Antibody-drug conjugates (ADC) that bind to 191p4d12 protein

Abstract

To provide antibody-drug conjugates (ADC) that bind to 191P4D12 protein and its variants for treating cancer.SOLUTION: Provided is an anti-191P4D12 antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, where the heavy chain variable region comprises a heavy chain variable region complementarity determining region (CDR) of a specific amino acid sequence, and the light chain variable region comprises a light chain CDR of a specified amino acid sequence. The antibody or antigen-binding fragment thereof may be conjugated to a cytotoxic agent.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to related applications This application is not a provisional patent application and claims the benefit of priority from U.S. Provisional Patent Application No. 61 / 387,933, filed on September 29, 2010. The content of each application listed in this paragraph is hereby incorporated by reference in its entirety into this specification.

[0002] Description of Sequence Listing Submitted via EFS - WEB As permitted and shown in MPEP§1730 II.B.2(a)(C), the entire content of the following Sequence Listing electronic filing via the USPTO EFS-WEB server is hereby incorporated by reference in its entirety into this specification for all purposes. The Sequence Listing is identified on the electronically filed text file as follows. TIFF2025113404000001.tif13128

[0003] Description of Rights to Inventions Made with Government Support Not applicable

[0004] Field of the Invention The invention described herein relates to antibodies that bind to a protein designated 191P4D12, binding fragments thereof, and antibody-drug conjugates (ADCs). The invention further relates to prognostic, prophylactic, and therapeutic methods, and compositions useful in the treatment of cancers that express 191P4D12.

Background Art

[0005] Background of the Invention Cancer is the second leading cause of human death after coronary artery disease. Worldwide, millions of people die from cancer every year. As reported by the American Cancer Society, in the United States alone, cancer causes more than half a million deaths annually and over 1.2 million new cases are diagnosed each year. While deaths from heart disease are declining significantly, deaths from cancer are generally increasing. It is predicted that cancer will be the leading cause of death early in the next century.

[0006] Worldwide, several cancers stand out as major causes of death. In particular, cancers of the lung, prostate, breast, colon, pancreas, ovary, and bladder are major cancer causes of death. These and virtually all other cancers share a common lethal feature. Almost without exception, metastatic disease from cancer is fatal. Furthermore, it has been shown that even cancer patients who survive the initial stages of their primary cancer experience a dramatic change in their lives as a common experience. Many cancer patients are driven by a strong sense of anxiety by the recognition of the possibility of recurrence or treatment failure. Many cancer patients experience physical debilitation after treatment. Furthermore, many cancer patients experience recurrence.

[0007] Worldwide, prostate cancer is the fourth most common cancer in men. In North America and Northern Europe, prostate cancer is the overwhelmingly most common cancer in men and the second leading cause of cancer death in men. In the United States alone, more than 30,000 men die from this disease each year. It is second only to lung cancer. Despite the magnitude of these numbers, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration, and chemotherapy continue to be the main treatment modalities. Unfortunately, these treatments are often not effective and often have undesirable consequences.

[0008] At the forefront of diagnosis, the absence of a prostate tumor marker that can accurately detect early-stage localized tumors remains a significant limitation in the diagnosis and management of the disease. Serum prostate-specific antigen (PSA) assays are very useful tools, but their specificity and general utility are widely regarded as lacking several important aspects.

[0009] Progress in the identification of additional prostate cancer-specific markers has been improved by generating prostate cancer xenografts that can recapitulate different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immunodeficient (SCID) mice and have shown the ability to mimic the transition from androgen dependence to androgen independence (Klein, et al., 1997 Nat. Med. 3:402 (Non-Patent Document 1)). Recently identified prostate cancer markers include PCTA-1 (Su, et al., 1996 Proc. Natl. Acad. Sci. USA 93:7252 (Non-Patent Document 2)), prostate-specific membrane antigen (PSMA) (Pinto, et al., Clin. Cancer Res. 1996 Sep 2 (9):1445-51 (Non-Patent Document 3)), STEAP (Hubert, et al., Proc. Natl. Acad. Sci. USA 1999 Dec 7; 96(25):14523-8 (Non-Patent Document 4)), and prostate stem cell antigen (PSCA) (Reiter, et al., Proc. Natl. Acad. Sci. USA 1998 95:1735 (Non-Patent Document 5)).

[0010] Previously identified markers such as PSA have facilitated efforts for the diagnosis and treatment of prostate cancer, but for further improvement in diagnosis and treatment, the identification of additional markers and therapeutic targets for prostate cancer and related cancers is needed. In 2000 in the United States, an estimated 130,200 cases of colorectal cancer occurred, including 93,800 cases of colon cancer and 36,400 cases of rectal cancer.

[0011] Colorectal cancer is the third most common cancer in men and women. Incidence rates decreased significantly between 1992 and 1996 (-2.1% per year). Studies suggest that these decreases are due to increased screening and polyp removal, preventing progression from polyp to invasive cancer. In 2000, an estimated 56,300 people died (47,700 from colon cancer and 8,600 from rectal cancer), accounting for approximately 11% of cancer deaths in the United States.

[0012] Currently, surgery is the most common form of treatment for colorectal cancer and non-invasive cancer, and is often curable. Chemotherapy or chemotherapy + radiation is given to most patients with cancer that has penetrated deeply into the intestinal wall or spread to lymph nodes, either before or after surgery. Permanent colostomy (creation of an abdominal opening for excretion of body wastes) is sometimes necessary for colon cancer and rarely for rectal cancer. Effective diagnostic and treatment methods for colorectal cancer are still needed.

[0013] Among all new cases of cancer in the United States, bladder cancer accounts for approximately 5% in men (the fifth most common neoplasm) and 3% in women (the eighth most common neoplasm). Incidence rates are increasing slowly, along with the increasing elderly population. In 1998, there were an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence rate in the United States is 32 per 100,000 in men and 8 per 100,000 in women. The past male / female ratio was 3:1, but this may be decreasing in relation to the smoking trend in women. In 1998, there were an estimated 11,000 deaths from bladder cancer (7,800 in men and 3,900 in women). The incidence and mortality rates of bladder cancer increase sharply with age and are a problem that increases as the population ages.

[0014] Most bladder cancers recur in the bladder. Bladder cancer is managed by a combination of transurethral resection (TUR) of the bladder and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer demonstrates 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 eradicating the cancer, but have an undeniable impact on urinary and sexual function. There remains a great need for beneficial treatments for patients with bladder cancer.

[0015] In 2000, there were an estimated 164,100 new cases of lung and bronchial cancer, representing 14% of all cancer diagnoses in the United States. The incidence of lung and bronchial cancer has decreased significantly in men, from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of increase in women began to slow. In 1996, the incidence in women was 42.3 per 100,000.

[0016] Lung and bronchial cancer resulted in an estimated 156,900 deaths in 2000, representing 28% of all cancer deaths. Between 1992 and 1996, the mortality rate for lung cancer decreased significantly in men (–1.7% per year). In contrast, the mortality rate in women was still increasing significantly (0.9% per year). Every year since 1987, more women have died from lung cancer than from breast cancer, which was the leading cause of cancer death in women for more than 40 years. The decrease in the incidence and mortality of lung cancer is most likely due to the decline in smoking rates over the past 30 years. However, the decrease in smoking prevalence in women lags significantly behind that in men. Although the decline in adult tobacco use has slowed, there is concern that tobacco use is increasing again among young people.

[0017] Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice. Since the disease has usually spread by the time it is detected, radiation therapy and chemotherapy are often needed in combination with surgery. Chemotherapy alone or in combination with radiation is the treatment of choice for small cell lung cancer. In this regimen, a large percentage of patients experience remission, which in some cases lasts a long time. However, there is still a need for effective treatment and diagnostic approaches for lung and bronchial cancer.

[0018] An estimated 182,800 new cases of invasive breast cancer were predicted to occur among women in the United States in 2000. Additionally, about 1,400 new cases of breast cancer were predicted to be diagnosed in men in 2000. The incidence rate of breast cancer in women increased by about 4% per year in the 1980s and then decreased to about 110.6 per 100,000 by the 1990s.

[0019] In the United States alone, there were an estimated 41,200 deaths (40,800 in women and 400 in men) due to breast cancer in 2000. Breast cancer is the second leading cause of cancer death among women. According to the most recent data, the mortality rate has decreased significantly in both white and black women between 1992 and 1996, with the greatest decrease among younger women. These decreases were probably the result of improved early detection and treatment.

[0020] Taking into account the medical situation and the patient's preferences, the treatment of breast cancer may involve lumpectomy (local removal of the tumor) and removal of the underarm lymph nodes; mastectomy (surgical removal of the breast) and removal of the underarm lymph nodes; radiation therapy; chemotherapy; or hormone therapy. Often, two or more methods are used in combination. From many studies, it has been found that for early-stage disease, the long-term survival rate after lumpectomy + radiation therapy is similar to the survival rate after modified radical mastectomy. Significant advances in reconstructive techniques offer several options for breast reconstruction after mastectomy. In recent years, such reconstruction has been done at the same time as mastectomy.

[0021] Local excision of non-invasive ductal carcinoma in situ (DCIS) with a sufficient amount of surrounding normal breast tissue can prevent local recurrence of DCIS. Radiation to the breast and / or tamoxifen can reduce the probability of the development of DCIS in the remaining breast tissue. This is important because DCIS, if left untreated, may progress to invasive breast cancer. Nevertheless, these treatments have serious side effects or sequelae. Therefore, effective breast cancer treatment is needed.

[0022] An estimated 23,100 new cases of ovarian cancer existed in the United States in 2000. This represents 4% of all cancers among women and is the second leading rank among gynecological cancers. Between 1992 and 1996, the incidence rate of ovarian cancer decreased significantly. As a result of ovarian cancer, an estimated 14,000 people died in 2000. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.

[0023] Surgery, radiation therapy, and chemotherapy are options for the treatment of ovarian cancer. Surgery usually involves the removal of one or both ovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus (hysterectomy). In some very early tumors, especially in young women who wish to have children, only the affected ovary is removed. In advanced disease, attempts are made to remove all intra-abdominal disease to enhance the effectiveness of chemotherapy. There remains an important need for effective treatment options for ovarian cancer.

[0024] In 2000, an estimated 28,300 new cases of pancreatic cancer existed in the United States. Over the past 20 years, the rate of pancreatic cancer has declined in men. The rate in women has remained approximately constant but may be beginning to decline. Pancreatic cancer caused an estimated 28,200 deaths in the United States in 2000. Over the past 20 years, the mortality rate in men has declined slightly but significantly (about -0.9% per year), while the rate in women has increased slightly.

[0025] Surgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer. These treatment options can extend survival and / or relieve symptoms in many patients, but in most cases, are unlikely to result in a cure. There is a significant need for additional treatment and diagnostic options for cancer. These options include the use of antibodies, vaccines, and small molecules as treatment methods. Additionally, it is necessary to use these methods as research tools for diagnosis, detection, monitoring, and as state-of-the-art technology in all areas of cancer treatment and research.

[0026] The therapeutic utility of monoclonal antibodies (mAbs) (G. Kohler and C. Milstein, Nature 256:495-497 (1975) (Non-Patent Document 6)) has been recognized. Monoclonal antibodies are currently recognized as treatments in transplantation, cancer, infectious diseases, cardiovascular diseases, and inflammation. Different isotypes have different effector functions. Such functional differences are reflected in the different three-dimensional structures of various immunoglobulin isotypes (P. M. Alzari, et al., Annual Rev. Immunol. 6:555-580 (1988) (Non-Patent Document 7)).

