e., fk). In yet another aspect of the amino acid moiety, the amino acid moiety is N-methylvaline-citrulline. In yet another aspect, the amino acid moiety is 5-aminovaleric acid, homophenylalanine lysine, tetraisoquinoline carboxylate lysine, cyclohexyl alanine lysine, isonepecotic acid lysine, beta-alanine lysine, glycine serine valine glutamine and isonepecotic acid.
[00270] The spacer unit
[00271] The spacer unit (-Y-), when present, links an amino acid unit to the drug unit when an amino acid unit is present. Alternatively, the spacer unit connects the stretcher unit to the drug unit when the amino acid unit is absent. The spacer unit also connects the drug unit to the antibody unit when the amino acid unit and stretcher unit are absent.
[00272] Spacer units are of two general types: non-self-immolative or self-immolative. A non-self-immolative spacer unit is one in which part or all of the spacer unit remains attached to the drug moiety after cleavage, particularly enzymatic, of an amino acid unit of the antibody-drug conjugate. Examples of a non-self-immolative spacer unit include, but are not limited to, a spacer unit (glycine-glycine) and a glycine spacer unit (both shown in scheme 1) (below). When a conjugate containing a glycine-glycine spacer unit or a glycine spacer unit undergoes enzymatic cleavage by an enzyme (eg, a tumor cell-associated protease, a cancer cell-associated protease, or a lymphocyte-associated protease), the glycine-glycine drug moiety or a glycine drug moiety is cleaved from L-Aa-Ww-. In one embodiment, an independent hydrolysis reaction occurs within the target cell, cleaving the drug-glycine moiety linkage and releasing the drug. Scheme 1

[00273] In some embodiments, a non-self-immolative spacer unit (-Y-) is -Gly-. In some embodiments, the non-self-immolative spacer unit (-Y-) is -Gly-Gly-.
[00274] In one embodiment, a drug-linker conjugate is provided, in which the spacer unit is absent (-Yy - where y=0), or a pharmaceutically acceptable salt or solvate thereof.
[00275] Alternatively, a conjugate containing a self-immolative spacer unit may release -D. As used herein, the term "self-immolative spacer" refers to a bifunctional chemical moiety that is capable of covalently linking together two spaced chemical moieties in a stable tripartite molecule. It will spontaneously separate from the second chemical moiety if its bond to the first moiety is cleaved.
[00276] In some embodiments, -Yy- is a p-aminobenzyl alcohol (PAB) unit (see schemes 2 and 3) whose phenylene portion is substituted with Qm, where Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro or -cyano; and m is an integer in the range 0 to 4. Alkyl, alkenyl and alkynyl groups, either alone or as part of another group, may optionally be substituted.
[00277] In some embodiments, -Y- is a PAB group that is linked to -Ww - through the amino nitrogen atom of the PAB group, and is directly connected to -D through a carbonate, carbamate or ether group. Without sticking to a particular theory or mechanism, scheme 2 represents a possible drug release mechanism from a PAB group, which is fixed directly to -D through a carbamate or carbonate group, as described by Toki et al., 2002 , J. Org. Chem. 67:1866-1872. Scheme 2

[00278] In scheme 2, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O- (C1-C8 alkynyl), - halogen, -nitro or -cyano; m is an integer in the range 0 to 4; and p in the range of 1 to about 20. Alkyl, alkenyl and alkynyl groups, either alone or as part of another group, may be optionally substituted.
[00279] Without sticking to a particular theory or mechanism, scheme 3 represents a possible drug release mechanism from a PAB group, which is directly linked to -D through an ether or amine bond, where D includes the group oxygen or nitrogen that is part of the drug unit. Scheme 3

[00280] In scheme 3, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O- (C1-C8 alkynyl), - halogen, -nitro or -cyano; m is an integer in the range 0 to 4; and p in the range of 1 to about 20. Alkyl, alkenyl and alkynyl groups, either alone or as part of another group, may be optionally substituted.
[00281] Other Examples of self-immolative spacers include, but are not limited to aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazol-5-methanol derivatives (Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho or para-aminobenzylacetals. Spacers that undergo cyclization upon 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), bicyclo[2.2.1] and ring systems suitably substituted bicyclo[2.2.2] (Storm et al., 1972, J. Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acid amides (Amsberry et al., 1990, J. Org. Chem. 55) :5867). The elimination of amine-containing drugs that are substituted at the α position of glycine (Kingsbury et al., 1984, J. Med. Chem. 27:1447) is also an example of self-immolative spacers.
[00282] In one embodiment, the spacer unit is a branched bis(hydroxymethyl)-styrene (BHMS) unit, as shown in scheme 4, which can be used to incorporate and release multiple drugs. Scheme 4

[00283] In scheme 4, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O- (C1-C8 alkynyl), - halogen, -nitro or -cyano; m is an integer in the range 0 to 4; n is 0 or 1; and p ranges from 1 to about 20. Alkyl, alkenyl and alkynyl groups, either alone or as part of another group, may be optionally substituted.
[00284] In some embodiments, the -D portions are the same. In yet another embodiment, the -D portions are different.
[00285] In one aspect, the spacer units (-Yy-) are represented by the formulas (X)-(XII):

[00286] wherein Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1 -C8 alkynyl), - halogen, -nitro or -cyano; and em is an integer in the range 0 to 4. Alkyl, alkenyl and alkynyl groups, either alone or as part of another group, may optionally be substituted.

[00287] The modalities of formulas I and II comprising antibody-drug conjugate compounds may include:

[00288] where w and y are each 0, 1 or 2, and,

[00289] where w and y are each 0,

[00290] The drug unit
[00291] The drug portion (D) can be any cytotoxic, cytostatic or immunomodulatory drug (eg immunosuppressant). D is a drug unit (moiety) that has an atom that can form a bond with the spacer unit, amino acid unit, stretcher unit, or antibody unit. In some embodiments, the drug unit D has a nitrogen atom that can form a bond with the spacer unit. As used herein, the terms "drug unit" and "drug portion" are synonymous and used interchangeably.
Useful classes of cytotoxic, cytostatic, or immunomodulatory agents include, for example, anti-tubulin agents, DNA minor groove binders, DNA replication inhibitors, and alkylating agents.
[00293] In some embodiments, the drug is an auristatin, such as auristatin E (also known in the art as a derivative of dolastatin-10) or a derivative thereof. Auristatin can be, for example, an ester formed between auristatin E and an acid keto. For example, auristatin E can be reacted with paraacetyl benzoic acid or benzoyl valeric acid to produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. The synthesis and structure of exemplary auristatins are described in U.S. Patent Application Publication No. 2003-0083263; in International Patent Publication No. WO 04/010957, in International Patent Publication No. WO 02/088172, and in US patents No. 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; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, each of which is incorporated herein by reference in its entirety and for all purposes.
[00294] Auristatins have been shown to interfere with microtubule dynamics and with nuclear and cell division and have anti-cancer activity. Auristatins bind to tubulin and can exert a cytotoxic or cytostatic effect on a cell that expresses 191P4D12. There are a number of different assays known in the art that can be used to determine whether an auristatin or a resulting antibody-drug conjugate exerts a cytostatic or cytotoxic effect on a desired cell lineage.
[00295] Methods to determine whether a compound binds to tubulin are known in the art. See, for example, Muller et al., Anal. Chem 2006, 78, 4390-4397; Hamel et al., Molecular Pharmacology, 1995 47:965-976; and Hamel et al., The Journal of Biological Chemistry, 1990 265:28, 17141-17149. For purposes of the present invention, the relative affinity of a compound for tubulin can be determined. Some preferred auristatins of the present invention bind tubulin with an affinity in the range 10 times lower (lower affinity) than the binding affinity of MMAE to tubulin up to 10 times, 20 times or even 100 times higher (higher affinity) than binding affinity of MMAE to tubulin.
[00296] In some modalities, -D is an auristatin with the formula DE or DF:

[00297] or a pharmaceutically acceptable salt form or solvate thereof;
[00298] in which, independently, at each point:
[00299] the wavy line indicates a connection;
[00300] R2is -C1-C20 alkyl, -C2-C20 alkenyl, or -C2-C20 alkynyl;
[00301] R3is -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl, -carbocycle, -C1-C20 alkylene (carbocycle), -C2-C20 alkenylene(carbocycle), -C2-C20 alkynylene(carbocycle), -aryl, -C1-C20 alkylene(aryl), -C2-C20 alkenylene(aryl), -C2-C20 alkynylene(aryl), heterocycle, -C1-C20 alkylene(heterocycle), -C2-C20 alkenylene (heterocycle), or -C 2 -C 20 alkynylene (heterocycle);
[00302] R4is -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl, carbocycle, -C1-C20 alkylene (carbocycle), -C2-C20 alkenylene(carbocycle), -C2-C20 alkynylene (carbocycle), aryl, -C1-C20 alkylene(aryl), -C2-C20 alkenylene (aryl), -C2-C20 alkynylene(aryl), - heterocycle, -C1-C20 alkylene(heterocycle), -C2-C20 alkenylene (heterocycle), or -C 2 -C 20 alkynylene (heterocycle);
[00303] R5is -H or -C1-C8 alkyl;
[00304] or R4 and R5 together form a carbocyclic ring and have the formula -(CRaRb)s- wherein Ra and Rb are, independently, -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl, or -carbocycle es is 2, 3, 4, 5 or 6,
[00305] R6 is -H, -C1-C20 alkyl, -C2-C20 alkenyl, or -C2-C20 alkynyl;
[00306] R7is -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl, carbocycle, -C1-C20 alkylene (carbocycle), -C2-C20 alkenylene(carbocycle), -C2-C20 alkynylene (carbocycle), -aryl, -C1-C20 alkylene(aryl), -C2-C20 alkenylene(aryl), -C2-C20 alkynylene(aryl), heterocycle, -C1-C20 alkylene(heterocycle), -C2-C20 alkenylene (heterocycle), or -C 2 -C 20 alkynylene (heterocycle);
[00307] each R8 is independently -H, -OH, -C1-C20 alkyl, -C2C20 alkenyl, -C2-C20 alkynyl, -O-(C1-C20 alkyl), -O-(C2-C20 alkenyl), -O -(C1-C20 alkynyl), or -carbocycle;
[00308] R9 is -H, -C1-C20 alkyl, -C2-C20 alkenyl, or -C2-C20 alkynyl;
[00309] R24 is -aryl, -heterocycle, or -carbocycle;
R25is -H, C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl, -carbocycle, -O-(C1-C20 alkyl), -O-(C2-C20 alkenyl), -O- (C2-C20 alkynyl), or OR18 where R18 is -H, a hydroxyl protecting group, or a direct bond where OR18 is =O;
[00311] R26 is -H, -C1-C20 alkyl, -C2-C20 alkenyl, or -C2-C20 alkynyl, -aryl, -heterocycle, or -carbocycle;
[00312] R10 is -aryl or -heterocycle;
Z is -O, -S, -NH, or -NR12, wherein R12 is -C1-C20 alkyl, -C2C20 alkenyl, or -C2-C20 alkynyl;
[00314] R11 is -H, -C1-C20 alkyl, --C2-C20 alkenyl, -C2-C20 alkynyl, -aryl, -heterocycle, -(R13O)m-R14, or -(R13O)m-CH(R15 )two;
[00315] m is an integer in the range 1 to 1000 or m=0 to 1000;
[00316] R13 is -C2-C20 alkylene, -C2-C20 alkenylene, or -C2-C20 alkynylene;
[00317] R14 is -H, -C1-C20 alkyl, -C2-C20 alkenyl, or -C2-C20 alkynyl;
[00318] each occurrence of R15 is independently -H, -COOH, -(CH2)nN(R16)2, -(CH2)n-SO3H, -(CH2)n-SO3-C1-C20 alkyl, -(CH2)n - SO3-C2-C20 alkenyl, or -(CH2)n-SO3-C2-C20 alkynyl;
[00319] each occurrence of R16 is independently -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl, or -(CH2)n-COOH; and
[00320] n is an integer in the range 0 to 6;
[00321] wherein said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, and heterocycle radicals, either alone or as part of another group, are optionally substituted.
Auristatins of formula DE include those in which said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, and heterocycle radicals are unsubstituted.
Auristatins of formula DE include those in which the groups of R2, R3, R4, R5, R6, R7, R8, and R9 are unsubstituted and the groups of R19, R20 and R21 are optionally substituted, as described herein .
[00324] Auristatins with the formula DE include those in which
[00325] R2is C1-C8 alkyl;
[00326] R3, R4and R7are independently selected from -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl, C3-C6 monocyclic carbocycle, -C1-C20 alkylene(C3-C6 monocyclic carbocycle ), -C2-C20 alkenylene(C3-C6 monocyclic carbocycle), -C2C20 alkynylene(C3-C6 monocyclic carbocycle), C6-C10 aryl, -C1-C20 alkylene(C6-C10 aryl), -C2-C20 alkenylene (C6 -C10 aryl), -C2-C20 alkynylene(C6-C10 aryl), heterocycle, -C1-C20 alkylene (heterocycle), -C2-C20 alkenylene(heterocycle), or -C2-C20 alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, carbocycle, aryl and heterocycle radicals are optionally substituted;
[00327] R5 is -H;
[00328] R6 is -C1-C8 alkyl;
[00329] each R8 is independently selected from -OH, -O-(C1-C20 alkyl), -O-(C2-C20 alkenyl), or -O-(C2-C20 alkynyl) wherein said alkyl radicals, alkenyl, and alkynyl are optionally substituted;
[00330] R9 is -H or -C1-C8 alkyl;
[00331] R24is -optionally substituted phenyl;
[00332] R25 is -OR18; where R18is H, a hydroxyl protecting group, or a direct bond where OR18represents =O;
[00333] R26 is selected from -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl, or -carbocycle; wherein said alkyl, alkenyl, alkynyl and carbocycle radicals are optionally substituted; or a pharmaceutically acceptable salt form or solvate thereof.
[00334] Auristatins with the formula DE include those in which
[00335] R2 is methyl
[00336] R3 is -H, -C1-C8 alkyl, -C2-C8 alkenyl, or C2-C8 alkynyl, wherein said alkyl, alkenyl and alkynyl radicals are optionally substituted;
[00337] R4is -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, C3-C6 monocyclic carbocycle, -C6-C10 aryl, -C1-C8 alkylene(C6-C10 aryl), - C2-C8 alkenylene(C6-C10 aryl), -C2-C8 alkynylene(C6-C10 aryl), -C1-C8 alkylene (C3-C6 monocyclic carbocycle), -C2-C8 alkenylene (C3-C6 monocyclic carbocycle), - C2-C8 alkynylene(C3-C6 monocyclic carbocycle); wherein said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl and carbocycle radicals, either alone or as part of another group, are optionally substituted;
[00338] R5 is -H;
[00339] R6 is methyl
[00340] R7is -C1-C8 alkyl, -C2-C8 alkenyl or -C2-C8 alkynyl;
[00341] each R8 is methoxy;
[00342] R9 is -H or -C1-C8 alkyl;
[00343] R24is -phenyl;
[00344] R25 is -OR18; where R18is H, a hydroxyl protecting group, or a direct bond where OR18represents =O;
[00345] R26 is methyl
[00346] or a pharmaceutically acceptable salt form thereof.
[00347] Auristatins with the formula DE include those in which:
[00348] R2is methyl R3is -H or -C1-C3 alkyl; R4is -C1-C5 alkyl; R5 is -H; R6is methyl R7is isopropyl or sec-butyl; R8 is methoxy; R9 is -H or -C1-C8 alkyl; R24is phenyl; R25 is -OR18; wherein R18 is -H, a hydroxyl protecting group, or a direct bond in which OR18 is =O; and R26 is methyl or a pharmaceutically acceptable salt or solvate form thereof.
[00349] Auristatins with the formula DE include those in which:
[00350] R2is methyl or C1-C3 alkyl,
[00351] R3 is -H or -C1-C3 alkyl;
[00352] R4 is -C1-C5 alkyl;
[00353] R5is H;
[00354] R6 is C1-C3 alkyl;
[00355] R7 is -C1-C5 alkyl;
[00356] R8 is -C1-C3 alkoxy;
[00357] R9 is -H or -C1-C8 alkyl;
[00358] R24 is phenyl;
[00359] R25 is -OR18; where R18is -H, a hydroxyl protecting group, or a direct bond where OR18represents =O; and
[00360] R26 is -C1-C3 alkyl;
[00361] or a pharmaceutically acceptable salt form thereof.
[00362] Auristatins with the DF formula include those in which:
[00363] R2 is methyl
[00364] R3, R4, and R7 are independently selected from -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl, C3-C6 monocyclic carbocycle, -C1-C20(C3-C6) alkylene monocyclic carbocycle), -C2-C20 alkenylene(C3-C6 monocyclic carbocycle), -C2C20alkynylene(C3-C6 monocyclic carbocycle), -C6-C10 aryl, -C1-C20 alkylene(C6-C10 aryl), -C2-C20 alkenylene (C6-C10 aryl), -C2-C20 alkynylene(C6-C10 aryl), heterocycle, -C1-C20 alkylene(heterocycle), -C2-C20 alkenylene(heterocycle), or -C2-C20 alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, carbocycle, aryl and heterocycle radicals, either alone or as part of another group, are optionally substituted;
[00365] R5 is -H;
[00366] R6 is methyl
[00367] each R8 is methoxy;
[00368] R9 is -H, -C1-C20 alkyl, -C2-C20 alkenyl, or -C2-C20 alkynyl; wherein said alkyl, alkenyl and alkynyl radicals are optionally substituted;
[00369] R10 aryl is optionally substituted or optionally substituted heterocycle;
Z is -O-, -S-, -NH-, or -NR12, where R12 is -C1-C20 alkyl, -C2-C20 alkenyl, or -C2-C20 alkynyl, each of which is optionally substituted ;
R11 is -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl, -aryl, -heterocycle, -(R13O)m-R14, or -(R13O)m-CH(R15) 2, wherein said alkyl, alkenyl, alkynyl, aryl and heterocycle radicals are optionally substituted;
[00372] m is an integer in the range 1 to 1000 or m = 0;
[00373] R13 is -C2-C20 alkylene, -C2-C20 alkenylene, or -C2-C20 alkynylene, each of which is optionally substituted;
R14 is -H, -C1-C20 alkyl, -C2-C20 alkenyl, or -C2-C20 alkynyl wherein said alkyl, alkenyl and alkynyl radicals are optionally substituted;
[00375] each occurrence of R15 is independently -H, -COOH, -(CH2)nN(R16)2, -(CH2)n-SO3H, -(CH2)n-SO3-C1-C20 alkyl, -(CH2)n - SO3-C2-C20 alkenyl, or -(CH2)n-SO3-C2-C20 alkynyl, said alkyl, alkenyl and alkynyl radicals being optionally substituted;
[00376] each occurrence of R16 is independently -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl or -(CH2)n-COOH, wherein said alkyl, alkenyl and alkynyl radicals are optionally substituted ;
[00377] n is an integer in the range 0 to 6;
[00378] or a pharmaceutically acceptable salt thereof.
[00379] In some of these embodiments, R10 is optionally substituted phenyl.
Auristatins of formula DF include those in which the groups of R2, R3, R4, R5, R6, R7, R8, and R9 are unsubstituted and the groups of R10 and R11 are as described herein.
Auristatins of formula DF include those in which said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, and heterocycle radicals are unsubstituted.
[00382] Auristatins with the DF formula include those in which
[00383] R2is -C1-C3 alkyl; R3is -H or -C1-C3 alkyl; R4is -C1-C5 alkyl; R5 is -H; R6 is -C1-C3 alkyl; R7is -C1-C5 alkyl; R8 is -C1-C3 alkoxy; R9 is -H or -C1-C8 alkyl; R10 is optionally substituted phenyl; Z is -O-, -S-, or -NH-; R11 is as defined here; or a pharmaceutically acceptable salt thereof.
[00384] Auristatins with the DF formula include those in which
[00385] R2is methyl R3is -H or -C1-C3 alkyl; R4is -C1-C5 alkyl; R5 is -H; R6is methyl R7is isopropyl or sec-butyl; R8 is methoxy; R9 is -H or -C1-C8 alkyl; R10 is optionally substituted phenyl; Z is -O-, -S-, or -NH-; and R11 is as defined in the present invention; or a pharmaceutically acceptable salt thereof.
[00386] Auristatins with the DF formula include those in which
[00387] R2is methyl R3is -H or -C1-C3 alkyl; R4is -C1-C5 alkyl; R5 is -H; R6 is methyl; R7is isopropyl or sec-butyl; R8 is methoxy; R9 is -H or C1-C8 alkyl; R10 is phenyl; and Z is -O- or -NH- and R11 is as defined herein, preferably hydrogen; or a pharmaceutically acceptable salt form thereof.
[00388] Auristatins with the DF formula include those in which
[00389] R2is -C1-C3 alkyl; R3is -H or -C1-C3 alkyl; R4is -C1-C5 alkyl; R5 is -H; R6 is -C1-C3 alkyl; R7is -C1-C5 alkyl; R8 is -C1-C3 alkoxy; R9 is -H or -C1-C8 alkyl; R10 is phenyl; and Z is -O- or -NH- and R11 is as defined herein, preferably hydrogen; or a pharmaceutically acceptable salt form thereof.
Auristatins of formula DE or DF include those in which R3, R4 and R7 are independently isopropyl or sec-butyl and R5 is -H. In an exemplary embodiment, R3 and R4 are each isopropyl, R5 is H, and R7 is sec-butyl. The remainder of the substituents are as defined herein.
Auristatins of formula DE or DF include those in which R2 and R6 are each methyl, and R9 is H. The remainder of the substituents are as defined herein.
Auristatins of formula DE or DF include those in which each occurrence of R8 is -OCH3. The remainder of the substituents are as defined herein.
[00393] Auristatins of formula DE or DF include those in which R3 and R4 are each isopropyl, R2 and R6 are each methyl, R5 is H, R7 is sec-butyl, each occurrence of R8 is -OCH3, and R9 is H. The remainder of the substituents are as defined herein.
Auristatins of formula DF include those in which Z is -O- or -NH-. The remainder of the substituents are as defined herein.
[00395] Auristatins of formula DF include those in which R10 is aryl. The remainder of the substituents are as defined herein.
[00396] Auristatins of formula DF include those in which R10 is -phenyl. The remainder of the substituents are as defined herein.
Auristatins of formula DF include those in which Z is -O-, and R11 is H, methyl or t-butyl. The remainder of the substituents are as defined herein.
[00398] Auristatins of formula DF include those in which, when Z is -NH-, R11 is -(R13O)m-CH(R15)2, wherein R15 is -(CH2)nN(R16)2, and R16 is -C1-C8 alkyl or -(CH2)n-COOH. The remainder of the substituents are as defined herein.
Auristatins of formula DF include those in which when Z is -NH-, R11 is -(R13O)m-CH(R15)2, wherein R15 is -(CH2)n-SO3H. The remainder of the substituents are as defined herein.
[00400] In preferred embodiments, when D is an auristatin of Formula DE, 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.
[00401] In some modalities, where D is an auristatin of Formula DF, a is 1 and w and y are 0.
[00402] Illustrative drug units (-D) include drug units that have the following structures:


or pharmaceutically acceptable salts or solvates thereof.
[00403] In one aspect, hydrophilic groups, for example but not limited to triethylene glycol esters (TEG) may be attached to the drug moiety in R11. Without sticking to theory, hydrophilic groups help in the internalization and non-agglomeration of the drug unit.
[00404] In some embodiments, the drug unit is not TZT-1027. In some embodiments, the drug unit is not auristatin E, dolastatin 10, or auristatin PE.
Exemplary antibody-drug conjugate compounds have the following structures, wherein "L" or "mAb-s-" represents a MAb 191P4D12 designated Ha22-2(2,4)6.1 shown herein:

or a pharmaceutically acceptable salt thereof.
[00406] In some embodiments, the drug unit is a calicheamicin, camptothecin, a maytansinoid, or an anthracycline. In some embodiments, the drug is a taxane, a topoisomerase inhibitor, a vinca alkaloid, or the like.
In some typical embodiments, suitable cytotoxic agents include, for example, DNA minor groove ligands (eg, enediins and lexitropsins, a CBI compound; see also US patent 6,130,237), duocarmycins, taxanes (eg. , paclitaxel and docetaxel), puromycins, and vinca alkaloids. Other cytotoxic agents include, for example, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotine, eleuterodermolinosin , and mitoxantrone.
[00408] In some embodiments, the drug is an anti-tubulin agent. Examples of anti-tubulin agents include, auristatins, taxanes (for example, Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik) and vinca alkaloids (for example, vincristine, vinblastine, vindesine, and vinorelbine). Other anti-tubulin agents include, for example, baccatin derivatives, taxane analogues (eg, epothilone A and B), nocodazole, colchicine and colcimide, estramustine, cryptophycins, cemadotine, maytansinoids, combretastatins, discodermolide, and eleuterobin.
[00409] In certain embodiments, the cytotoxic agent is a maytansinoid, another group of anti-tubulin agents. For example, in specific embodiments, the maytansinoid is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res. 52:127-131).
[00410] In certain embodiments, the cytotoxic or cytostatic agent is a dolastatin. In certain embodiments, the cytotoxic or cytostatic agent is of the auristatin class. Thus, in a specific modality, the cytotoxic or cytostatic agent is MMAE (formula XI). In another specific embodiment, the cytotoxic or cytostatic agent is AFP (formula XVI).