[0027] Mice are convenient for immunization and recognize most human antigens as foreign substances, so mAbs against therapeutically relevant human targets are typically of murine origin. However, murine mAbs have inherent drawbacks as human therapeutics. Murine mAbs require frequent dosing because the circulating half-life of mAbs in humans is shorter than that of human antibodies. More importantly, repeated administration of murine antibodies to the human immune system elicits a response by the human immune system recognizing the murine proteins as foreign substances, resulting in a human anti-mouse antibody (HAMA) response. Such a HAMA response can lead to allergic reactions and rapid clearance of the murine antibodies from the system, thereby rendering treatment with murine antibodies ineffective. To avoid such effects, attempts have been made to create a human immune system within mice.

[0028] The initial attempts aimed to create transgenic mice that could respond to antigens with antibodies having human sequences (see Bruggemann, et al., Proc. Nat'l. Acad. Sci. USA 86:6709-6713(1989)(Non-Patent Document 8)), but there were limitations to the amount of DNA that could be stably maintained by available cloning vehicles. The use of yeast artificial chromosome (YAC) cloning vectors opened the way to introduce large germline fragments of the human Ig locus into transgenic animals. Essentially, most of the human V-region genes, D-region genes, and J-region genes, as well as the human constant regions, arranged at the same intervals found in the human genome, were introduced into mice using YACs. One such transgenic mouse line is known as the XenoMouse® mouse and is commercially available from Amgen Fremont, Inc. (Fremont CA).

Prior Art Documents

Non-Patent Documents

[0029]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

[0030] The present invention provides antibodies, binding fragments thereof, and antibody-drug conjugates (ADCs) that bind to the 191P4D12 protein and polypeptide fragments of the 191P4D12 protein. In some embodiments, the present invention includes a fully human antibody conjugated to a therapeutic agent. In certain embodiments, there are conditions such that the entire nucleic acid sequence of FIG. 3 is not encoded and / or the entire amino acid sequence of FIG. 2 is not prepared. In certain embodiments, the entire nucleic acid sequence of FIG. 3 is encoded and / or the amino acid sequence of FIG. 2 is prepared, both within their respective human unit dosage forms.

[0031] The present invention further provides an immunogenic composition or a therapeutic composition, such as an antibody-drug conjugate, and a strategy for treating cancers expressing 191P4D12, such as cancers of the tissues listed in Table I. [Invention 1001] A 191P4D12 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising a CDR having the amino acid sequence of the heavy chain variable region complementarity-determining region (CDR) shown in SEQ ID NO:7 and a light chain variable region comprising a CDR having the amino acid sequence of the light chain variable region CDR shown in SEQ ID NO:8. [Invention 1002] The antibody or antigen-binding fragment of the present invention 1001, comprising a heavy-chain variable region consisting of the amino acid sequence in the range from the 20th amino acid (glutamic acid) to the 136th amino acid (serine) of SEQ ID NO:7 and a light-chain variable region consisting of the amino acid sequence in the range from the 23rd amino acid (aspartic acid) to the 130th amino acid (arginine) of SEQ ID NO:8. [The present invention 1003] The antibody of the present invention 1002, comprising a heavy chain consisting of the amino acid sequence in the range from the 20th amino acid (glutamic acid) to the 466th amino acid (lysine) of SEQ ID NO:7 and a light chain consisting of the amino acid sequence in the range from the 23rd amino acid (aspartic acid) to the 236th amino acid (cysteine) of SEQ ID NO:8. [The present invention 1004] The antigen-binding fragment of the present invention 1001 or 1002, which is a Fab, F(ab')2, Fv, or scfv fragment. [The present invention 1005] The antibody of the present invention 1001 or 1002, which is a fully human antibody. [The present invention 1006] The antibody of any one of the present inventions 1001 to 1005, which is produced recombinantly. [The present invention 1007] An antibody or its antigen-binding fragment, comprising a heavy-chain variable region consisting of the amino acid sequence of the heavy-chain variable region of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267, and a light-chain variable region consisting of the amino acid sequence of the light-chain variable region of the antibody produced by the hybridoma deposited under ATCC accession number PTA-11267. [The present invention 1008] The antibody of the present invention 1007, comprising a heavy chain consisting of the amino acid sequence of the heavy chain of the antibody produced by the hybridoma deposited under ATCC accession number PTA-11267, and a light chain consisting of the amino acid sequence of the light chain of the antibody produced by the hybridoma deposited under ATCC accession number PTA-11267. [The present invention 1009] An antibody-drug conjugate comprising any of the antibodies or antigen-binding fragments of the present invention conjugated to a cytotoxic agent. [Invention 1010] The antibody-drug conjugate of Invention 1009, wherein the cytotoxic agent is monomethyl auristatin E. [Invention 1011] The antibody-drug conjugate of Invention 1009 or Invention 1010 for use in cancer treatment. [Invention 1012] The antibody-drug conjugate of Invention 1011, wherein the cancer is pancreatic cancer, lung cancer, bladder cancer, or breast cancer. [Invention 1013] The antibody-drug conjugate of Invention 1009 or Invention 1010 for use in cancer treatment in combination with radiation or a chemotherapeutic agent. [Invention 1014] A pharmaceutical composition comprising the antibody-drug conjugate of Invention 1009 or Invention 1010 in a unit dosage form for humans. [Invention 1015] The pharmaceutical composition of Invention 1014 for use in cancer treatment. [Invention 1016] The pharmaceutical composition of Invention 1015, wherein the cancer is pancreatic cancer, lung cancer, bladder cancer, or breast cancer. [Invention 1017] A method for treating cancer in a subject, comprising the step of administering the antibody-drug conjugate of Invention 1009 or Invention 1010 to the subject.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0033] Detailed Description of the Invention Summary of Sections I.) Definitions II.) 191P4D12 Antibody III.) General Introduction to Antibody-Drug Conjugates III(A). Maytansinoids III(B). Auristatins and Dolastatins III(C). Calicheamicin III(D). Other Cytotoxic Agents IV.) Antibody-Drug Conjugates That Bind to 191P4D12 V.) Linker Unit VI.) Stretcher Unit VII.) Amino Acid Unit VIII.) Spacer Unit IX.) Drug Unit X.) Drug Loading XI) Methods for Confirming the Cytotoxic Effect of ADCs XII.) Treatment of Cancers Expressing 191P4D12 XIII.) 191P4D12 as a Target for Antibody-Based Therapies XIV.) 191P4D12 ADC Cocktails XV.) Combination Therapies XVI.) Kits / Manufactured Articles

[0034] I.) Definitions Unless otherwise defined, all technical terms, symbols, and other scientific or technical terms used in this specification are intended to have the meanings commonly understood by those skilled in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined in this specification for clarity and / or for ready reference. Inclusion of such definitions in this specification should not necessarily be construed as indicating a substantial difference from what is commonly understood in the art. Many of the techniques and procedures described or referenced in this specification are well understood by those skilled in the art and are commonly used by them using conventional methodologies, such as the widely used molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Procedures generally include the use of commercially available kits and reagents, and are carried out according to the protocols and / or parameters specified by the manufacturer unless otherwise described.

[0035] When a trade name is used in this specification, the reference to the trade name refers to the product formulation, generic pharmaceuticals, and the active pharmaceutical ingredient of the product of the trade name, unless otherwise specified by the context.

[0036] The terms "advanced cancer", "locally advanced cancer", "advanced disease" and "locally advanced disease" mean cancer that has spread beyond the associated tissue capsule, and this term is intended to include diseases at stage C in the American Urological Association (AUA) system, diseases at stages C1 - C2 in the Whitmore-Jewett system, and diseases at stages T3 - T4 and N+ in the TNM (tumor, node, metastasis) system. Generally, surgery is not recommended for patients with locally advanced disease. These patients have substantially less favorable outcomes compared to patients with clinically localized (organ-confined) cancer.

[0037] The abbreviation "AFP" refers to dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylenediamine (see formula XVI below).

[0038] The abbreviation "MMAE" refers to monomethyl auristatin E (see formula XI below).

[0039] The abbreviation "AEB" refers to an ester produced by reacting auristatin E with para-acetylbenzoic acid (see formula XX below).

[0040] The abbreviation "AEVB" refers to an ester produced by reacting auristatin E with benzoylvaleric acid (see formula XXI below).

[0041] The abbreviation "MMAF" refers to dovaline-valine-dolaisoleuine-dolaproine-phenylalanine (see formula XVIV below).

[0042] Unless otherwise specified, the term "alkyl" refers to straight-chain or branched-chain saturated hydrocarbons having from about 1 to about 20 carbon atoms (and all combinations and sub-combinations of ranges and specific numbers of carbon atoms 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.

[0043] The alkyl group, whether alone or as part of another group, may be substituted by one or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogen), including but not limited to -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, =O, -N3, -NH2, -NH(R'), -N(R')2, and -CN. Each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl. The -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl groups may be further substituted by one or more groups, including but not limited to -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)N(R'')2, -NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2, and -CN. Each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.

[0044] Unless otherwise specified, the terms "alkenyl" and "alkynyl" refer to straight-chain and branched carbon chains having from about 2 to about 20 carbon atoms (as well as all combinations and subcombinations of ranges and specific numbers of carbon atoms within this), 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, -isobutenylenyl, -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, ethynyl, propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1-butynyl.

[0045] The alkenyl and alkynyl groups, whether alone or as part of another group, may be substituted by one or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogen), including but not limited to -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, =O, -N3, -NH2, -NH(R'), -N(R')2, and -CN. Each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl. The -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl groups may be further substituted by one or more substituents, including but not limited to -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)N(R'')2, -NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2, and -CN. Each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.

[0046] Unless otherwise specified, the term "alkylene" refers to a branched or straight-chain saturated hydrocarbon radical having from about 1 to about 20 carbon atoms (and all combinations and sub-combinations of ranges and specific numbers of carbon atoms therein), with two monovalent radical centers obtained by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, and from about 1 to about 8 carbon atoms being preferred. Representative alkylene includes, but is not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decalene, 1,4-cyclohexylene, and the like.The alkylene group, whether alone or as part of another group, may be substituted by one or more groups 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, preferably one to three groups (and any additional substituents selected from halogen). Each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl. The -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl groups may be further substituted by one or more substituents including, but not limited to, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)N(R'')2, -NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2, and -CN. Each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.

[0047] 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-).

[0048] 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-).

[0049] Unless otherwise specified, the term "aryl" refers to a monovalent aromatic hydrocarbon radical consisting of 6 to 20 carbon atoms (and all combinations and sub-combinations of ranges and specific numbers of carbon atoms within this range), obtained by removing one hydrogen atom from one carbon atom of the parent aromatic ring structure. Some aryl groups are represented by "Ar" in exemplary structures. Representative aryl groups include, but are not limited to, radicals obtained from benzene, substituted benzene, phenyl, naphthalene, anthracene, biphenyl, etc.

[0050] The aryl group, whether alone or as part of another group, may be substituted by one or more, preferably 1 to 5, more preferably 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, wherein each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl, and the -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), and -aryl groups may be further substituted by one or more substituents including, but not limited to, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)N(R'')2, -NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2, and -CN, wherein each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.

[0051] Unless otherwise specified, the term "aryl" refers to an optionally substituted aryl group that is divalent (i.e., obtained by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent aromatic ring structure), and as shown in the following structure using phenyl as an exemplary aryl group, it may have an ortho, meta, or para structure. TIFF2025113404000002.tif30128 Representative "-(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-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl, and the like.

[0052] Unless otherwise specified, the term "heterocyclic ring" refers to a monocyclic, bicyclic, or polycyclic ring structure having 3 to 14 ring atoms (also called ring members). Here, at least one ring atom in at least one ring is a heteroatom selected from N, O, P, or S (as well as all combinations and partial combinations of a range and specific numbers of carbon atoms and heteroatoms therein). The heterocyclic ring may have 1 to 4 ring heteroatoms independently selected from N, O, P, or S. One or more N, C, or S atoms in the heterocyclic ring may be oxidized. The monocyclic heterocyclic ring preferably has 3 to 7 ring members (e.g., 2 to 6 carbon atoms and 1 to 3 heteroatoms independently selected from N, O, P, or S), and the bicyclic heterocyclic ring preferably has 5 to 10 ring members (e.g., 4 to 9 carbon atoms and 1 to 3 heteroatoms independently selected from N, O, P, or S). The ring containing heteroatoms may be aromatic or non-aromatic. Unless otherwise specified, any heteroatom or carbon atom of the heterocyclic ring is attached to its pendant group to form a stable structure.

[0053] Heterocyclic rings 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 - present), particularly in Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. 82:5566 (1960).

[0054] Examples of "heterocyclic ring" groups include, by way of example and without 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, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiadiazinyl, thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathiinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4H-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phthalazinyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxyindolyl, benzoxazolinyl, and isatinoyl. Preferred "heterocyclic ring" groups include, but are not limited to, benzofuranyl, benzothiophenyl, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, and tetrazolyl.

[0055] Heterocyclic groups, whether alone or as part of another group, include -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', -S3R', -S(O)2R', and -CN, each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl, and is substituted with one or more groups, preferably one to two groups, including, but not limited to, -S(O)R', -OH, -N3, -NH2, -NH(R'), -N(R')2, and -CN, and ... The groups -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -C2-C8 alkynyl, and -aryl are -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 It may be further substituted by one or more substituents including, but not limited to, (O)N(R'')2, -NHC(O)R'', -SR'', -S03R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2, and -CN, where each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or aryl.

[0056] By way of example, and not limitation, the carbon-bonded heterocycle may be bonded at the 2-, 3-, 4-, 5-, or 6-position of pyridine; the 3-, 4-, 5-, or 6-position of pyridazine; the 2-, 4-, 5-, or 6-position of pyrimidine; the 2-, 3-, 5-, or 6-position of pyrazine; the 2-, 3-, 4-, or 5-position of furan, tetrahydrofuran, thiophene, thienyl, pyrrole, or tetrahydropyrrole; the 2-, 4-, or 5-position of oxazole, imidazole, or thiazole; the 3-, 4-, or 5-position of isoxazole, pyrazole, or isothiazole; the 2- or 3-position of aziridine; the 2-, 3-, or 4-position of azetidine; the 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of quinoline; or the 1-, 3-, 4-, 5-, 6-, 7-, or 8-position of isoquinoline. Even more typically, the carbon-bonded heterocycle includes 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

[0057] By way of example, and not limitation, the nitrogen-bonded heterocycle may be bonded at the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, or  1H-indazole; the 2-position of isoindole or isoindoline; the 4-position of morpholine; and the 9-position of carbazole or β-carboline. Even more typically, the nitrogen-bonded heterocycle includes 1-aziridyl, 1-azetyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0058] Unless otherwise specified, the term "carbocycle" refers to a saturated or unsaturated, non-aromatic, monocyclic, bicyclic, or polycyclic ring structure having from 3 to 14 ring atoms (and all combinations and sub-combinations of ranges and specific numbers of carbon atoms within this range), all of the ring atoms being carbon atoms. The monocyclic carbocycle preferably has from 3 to 6 ring atoms, more preferably 5 or 6 ring atoms. The bicyclic carbocycle preferably has from 7 to 12 ring atoms arranged, for example, as a bicyclo[4,5], [5,5], [5,6], or [6,6] structure, or has 9 or 10 ring atoms arranged as a bicyclo[5,6] or [6,6] structure. The term "carbocycle" includes, for example, a monocyclic carbocycle fused to an aryl ring (e.g., a monocyclic carbocycle fused to a benzene ring). The carbocycle preferably has from 3 to 8 carbocycle atoms.

[0059] The carbocyclic group, whether alone or as part of another group, for example, is substituted by one or more 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, =O, -N3, -NH2, -NH(R'), -N(R')2, and -CN, preferably one or two groups (and any additional substituents selected from halogen), and each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl. The -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), and -aryl groups may be further substituted by one or more substituents including, but not limited to, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)N(R'')2, -NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2, and -CN, and each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.

[0060] 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.

[0061] "Carbocyclo", whether alone or as part of another group, refers to a substituted carbocyclic group as defined above that is divalent (i.e., obtained by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent carbocyclic structure).

[0062] Unless otherwise specified by the context, a hyphen (-) designates the point of attachment to the pendant molecule. Thus, the terms "-(C1 - C8 alkylene)aryl" or "-C1 - C8 alkylene(aryl)" refer to a C1 - C8 alkylene radical as defined herein, wherein any carbon atom of the alkylene radical is attached to the pendant molecule and one of the hydrogen atoms bonded to the carbon atom of the alkylene radical is replaced by an aryl radical as defined herein.

[0063] When a particular group is "substituted", the group may have one or more substituents selected independently from the list of substituents, preferably 1 to 5 substituents, more preferably 1 to 3 substituents, and most preferably 1 to 2 substituents. However, the group may generally have any number of substituents selected from halogens. Multiple groups that are substituted are also indicated in this manner.

[0064] The definition of any substituent or variation at a particular location within a molecule is intended to be independent of definitions elsewhere in that molecule. One of ordinary skill in the art will understand that the substituents and substitution patterns on the compounds of the present invention can be selected to provide compounds that are chemically stable and can be readily synthesized by techniques known in the art and methods shown herein.

[0065] As used herein, a protecting group refers to a group that selectively blocks one reactive site in a polyfunctional compound, either transiently or persistently. 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 a subject in order for the compound to become active. Cleavage occurs via normal metabolic processes in the body. Hydroxy protecting groups are well known in the art and include, for example, ethers (including alkyl ethers and silyl ethers such as dialkylsilyl ethers, trialkylsilyl ethers, and dialkylalkoxysilyl ethers), esters, carbonates, carbamates, sulfonates, as well as phosphate protecting groups. The entirety of Protective Groups in Organic Synthesis by T. W. Greene and P. G. M. Wuts (John Wiley & sons, 3 rdSee (Edition). Examples of hydroxy protecting groups include methyl ether; methoxymethyl ether, methylthiomethyl ether, (phenyldimethylsilyl)methoxymethyl ether, benzyloxymethyl ether, p-methoxybenzyloxymethyl ether, p-nitrobenzyloxymethyl ether, o-nitrobenzyloxymethyl ether, (4-methoxyphenoxy)methyl ether, guaiacol methyl ether, t-butoxymethyl ether, 4-pentenyl oxymethyl ether, siloxymethyl ether, 2-methoxyethoxymethyl ether, 2,2,2-trichloroethoxymethyl ether, bis(2-chloroethoxy)methyl ether, 2-(trimethylsilyl)ethoxymethyl ether, methoxymethyl 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 ether, 1,4-dioxan-2-yl ether, tetrahydrofuranyl ether, tetrahydrothiofuranyl ether;Replacement 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 ether, p-chlorophenyl ether, p-methoxyphenyl ether, benzyl ether, p-methoxybenzyl ether, 3,4-dimethoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, tripropylsilyl ether, dimethylisopropylsilyl ether, diethylisopropylsilyl ether, dimethylhexylsilyl ether, t-butyldimethylsilyl ether, diphenylmethylsilyl ether, benzoylformate ester, acetate ester, chloroacetate ester, dichloroacetate ester, trichloroacetate ester, trifluoroacetate ester, methoxyacetate ester, triphenylmethoxyacetate ester, phenylacetate ester, benzoate ester, alkylmethyl carbonate, alkyl 9-fluorenylmethyl carbonate, alkylethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate, 1,1-dimethyl-2,2,2-trichloroethyl carbonate, alkyl sulfonate, methanesulfonate, benzyl sulfonate, tosylate, methylene acetal, ethylidene acetal, and t-butylmethylidene ketal are included, but not limited thereto. Preferred protecting groups are of the formula -R; a , -Si(R a )(R a )(R a ), -C(O)R a , -C(O)OR a , -C(O)NH(R a ), -S(O)2R a , -S(O)2OH, P(O)(OH)2, and -P(O)(OH)OR a , where in the formula, R a is C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20Alkynyl, -C1-C 20 Alkylene (carbocycle), -C2-C 20 Alkenylene (carbocycle), -C2-C 20 Alkynylene (carbocycle), -C6-C 10 Aryl, -C1-C 20 Alkylene (aryl), -C2-C 20 Alkenylene (aryl), -C2-C 20 Alkynylene (aryl), -C1-C 20 Alkylene (heterocycle), -C2-C 20 Alkenylene (heterocycle), or -C2-C 20 Alkynylene (heterocycle), and the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, and heterocyclic radicals are either alone or as part of another group, and may be substituted.

[0066] For the purposes of this specification, "change in the native glycosylation pattern" is intended to mean deleting one or more carbohydrate moieties found in the native sequence 191P4D12 (either by removing the underlying glycosylation site or by deleting glycosylation by chemical and / or enzymatic means), and / or adding one or more glycosylation sites not present in the native sequence 191P4D12. Further, the foregoing phrase includes qualitative changes in the glycosylation of the native protein, including changes in the nature and ratio of the various carbohydrate moieties present.

[0067] The term "analog" refers to a molecule that is structurally similar to another molecule (e.g., a 191P4D12-related protein) or shares similar or corresponding attributes. For example, an analog of the 191P4D12 protein can specifically bind to an antibody that specifically binds to 191P4D12 or to a T cell.

[0068] The term "antibody" is used in its broadest sense unless otherwise specifically defined. Thus, an "antibody" may be natural or may be artificially produced, such as a monoclonal antibody produced by conventional hybridoma technology. The 191P4D12 antibody includes monoclonal and polyclonal antibodies, as well as fragments containing the antigen-binding domains of these antibodies and / or one or more complementarity-determining regions. As used herein, the term "antibody" refers to any form of antibody or fragment thereof that specifically binds to 191P4D12 and / or exhibits a desired biological activity. The term "antibody" specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they specifically bind to 191P4D12 and / or exhibit a desired biological activity. Any specific antibody can be used in the methods and compositions provided herein. Thus, in one aspect, the term "antibody" includes a molecule comprising at least one variable region derived from a light chain immunoglobulin molecule and at least one variable region derived from a heavy chain molecule that, when combined, form a specific binding site for the target antigen. In one aspect, the antibody is an IgG antibody. For example, the antibody is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. Antibodies useful in the present methods and compositions can be made in a variety of animals including, but not limited to, cell cultures, phage, or cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. Thus, in one aspect, the antibodies of the present invention are mammalian antibodies. Phage technology can be used to isolate the initial antibody or to generate variants with altered specificity or avidity characteristics. Such techniques are routine and well known in the art. In one aspect, 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 containing a DNA sequence encoding the antibody.One or more vectors can be used to transfect a host cell with a DNA sequence that expresses at least one VL region and one VH region. Exemplary descriptions of recombinant means for antibody production and antibody generation 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); CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons, latest edition). The antibodies of the present invention can be modified by recombinant means to enhance the efficacy of the antibody in mediating the desired function. Thus, it is within the scope of the present invention that antibodies can be modified by substitution using recombinant means. Typically, the substitution is a conservative substitution. For example, at least one amino acid in the constant region of the antibody can be substituted with a different residue. See, for example, U.S. Patent No. 5,624,821, U.S. Patent No. 6,194,551, Application No. WO9958572; and Angal, et al., Mol. Immunol. 30:105-08 (1993). Modifications in amino acids include deletions, additions, and substitutions of amino acids. In some cases, such changes are made to reduce undesirable activities, such as complement-dependent cytotoxicity. Frequently, antibodies are labeled by binding a substance that provides a detectable signal either covalently or non-covalently. A wide variety of labels and conjugation techniques are known and widely reported in both scientific and patent literature. These antibodies can be screened for binding to normal 191P4D12 or defective 191P4D12. For example, Antibody Engineering: A Practical ApproachSee (Oxford University Press, 1996). Suitable antibodies having desirable biological activities can be identified using the following in vitro assays, including but not limited to proliferation, migration, adhesion, soft agar growth, angiogenesis, cell - cell communication, apoptosis, transport, signal transduction, and the following in vivo assays, such as inhibition of tumor growth. The antibodies provided herein may also be useful in diagnostic applications. As capture antibodies or non - neutralizing antibodies, these antibodies can be screened for their ability to bind to a particular antigen without inhibiting the antigen's receptor binding or biological activity. As neutralizing antibodies, the antibodies may be useful in competitive binding assays. These antibodies can also be used to quantify 191P4D12 or its receptor.