[00411] In certain embodiments, the cytotoxic or cytostatic agent is a compound of the formulas XII-XXI or a pharmaceutically acceptable salt thereof:


X.) Drug Loading
[00412] Drug loading is represented by p and is the average number of drug moieties per antibody in a molecule. Drug loading can range from 1 to 20 drug portions (D) per antibody. The ADCs of the invention include collections of antibodies conjugated to a range of drug moieties, from 1 to 20. The average number of drug moieties per antibody in ADC preparations from conjugation reactions can be characterized by conventional means such as mass spectroscopy and ELISA assay. The quantitative distribution of the ADC in terms of p can also be determined. In some cases, separation, purification, and characterization of homogeneous ADCs, where p is a given value from ADCs with other drug loadings, can be achieved by means such as electrophoresis.
[00413] For some antibody-drug conjugates, p may be limited by the number of binding sites on the antibody. For example, when the linkage is a cysteine thiol, as in the exemplary embodiments above, an antibody may have only one or more cysteine thiol groups, or it may have only one or more thiol groups sufficiently reactive through which a linker can be attached. In certain embodiments, increased drug loading, eg, p >5, can cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. In certain embodiments, drug loading for an ADC of the invention ranges from 1 to about 8; from about 2 to about 6; from about 3 to about 5; from about 3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8; from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3 to about 3.8; or from about 3.3 to about 3.7. In fact, it has been shown that for certain ADCs, the optimal ratio of drug to antibody portions may be less than 8, and may be from about 2 to about 5. See US Patent No. 7,498,298 (herein incorporated in reference title in its entirety).
[00414] In certain embodiments, less than the theoretical maximum drug moieties are conjugated to an antibody during a conjugation reaction. An antibody can contain, for example, lysine residues that do not react with the drug-linker intermediate or linking reagent, as discussed below. In general, antibodies do not contain many free and reactive cysteine thiol groups, which can be attached to a drug moiety; in fact, most cysteine thiol residues in antibodies exist as disulfide bridges. In certain embodiments, an antibody can be reduced with a reducing agent, such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups. In certain embodiments, an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups, such as lysine or cysteine.
[00415] The loading (drug/antibody ratio) of an ADC can be controlled in different ways, for example, by: (i) limiting the molar excess of drug-linker intermediate or binding reagent relative to the antibody, (ii) limiting the time or temperature of the conjugation reaction, (iii) partial reducing or limiting conditions for the modification of the cysteine thiol, (iv) manipulating by recombinant techniques the amino acid sequence of the antibody so that the number and position of the cysteine residues is modified to control the number and/or position of linker-drug bonds (such as thioMab or thioFab prepared as described herein and in WO2006/034488 (incorporated by reference herein in its entirety)).
[00416] It should be understood that when more than one nucleophilic group reacts with a drug-linker intermediate or linking reagent followed by the drug portion reagent, then the resulting product is a mixture of the ADC compounds with a distribution of one or more drug moieties linked to an antibody. The average number of drugs per antibody can be calculated from the mixture by a dual antibody ELISA assay, which is antibody-specific and drug-specific. Individual ADC molecules can be identified in the mixture by mass spectroscopy and separated by HPLC, eg, 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 #624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, SC, et al., "Controlling the location of drug attachments in antibody-drug conjugates," Abstract #627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45 , March 2004). In certain embodiments, a homogeneous ADC with a single loading value can be isolated from the conjugation mixture by electrophoresis or chromatography. XI .) Methods for Determining the Cytotoxic Effect of ADCs
[00417] Methods to determine whether a drug or antibody-drug conjugate exerts a cytostatic and/or cytotoxic effect on a cell are known. In general, the cytotoxic or cytostatic activity of an antibody-drug conjugate can be measured by: exposing mammalian cells that express a target protein of the antibody-drug conjugate in a cell culture medium; culturing the cells for a period of about 6 hours to about 5 days; and measure cell viability. In vitro cell-based tests can be used to measure the viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the antibody-drug conjugate.
[00418] To determine whether an antibody-drug conjugate exerts a cytostatic effect, a thymidine incorporation assay can be used. For example, cancer cells expressing a target antigen at a density of 5,000 cells/well of a 96-well plate can be cultured for a period of 72 hours and exposed to 0.5 μCi of 3H-thymidine for the final 8 hours of the 72 hour period. 3 H-thymidine incorporation into cultured cells is measured in the presence and absence of the antibody-drug conjugate.
[00419] To determine cytotoxicity, necrosis or apoptosis (programmed cell death) can be measured. Necrosis is typically accompanied by increased plasma membrane permeability; cell swelling, and plasma membrane rupture. Apoptosis is typically characterized by membrane blebbing, cytoplasmic condensation, and activation of endogenous endonucleases. Determining any of these effects on cancer cells indicates that an antibody-drug conjugate is useful in treating cancers.
Cell viability can be measured by determining in a cell the uptake of a dye such as neutral red, trypan blue, or ALAMAR® blue (see, for example, Page et al., 1993, Intl. J. Oncology 3:473-476). In such an assay, cells are incubated in dye-containing media, the cells are washed, and the remaining dye, which reflects the cellular uptake of the dye, is measured spectrophotometrically. The protein-binding sulphorodamine B (SRB) dye can also be used to measure cytotoxicity (Skehan et al., 1990, J. Natl. Cancer Inst. 82:1107-12).
[00421] Alternatively, a tetrazolium salt, such as MTT, is used in a quantitative colorimetric assay for mammalian cell survival and proliferation by detecting living but not dead cells (see, for example, Mosmann, 1983, J. Immunol Methods 65:55-63).
Apoptosis can be quantified by measuring, for example, DNA fragmentation. Commercial photometric methods for quantitative in vitro determination of DNA fragmentation are available. Examples of these assays, including TUNEL (which detects the incorporation of labeled nucleotides into fragmented DNA) and ELISA-based assays, are described in Biochemica, 1999, no. 2, p. 34 to 37 (Roche Molecular Biochemicals).
[00423] Apoptosis can also be determined by measuring morphological changes in a cell. For example, as with necrosis, loss of plasma membrane integrity can be determined by measuring the absorption of certain dyes (eg a fluorescent dye such as acridine orange or ethidium bromide). A method for measuring the number of apoptotic cells has been described by Duke and Cohen, Current Protocols in Immunology (Coligan et al. eds., 1992, pp. 3.17.1-3.17.16). Cells can also be labeled with a DNA dye (eg, acridine orange, ethidium bromide, or propidium iodide) and observed for chromatin condensation and margination along the inner nuclear membrane. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane bulging, and cell retraction.
[00424] The presence of apoptotic cells can be measured in both the adhered and "floating" compartments of the cultures. For example, both compartments can be collected by removing the supernatant, trypsinizing adhered cells, combining the preparations after a washing step by centrifugation (eg, 10 minutes at 2000 rpm), and detecting apoptosis (eg, by means of measurement of DNA fragmentation). (See, for example, Piazza et al., 1995, Cancer Research 55:3110-16).
In vivo, the effect of a therapeutic composition of 191P4D12 can be evaluated in a suitable animal model. For example, xenogenic cancer models can be used, whereby cancer explants or subcultured xenograft tissues are introduced into immunocompromised animals such as nude or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-408) . For example, PCT patent application WO98/16628 and US patent 6,107,540 describe various human prostate cancer xenograft models capable of recapitulating the development of primary tumors, micrometastases, and osteoblastic metastasis formation characteristic of stage disease late. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.
[00426] In vivo assays that assess the promotion of apoptosis are useful in evaluating therapeutic compositions. In one embodiment, xenografts from tumor-bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to mice carrying the untreated control xenograft. The extent to which apoptotic foci are found in tumors from treated mice provides an indication of the therapeutic efficacy of the composition.
Therapeutic compositions used in the practice of the aforementioned methods may be formulated into pharmaceutical compositions comprising a vehicle suitable for the desired method of application. Suitable carriers include any material which, when combined with the therapeutic composition, retains the antitumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to any of a number of standard pharmaceutical carriers such as sterile phosphate-buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16th edition, A. Osal., Ed., 1980).
[00428] Therapeutic formulations can be solubilized and administered through any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intraorganic, orthotopic, and the like. A preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinyl chloride or polyethylene bags containing 0.9% sterile sodium chloride for injection, USP. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing, for example, benzyl alcohol preservative) or in sterile water prior to injection.
Dosages and administration protocols for treating cancers using the aforementioned methods will vary with the method and the target cancer, and will generally depend on various other factors understood in the art. XII .) Treatment of cancer(s) expressing 191P4D12
[00430] The identification of 191P4D12 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in cancers, such as those mentioned in table I, opens up numerous therapeutic approaches for the treatment of these cancers.
Of note, targeted antitumor therapies have been useful even when the targeted protein is expressed in normal tissues, even normal vital organ tissues. A vital organ is one that is needed to sustain life, such as the heart or colon. A non-vital organ is one that can be removed and as a consequence, the individual can still survive. Examples of non-vital organs are ovary, breast, and prostate.
[00432] The expression of a target protein in normal tissue, even in vital normal tissue, does not negate the usefulness of a targeting agent for the protein as a therapeutic for certain tumors in which the protein is also overexpressed. For example, expression in vital organs is not in itself harmful. Furthermore, organs considered expendable, such as the prostate and ovary, can be removed without affecting mortality. Finally, some vital organs are unaffected by normal organ expression because of an immunological privilege. Immunoprivileged organs are organs that are protected from the blood by a blood-organ barrier and thus not accessible for immunotherapy. Examples of immune-privileged organs are the brain and testicles.
Consequently, therapeutic approaches that inhibit the activity of a 191P4D12 protein are useful for patients who have a cancer that expresses 191P4D12. These therapeutic approaches generally fall into three classes. The first class modulates the function of 191P4D12 as it refers to tumor cell growth leading to inhibition or delay of tumor cell growth or induction of its death. The second class comprises various methods for inhibiting the binding or association of a 191P4D12 protein with its binding partner or with other proteins. The third class comprises a variety of methods for inhibiting the transcription of a 191P4D12 gene or the translation of 191P4D12 mRNA.
[00434] Consequently, cancer patients can be evaluated for the presence and level of expression of 191P4D12, preferably using immunohistochemical assessments of tumor tissue, quantitative imaging of 191P4D12, or other techniques that reliably indicate the presence and degree of expression of 191P4D12. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissues are well known in the art. XIII .) 191P4D12 as a target for antibody-based therapy
[00435] 191P4D12 is an attractive target for antibody-based therapeutic strategies. Several antibody strategies are known in the art to target extracellular and intracellular molecules (see, for example, complement-mediated killing and ADCC as well as the use of intrabodies). Because 191P4D12 is expressed by cancer cells of various lineages relative to the corresponding normal cells, systemic administration of 191P4D12 immunoreactive compositions is prepared, which have excellent sensitivity without toxic, non-specific and/or non-target effects caused by binding of the immunoreactive composition to non-target organs and tissues. Antibodies specifically reactive with 191P4D12 domains are useful to treat cancers expressing 191P4D12 systemically, preferably as antibody-drug conjugates (i.e. ADCs) where the conjugate is with a toxin or therapeutic agent.
[00436] Those of skill in the art understand that antibodies can be used to specifically target and bind to immunogenic molecules, such as an immunogenic region of a 191P4D12 sequence shown in Figure 1. Furthermore, those of skill in the art understand that it is routine to conjugate antibodies to cytotoxic agents (see, for example, Slevers et al. Blood 93:11 3678-3684 (June 1, 1999)). When cytotoxic and/or therapeutic agents are applied directly to cells, such as by conjugating them to specific antibodies for a molecule expressed by that cell (eg, 191P4D12), the cytotoxic agent will exert its known biological effect (i.e., cytotoxicity) in these cells.
A wide variety of compositions and methods for using antibody-cytotoxic agent conjugates to kill cells are known in the art. In the context of cancers, typical methods involve administering to a mammal having a tumor, a biologically effective amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent linked to a targeting agent (e.g., a MAb 191P4D12, of preferably, Ha22-2(2,4)6.1) that binds to an antigen (eg, 191P4D12) expressed, accessible for binding, or located on cell surfaces. A typical embodiment is a method for delivering a cytotoxic and/or therapeutic agent to a cell expressing 191P4D12, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 191P4D12 epitope, and exposing the cell to the antibody-drug conjugate ( ADC). Another illustrative embodiment is a method of treating an individual suspected of suffering from metastatic cancer, comprising a step of parenterally administering to said individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.
[00438] Immunotherapy for cancer using 191P4D12 antibodies can be done according to several approaches that have been successfully employed in the treatment of 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 90:24372444), gastric cancer (Kasprzyk et al., al., 1992, Cancer Res. 52:27712776), 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), 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 therapeutic approaches involve conjugation of naked antibody to a toxin or radioisotope, such as conjugation of Y91 or I131 to anti-CD20 antibodies (eg, Zevalin®, IDEC Pharmaceuticals Corp. or Bexxar®, Coulter Pharmaceuticals) respectively, while others involve the co-administration of antibodies and other therapeutic agents, such as Herceptin® (trastuzu MAb) with paclitaxel (Genentech, Inc.). In a preferred embodiment, the antibodies will be conjugated to a cytotoxic agent, above preferably an auristatin derivative designated MMAE (Seattle Genetics, Inc).
Although 191P4D12 antibody therapy is useful for all stages of cancer, antibody therapy may be particularly suitable in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more cycles of chemotherapy. Alternatively, the antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy may allow the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent 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 chemotherapeutic agents.
191P4D12 monoclonal antibodies that treat the cancers shown in Table I include those that initiate a potent immune response against the tumor or those that are directly cytotoxic. In this sense, 191P4D12 monoclonal antibodies (MAbs) can generate tumor cell lysis by complement-mediated or antibody-dependent cell cytotoxicity (ADCC) mechanisms, which require an intact Fc portion of the immunoglobulin molecule to interact with Fc receptor sites of the effector cell into complement proteins. In addition, 191P4D12 MAbs that exert a direct biological effect on tumor growth are useful for treating cancers that express 191P4D12. The mechanisms by which directly cytotoxic MAbs act include: inhibition of cell growth, modulation of cell differentiation, modulation of tumor angiogenesis factor profiles, and induction of apoptosis. The mechanism(s) by which a particular 191P4D12 MAb exerts an antitumor effect is/are evaluated using any number of in vitro assays that assess cell death, such as ADCC, cell lysis complement-mediated, and so on, as is well known in the art.
Consequently, the preferred monoclonal antibodies used in the therapeutic methods of the invention are those that are fully human and that specifically bind to the target 191P4D12 antigen with high affinity. XIV.) 191P4D12 ADC Cocktails