[0069] The term "antigen - binding portion" or "antibody fragment" (or simply "antibody portion") of an antibody as used herein means one or more fragments of a 191P4D12 antibody that retain the ability to specifically bind to an antigen (e.g., 191P4D12 and variants; Figure 1). It has been shown that the antigen - binding function of an antibody can be exerted by fragments of the full - length antibody. Examples of binding fragments included within the term "antigen - binding portion" of an antibody are: (i) a monovalent fragment consisting of the V L , V H , C L and C H1 domains; (ii) an F(ab')2 fragment, a bivalent fragment in which two Fab fragments are linked in the hinge region by a disulfide bridge; (iii) an Fd fragment consisting of the V H and C H1 domains; (iv) an Fv fragment consisting of the V L and V H domains of one arm of the antibody; (v) a V HdAb fragments consisting of domains (Ward, et al., (1989) Nature 341:544-546); and (vi) isolated complementarity determining regions (CDRs) are included. Further, the two domains of the Fv fragment, V L and V H are encoded by separate genes, but using recombinant methods, they can be joined by a synthetic linker that enables them to be made as a single protein chain in which the V L region and the V H region pair up (see, for example, Bird, et al., (1988) Science 242:423-426; and Huston, et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883), forming a monovalent molecule (known as a single-chain Fv (scFv)). Such single-chain antibodies are also intended to be included within the term "antigen-binding portion" of an antibody. These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0070] As used herein, any form of "antigen" can be used to generate an antibody specific for 191P4D12. Thus, the eliciting antigen can be one epitope, multiple epitopes, or the entire protein, which can be alone or in combination with one or more immunogenic enhancing agents known in the art. The eliciting antigen can be an isolated full-length protein, a cell surface protein (e.g., immunizing with cells transfected with at least a portion of the antigen), or a soluble protein (e.g., immunizing with only the extracellular domain portion of the protein). The antigen can be produced in a recombinant cell. The DNA encoding the antigen can be genomic DNA or non-genomic DNA (e.g., cDNA) and encodes at least a portion of the extracellular domain. As used herein, the term "portion" refers, as appropriate, to the minimum number of amino acids or nucleic acids that constitute an immunogenic epitope of the antigen of interest. Any gene vector suitable for transformation of cells of interest can be used. Gene vectors include, but are not limited to, adenoviral vectors, plasmids, and non-viral vectors such as cationic lipids. In one aspect, the antibodies of the methods and compositions herein specifically bind to at least a portion of the extracellular domain of 191P4D12 of interest.

[0071] The antibodies or antigen-binding fragments thereof provided herein can conjugate to a "bioactive agent". As used herein, the term "bioactive agent" refers to any synthetic or natural compound that binds to an antigen and enhances or mediates a desired biological effect to enhance and / or potentiate a cell-killing toxin. In one aspect, 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 can bind to a desired antigenic epitope and can directly or indirectly exert a biological effect. Direct effects include, but are not limited to, modulation, stimulation, and / or inhibition of growth signals; modulation, stimulation, and / or inhibition of anti-apoptosis signals; modulation, stimulation, and / or inhibition of apoptosis or necrosis signals; modulation, stimulation, and / or inhibition of the ADCC cascade; and modulation, stimulation, and / or inhibition of the CDC cascade.

[0072] "Bispecific" antibodies are also useful in the present methods and compositions. As used herein, the term "bispecific antibody" refers to an antibody having binding specificity for at least two different antigenic epitopes, typically a monoclonal antibody. In one aspect, the epitopes are derived from the same antigen. In another aspect, the epitopes are derived from two different antigens. Methods for generating bispecific antibodies are known in the art. For example, bispecific antibodies can be recombinantly produced using co-expression of two immunoglobulin heavy chain / light chain pairs. See, e.g., Milstein, et al., Nature 305:537-39 (1983). Alternatively, bispecific antibodies can be prepared using chemical linkage. See, e.g., Brennan, et al., Science 229:81 (1985). Bispecific antibodies include bispecific antibody fragments. See, e.g., Hollinger, et al., Proc. Natl. Acad. Sci. U.S.A. 90:6444-48 (1993), Gruber, et al., J. Immunol. 152:5368 (1994).

[0073] 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 the corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they specifically bind to the target antigen and / or exhibit the desired biological activity. (U.S. Patent No. 4,816,567; and Morrison, et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

[0074] The term "chemotherapeutic agent" refers to all compounds effective in inhibiting tumor growth. Non-limiting examples of chemotherapeutic agents include alkylating agents such as nitrogen mustard, ethyleneimine compounds, and alkyl sulfonates; antimetabolites such as folic acid, purine antagonists or pyrimidine antagonists; mitotic inhibitors such as antimitotic agents including vinca alkaloids, auristatins, and podophyllotoxin derivatives; cytotoxic antibiotics; compounds that impair or inhibit DNA expression or replication such as DNA minor groove binders; and growth factor receptor antagonists. Further, chemotherapeutic agents include cytotoxic agents (as defined herein), antibodies, biological molecules and small molecules.

[0075] The term "compound" refers to and includes the compound itself, and, whether or not expressly stated and unless the context clearly dictates otherwise, also refers to and includes the following: amorphous and crystalline forms of the compound, including polymorphs; these forms may be part of a mixture or may be isolated; free acid and free base forms of the compound, typically in the form shown by the structures provided herein; isomers of the compound, which refer to optical isomers and tautomers; optical isomers include enantiomers and diastereomers, chiral and achiral isomers; optical isomers include isolated optical isomers as well as mixtures of optical isomers including racemic and non-racemic mixtures; isomers may be in isolated form or may be included in a mixture with one or more other isomers; isotopes of the compound, including compounds containing deuterium and compounds containing tritium, as well as compounds containing radioisotopes including radioisotopes that are therapeutically and diagnostically effective; multimers of the compound, including dimers, trimers, etc.; salts of the compound, including acid addition salts and base addition salts, preferably pharmaceutically acceptable salts; salts of the compound with organic and inorganic counterions, preferably pharmaceutically acceptable salts; salts of the compound, including zwitterionic forms, preferably pharmaceutically acceptable salts; in this case, if the compound binds to more than one type of counterion, the more than one type of counterion may be the same or different; and solvates of the compound, including hemisolvates, monosolvates, disolvates, etc.; solvates include organic solvates and inorganic solvates, and the inorganic solvates include hydrates; if the compound binds to more than one type of solvent molecule, the more than one type of solvent molecule may be the same or different. In some cases, references made herein to the compounds of the invention include explicit reference to one of the above forms, e.g., a salt and / or solvate. However, this reference is for emphasis only and should not be construed to exclude the other forms as specified above.

[0076] As used herein, the term "conservative substitution" refers to amino acid substitutions that are known to those of skill in the art and generally can be made without altering the biological activity of the resulting molecule. Those of skill in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson, et al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin / Cummings Pub. Co., p.224 (4th Edition 1987)). Such exemplary substitutions are preferably made according to the substitutions shown in Tables II and III(a-b). For example, such changes include substituting any of isoleucine (I), valine (V), and leucine (L) with any of the other of these hydrophobic amino acids; substituting glutamic acid (E) with aspartic acid (D) and vice versa; substituting asparagine (N) with glutamine (Q) and vice versa; and substituting threonine (T) with serine (S) and vice versa. Other substitutions may also be considered conservative depending on the environment of a particular amino acid and its role in the protein's tertiary structure. For example, glycine (G) and alanine (A) are frequently interchangeable. Alanine (A) and valine (V) may be as well. The relatively hydrophobic methionine (M) is frequently interchangeable with leucine and isoleucine and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable at positions where the large feature of the amino acid residue is charge and the different pKs of these two amino acid residues are not important. Still other changes may be considered "conservative" in a particular environment (see, e.g., Table III(a) herein; pages 13-15 of "Biochemistry" 2nd ED. edited by Lubert Stryer (Stanford University); Henikoff, et al., PNAS 1992 Vol 89 10915-10919; Lei, et al., J Biol Chem 1995 May19;270(20):11882-11886).Other substitutions are also acceptable and can be determined empirically or according to known conservative substitutions.

[0077] The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression activity and function of cells and / or causes cell destruction. This term is intended to include radioisotopes, chemotherapeutic agents, and toxins, such as low molecular weight toxins or enzyme-active toxins of bacterial origin, fungal origin, plant origin, or animal origin, including their fragments and / or variants. Examples of cytotoxic agents include auristatin (e.g., auristatin E, auristatin F, MMAE, and MMAF), auromycin, maytansinoid, ricin, ricin A chain, combrestatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracinedione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, α-sarcin, gelonin, mitogellin, restrictocin, phenomycin, enomycin, curicin, crotonin, calicheamicin, Sapaonaria officinalis inhibitor, and glucocorticoids, as well as other chemotherapeutic agents, and radioisotopes, such as At 211 、I 131 、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 or Bi 213 、P 32 、and Lu 177It includes, but is not limited to, radioactive isotopes of Lu. The antibody can also be conjugated to an anti-cancer prodrug activating enzyme capable of converting a prodrug into its active form.

[0078] As used herein, the term "diabody" refers to a small antibody fragment containing two antigen-binding sites, and this fragment contains a heavy-chain variable domain (V L ) connected to a light-chain variable domain (V H ) in the same polypeptide chain (V H -V L ). By using a linker that is too short to allow pairing between the two domains of the same chain, those domains are paired with the complementary domains of another chain to create two antigen-binding sites. Diabodies are more fully described, for example, in EP404,097; WO93 / 11161; and Hollinger, et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993).

[0079] The term "deplete" with respect to the action of a 191P4D12 binder on 191P4D12-expressing cells refers to a decrease in the number of 191P4D12-expressing cells or the elimination of 191P4D12-expressing cells.

[0080] The term "gene product" is used herein to refer to a peptide / protein or mRNA. For example, the "gene product of the present invention" is sometimes also referred to herein as the "cancer amino acid sequence", the "cancer protein", the "protein of cancer listed in Table I", the "cancer mRNA", the "mRNA of cancer listed in Table I", etc. In one aspect, the cancer protein is encoded by the nucleic acid of FIG. 1. The cancer protein may be a fragment or the full-length protein encoded by the nucleic acid of FIG. 1. In one aspect, the cancer amino acid sequence is used to determine sequence identity or sequence similarity. In another aspect, the sequence is a natural allelic variant of the protein encoded by the nucleic acid of FIG. 1. In another aspect, the sequence is a sequence variant as further described herein.

[0081] "Heteroconjugate" antibodies are useful in the methods and compositions described herein. As used herein, the term "heteroconjugate antibody" refers to two antibodies that are covalently bound. Such antibodies can be prepared using known methods in synthetic protein chemistry, including the use of cross-linking agents. See, for example, U.S. Patent No. 4,676,980.

[0082] The term "homolog" refers to a molecule that exhibits homology to another molecule, for example, by having a sequence of chemical residues that are the same or similar at corresponding positions.

[0083] In one aspect, the antibodies provided herein are "human antibodies." As used herein, the term "human antibody" refers to an antibody in which essentially all of the sequences of the light and heavy chain sequences, including the complementarity determining regions (CDRs), are derived from human genes. In one aspect, human monoclonal antibodies are prepared by trioma technology, human B cell technology (see, e.g., Kozbor, et al., Immunol. Today 4:72 (1983)), EBV transformation technology (see, e.g., Cole et al., Monoclonal Antibodies And Cancer Therapy 77-96 (1985)), or phage display (see, e.g., Marks, et al., J. Mol. Biol. 222:581 (1991)). In certain aspects, human antibodies are produced in transgenic mice. Techniques for making such partially or fully human antibodies are known in the art, and any such technique can be used. According to one particular preferred aspect, full-length human antibody sequences are made in transgenic mice engineered to express human heavy chain antibody genes and human light chain antibody genes. Exemplary descriptions regarding the preparation of transgenic mice that produce human antibodies and their progeny are found in application number WO02 / 43478 and U.S. Patent No. 6,657,103 (Abgenix). B cells from transgenic mice that produce the desired antibody can then be fused to create a hybridoma cell line for the continuous production of that antibody. See, e.g., U.S. Patent Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; as well as Jakobovits, Adv. Drug Del. Rev. 31:33-42 (1998); Green, et al., J. Exp. Med. 188:483-95 (1998).

[0084] As used herein, the term "humanized antibody" refers to an antibody form that includes sequences derived from non-human (e.g., murine) antibodies as well as sequences derived from human antibodies. Such an antibody is a chimeric antibody that includes a minimal sequence derived from a non-human immunoglobulin. Generally, a humanized antibody includes substantially all of at least one, typically two, variable domains, all or substantially all of its hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of its FR regions being those of a human immunoglobulin sequence. A humanized antibody also optionally includes at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, for example, Cabilly, U.S. Patent No. 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).

[0085] As used herein, the term "inhibit" or "inhibiting" means to reduce by a measurable amount or to prevent completely.

[0086] The terms "isolated" or "biologically pure" refer to a substance that is substantially or essentially free of the components that are normally associated with that substance in its native state. Thus, an isolated peptide according to the present invention preferably does not contain the substances that are normally associated with that peptide in its in situ environment. For example, a polynucleotide is said to be "isolated" when it is substantially separated from contaminating polynucleotides that may correspond to or be complementary to genes other than the 191P4D12 gene, or that encode polypeptides or fragments thereof other than the 191P4D12 gene product. One of ordinary skill in the art can readily use nucleic acid isolation procedures to obtain an isolated 191P4D12 polynucleotide. A protein is said to be "isolated" when, for example, physical, mechanical, or chemical methods are used to remove the 191P4D12 protein from the cellular components that are normally associated with the 191P4D12 protein. One of ordinary skill in the art can readily use standard purification methods to obtain an isolated 191P4D12 protein. Alternatively, an isolated protein can be prepared by chemical means.

[0087] Suitable "labels" include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents disclosing the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Further, the antibodies provided herein may be useful as the antigen-binding component of a fluorobody. See, for example, Zeytun et al., Nat. Biotechnol. 21:1473-79 (2003).

[0088] The term "mammal" refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses, and humans. In one aspect of the invention, the mammal is a mouse. In another aspect of the invention, the mammal is a human.

[0089] The terms "metastatic cancer" and "metastatic disease" mean cancer that has spread to regional lymph nodes or distant sites and are meant to include diseases of stage D in the AUA system and stage TxNxM+ in the TNM system.

[0090] The term "modulator" or "test compound" or "drug candidate" or grammatical equivalents, as used herein, refers to any molecule, such as a protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., that is being tested for its ability to directly or indirectly alter the phenotype of cancer or the expression of a cancer sequence, e.g., a nucleic acid sequence or protein sequence, or the effect of a cancer sequence, e.g., signal transduction, gene expression, protein interaction, etc. In one aspect, the modulator neutralizes the effect of the cancer protein of the invention. "Neutralize" means that the activity of the protein is inhibited or blocked along with the resulting effect on the cell. In another aspect, the modulator neutralizes the effect of the gene of the invention and its corresponding protein by normalizing the level of that protein. In a preferred aspect, the modulator alters the expression profile, the expression profile of the nucleic acid or protein provided herein, or the downstream effector pathway. In one aspect, the modulator suppresses the cancer phenotype, e.g., to a normal tissue fingerprint. In another aspect, the modulator induces the cancer phenotype. Generally, multiple assay mixtures are performed in parallel using various drug concentrations to obtain a difference in response to those various concentrations. Typically, one of these concentrations serves as a negative control, i.e., a zero concentration or below the detection level.

[0091] The modulator, drug candidate, or test compound includes a number of chemical classes, but typically these are organic molecules, preferably small organic compounds having a molecular weight greater than 100 Daltons and less than about 2,500 Daltons. Preferred small molecules are less than 2000 D or less than 1500 D or less than 1000 D or less than 500 D. The candidate agent contains functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically includes at least an amine group, a carbonyl group, a hydroxyl group, or a carboxyl group, preferably including at least two of these functional chemical groups. This candidate factor often includes a cyclic carbon structure or a heterocyclic structure substituted with one or more of the above functional groups, and / or an aromatic ring structure or a polyaromatic ring structure. The modulator also includes biomolecules such as peptides, sugars, fatty acids, steroids, purines, pyrimidines, their derivatives, their structural analogs, or combinations thereof. Particularly preferred are peptides. Certain types of modulators are peptides, for example, from about 5 amino acids to about 35 amino acids, preferably from about 5 amino acids to about 20 amino acids, and particularly preferably from about 7 amino acids to about 15 amino acids. Preferably, the cancer regulatory protein is soluble, contains a non-transmembrane region, and / or has an N-terminal Cys to assist solubility. In one aspect, the C-terminus of the fragment is maintained as a free acid and its N-terminus is a free amine to assist coupling, i.e., coupling to cysteine. In one aspect, the cancer protein of the present invention is conjugated to an immunogenic agent as contemplated herein. In one aspect, the cancer protein is conjugated to BSA. For example, peptides of the present invention of a preferred length can be linked to each other or to other amino acids to create longer peptides / proteins. The regulatory peptide may be a digest of a natural protein, a random peptide, or a "biased" random peptide as outlined above. In a preferred aspect, the peptide / protein-based modulator is an antibody and its fragments as defined herein.

[0092] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against one antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically contain multiple antibodies directed against different epitopes (i.e., specific therefor). In one aspect, a polyclonal antibody comprises multiple monoclonal antibodies having different epitope specificities, affinities, or avidities for different epitopes of one antigen comprising multiple antigenic epitopes. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler, et al., Nature 256:495 (1975), or by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson, et al., Nature 352:624-628 (1991) and Marks, et al., J. Mol. Biol. 222:581-597 (1991). These monoclonal antibodies bind with a Kd of usually at least about 1 μM, more usually at least about 300 nM, typically at least about 30 nM, preferably at least about 10 nM, more preferably at least about 3 nM or greater, as determined usually by ELISA.

[0093] "Pharmaceutical excipient" includes substances such as adjuvants, carriers, pH adjusters, and buffers, tonicity adjusters, wetting agents, preservatives, and the like.

[0094] "Pharmaceutically acceptable" refers to a composition that is physiologically compatible with humans or other mammals, being non-toxic and / or inert.

[0095] The term "polynucleotide" means a polymeric form of nucleotides, ribonucleotides or deoxyribonucleotides, each of at least 10 bases or 10 base pairs in length, or a modified form of any type of nucleotide, which includes single-stranded and double-stranded forms of DNA and / or RNA. In the art, this term is often used synonymously with "oligonucleotide". A polynucleotide may contain the sequences disclosed herein. For example, as shown in FIG. 1, thymidine (T) may also be uracil (U). This definition relates to the chemical structure differences between DNA and RNA, particularly the observation that one of the four major bases in RNA is uracil (U) instead of thymidine (T).

[0096] The term "polypeptide" means a polymer of at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, or at least about 8 amino acids. Throughout this specification, standard three-letter or one-letter designations for amino acids are used. In the art, this term is often synonymous with "peptide" or "protein".

[0097] A "recombinant" DNA molecule or RNA molecule is a DNA molecule or RNA molecule that has been subjected to molecular manipulation in vitro.

[0098] As used herein, the terms "single-chain Fv", "scFv", or "single-chain" antibody refer to an antibody fragment that contains the V H domain and the V L domain, and these domains are present in a single polypeptide chain. Generally, an Fv polypeptide consists of the V H domain and the V LIt further includes a polypeptide linker that enables the sFv to form a desirable structure for antigen binding between the domains. For an overview of sFv, see Pluckthun, The Pharmacology Of Monoclonal Antibodies, vol.113, edited by Rosenberg and Moore, Springer-Verlag, New York, pp.269-315 (1994).

[0099] As used herein, the terms "specific," "specifically binds," and "binds specifically" refer to the selective binding of an antibody to a target antigen epitope. An antibody can be tested for binding specificity by comparing its binding to the appropriate antigen to its binding to an irrelevant antigen or antigen mixture under a given set of conditions. An antibody is considered specific if it binds at least 2-fold, at least 5-fold, at least 7-fold, preferably at least 10-fold more to its appropriate antigen than to an irrelevant antigen or antigen mixture. In one aspect, a specific antibody is an antibody that binds only to the 191P4D12 antigen and not to an irrelevant antigen. In another aspect, a specific antibody is an antibody that binds to the human 191P4D12 antigen but does not bind to a non-human 191P4D12 antigen having 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 aspect, a specific antibody is an antibody that binds to the human 191P4D12 antigen and also binds to the mouse 191P4D12 antigen, but binds more strongly to the human antigen. In another aspect, a specific antibody is an antibody that binds to the human 191P4D12 antigen and also binds to the primate 191P4D12 antigen, but binds more strongly to the human antigen. In another aspect, a specific antibody is an antibody that binds to the human 191P4D12 antigen and any non-human 191P4D12 antigen, but binds more strongly to the human antigen or any combination thereof.

[0100] As used herein, the terms "for treating" or "therapeutic" and grammatically related terms refer to any improvement in any result of a disease, e.g., extension of survival, reduction of morbidity, and / or attenuation of side effects that are a byproduct of an alternative treatment modality. As will be readily appreciated in the art, complete eradication of a disease is preferred, although not a requirement for an act of treatment.

[0101] The term "variant" refers to a molecule that shows a change from a described type or reference, e.g., a protein having one or more different amino acid residues at corresponding positions of a specifically described protein (e.g., the 191P4D12 protein shown in FIG. 1). An analog is an example of a variant protein. Splice isoforms and single nucleotide polymorphisms (SNPs) are further examples of variants.