The therapeutic methods of the invention contemplate the administration of single 191P4D12 ADCs as well as combinations, or cocktails, of different MAbs (i.e., 191P4D12 MAbs or Mabs that bind another protein). Such MAb cocktails may have certain advantages in that they contain MAbs that target different epitopes, exploit different effector mechanisms, or combine directly cytotoxic MAbs with MAbs that rely on immune effector functionality. Such MAbs in combination can exhibit synergistic therapeutic effects. In addition, MAbs 191P4D12 may be administered concurrently with other therapeutic modalities, including, but not limited to, various chemotherapeutic and biological agents, androgen blockers, immune modulators (eg, IL-2, GM-CSF), surgery or radiation . In a preferred embodiment, MAbs 191P4D12 are administered in conjugated form.
191P4D12 ADC formulations are administered via any route capable of delivering the antibodies to a tumor cell. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumoral, intradermal, and the like. Treatment generally involves repeated administration of the 191P4D12 ADC preparation, via an acceptable route of administration, such as intravenous (IV) injection, typically at a dose in the range that includes but is not limited to 0.1, 0, 2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of 10 to 1000 mg of MAb per week are effective and well tolerated.
[00444] Based on clinical experience with Herceptin® (Trastuzumab) in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg/kg of patient body weight IV, followed by weekly doses of approximately 2 mg/ kg IV of the MAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as an infusion of 90 minutes or longer. The periodic maintenance dose is given as an infusion of 30 minutes or more, provided the starting dose has been well tolerated. As understood by those skilled in the art, several factors can influence the optimal dosage regimen in a particular case. Such factors include, for example, the binding affinity and half-life of the MAbs used, the degree of expression of 191P4D12 in the patient, the extent of circulating dispersed 191P4D12 antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic agents or other agents used in combination with the method of treatment of the invention, as well as the health status of a specific patient.
[00445] Optionally, patients should be evaluated for 191P4D12 levels in a given sample (eg, circulating 191P4D12 antigen levels and/or 191P4D12 expressing cells) in order to aid in determining the most effective dosing regimen, etc. Such assessments are also used for monitoring purposes throughout therapy, and are useful for measuring therapeutic success in combination with the assessment of other parameters (eg urine cytology and/or ImmunoCyt levels in bladder cancer therapy , or by analogy, serum PSA levels in prostate cancer therapy).
[00446] An object of the present invention is to provide 191P4D12 ADCs, which inhibit or delay the growth of tumor cells expressing 191P4D12. A further object of this invention is to provide methods for inhibiting angiogenesis and other biological functions and thus reducing tumor growth in mammals, preferably humans, using such 191P4D12 ADCs, and in particular using such combined 191P4D12 ADCs with other immunologically active drugs or treatments. XV.) Combination Therapy
[00447] In one embodiment, there is synergy when tumors, including human tumors, are treated with 191P4D12 ADCs in conjunction with chemotherapy agents or radiation or combinations thereof. In other words, the inhibition of tumor growth by a 191P4D12 ADC is enhanced more than expected when combined with chemotherapy agents or radiation or combinations thereof. Synergy can be shown, for example, by greater inhibition of tumor growth with combined treatment than would be expected from treatment with 191P4D12 ADC alone or the additive effect of treatment with a 191P4D12 ADC and a chemotherapeutic agent or radiation . Preferably, synergy is demonstrated by cancer remission, when remission is not expected from treatment with 191P4D12 ADC or with treatment using an additive combination of a 191P4D12 ADC and a chemotherapeutic agent or radiation.
[00448] The method of inhibiting tumor cell growth using a 191P4D12 ADC and a combination of chemotherapy or radiation, or both, comprises administering the 191P4D12 ADC before, during, or after starting chemotherapy or radiotherapy, as well as any combination of the same (ie, before and during, before and after, during and after, or before, during, and after starting chemotherapy and/or radiotherapy). For example, 191P4D12 ADC is typically administered between 1 and 60 days, preferably between 3 and 40 days, more preferably between 5 and 12 days prior to initiating radiotherapy and/or chemotherapy. However, depending on the treatment protocol and the specific needs of the patient, the method is designed to provide the most effective treatment and, ultimately, to prolong the patient's life.
[00449] The administration of chemotherapeutic agents can be done in a variety of ways, including systemically by parenteral and enteral routes. In one embodiment, the 191P4D12 ADCs and the chemotherapeutic agent are administered as separate molecules. Specific examples of chemotherapeutic agents or chemotherapy include cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarazine , etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleuquin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, alpha hydroxyurea megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, gemcitabine, taxozocine, and same combinations.
[00450] The radiation source, used in combination with a 191P4D12 ADC, can be external or internal to the patient being treated. When the source is external to the patient, the therapy is known as external beam radiotherapy (EBRT). When the radiation source is internal to the patient, the treatment is called brachytherapy (BT).
The therapeutic regimens described above can be further combined with additional cancer treatment agents and/or regimens, e.g., additional chemotherapy, cancer vaccines, signal transduction inhibitors, agents useful in the treatment of abnormal cell growth or cancer, antibodies (for example anti-CTLA-4 antibodies as described in WO/2005/092380 (Pfizer)) or other ligands that inhibit tumor growth by binding to IGF-1R, and cytokines.
When the mammal undergoes additional chemotherapy, the chemotherapeutic agents described above can be used. Additionally, growth factor inhibitors, biological response modifiers, anti-hormonal therapy, selective estrogen receptor modulators (SERMs), angiogenesis inhibitors, and antiandrogens can be used. For example, anti-hormones, for example anti-estrogens like Nolvadex (tamoxifen) or, anti-androgens like Casodex (4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3'-(trifluoromethyl) propionanilide) can be used.
The above therapeutic approaches can be combined with any of a wide variety of surgical, chemotherapy or radiotherapy regimens. The therapeutic approaches of the invention may allow the use of reduced dosages of chemotherapy (or other therapies) and/or less frequent administration, an advantage for all patients and particularly for those who do not tolerate the toxicity of the chemotherapeutic agent well. Kits/articles of manufacture
[00454] For use in the laboratory, prognostic, prophylactic, diagnostic and therapeutic applications described herein, kits are within the scope of the invention. Such kits may comprise a vehicle, a package, or container that is compartmentalized to receive one or more containers, such as vials, tubes, and the like, each of the containers comprising one of the separate elements to be used in the method, along with a label or package insert comprising instructions for use, such as a use described herein. For example, the recipient(s) may comprise an antibody which is or may be detectably labeled. Kits can comprise a container comprising a drug unit. The kit may include all or part of the amino acid sequences in Figure 2, or Figure 3 or analogs thereof, or a nucleic acid molecule encoding such amino acid sequences.
[00455] The kit of the invention will typically comprise the container described above and one or more other containers associated with it, which comprise materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; labels for the vehicle, packaging, container, bottle and/or tube listing contents and/or instructions for use, and package inserts with instructions for use.
[00456] A label may be present on or with the container to indicate that the composition is used for a specific therapy or non-therapeutic application, such as a prognostic, prophylactic, diagnostic or laboratory application, and may also indicate instructions for in vivo or in vitro use, such as those described herein. Instructions and other information may also be included on a package insert or label included with or on the kit. The label can be on or associated with the container. A label can be on top of a container when letters, numbers, or other characters that make up the label are molded or engraved on the container itself; a label may be associated with a container when it is present within a receptacle or carrier that also holds the container, for example, as a package insert. The label may indicate that a composition is used to diagnose, treat, prevent or predict a condition, such as cancer of a tissue shown in Table I.
[00457] The terms "kit" and "article of manufacture" may be used interchangeably.
[00458] In another embodiment of the invention, an article of manufacture is provided containing compositions, such as antibody(s), or antibody-drug conjugates (ADCs) for example, materials useful for the diagnosis, prognosis, prophylaxis and/or treatment of cancers of fabrics, such as those shown in Table I. The article of manufacture typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, such as glass, metal or plastic. The container may hold amino acid sequence(s), small molecule(s), nucleic acid sequence(s), cell population(s) and/or antibody(s). In another embodiment, a container comprises an antibody, a binding fragment thereof, or a specific binding protein for use in assessing 191P4D12 protein expression in cells and tissues, or for laboratory, prognostic, diagnostic, prophylactic and therapeutic purposes. relevant; indications and/or instructions for use may be included on or with such container, as well as reagents and other compositions or tools used for these purposes.
[00459] The container may alternatively hold a composition that is effective to treat, diagnose, predict or prevent a condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a bottle that has a cap pierceable by a hypodermic injection needle). The active agents in the composition can be an antibody capable of specifically binding 191P4D12 or an antibody-drug conjugate that specifically binds 191P4D12.
The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution and/or dextrose solution. It may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with directions and/or instructions for use. Examples:
[00461] Various aspects of the invention are further described and illustrated by means of the various examples that follow, none of which are intended to limit the scope of the invention. Example 1 The 191P4D12 antigen
The 191P4D12 gene sequence was discovered using suppressive subtractive hybridization (SSH) methods known in the art. The 223 bp 191P4D12 SSH sequence was identified from negative (-) bladder tumor cDNAs derived from a pool of nine (9) normal tissues using standard methods. A full-length cDNA clone for 191P4D12 was isolated from a bladder cancer cDNA library. The cDNA is 3464 bp long and encodes a 510 amino acid ORF (see Figure 1). The 191P4D12 gene shows homology to the Nectin-4 gene. For additional reference, see, US2004/0083497 (Agensys, Inc., Santa Monica, CA) and PCT publication WO2004/016799 (Agensys, Inc., Santa Monica, CA). For exemplary modalities of the 191P4D12 antigen, see Figure 1. Example 2 Generation of monoclonal antibodies (MAbs) 191P4D12
In one embodiment, therapeutic monoclonal antibodies ("MAbs") to 191P4D12 and 191P4D12 variants comprise those that react with specific epitopes for each protein or are specific for sequences in common between the variants that could bind, internalize, perturb or modulate the biological function of 191P4D12 or 191P4D12 variants, for example, those that would disrupt interaction with ligands, substrates, and binding partners. Immunogens for generating such MAbs include those designed to encode or contain extracellular domains or the entire 191P4D12 protein sequence, regions predicted to contain functional motifs, and regions of 191P4D12 protein variants predicted to be antigenic from computer analysis of the amino acid sequence. Immunogens include recombinant peptides and proteins such as tag5-191P4D12, a purified mammalian cell-derived His-tagged protein. In addition, cells engineered to express high levels of 191P4D12, such as RAT1-191P4D12 or 300.19-191P4D12, are used to immunize mice.
MAbs for 191P4D12 were generated using XenoMouse® technology (Amgem Fremont) in which the murine heavy and kappa light chain loci were inactivated and most of the human heavy chain and kappa light chain immunoglobulin loci were inserted. The MAb designated Ha22-2(2,4)6.1 was generated from the immunization of human Y1 producing XenoMice mice with pTag5/mychis-191P4D12 (amino acids 23 to 351).
MAb 191P4D12 Ha22-2(2,4)6.1 specifically binds to pTag5/mychis-191P4D12 protein by ELISA as well as to recombinant cells expressing 191P4D12 and multiple cancer cell lines expressing 191P4D12.
The hybridoma producing an antibody designated Ha22-2(2,4)6.1 was shipped (via Federal Express) to the American Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108 on August 18, 2010 and the access number PTA-11267 was assigned.
The DNA coding sequences for MAb 191P4D12 Ha22-2(2,4)6.1 were determined after isolation of mRNA from the respective hybridoma cells with Trizol reagent (Life Technologies, Gibco BRL).
The anti-191P4D12 Ha22-2(2,4)6.1l heavy and light chain variable nucleic acid sequences were sequenced from the hybridoma cells using the following protocol. Hybridoma cells secreting Ha22-2(2,4)6.1 were lysed with Trizol reagent (Life Technologies, Gibco BRL). Total RNA was purified and quantified. First strand cDNAs were generated from total RNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system. First strand cDNA was amplified using human immunoglobulin variable heavy chain primers and human immunoglobulin variable light chain primers. PCR products were sequenced and heavy and light chain variable regions were determined.
The nucleic acid and amino acid sequences of the heavy and light chain variable regions are mentioned in figure 2 and figure 3. The alignment of MAb Ha22-2(2,4)6.1 to the human Ig germline is shown in Figure 4A-4B.
[00470] Example 3 Expression of Ha22-2(2,4)6.1 using recombinant DNA methods
To express the Mab Ha22-2(2,4)6.1 recombinantly in the transfected cells, the heavy and light chain variable sequences of the MAb Ha22-2(2,4)6.1 were cloned upstream of the constant regions of Human heavy chain IgG1 and human light chain IgK, respectively. The complete human heavy chain and light chain cassettes of MAb Ha22-2(2,4)6.1 were cloned downstream of the CMV promoter/enhancer in a cloning vector. A polyadenylation site was included downstream of the MAb coding sequence. Constructs expressing recombinant Mab Ha22-2(2,4)6.1 were transfected into CHO cells. MAb Ha22-2(2,4)6.1 secreted from the recombinant cells was evaluated for binding to cell surface 191P4D12 by flow cytometry (Figure 5A). RAT-control and RAT-191P4D12 cells were stained with MAb Ha22-2(2,4)6.1 from hybridoma or from CHO cells transfected with the Ha22-2(2,4)6.1 heavy and light chain vector constructs. Binding was detected by flow cytometry.
The results show that recombinantly expressed Ha22-2(2,4)6.1 expressed in CHO cells binds 191P4D12 in a similar manner to Ha22-2(2,4)6.1 purified from the hybridoma. MAb Ha22-2(2,4)6.1 secreted from the recombinant cells was also evaluated for binding to recombinant 191P4D12 protein by ELISA. As shown in Figure 5B, the binding of Ha22-2(2,4)6.1 to the 191P4D12 protein was identical between MAb material derived from CHO and from hybridoma cells. Example 4 Antibody-drug conjugation of MAb Ha22-2(2,4)6.1
MAb Ha22-2(2,4)6.1 (figure 2) was conjugated to an auristatin derivative designated MMAE (formula XI) using a vc linker (Val-Cit) described herein to create the antibody-drug conjugate ( ADC) of the invention designated Ha22-2(2,4)6.1vcMMAE using the following protocols. Conjugation of the vc linker (Val-Cit) to the MMAE (Seattle Genetics, Inc., Seattle, WA) was completed using the general method shown in Table IV to create the cytotoxic vcMMAE (see, US Patent No. 7,659,241).
Next, the antibody-drug conjugate (ADC) of the invention, designated Ha22-2(2,4)6.1vcMMAE, was made using the following protocols.
Briefly, a 15 mg/ml solution of Ha22-2(2,4)6.1MAb in 10 mM nitrate at pH 5.0, 1% sorbitol, 3% L-arginine is added with one volume of 20% 0.1M TrisCl pH 8.4, 25mM EDTA and 750mM NaCl to adjust the solution pH to 7.5, 5mM EDTA and 150mM sodium chloride. The mAb is then partially reduced by adding 2.3 molar equivalents of TCEP (relative to moles of MAb) and then stirred at 37°C for 2 hours. The partially reduced MAb solution is then cooled to 5°C and 4.4 molar equivalents of vcMMAE (relative to moles of antibody) are added as a 6% (v/v) DMSO solution. The mixture is stirred for 60 minutes at 5°C and then for an additional 15 minutes after the addition of 1 molar equivalent of N-acetylcysteine relative to vcMMAE. Excess suppressed vcMMAE and other reaction components are removed by ultrafiltration/diafiltration of the antibody-drug conjugate (ADC) with 10 volumes of 20 mM histidine, pH 6.0.
[00476] The resulting antibody-drug conjugate (ADC) is designated Ha22-2(2,4)6.1vcMMAE and has the following formula:

[00477] where the MAb is Ha22-2(2,4)6.1 (figure 2 and figure 3) and p is from 1 to 8. The p value of the antibody-drug conjugate shown in this example was about 3.8 . Example 5 Characterization of Ha22-2(2,4)6.1vcMMAE
[00478] Antibody-drug conjugates that bind to 191P4D12 were generated using the procedures presented in the example titled "Antibody-drug conjugation of MAb Ha22-2(2,4)6.1" and were selected, identified, and characterized using a combination of assays known in the art. A. FACS Affinity Determination
Ha22-2(2,4)6.1vcMMAE was tested for its binding affinity to 191P4D12 expressed on the surface of PC3-human-191P4D12, PC3-cynomolgus-191P4D12, and PC3-mouse-191P4D12 cells respectively. Briefly, eleven (11) dilutions of Ha22-2(2,4)6.1vcMMAE were incubated with each cell type (50,000 cells per well) overnight at 4°C at a final concentration of 160 nM at 0.011 nM. At the end of the incubation, cells are washed and incubated with anti-hIgG-PE detection antibody for 45 min at 4°C. After washing off unbound detection antibodies, cells are analyzed by FACS. The mean fluorescence intensity (MFI) values were obtained as mentioned in Figures 6 to 8. The MFI values were entered into the Graphpad Prism program and analyzed using an equation for binding to a site (hyperbola) of Y=Bmax*X /(Kd+X) to generate the Ha22-2(2,4)6.1vcMMAE saturation curves shown also in figures 6 to 8, respectively. Bmax is the MFI value at the maximum binding of Ha22-2(2,4)6.1vcMMAE to 191P4D12; Kd is the binding affinity of Ha22-2(2,4)6.1vcMMAE which is the concentration of Ha22-2(2,4)6.1vcMMAE required to achieve half maximal binding.
The calculated affinity (Kd) of Ha22-2(2,4)6.1vcMMAE to 191P4D12 expressed on the surface of PC3-human-191P4D12, PC3-cynomolgus-191P4D12, and PC3-mouse-191P4D12 cells, respectively, is 0.69 nM (Figure 6), 0.34 nM (Figure 7), and 1.6 nM (Figure 8). B. Determination of affinity by SPR
The affinity of MAb Ha22-2(2,4)6.1 and Ha22-2(2,4)6.1vcMMAE to purified recombinant 191P4D12 (ECD amino acids 1 to 348) was done by surface plasmon resonance (SPR) (BIAcore). Briefly, goat anti-human FcY polyclonal antibodies (Jackson Immuno Research Labs, Inc.) were covalently immobilized onto the surface of a CM5 sensor chip (Biacore). Purified Ha22-2(2,4)6.1 MAb or Ha22-2(2,4)6.1vcMMAE were then captured on the surface of said chip. On average, approximately 300 RUs of test MAb Ha22-2(2,4)6.1 or Ha22-2(2,4)6.1vcMMAE were captured in each cycle. Subsequently, a series of five (5) to six (6) dilutions of recombinant 191P4D12 (ECD amino acids 1 to 348) in the range of 1 nM to 100 nM was injected onto such a surface to generate binding curves (sensograms) which were processed and globally adjusted to a 1:1 interaction model using the BIAevaluation 3.2 and CLAMP programs (Myszka and Morton, 1998) (figure 22). Table V summarizes the association and dissociation rate constants as well as the affinities of MAb Ha22-2(2,4)6.1 and Ha22-2(2,4)6.1vcMMAE to recombinant 191P4D12 (ECD amino acids 1 to 348). C. Domain mapping of MAb Ha22-2(2,4)6.1
To map the binding site of MAb Ha22-2(2,4)6.1 to a specific domain of the 191P4D12 protein, several recombinant Rat1(E) cell lines expressing such domains (or a combination thereof) were generated (table SAW). The binding of Ha22-2(2,4)6.1 to the cell surface was assessed by FACS using standard protocols. As shown in Figure 10, MAb Ha22-2(2,4)6.1 binds to cells expressing the VC1 domain as well as wild-type 191P4D12, but not to cells expressing the C1C2 domain. Additionally, another MAb 191P4D12 titled Ha22-8e6.1 recognizes the C1C2 domain of 191P4D12 on the cell surface, but not the VC1 domain. This suggests that the binding site for MAb Ha22-2(2,4)6.1 is located in the 1 to 147 aa domain of 191P4D12, but that not all MAbs that bind to 191P4D12 recognize this domain.
[00483] To further corroborate the results presented in Figure 10, a Western Blot analysis was performed. Briefly, the entire extracellular portion of 191P4D12 (full length) as well as the specific domains shown in Table VI were expressed in 293T cells as purified, murine Fc fusion proteins. Goat mouse anti-HRP antibody was used as a control. As shown in Figure 11, when separated on SDS-PAGE (unreduced) and labeled with Ha22-2(2,4)6.1-biotin followed by streptavidin-HRP, the bands that correspond to the full-length 191P4D12 fusion constructs ( column 1), V (column 2) and VC1 (column 3), but not the fusion construct of C1C2 (column 4) are recognized. This further suggests that the binding epitope for MAb Ha22-2(2,4)6.1 is located within the 1 to 147 aa domain of 191P4D12. Example 6 Ha22-2(2,4)6.1vcMMAE-mediated cell cytotoxicity
The ability of Ha22-2(2,4)6.1vcMMAE to mediate 191P4D12-dependent cytotoxicity was evaluated in PC3 cells engineered to express human 191P4D12, cynomolgus 191P4D12 and rat 191P4D12. Briefly, PC3-Neo, PC3-human-191P4D12, PC3-cynomolgus-191P4D12 or PC3-mouse-191P4D12 cells (1500 cells/well) were seeded in a 96-well plate on day 1. The next day an equal volume of medium containing the indicated concentration of Ha22-2(2,4) 6.1vcMMAE or a control MAb conjugated to vcMMAE (ie, control-vcMMAE) was added to each well. Cells were allowed to incubate for 4 days at 37 degrees C. At the end of the incubation period, Alamar blue was added to each well and the incubation was continued for a further 4 hours. The resulting fluorescence was detected using a Biotek plate reader with an excitation wavelength of 620 nM and an emission wavelength of 540 nM.
The results in Figure 9A-9D show that Ha22-2(2,4)6.1vcMMAE mediated cytotoxicity in PC3-human-191P4D12 (figure 9A), PC3-cynomolgus-191P4D12 (figure 9B), and PC3- cells mouse-191P4D12 (Fig. 9C) while a control human IgG conjugated to vcMMAE had no effect. The specificity of Ha22-2(2,4)6.1vcMMAE was further demonstrated by the lack of toxicity for PC3-Neo cells that do not express 191P4D12 (Fig. 9D). Thus, these results indicate that Ha22-2(2,4)6.1vcMMAE can selectively release a cytotoxic drug to cells expressing 191P4D12, leading to its death.
[00486] Example 7 Ha22-2(2,4)6.1vcMMAE Inhibits Tumor Growth In Vivo
The significant expression of 191P4D12 on the cell surface of tumor tissues, together with its restrictive expression in normal tissues makes 191P4D12 a good target for antibody therapy and similarly, ADC therapy. In this way, the therapeutic efficacy of Ha22-2(2,4)6.1vcMMAE in xenograft mouse models of human bladder, lung, breast, and pancreatic cancer is evaluated.
[00488] The efficacy of antibody-drug conjugate on tumor growth and metastasis formation is studied in mouse cancer xenograft models (eg, subcutaneously and orthotopically).
Subcutaneous (s.c.) tumors are generated by injecting 5 x 104-106 cancer cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) into the right flank of male SCID mice. To test the effectiveness of ADC on tumor formation, ADC injections are started on the same day as tumor cell injections. As a control, mice are injected with purified human IgG or PBS; or a purified MAb that recognizes an irrelevant antigen not expressed on human cells. In preliminary studies, no difference is found between control IgG or PBS in tumor growth. Tumor sizes are determined by caliper measurements, and tumor volume is calculated as width2 x length/2, with width being the smallest dimension and length being the largest dimension. Mice with subcutaneous tumors larger than 1.5 cm in diameter are sacrificed.
Ovarian tumors often metastasize and grow within the peritoneal cavity. Consequently, intraperitoneal growth of ovarian tumors in mice is accomplished by injecting 2 million cells directly into the peritoneum of female mice. The mice are monitored for general health, physical activity, and appearance until almost time of death. At the time of sacrifice, the peritoneal cavity can be examined to determine tumor burden and the lungs are collected to assess for metastases to distant sites. Alternatively, death can be used as an outcome. Mice are then segregated into groups for appropriate treatments, with 191P4D12 or control MAbs being injected i.p.
[00491] An advantage of xenograft cancer models is the ability to study neovascularization and angiogenesis. Tumor growth is partially dependent on the development of new blood vessels. Although the developing capillary system and blood network are of host origin, the initiation and architecture of the neovasculature is 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). The effect of antibody and small molecules on neovascularization is studied according to procedures known in the art, such as by IHC analysis of tumor tissues and their surrounding environment.
[00492] Ha22-2(2,4)6.1ADC inhibits the formation of lung, bladder, breast, and pancreatic cancer xenografts. These results indicate the usefulness of Ha22-2(2,4)6.1ADC in the treatment of local and advanced stages of cancer, and preferably the cancers shown in Table I. 191P4D12 ADCs:
[00493] Monoclonal antibodies were raised against 191P4D12, as described in the example entitled "Generation of the 191P4D12 monoclonal antibodies (MAbs)." Additionally, the MAbs are conjugated to a toxin as described in the example entitled "Antibody drug conjugation of MAb Ha22-2(2,4)6.1" to form Ha22-2(2,4)6.1vcMMAE. Ha22-2(2,4)6.1vc MMAE is characterized by FACS, and other methods known in the art to determine its ability to bind 191P4D12. Cell lines and xenografts:
BT-483 and HPAC cells are maintained in DMEM, supplemented with L-glutamine and 10% FBS, as known in the art. AG-B8, AG-Panc4, AG-Panc2, AG-B1, AG-L4, and AG-Panc3 xenografts are maintained by serial propagation in SCID mice. Evaluation of MAb Ha22-2(2,4)6.1vcMMAE in the model of subcutaneous tumor formation of human AG-L4 lung cancer xenograft in SCID mice.
[00495] In this experiment, the AG-L4 patient-derived lung cancer xenograft was maintained by serial passage in SCID mice. Stock tumors were collected in a sterile manner and enzymatically digested in single cell suspensions. Two (2) million cells were implanted in the flank of individual SCID mice. The animals were then randomly assigned to seven groups: six (6) groups treated with antibody 191P4D12 and one group with control antibody H3-1.10.1.2 (n=10). All antibodies were administered intraperitoneally at 750 μg/animal twice a week until the end of the study. Tumor growth was monitored using caliper measurements every 3 to 4 days. Tumor volume was calculated as width2 x length/2, where width is the smallest dimension and length is the largest dimension.
[00496] The results show that MAb 191P4D12 did not significantly inhibit tumor growth in AG-L4 human lung cancer xenograft in SCID mice. Additionally, other MAbs 191P4D12 were used in this study. Results are not shown. (figure 12). Evaluation of MAb Ha22-2(2,4)6.1 in the HPAC human pancreatic cancer xenograft subcutaneous tumor formation model in SCID mice.