[0102] The "191P4D12 protein" and / or "191P4D12-related protein" of the present invention includes allelic variants, conservative substitution variants, analogs, and homologs that are specifically identified herein (see Figure 1), and can be isolated / created and characterized without undue experimentation according to the methods outlined herein or methods readily available in the art. Also included are fusion proteins formed by combining portions or fragments of different 191P4D12 proteins, as well as fusion proteins of 191P4D12 proteins and heterologous polypeptides. Such 191P4D12 proteins are collectively referred to as 191P4D12-related proteins, the proteins of the present invention, or 191P4D12.The term "191P4D12-related protein" refers to a polypeptide fragment of the 191P4D12 protein sequence that is 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 a polypeptide fragment of the 191P4D12 protein sequence that is at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 330, at least 335, at least 339, or more amino acids.

[0103] II.) 191P4D12 antibody Another aspect of the invention provides an antibody that binds to a 191P4D12-related protein (see Figure 1). In one embodiment, the antibody that binds to a 191P4D12-related protein is an antibody that specifically binds to a 191P4D12 protein comprising the amino acid sequence of SEQ ID NO.:2. Antibodies that specifically bind to a 191P4D12 protein comprising the amino acid sequence of SEQ ID NO.:2 include antibodies that can bind to other 19P4D12-related proteins. For example, an antibody that binds to a 191P4D12 protein comprising the amino acid sequence of SEQ ID NO.:2 can bind to 191P4D12-related proteins such as 191P4D12 variants and its homologs or analogs.

[0104] The 191P4D12 antibodies of the invention are particularly useful in prognostic assays, imaging and treatment methodologies for cancer (see, for example, Table I). Similarly, such antibodies are useful to the extent that 191P4D12 is expressed or overexpressed in these other cancers in the treatment and / or prognosis of colon cancer and other cancers. Furthermore, intracellularly expressed antibodies (e.g., single-chain antibodies) are therapeutically useful in treating cancers in which the expression of 191P4D12 is involved, such as advanced or metastatic colon cancer or other advanced or metastatic cancers.

[0105] A variety of methods for preparing antibodies, particularly monoclonal antibodies, are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host with an isolated or immunoconjugated 191P4D12-related protein, peptide, or fragment (Antibodies: A Laboratory Manual, edited by CSH Press, Harlow and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). Additionally, 191P4D12 fusion proteins such as 191P4D12 GST-fusion proteins can also be used. In certain embodiments, a GST fusion protein containing all or most of the amino acid sequence of FIG. 1 is generated and then used as an immunogen to generate appropriate antibodies. In another embodiment, a 191P4D12-related protein is synthesized and used as an immunogen.

[0106] Furthermore, DNA-based immunization techniques known in the art are used (with or without purified 191P4D12-related protein or 191P4D12-expressing cells) to generate an immune response against the encoded immunogen (see Donnelly et al., 1997, Ann. Rev. Immunol. 15:617-648 for a review).

[0107] The amino acid sequence of the 191P4D12 protein shown in Figure 1 can be analyzed to select specific regions of the 191P4D12 protein for antibody production. For example, hydrophobicity and hydrophilicity analyses of the 191P4D12 amino acid sequence are used to identify hydrophilic regions in the 191P4D12 structure. Regions of the 191P4D12 protein that exhibit immunogenic structures, as well as other regions and domains, can be readily identified using a variety of other methods known in the art, such as Chou-Fasman analysis, Garnier-Robson analysis, Kyte-Doolittle analysis, Eisenberg analysis, Karplus-Schultz analysis, or Jameson-Wolf analysis. Hydrophilic profiles can be generated using the method of Hopp, T.P. and Woods, K.R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydrophobicity profiles can be generated using the method of Kyte, J. and Doolittle, R.F., 1982, J. Mol. Biol. 157:105-132. Percent (%) accessible residue 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. β-turn profiles can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294. Accordingly, each region identified by any of these programs or methods is within the scope of the present invention. Preferred methods for the production of 191P4D12 antibodies are further exemplified by the examples provided herein. Methods for preparing proteins or polypeptides for use as immunogens are well known in the art. Methods for preparing immunogenic conjugates of proteins with carriers, such as BSA, KLH, or other carrier proteins, are also well known in the art.In certain situations, for example, direct coupling using a carbodiimide reagent is employed. In other cases, a linking reagent, such as those supplied by Pierce Chemical Co., Rockford, IL, is effective. Administration of the 191P4D12 immunogen is often carried out by injection over an appropriate period, often with the use of a suitable adjuvant, as understood in the art. Antibody titers can be assayed to confirm the validity of antibody formation during the immunization schedule.

[0108] The 191P4D12 monoclonal antibody can be generated by various means well known in the art. For example, an immortalized cell line secreting the desired monoclonal antibody is prepared using the standard hybridoma technique of Kohler and Milstein or a modified method of immortalizing antibody-producing B cells, as is generally known. The immortalized cell line secreting the desired antibody is screened by an immunoassay in which the antigen is the 191P4D12-related protein. If a suitable immortalized cell culture is identified, the cells can be grown and the antibody can be produced from either an in vitro culture or ascites.

[0109] The antibodies or fragments of the present invention can also be produced by recombinant means. In the situation of antibodies derived from multiple species with chimeric regions or complementarity determining regions (CDRs) spliced together, regions that specifically bind to the desired regions of the 191P4D12 protein can also be generated. Humanized or human 191P4D12 antibodies can also be produced and are preferred for use in therapeutic situations. Methods for humanizing mouse 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. Acad. Sci. USA 89:4285 and Sims et al., 1993, J. Immunol. 151:2296.

[0110] In a preferred embodiment, the antibodies of the present invention include fully human 191P4D12 antibodies (191P4D12 MAb). Various methods in the art provide means for generating fully human 191P4D12 MAb. For example, a preferred embodiment provides a technique using a transgenic mouse called Xenomouse (Amgen Fremont, Inc.), which has inactivated antibody production and is engineered using human heavy and light chain loci. An exemplary description of making a transgenic mouse that produces human antibodies can be found in U.S. Patent No. 6,657,103. See also U.S. Patent Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; as well as Mendez, et. al., Nature Genetics 15:146-156 (1998); Kellerman, S. A. & Green, L. L., Curr. Opin. Biotechnol. 13:593-597 (2002).

[0111] Furthermore, the human antibodies of the present invention can be produced using HuMAb mice (Medarex, Inc.) containing rearranged human heavy chains (μ and γ) and κ light chain immunoglobulin sequences, as well as human immunoglobulin gene miniloci encoding targeting mutations that inactivate the endogenous μ and κ chain loci (see, for example, Lonberg, et al. (1994) Nature 368(6474):856-859).

[0112] In another aspect, the fully human antibodies of the present invention can be produced using mice having human immunoglobulin sequences on the transgene and transchromosome, such as mice having a human heavy chain transgene and a human light chain transchromosome. Such mice are referred to herein as "KM mice" and are described in Tomizuka, et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727 and PCT publication WO02 / 43478 to Tomizuka et al.

[0113] The human monoclonal antibodies of the present invention can also be prepared using phage display methods to screen libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are well established in the art. See, for example, U.S. Patent Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner, et al.; U.S. Patent Nos. 5,427,908 and 5,580,717 to Dower, et al.; U.S. Patent Nos. 5,969,108 and 6,172,197 to McCafferty, et al.; and U.S. Patent Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915; and 6,593,081 to Griffiths, et al.

[0114] The human monoclonal antibodies of the present invention can also be made using SCID mice in which human immune cells have been reconstituted so that a human antibody response occurs upon immunization. Such mice are described, for example, in U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson, et al.

[0115] In a preferred embodiment, the 191P4D12 MAb of the present invention is produced by a hybridoma deposited with the American Type Culture Collection (ATCC) accession number: PTA-11267, and comprises a heavy chain variable region and a light chain variable region of an antibody named Ha22-2(2,4)6.1 (see FIG. 3), or a heavy chain variable region and a light chain variable region comprising an amino acid sequence homologous to the amino acid sequences of the heavy chain variable region and the light chain variable region of Ha22-2(2,4)6.1, wherein the antibody retains the desired functional properties of the 191P4D12 MAb of the present invention. The heavy chain variable region of Ha22-2(2,4)6.1 consists of 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 D residue to the 130th R residue of SEQ ID NO:8. As the constant region of the antibody of the present invention, any constant region subclass can be selected. In one embodiment, a human IgG1 constant region can be used as the heavy chain constant region and a human Igκ constant region can be used as the light chain constant region.

[0116] For example, the present invention provides an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region: (a) the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to the heavy chain variable region amino acid sequence shown in FIG. 3, and (b) the light chain variable region comprises an amino acid sequence that is at least 80% homologous to the light chain variable region amino acid sequence shown in FIG. 3.

[0117] In other embodiments, V H and / or V LThe amino acid sequence may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the V H and V L sequences shown in Figure 3.

[0118] In another aspect, the invention provides an isolated monoclonal antibody, or an antigen-binding portion thereof, comprising a humanized heavy chain variable region and a humanized light chain variable region, wherein (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, (b) the light chain variable region comprises CDRs having the amino acid sequences of the light chain variable region CDRs shown in Figure 3.

[0119] The engineered antibodies of the invention include those in which the framework residues in V H and / or V L have been modified (e.g., to improve the properties of the antibody). Typically, such framework modifications are made to reduce 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 are different from the germline sequence from which the antibody originated. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody originated. To return the framework region sequence to its germline configuration, somatic mutations can be "backmutated" to the germline sequence, for example, by site-directed mutagenesis or mutagenesis via PCR (e.g., "backmutating" leucine to methionine). Such "backmutated" antibodies are also intended to be included within the scope of the present invention.

[0120] Another type of framework modification involves mutating one or more residues within a framework region, or even one or more residues within one or more CDR regions, to remove T cell epitopes and reduce the potential immunogenicity of the antibody. This approach, also referred to as "deimmunization," is further described in U.S. Patent Publication No. 2003 / 0153043 by Carr, et al.

[0121] In addition to, or instead of, the modifications made in the framework or CDR regions, the antibodies of the present invention may typically be engineered to include modifications within the Fc region to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antibody-dependent cell cytotoxicity. Further, the 191P4D12 MAb of the present invention may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody), modified to alter glycosylation, or modified to alter one or more functional properties of the MAb. These aspects are further described in more detail below.

[0122] In one aspect, the hinge region of CH1 is modified such that the number of cysteine residues within the hinge region is changed, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 to Bodmer, et al. The number of cysteine residues within the hinge region of CH1 may be changed, for example, to facilitate assembly of the light and heavy chains, or to increase or decrease the stability of the 191P4D12 MAb.

[0123] In another aspect, the Fc hinge region of the antibody is mutated to shorten the biological half-life of 191P4D12 MAb. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the Staphylococcyl protein A (SpA) binding of the antibody is reduced compared to the native Fc-hinge domain SpA binding. This approach is described in more detail in U.S. Patent No. 6,165,745 by Ward, et al.

[0124] In another aspect, 191P4D12 MAb is modified to have an increased biological half-life. Various 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 CH1 region or the CL region of the antibody can be altered to contain salvage receptor binding epitopes taken from two loops of the CH2 domain of the Fc region of IgG as described in U.S. Patents No. 5,869,046 and 6,121,022 by Presta, et al.

[0125] In yet another aspect, the Fc region is altered by substituting at least one amino acid residue with a different amino acid residue to change the effector function of 191P4D12 MAb. For example, one or more amino acids selected from amino acid specific residues can be substituted with different amino acid residues such that the affinity of the antibody for an effector ligand is changed while retaining the antigen binding ability of the parental antibody. The effector ligand whose affinity is changed can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patents No. 5,624,821 and 5,648,260 by Winter, et al.

[0126] The reactivity between the 191P4D12 antibody and the 191P4D12-related protein can be established by many well-known means including Western blot, immunoprecipitation, ELISA, and FACS analysis, as appropriate, using the 191P4D12-related protein, 191P4D12-expressing cells or their extracts. The 191P4D12 antibody or its fragment may be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelating agents or enzymes. Furthermore, bispecific antibodies specific for two or more 191P4D12 epitopes are prepared using methods generally known in the art. Homodimeric antibodies can also be prepared by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res. 53:2560-2565).

[0127] In yet another preferred embodiment, the 191P4D12 MAb of the present invention is an antibody comprising the heavy and light chains of an antibody named 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 E residue to the 466th K residue of SEQ ID NO:7, and the light chain of Ha22-2(2,4)6.1 consists of the amino acid sequence from the 23rd D residue to the 236th C residue of SEQ ID NO:8. This sequence is shown in FIGS. 2 and 3. In a preferred embodiment, Ha22-2(2,4)6.1 is conjugated to a cytotoxic agent.

[0128] Ha22 - 2(2,4)6.1 The hybridoma producing the antibody named August 18, 2010 was sent (via Federal Express) to American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, and accession number PTA - 11267 was assigned.

[0129] III.) Antibody-Drug Conjugates General Discussion In another aspect, the invention provides an antibody-drug conjugate (ADC) in which the antibody is conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., a toxin having enzymatic activity derived from a bacterium, fungus, plant, or animal, or a fragment thereof), or is conjugated to a radioisotope (i.e., a radioconjugate). In another aspect, the invention further provides methods of using the ADCs. In one aspect, the ADC comprises the 191P4D12 MAb described herein covalently bound to a cytotoxic agent or a detectable agent.

[0130] In cancer treatment, when an antibody-drug conjugate is used to locally deliver a cytotoxic or cytostatic agent, i.e., a drug that kills or inhibits tumor cells (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151-172; U.S. Patent No. 4,975,278), it becomes possible to target the drug moiety to the tumor and accumulate it intracellularly within the tumor. In this case, systemic delivery of these unconjugated drugs can result in unacceptable levels of toxicity to normal cells, and tumor cells are also required to be eliminated (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. 475-506). Thereby, the minimum toxicity and the maximum efficacy are required. In these strategies, both polyclonal and monoclonal antibodies have been reported to be useful (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-drug 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), maytansinoids (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. 53:3336-3342). These toxins can exert cytotoxic and cell division inhibitory effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to become inactive or have reduced activity when conjugated to large antibodies or protein receptor ligands.

[0131] Examples of antibody-drug conjugates include a murine IgG1κ monoclonal antibody against the CD20 antigen found on the surface of normal and malignant B lymphocytes and 111 In or 90ZEVALIN® (ibritumomab tiuxetan, Biogen / Idec) is an antibody-radioligand conjugate in which a Y-radioligand is bound by a thiourea linker-chelate agent (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).

[0132] Furthermore, in 2000, MYLOTARG® (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody-drug conjugate in which a huCD33 antibody is linked to calicheamicin, was approved for the treatment of acute myeloid leukemia by injection (Drugs of the Future (2000) 25(7):686; U.S. Patent Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116; 5,767,285; 5,773,001).

[0133] Furthermore, for the treatment of cancers expressing CanAg, such as colon cancer, pancreatic cancer, gastric cancer, and other cancers, Cantuzumab mertansine (Immunogen, Inc.), an antibody-drug conjugate in which a huC242 antibody is linked to a maytansinoid drug moiety, DM1, via a disulfide linker, SPP, is in Phase II clinical trials.

[0134] Furthermore, for the promising treatment of prostate tumors, MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), an antibody-drug conjugate in which an anti-prostate specific membrane antigen (PSMA) monoclonal antibody is linked to a maytansinoid drug moiety, DM1, is under development.

[0135] Finally, auristatin peptides, auristatin E (AE), and monomethyl auristatin (MMAE), which are synthetic analogs of dolastatin, have been conjugated to the chimeric monoclonal antibodies cBR96 (specific for Lewis Y on carcinomas) and cAC10 (specific for CD30 on hematological malignancies) (Doronina et al (2003) Nature Biotechnology 21(7):778-784) and are in therapeutic development.

[0136] Furthermore, chemotherapeutic agents useful in the preparation of ADCs are described herein. Toxins having enzymatic activity and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin, as well as trichothecene. See, for example, WO93 / 21232, published October 28, 1993. A variety of radionuclides can be utilized to produce radiolabeled antibodies. Examples include 212 Bi, 131 I, 131 In, 90 Y, and 186It contains Re. Conjugates of antibodies and cytotoxic agents can be made using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described by Vitetta et al (1987) Science, 238:1098. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies (WO94 / 11026).

[0137] Also contemplated herein are conjugates of antibodies with one or more low molecular weight toxins, such as calicheamicin, maytansinoid, dolastatin, auristatin, trichothecene, and CC1065, and derivatives of these toxins having toxin activity.

[0138] III(A). Maytansinoid Maytansinoid 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 or prepared using genetic engineering techniques (see Yu et al (2002) PNAS 99:7968-7973). Alternatively, maytansinol and maytansinol analogs can be prepared synthetically according to known methods.

[0139] Exemplary mitansinoid drug moieties include those having a modified aromatic ring, such as C-19-dichloro (US4256746) (prepared by lithium aluminum hydride reduction of ansamytocin P2); C-20-hydroxy (or C-20-demethyl) + / - C-19-dichloro (U.S. Patent 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-acyl-oxy (-OCOR), + / - dichloro (U.S. Patent No. 4,294,757) (prepared by acylation using acyl chloride), as well as those having modifications at other positions.

[0140] Exemplary mitansinoid drug moieties include those having modifications, such as C-9-SH (US4,424,219) (prepared by reaction of mitansinol with H2S or P2S5); C-14-alkoxymethyl (demethoxy / CH2OR) (US4331598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (US4450254) (prepared from Nocardia); C-15-hydroxy / acyloxy (US4,364,866) (prepared by mitansinol conversion by Streptomyces); C-15-methoxy (U.S. Patent Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudlflora); C-18-N-demethyl (U.S. Patent Nos. 4,362,663 and 4,322,348) (prepared by demethylation of mitansinol by Streptomyces); and 4,5-deoxy (US4,371,533) (prepared by titanium trichloride / LAH reduction of mitansinol) are also included.

[0141] ADCs containing maytansinoids, methods for making the same, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020; 5,416,064; 6,441,163 and European Patent No. EP0425235 B1. These disclosures are hereby expressly incorporated herein by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described an ADC in which a maytansinoid named DM1 was conjugated to a monoclonal antibody C242 against human colorectal cancer. This conjugate was found to be highly cytotoxic to cultured colon cancer cells and showed antitumor activity in in vivo tumor growth assays. Chari et al., Cancer Research 52:127-131 (1992) described an ADC in which a maytansinoid was conjugated via a disulfide linker to a mouse antibody A7 that binds to an antigen on a human colon cancer cell line or to another mouse monoclonal antibody TA.1 that binds to the HER-2 / neu oncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was tested in vitro against the human breast cancer cell line SK-BR-3 that expresses 3x10 5 HER-2 surface antigens per cell. This drug conjugate achieved cytotoxicity at approximately the same level as the free maytansinoid drug. Cytotoxicity could be enhanced by increasing the number of maytansinoid molecules per antibody molecule. The systemic cytotoxicity of the A7-maytansinoid conjugate in mice was low.

[0142] III(B). Auristatin and dolastatin In one aspect, the ADC comprises an antibody of the invention conjugated to an auristatin which is dolastatin or a peptidic mimetic and derivative of dolastatin (U.S. Patent Nos. 5,635,483; 5,780,588). Dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and mitosis and cell division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), and have anticancer activity (US5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob. Agents Chemother.42:2961-2965). The dolastatin or auristatin drug moiety can be attached to the antibody via the N (amino) - or C (carboxyl) - terminus of the peptidic drug moiety (WO02 / 088172).

[0143] Exemplary auristatin embodiments include N - terminal conjugated monomethyl auristatin drug moieties DE and DF, disclosed in Senter et al., Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, published May 28, 2004, and described in U.S. Patent Application Publication No. 2005 / 0238649. These disclosures are hereby incorporated by reference in their entirety.

[0144] An exemplary auristatin embodiment is MMAE (where the wavy line indicates a covalent bond with the linker (L) of the antibody - drug conjugate). TIFF2025113404000003.tif29146

[0145] Another exemplary auristatin embodiment is MMAF. Where the wavy line indicates a covalent bond with the linker (L) of the antibody - drug conjugate (US2005 / 0238649). TIFF2025113404000004.tif29142

[0146] Further exemplary embodiments that include MMAE or MMAF and various linker components (further described herein) have the following structures and abbreviations (Ab means antibody, S is the sulfur of the antibody, and p is from 1 to about 8). TIFF2025113404000005.tif103151

[0147] Typically, peptide-based drug moieties can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid phase synthesis methods well known in the field of peptide chemistry (see E. Schroder and K. Lubke, 「The Peptides」, volume 1, pp 76-136, 1965, Academic Press). The auristatin / dolastatin drug moieties may be prepared according to the methods of US5635483; US5780588; 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. Soc. Perkin Trans. 15:859-863; and Doronina (2003) Nat Biotechnol 21(7):778-784.

[0148] III(C). Calicheamicin In other embodiments, the ADC comprises an antibody of the invention conjugated to one or more calicheamicin molecules. Calicheamicin-based antibiotics can cleave double-stranded DNA at sub-pM concentrations. For the preparation of calicheamicin-based conjugates, see U.S. Pat. Nos. 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 analogs of calicheamicin that can be used include, but are not limited to, γ1 I , α2 I , α3 I , N-acetyl-γ1 I , PSAG, and θ I 1 (Hinman et al., Cancer Research 53:3336-3,342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the above U.S. patents to American Cyanamid). Another antitumor drug that can be conjugated to the antibody is QFA, an antimetabolite of folic acid. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Thus, when these agents are taken up by cells via antibody-mediated internalization, their cytotoxic effects are greatly enhanced.

[0149] III(D). Other cytotoxic agents Other anti-cancer agents that can be conjugated to the antibody of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, a family of agents collectively referred to as the LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, and esperamicin (U.S. Pat. No. 5,877,296).

[0150] Toxins having enzymatic activity and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, saporin, dianthin protein, pokeweed antiviral protein (PAPI, PAPII, and PAP-S), cucumber inhibitor, curcin, crocin, pumpkin inhibitor, gelonin, mitogelin, restrictocin, phenomycin, and enomycin, and trichothecene. See, for example, WO93 / 21232 published on October 28, 1993.

[0151] Furthermore, the present invention contemplates an ADC formed between an antibody and a compound having nuclease activity (e.g., ribonuclease or DNA endonuclease, e.g., deoxyribonuclease; DNase).

[0152] To selectively destroy tumors, the antibody may contain a highly radioactive atom. Various radioisotopes can be utilized to produce radiolabeled antibodies. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioisotopes of Lu. When the conjugate is used for detection, it may contain a radioactive atom for scintigraphy studies, e.g., tc 99m or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as nuclear magnetic resonance imaging, MRI), e.g., again, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0153] A radioactive label or other label can be incorporated into the conjugate in a known manner. For example, instead of hydrogen, a peptide may be biosynthesized or synthesized by amino acid chemical synthesis using an appropriate amino acid precursor containing, for example, fluorine-19. Through cysteine residues in the peptide, tc 99m or I 123 、Re 186 、Re 188 、and In 111 and other labels can be attached. Iodine-123 can be incorporated using the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57) to attach yttrium-90 via lysine residues. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) details other methods.