[00497] In another experiment, human pancreatic cancer HPAC cells (2.0 million/mouse) were injected into the flank of individual SCID mice. The animals were then randomly assigned to eight groups: seven (7) groups treated with 191P4D12 antibody and one group with control antibody H3-1.4.1.2 (n=10). All antibodies were administered intraperitoneally at 500 μg/animal twice a week until the end of the study. Tumor growth was monitored using caliper measurements every 3 to 4 days. Tumor volume was calculated as width2 x length/2, where width is the smallest dimension and length is the largest dimension.
[00498] The results show that MAb 191P4D12 did not inhibit tumor growth in a human pancreatic xenograft in SCID mice when compared to the control antibody. Additionally, other MAbs 191P4D12 were used in this study. Results are not shown. (figure 13).
[00499] Evaluation of MAb Ha22-2(2,4)6.1 in the AG-Panc3 human pancreatic cancer xenograft subcutaneous tumor formation model in SCID mice.
[00500] In another experiment, the AG-Panc3 patient-derived pancreatic cancer xenograft was maintained by serial passages in SCID mice. Stock tumors were collected in a sterile manner and crushed into 1 mm3 pieces. Six pieces were implanted in the flank of individual SCID mice. The animals were then randomly assigned to the following cohorts (n=10): two (2) groups treated with MAb 191P4D12 and one group with control antibody H3-1.4.1.2. All antibodies were administered intraperitoneally at 500 μg/animal twice a week until the end of the study. Tumor growth was monitored using caliper measurements every 3 to 4 days. Tumor volume was calculated as width2 x length/2, where width is the smallest dimension and length is the largest dimension.
[00501] The results show that MAb 191P4D12 did not inhibit tumor growth in a human pancreatic xenograft in SCID mice when compared to the control antibody. Additionally, other MAbs 191P4D12 were used in this study. Results are not shown. (figure 14). Efficacy of Ha22-2(2,4)6.1-vcMMAE in AG-L4 established subcutaneous human lung cancer xenograft in SCID mice.
[00502] In another experiment, the AG-L13 patient-derived lung cancer xenograft was maintained by serial passage in SCID mice. Stock tumors were collected in a sterile manner and crushed into 1 mm3 pieces. Six (6) pieces were implanted in the flank of individual SCID mice. Tumors were allowed to grow without treatment until they reached an approximate volume of 200 mm3. Ha22-2(2,4)6.1vcMMAE and control ADC were administered at 10 mg/kg every seven (7) days for two doses by intravenous bolus injection. The amount of ADC administered was based on each animal's individual body weight obtained immediately prior to dosing. Tumor growth was monitored using caliper measurements every 3 to 4 days. Tumor volume was calculated as width2 x length/2, where width is the smallest dimension and length is the largest dimension.
[00503] The results show that Ha22-2(2,4)6.1-vcMMAE treatment significantly inhibited the growth of subcutaneously implanted AG-L4 lung cancer xenografts in nude mice compared to the control ADC. Additionally, other MAbs 191P4D12 were used in this study. Results are not shown. (figure 15). Efficacy of Ha22-2(2,4)6.1-vcMMAE in established BT-483 subcutaneous human breast cancer xenograft in SCID mice.
[00504] In this experiment, BT-483 human breast cancer cells were used to generate stock xenografts, which were maintained by serial passage in SCID mice. Stock tumors were collected in a sterile manner and crushed into 1 mm3 pieces. Six (6) pieces were implanted in the flank of individual SCID mice. Tumors were allowed to grow without treatment until they reached an approximate volume of 100 mm3. Ha22-2(2,4)6.1vcMMAE and control ADC were administered at 5 mg/kg every four (4) days for four (4) doses by intravenous bolus injection. The amount of ADC administered was based on each animal's individual body weight obtained immediately prior to dosing. Tumor growth was monitored using caliper measurements every 3 to 4 days. Tumor volume was calculated as width2 x length/2, where width is the smallest dimension and length is the largest dimension.
[00505] The results show that Ha22-2(2,4)6.1-vcMMAE treatment significantly inhibited the growth of BT-483 breast tumor xenografts implanted subcutaneously in SCID mice compared to the control ADC. Additionally, other MAbs 191P4D12 were used in this study. Results are not shown. (figure 16). Efficacy of Ha22-2(2,4)6.1-vcMMAE in AG-B1 established subcutaneous human bladder cancer xenograft in SCID mice.
[00506] In another experiment, the AG-B1 patient-derived bladder cancer xenograft was maintained by serial passage in SCID mice. Stock tumors were collected in a sterile manner and crushed into 1 mm3 pieces. Six (6) pieces were implanted in the flank of individual SCID mice. Tumors were allowed to grow without treatment until they reached an approximate volume of 230 mm3. Ha22-2(2,4)6.1vcMMAE and control ADC were administered at 4 mg/kg once by intravenous bolus injection. The amount of ADC administered was based on each animal's individual body weight obtained immediately prior to dosing. Tumor growth was monitored using caliper measurements every 3 to 4 days. Tumor volume was calculated as width2 x length/2, where width is the smallest dimension and length is the largest dimension.
[00507] The results show that Ha22-2(2,4)6.1-vcMMAE treatment significantly inhibited the growth of AG-B1 bladder cancer xenografts compared to control ADC. Additionally, other MAbs 191P4D12 were used in this study. Results are not shown. (figure 17). Efficacy of Ha22-2(2,4)6.1-vcMMAE in AG-Panc2 established subcutaneous human pancreatic cancer xenograft in SCID mice.
[00508] In another experiment, the AG-Panc2 patient-derived pancreatic cancer xenograft was maintained by serial passages in SCID mice. Stock tumors were collected in a sterile manner and crushed into 1 mm3 pieces. Five (5) pieces were implanted in the flank of individual SCID mice. Tumors were allowed to grow without treatment until they reached an approximate volume of 100 mm3. Ha22-2(2,4)6.1vcMMAE and control ADC were administered at 5 mg/kg every four (4) days for four (4) doses by intravenous bolus injection. The amount of ADC administered was based on each animal's individual body weight obtained immediately prior to dosing. Tumor growth was monitored using caliper measurements every 3 to 4 days. Tumor volume was calculated as width2 x length/2, where width is the smallest dimension and length is the largest dimension.
[00509] The results show that Ha22-2(2,4)6.1-vcMMAE treatment significantly inhibited the growth of AG-Panc2 pancreatic cancer xenografts compared to the control ADC. Additionally, other MAbs 191P4D12 were used in this study. Results are not shown. (figure 18). Efficacy of Ha22-2(2,4)6.1-vcMMAE in AG-Panc4 established subcutaneous human pancreatic cancer xenograft in SCID mice.
[00510] In another experiment, the AG-Panc4 patient-derived pancreatic cancer xenograft was maintained by serial passages in SCID mice. Stock tumors were collected in a sterile manner and crushed into 1 mm3 pieces. Six (6) pieces were implanted in the flank of individual SCID mice. Ha22-2(2,4)6.1vcMMAE and control ADC were administered at 5 mg/kg every seven (7) days for three doses by intravenous bolus injection. The amount of ADC administered was based on each animal's individual body weight obtained immediately prior to dosing. Tumor growth was monitored using caliper measurements every 3 to 4 days. Tumor volume was calculated as width2 x length/2, where width is the smallest dimension and length is the largest dimension.
[00511] The results show that Ha22-2(2,4)6.1-vcMMAE treatment significantly inhibited the growth of AG-Panc4 pancreatic cancer xenografts compared to control ADC. Additionally, other MAbs 191P4D12 were used in this study. Results are not shown. (figure 19).
[00512] Efficacy of Ha22-2(2,4)6.1-vcMMAE at comparative dosage in AG-B8 established subcutaneous human bladder cancer xenograft in SCID mice.
[00513] In this experiment, the AG-B8 patient-derived bladder cancer xenograft was maintained by serial passage in SCID mice. Stock tumors were collected in a sterile manner and crushed into 1 mm3 pieces. Six (6) pieces were implanted in the flank of individual SCID mice. Tumors were allowed to grow without treatment until they reached an approximate volume of 200 mm3. The animals were then randomly assigned to the following three cohorts (n=6): two (2) groups treated with Ha22-2(2,4)6.1-vcMMAE and one control group VCD37-5ce5p-vcMMAE ADC. Ha22-2(2,4)6.1-vcMMAE was given at 5 mg/kg or 10 mg/kg and control ADC was given at 5 mg/kg. All ADCs were administered once by intravenous bolus injection. The amount of ADC administered was based on each animal's individual body weight obtained immediately prior to dosing. Tumor growth was monitored using caliper measurements every 3 to 4 days. Tumor volume was calculated as width2 x length/2, where width is the smallest dimension and length is the largest dimension.
[00514] The results show that treatment with Ha22-2(2,4)6.1vc MMAE at 10mg/kg inhibited the growth of AG-B8 bladder cancer xenografts compared to Ha22-2(2,4)6.1vcMMAE at 5mg/kg. (figure 20). Conclusion
[00515] In summary, Figures 12 to 20, show that the 191P4D12 ADC called Ha22-2(2,4)6.1vcMMAE significantly inhibited the growth of tumor cells expressing 191P4D12 when compared to the control ADCs. Thus, Ha22-2(2,4)6.1vcMMAE can be used for therapeutic purposes to treat and manage the cancers presented in Table I. Example 8 Human clinical trials for the treatment and diagnosis of human carcinomas through the use of 191P4D12 ADCs
191P4D12 ADCs are used in accordance with the present invention, which specifically bind to 191P4D12, and are used in the treatment of certain tumors, preferably those mentioned in table I. With respect to each of these indications, two clinical approaches are successfully pursued.
[00517] I.) Adjunct Therapy: In adjunct therapy, patients are treated with 191P4D12 ADCs in combination with a chemotherapeutic or antineoplastic agent and/or radiation therapy or a combination thereof. Primary cancer targets, such as those mentioned in Table I, are treated under standard protocols by adding 191P4D12 ADCs to standard first- and second-line therapy. The protocol designs address efficacy, as assessed by the following examples, which include, but are not limited to, a reduction in tumor mass of primary or metastatic lesions, increased progression-free survival, overall survival, improved patient health, disease stabilization, as well as the ability to reduce the usual doses of standard chemotherapy and other biological agents. These dosage reductions allow for additional and/or prolonged therapy by reducing the dose-related toxicity of the chemotherapeutic or biological agent. 191P4D12 ADCs are used in several adjunct clinical trials in combination with chemotherapeutic or antineoplastic agents.
[00518] II.) Monotherapy: Regarding the use of 191P4D12 ADCs in tumor monotherapy, 191P4D12 ADCs are administered to patients without a chemotherapeutic or antineoplastic agent. In one modality, monotherapy is conducted clinically in end-stage cancer patients with extensive metastatic disease. The protocol designs address efficacy, as assessed by the following examples, which include, but are not limited to, a reduction in tumor mass of primary or metastatic lesions, increased progression-free survival, overall survival, improved patient health, disease stabilization, as well as the ability to reduce the usual doses of standard chemotherapy and other biological agents.
[00519] Dosage
[00520] Dosing regimens can be adjusted to provide the desired optimal response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be reduced or increased proportionately as indicated by the requirements of the therapeutic situation. It is specifically advantageous to formulate the parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form for use in the present invention refers to physically distinct units suitable as unitary dosages for the mammalian subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the antibody and/or ADC and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of composition of such active compound for the treatment of sensitivity in individuals.
An exemplary, non-limiting range for a therapeutically effective amount of a 191P4D12 ADC administered in combination according to the 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 exemplary non-limiting ranges are, for example, about 0.5 to about 5 mg/kg, or for example, about 0.8 to about 5 mg/kg, or, for example, about 1 to about 7.5 mg/kg. The high-dose embodiment of the invention relates to a dosage of more than 10 mg/kg. It should be noted that dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It should further be understood that for any specific individual, specific dosage regimens must be adjusted over time in accordance with the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges herein described are for exemplary purposes only and are not intended to limit the scope or practice of the claimed composition. Clinical Development Plan (CDP)
CDP follows and develops treatments of 191P4D12 ADCs with regard to adjunct therapy or monotherapy. Tests initially demonstrate safety and later confirm efficacy in repeated doses. Trials are open-label comparing standard chemotherapy with standard therapy plus 191P4D12 ADCs. As will be understood, a non-limiting criterion that can be used with regard to patient inclusion is the expression levels of 191P4D12 in their tumors, as determined by biopsy.
As with any protein or antibody infusion based therapeutic agent safety concerns are primarily related to (i) cytokine release syndrome ie hypotension, fever, agitation, chills; (ii) development of an immunogenic response to the material (ie, development of human antibodies by the patient against the antibody therapeutic agent, or HAMA response); and, (iii) toxicity to normal cells expressing 191P4D12. Standard testing and tracking is used to monitor each of these security concerns. 191P4D12 ADCs have been found to be safe for human administration. Example 9 Detection of 191P4D12 protein in cancer patient specimens by IHC.
[00524] The expression of 191P4D12 protein by immunohistochemistry was tested in tumor specimens from patients with (i) bladder, (ii) breast, (iii) pancreatic, (iv) lung, (v) ovary, (vi) cancer esophagus, and (vii) from head and neck patients. Briefly, tissues embedded in paraffin wax and fixed in formalin were cut into sections of four (4) microns and mounted on glass slides. Sections were dewaxed, rehydrated and treated with EDTA antigen retrieval solution (Biogenex, San Ramon, CA) in the EZ-Retriever microwave (Biogenex, San Ramon, CA) for 30 minutes at 95°C. The sections were then treated with a 3% hydrogen peroxide solution to inactivate endogenous peroxidase activity. The serum-free protein block (Dako, Carpenteria, CA) was used to inhibit non-specific binding prior to incubation with anti-191P4D12 mouse monoclonal antibody or an isotypic control. Subsequently, sections were treated with the Super Sensitive® Polymer-horseradish peroxidase (HRP) detection system which consists of an incubation in Super Enhancer® reagent followed by an incubation with polymer-secondary antibody HRP conjugate (BioGenex, San Ramon, CA ). The sections were then developed using the DAB kit (BioGenex, San Ramon, CA). Nuclei were stained using hematoxylin, and analyzed by brightfield microscopy. Specific labeling was detected in the patient specimens using the immunoreactive antibody 191P4D12, as indicated by the brown stain. (See, Figure 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 specimen. (See Figure 21(B), 21(D), 21(F), 21(H), 21(J), 21(L), and 21(N)).
[00525] The results show expression of 191P4D12 in tumor cells from tissues of patients with cancer of the bladder, breast, pancreatic, lung, ovary, esophagus, and head and neck. These results indicate that 191P4D12 is expressed in human cancers and that antibodies directed against this antigen and the antibody-drug conjugate designated Ha22-2(2,4)6.1vcMMAE) are useful for diagnostic and therapeutic purposes. (figure 21). Example 10 Determination of the binding epitope of MAb Ha22-2(2,4)6.1
[00526] The 191P4D12 protein of human, cynomolgus, rat and murine origin was recombinantly overexpressed in a PC3 cell line to determine the cross-reactivity of Ha22-2(2,4)6.1 to these orthologs. Ha22-2(2,4)6.1 has been shown to cross-react strongly with the cynomolgus and rat orthologs of 191P4D12 (figure 23). EC50 binding values are shown in table VII. Binding of Ha22-2(2,4)6.1 to the murine ortholog shows a significant reduction in the EC50 binding value, which shows that important amino acid substitutions in domain V (compared to human and rat sequences) affected the affinity of Ha22 - 2(2,4)6.1 to 191P4D12.
Table VIII shows the alignment of protein sequences from aa 1 to 180 of 191P4D12 orthologs containing the V domain. Only two amino acids in the rat ortholog sequence, Thr-75 and Ser-90, are substituted into the murine ortholog sequence by Ile and Asn, respectively (marked in bold text). It should be noted that the corresponding amino acids in the human sequence are Ala-76 and Ser-91. To determine whether these amino acids comprise the Ha22-2(2,4)6.1 binding epitope, several mutant constructs of 191P4D12 and their murine orthologs were generated and expressed in PC3 cells (Table IX). "Murine" amino acids were introduced instead of mutagenesis by standard alanine substitution in the human sequence and vice versa in the mouse sequence.
[00528] It has been shown that the Ser-91 to Asn mutation in 191P4D12 greatly impairs the binding of Ha22-2(2,4)6.1, confirming that this amino acid, Ser-91, is essential for binding and must understand the epitope recognized by MAb Ha22-2(2,4)6.1. An additional Ala mutation at position 76 (double mutant A76I, S91N) was also introduced into 191P4D12. The binding of Ha22-2(2,4)6.1 to the A76I double mutant, S91N was shown to be very similar to the murine ortholog binding (Figure 24). Conversely, mutating Asn-90 in the murine sequence to Ser dramatically improves the binding of Ha22-2(2,4)6.1 to the mutant murine ortholog, further confirming the importance of the amino acid at this position for the binding of Ha22-2( 2.4)6.1. The binding of Ha22-2(2,4)6.1 to the double mutant of murine ortholog A90S, I75A appears very similar to the human ortholog of 191P4D12.
Together, these data prove 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 are part of the epitope recognized by Ha22-2( 2.4)6.1 on the surface of 191P4D12.
[00530] To visualize this concept, we generated a computer model of the V domain of 191P4D12 based on published crystal structure data for 191P4D12 family members and Ig domain-containing proteins using PyMOL (figure 25). The positions of Ala-76 (dotted) and Ser-91 (shaded with crossed lines) are shown.
Additionally, to further refine the binding site of Ha22-2(2,4)6.1 in the 191P4D12 molecule, we designed and expressed a 191P4D12 fragment that corresponds to the V domain on the surface of Rato(1)E cells. The following construct was generated in a retroviral vector:
[00532] 191P4D12 (aa1-150.347-510)
The binding of MAb Ha22-2(2,4)6.1 was evaluated by FACS. As shown in Figure 26, Ha22-2(2,4)6.1 binds to cells expressing the V domain (A) as well as wild type 191P4D12 (B), but not to cells expressing the C1C2 domain generated earlier (C) . This proves that the binding site for this antibody is located in the V domain of 191P4D12 within the first 150 amino acids.
[00534] The results show that the MAb Ha22-2(2,4)6.1 binds to the v domain of the 191P4D12 protein from the position aa 1 to 150 and further shows that the specific epitope comprising the aa Ser-91 and aa Ala-76 are critical for the binding of MAb Ha22-2(2,4)6.1.
[00535] Throughout this application, various data from websites, publications, patent applications and patents are mentioned. (Websites are referred to by their Standard Resource Locator, or URL, addresses on the internet.) Descriptions of each of these references are incorporated herein by reference in their entirety.
[00536] The present invention should not have its scope limited by the embodiments presented in the present invention, which is intended to be only illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other modalities may be practiced without departing from the true scope and spirit of the invention. TABLES Table I: Tissues expressing 191P4D12 when malignant. Colon Pancreas Ovarian Breast Lung Bladder Table II: Amino Acid Abbreviations
Table III: Amino Acid Substitution Matrix
[00537] Adapted from the BLOSUM62 amino acid substitution matrix of the GCG 9.0 program (block substitution matrix). The higher the value, the more likely a replacement is found in related natural proteins.
Table IV: General method for vcMMAE synthesis Where: AA1 = amino acid 1 AA2 = amino acid 2 AA5 = amino acid 5 DIL = Dolaisoleucine DAP = Dolaproline Ligand = Val-Cit (vc)
Table V: Biacore Association and Dissociation Rates and Resulting Affinity Calculation
Table VI: 191P4D12 Constructs Used in the Domain Mapping Test
Table VII
Table IX

权利要求:
Claims (38)
[0001]
1. Antibody-drug conjugate, characterized in that it comprises an anti-191P4D12 antibody or its antigen-binding fragment conjugated to monomethyl auristatin E (MMAE), wherein the antibody or its antigen-binding fragment comprises a variable region of the heavy chain comprising complementarity determining regions (CDRs) comprising the amino acid sequences of the heavy chain variable region CDRs shown from the 20th amino acid (glutamic acid) to the 136th amino acid (serine) of SEQ ID NO:7 and a variable region of light chain comprising CDRs comprising the amino acid sequences of the light chain variable region CDRs shown from the 23rd amino acid (aspartic acid) to the 130th amino acid (arginine) in SEQ ID NO:8.
[0002]
2. Antibody-drug conjugate according to claim 1, characterized in that the antibody or its antigen-binding fragment comprises a heavy chain variable region CDR1 (CDRH1) comprising the amino acid sequence from 45 to 52 of the SEQ ID NO: 7, a CDRH2 comprising amino acid sequence 70 to 77 in SEQ ID NO: 7, a CDRH3 comprising amino acid sequence 116 to 125 of SEQ ID NO: 7, a light chain variable region CDR1 (CDRL1) comprising amino acid sequence 49 to 54 of SEQ ID NO: 8, a CDRL2 comprising amino acid sequence 72 to 74 of SEQ ID NO: 8 and a CDRL3 comprising amino acid sequence 111 to 119 of SEQ ID NO: 8.
[0003]
3. Antibody-drug conjugate according to claim 1 or 2, characterized in that the antibody or its antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of the 20th amino acid (glutamic acid) to the 136th amino acid (serine) of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence from the 23rd amino acid (aspartic acid) to the 130th amino acid (arginine) of SEQ ID NO: 8.
[0004]
4. Antibody-drug conjugate according to any one of claims 1 to 3, characterized in that the antibody comprises a heavy chain comprising the amino acid sequence from the 20th amino acid (glutamic acid) to the 466th amino acid (lysine) of the SEQ ID NO: 7 and a light chain comprising the amino acid sequence from the 23rd amino acid (aspartic acid) to the 236th amino acid (cysteine) of SEQ ID NO: 8.
[0005]
5. Antibody-drug conjugate, according to any one of claims 1 to 3, characterized in that the antigen-binding fragment is a Fab, F(ab')2, Fv or scFv fragment.
[0006]
6. Antibody-drug conjugate, according to any one of claims 1 to 3, characterized in that the antibody is a completely human antibody.
[0007]
7. Antibody-drug conjugate, according to any one of claims 1 to 6, characterized in that the antibody is produced recombinantly.
[0008]
8. Antibody-drug conjugate, characterized in that it comprises an anti-191P4D12 antibody or its antigen-binding fragment conjugated to monomethyl auristatin E (MMAE), wherein the antibody or antibody fragment thereof comprises a variable region of the chain a heavy chain comprising the amino acid sequence of the heavy chain variable region of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267, and a light chain variable region comprising the amino acid sequence of light chain variable region of an antibody produced by a hybridoma deposited under ATCC Accession No. PTA-11267.
[0009]
9. Antibody-drug conjugate, according to claim 8, characterized in that the antibody comprises a heavy chain comprising the amino acid sequence of the heavy chain of an antibody produced by a hybridoma deposited under ATCC accession number PTA- 11267, and a light chain comprising the light chain amino acid sequence of an antibody produced by a hybridoma deposited under ATCC Accession No. PTA-11267.
[0010]
10. Antibody-drug conjugate, according to any one of claims 1 to 9, characterized in that the antibody or antibody fragment thereof is conjugated to 1 to 20 units of MMAE.
[0011]
11. Antibody-drug conjugate, according to any one of claims 1 to 10, characterized in that the antibody or antibody fragment thereof is conjugated to 1 to 10 units of MMAE.
[0012]
12. Antibody-drug conjugate, according to any one of claims 1 to 11, characterized in that the antibody or antibody fragment thereof is conjugated to MMAE through a linker.
[0013]
13. Antibody-drug conjugate, according to claim 12, characterized in that the ligand comprises valine-citrulline.
[0014]
14. Antibody-drug conjugate according to claim 13, characterized in that the ligand is an enzyme cleavable ligand and in which the ligand forms a bond with a sulfur atom of the antibody or antigen-binding fragment thereof .
[0015]
15. Antibody-drug conjugate, according to claim 12, characterized in that the ligand has a formula of: -Aa-Ww-Yy-; where -A- is an extender unit, a is 0 or 1; -W- is an amino acid unit, w is an integer from 0 to 12; and -Y- is a spacer unit, y is 0, 1 or 2; where the extender unit has the structure of Formula (1) below; the amino acid unit is valine-citrulline; and the spacer unit is a PAB group having the structure of Formula (2) below;
[0016]
16. Antibody-drug conjugate according to any one of claims 1 to 15, characterized in that it comprises 2 to 8 MMAE units per antibody or antigen-binding fragment thereof.
[0017]
17. Antibody-drug conjugate, according to any one of claims 1 to 15, characterized in that it comprises 2 to 5 units of MMAE per antibody or antigen-binding fragment thereof.
[0018]
18. Antibody-drug conjugate, according to any one of claims 1 to 15, characterized in that the antibody-drug conjugate has the following structure:
[0019]
19. Antibody-drug conjugate according to claim 18, characterized in that P is from 2 to 5.
[0020]
20. Antibody-drug conjugate, according to claim 19, characterized in that the antibody is a fully human monoclonal antibody, in which the antibody is an IgG1 and in which p is from 3 to 5.
[0021]
21. Antibody-drug conjugate according to any one of claims 1 to 20, characterized in that it is for use in the treatment of cancer.
[0022]
22. Antibody-drug conjugate according to claim 21, characterized in that the cancer is pancreatic cancer, lung cancer, bladder cancer, or breast cancer.
[0023]
23. Antibody-drug conjugate according to claim 21 or 22, characterized in that it is for use in the treatment of cancer in combination with radiation or a chemotherapeutic agent.
[0024]
24. Pharmaceutical composition, characterized in that it comprises the antibody-drug conjugate as defined in any one of claims 1 to 20.
[0025]
25. Pharmaceutical composition according to claim 24, characterized in that it is for use in the treatment of cancer.
[0026]
26. Pharmaceutical composition according to claim 25, characterized in that the cancer is pancreatic cancer, lung cancer, bladder cancer, or breast cancer.
[0027]
27. Pharmaceutical composition according to claim 26, characterized in that the cancer is bladder cancer.
[0028]
28. Pharmaceutical composition according to claim 27, characterized in that bladder cancer is advanced bladder cancer.
[0029]
29. Pharmaceutical composition according to claim 28, characterized in that bladder cancer is metastatic bladder cancer.
[0030]
30. Pharmaceutical composition according to any one of claims 24 to 29, characterized in that the antibody-drug conjugate is formulated to provide the antibody-drug conjugate in an amount from about 0.5 to about 10 mg/ kg of the antibody-drug conjugate for the human subject.
[0031]
31. Pharmaceutical composition according to any one of claims 24 to 30, characterized in that the antibody-drug conjugate is formulated to provide the antibody-drug conjugate in an amount from about 0.5 to about 2 mg/ kg of the antibody-drug conjugate for the human subject.
[0032]
32. Use of an antibody-drug conjugate as defined in any one of claims 1 to 20, characterized in that it is for the preparation of a pharmaceutical composition to treat cancer in an individual.
[0033]
33. Use according to claim 32, characterized in that the cancer is pancreatic cancer, lung cancer, bladder cancer or breast cancer.
[0034]
34. Use according to claim 33, characterized by the fact that the cancer is bladder cancer.
[0035]
35. Use according to claim 34, characterized by the fact that bladder cancer is advanced bladder cancer.
[0036]
36. Use according to claim 34, characterized in that bladder cancer is metastatic bladder cancer.
[0037]
37. Use according to any one of claims 32 to 36, characterized in that the antibody-drug conjugate is formulated to provide the antibody-drug conjugate in an amount of from about 0.5 to about 10 mg/kg of the antibody-drug conjugate for the human subject.
[0038]
38. Use according to any one of claims 32 to 37, characterized in that the antibody-drug conjugate is formulated to provide the antibody-drug conjugate in an amount of about 0.5 to about 2 mg/kg of the antibody-drug conjugate for the human subject.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-07-02| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-09-10| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-09-01| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-05-18| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-06-01| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-07-06| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 29/09/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US38793310P| true| 2010-09-29|2010-09-29|

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US61/387,933|2010-09-29|

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PCT/US2011/054054|WO2012047724A1|2010-09-29|2011-09-29|Antibody drug conjugatesthat bind to 191p4d12 proteins|

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