[0154] IV.) Antibody-drug conjugate compounds that bind to 191P4D12 The present invention particularly provides an antibody-drug conjugate compound for targeted delivery of a drug. The inventors have discovered that this antibody-drug conjugate compound has strong cytotoxic activity and / or cell division inhibitory activity against 191P4D12-expressing cells. This antibody-drug conjugate compound includes an antibody unit covalently bonded to at least one drug unit. The drug unit may be covalently bonded directly or via a linker unit (LU).

[0155] In some embodiments, the antibody-drug conjugate compound has the following formula: L-(LU-D) P (I) or a pharmaceutically acceptable salt or solvate thereof, wherein, L is an antibody unit, for example, the 191P4D12 MAb of the present invention, (LU-D) is a linker unit-drug unit moiety, wherein, LU- is a linker unit, -D is a drug unit having cell division inhibitory activity or cytotoxic activity against target cells; p is an integer from 1 to 20.

[0156] In some embodiments, p is 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 embodiments, p is 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 embodiments, p is 1, 2, 3, 4, 5, or 6. In some embodiments, p is 2 or 4.

[0157] In some embodiments, the antibody-drug conjugate compound has the following formula: L-(A a -W w -Y y -D) p (II) or a pharmaceutically acceptable salt or solvate thereof, wherein, L is an antibody unit, such as 191P4D12 MAb; -A a -W W -Y y - is a linker unit (LU), wherein, -A- is a stretcher unit, a is 0 or 1, each -W- is independently an amino acid unit, w is an integer from 0 to 12, -Y- is a self-immolative spacer unit, y is 0, 1, or 2; -D is a drug unit having cell division inhibitory activity or cytotoxic activity against target cells; p is an integer from 1 to 20.

[0158] In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1, or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, p is 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 embodiments, p is from 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is 1, 2, 3, 4, 5, or 6. In some embodiments, p is 2 or 4. In some embodiments, when w is not 0, y is 1 or 2. In some embodiments, when w is from 1 to 12, y is 1 or 2. In some embodiments, w is from 2 to 12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0.

[0159] For a composition comprising a plurality of antibodies, the drug loading, which is the average number of drug molecules per antibody, is represented by p. The drug loading may be from 1 to 20 drugs (D) per antibody. The average number of drugs per antibody in the conjugation reaction preparation can be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of the antibody-drug conjugate with respect to p can also be determined. In some cases, the separation, purification, and characterization of a homogeneous antibody-drug conjugate with a particular value of p from antibody-drug conjugates having other drug loadings can be achieved by means such as reverse-phase HPLC or electrophoresis. In an exemplary embodiment, p is from 2 to 8.

[0160] The preparation of the antibody-drug conjugate compound can be achieved by any technique known to those skilled in the art. Briefly, the antibody-drug conjugate compound includes the 191P4D12 MAb as the antibody unit, the drug, and optionally a linker that connects the drug and the linker. In a preferred embodiment, the antibody is the 191P4D12 MAb that includes the heavy chain variable region and the light chain variable region of the antibody named Ha22-2(2,4)6.1 described above. In a more preferred embodiment, the antibody is the 191P4D12 MAb that includes the heavy chain and the light chain of the antibody named Ha22-2(2,4)6.1 described above. To covalently bind the drug and / or the linker to the linker, many different reactions can be utilized. This is often achieved by the reaction of the linker, such as the amino acid residues of the antibody molecule, which includes the amine group of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl group of cysteine, and various moieties of aromatic amino acids. One of the most commonly used non-specific covalent binding methods is the carbodiimide reaction that links the carboxy (or amino) group of the compound to the amino (or carboxy) group of the antibody. Further, to link the amino group of the compound to the amino group of the antibody molecule, a bifunctional agent, such as a dialdehyde or an imido ester, is used. The Schiff base reaction is also used to attach the drug to the linker. This method involves oxidizing the drug containing a glycol or hydroxy group with periodic acid to form an aldehyde, and then reacting this aldehyde with the linker. The attachment is carried out through the formation of a Schiff base having an amino group of the linker. Isothiocyanate can also be used as a coupling agent to covalently bind the drug to the linker. Other techniques are known to those skilled in the art and are within the scope of the present invention.

[0161] In a particular embodiment, an intermediate that is a precursor of the linker is reacted with the drug under appropriate conditions. In a particular embodiment, a reactive group present in the drug and / or the intermediate is used. Thereafter, the product of the reaction between the drug and the intermediate, i.e., the derivatized drug, is reacted with the 191P4D12 MAb under appropriate conditions.

[0162] Specific units of the antibody-drug conjugate compounds are detailed herein. The synthesis and structure of exemplary linker units, stretcher units, amino acid units, self-immolative spacer units, and drug units are also described in U.S. Patent Application Publication Nos. 2003-0083263, 2005-0238649, and 2005-0009751. Each of these is hereby incorporated by reference in its entirety and for all purposes.

[0163] V.) Linker Unit Typically, an antibody-drug conjugate compound includes a linker unit between the drug unit and the antibody unit. In some embodiments, the linker is cleavable under intracellular conditions such that the drug unit is released from the antibody when the linker is cleaved in the intracellular environment. In still other embodiments, the linker unit cannot be cleaved and the drug is released, for example, by antibody degradation.

[0164] In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within lysosomes or endosomes or caveolae). The linker may be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including but not limited to lysosomal protease or endosomal protease. In some embodiments, the peptidyl linker is at least 2 amino acids in length or at least 3 amino acids in length. The cleaving agents may include cathepsin B and cathepsin D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release the active drug within the target cell (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). The most representative one is a peptidyl linker cleavable by an enzyme present in 191P4D12-expressing cells. For example, a peptidyl linker cleavable by cathepsin-B, a thiol-dependent protease highly expressed in cancer tissues (e.g., Phe-Leu or Gly-Phe-Leu-Gly linker (SEQ ID NO:9)) can be used. Other examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, which is hereby incorporated by reference in its entirety and for all purposes. In certain embodiments, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin having a Val-Cit linker). One advantage of using intracellular proteolysis for therapeutic agent release is that the agent is typically attenuated when conjugated, and the serum stability of the conjugate is typically high.

[0165] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a particular pH value. Typically, the pH-sensitive linker is hydrolyzed under acidic conditions. For example, acid-labile linkers that are hydrolyzed within lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamide, orthoesters, acetals, ketals, etc.) can be used (see, e.g., U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, e.g., in blood at neutral pH, but are unstable at pH values less than pH 5.5 or pH 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to a therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Patent No. 5,622,929)).

[0166] In yet other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). For example, various disulfide linkers are known in the art, including 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-α-methyl-α-(2-pyridyl-dithio)toluene), SPDB, and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987). See also U.S. Patent No. 4,880,935).

[0167] In yet other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), 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).

[0168] In yet other embodiments, the linker unit is non-cleavable and the drug is released by antibody degradation (see U.S. Patent Application Publication No. 2005 / 0238649, which is hereby incorporated by reference in its entirety and for all purposes).

[0169] Typically, the linker is substantially insensitive to the extracellular environment. As used herein with respect to a linker, "substantially insensitive to the extracellular environment" means that when the antibody-drug conjugate compound is present in the extracellular environment (e.g., in plasma), about 20% or less, typically about 15% or less, more typically about 10% or less, even more typically about 5% or less, about 3% or less, or about 1% or less of the linker contained in a sample of the antibody-drug conjugate compound is cleaved. Whether a linker is substantially insensitive to the extracellular environment can be ascertained, for example, by incubating the antibody-drug conjugate compound with plasma for a predetermined time (e.g., 2 hours, 4 hours, 8 hours, 16 hours, or 24 hours) and then quantifying the amount of free drug present in the plasma.

[0170] In other non-exclusive embodiments, the linker facilitates cellular internalization. In certain embodiments, the linker facilitates cellular internalization when conjugated to a therapeutic agent (i.e., in the context of the linker-therapeutic agent moiety of the antibody-drug conjugate compounds described herein). In yet other embodiments, the linker facilitates cellular internalization when conjugated to an auristatin compound and 191P4D12 MAb.

[0171] Various exemplary linkers that can be used with the compositions and methods of the present invention are described in WO2004-010957, US Patent Application Publication No. 2006 / 0074008, US Patent Application Publication No. 20050238649, and US Patent Application Publication No. 2006 / 0024317 (each of which is hereby incorporated by reference in its entirety and for all purposes).

[0172] A "linker unit" (LU) is a bifunctional compound that can be used to link a drug unit and an antibody unit to form an antibody-drug conjugate compound. In some embodiments, the linker unit has the following formula: -A a -W w -Y y - wherein, -A- is a stretcher unit, a is 0 or 1, each -W- is independently an amino acid unit, w is an integer from 0 to 12, -Y- is a self-sacrificing spacer unit, y is 0, 1, or 2.

[0173] In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1, or  2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, when w is from 1 to 12, y is 1 or 2. In some embodiments, w is from 2 to 12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0.

[0174] VI.) Stretcher unit If a stretcher unit (A) is present, the stretcher unit (A) can link an antibody unit to an amino acid unit (-W-) if the amino acid unit (-W-) is present, to a spacer unit (-Y-) if the spacer unit (-Y-) is present, or to a drug unit (-D). Useful functional groups that may be present in 191P4D12 MAb (e.g., Ha22-2(2,4)6.1) include, but are not limited to, sulfhydryl groups, amino groups, hydroxyl groups, anomeric hydroxyl groups of carbohydrates, and carboxyl groups, whether natural or via chemical manipulation. Suitable functional groups are sulfhydryl and amino. In one example, a sulfhydryl group can be created by reducing the intramolecular disulfide bond of 191P4D12 MAb. In another embodiment, a sulfhydryl group can be created by reacting the amino group of the lysine portion of 191P4D12 MAb with 2-iminothiolane (Traut reagent) or other sulfhydryl-generating reagents. In certain embodiments, 191P4D12 MAb is a recombinant antibody and is engineered to have one or more lysines. In certain other embodiments, the recombinant 191P4D12 MAb is engineered to have additional sulfhydryl groups, e.g., additional cysteine.

[0175] In one embodiment, the stretcher unit forms a bond with a sulfur atom of the antibody unit. The sulfur atom may be derived from a sulfhydryl group of the antibody. Representative stretcher units of this embodiment are depicted within the square brackets of Formulas IIIa and IIIb. In the formulas, L-, -W-, -Y-, -D, w, and y are as defined above, and R 17 is -C1-C 10 alkylene-, -C1-C 10 alkenylene-, -C1-C 10 alkynylene-, carbocyclo-, -O-(C1-C8 alkylene)-, O-(C1-C8 alkenylene)-, -O-(C1-C8 alkynylene)-, -arylene-, -C1-C 10 alkylene-arylene-, -C2-C 10 alkenylene-arylene, -C2-C10 Alkynylene-arylene, -arylene-C1-C 10 Alkylene-, -arylene-C2-C 10 Alkenylene-, -arylene-C2-C 10 Alkynylene-, -C1-C 10 Alkylene-(carbocyclo)-, -C2-C 10 Alkenylene-(carbocyclo)-, -C2-C 10 Alkynylene-(carbocyclo)-, -(carbocyclo)-C1-C 10 Alkylene-, -(carbocyclo)-C2-C 10 Alkenylene-, -(carbocyclo)-C2-C 10 Alkynylene, -heterocyclo-, -C1-C 10 Alkylene-(heterocyclo)-, -C2-C 10 Alkenylene-(heterocyclo)-, -C2-C 10 Alkynylene-(heterocyclo)-, -(heterocyclo)-C1-C 10 Alkylene-, -(heterocyclo)-C2-C 10 Alkenylene-, -(heterocyclo)-C1-C 10 Alkynylene-, -(CH2CH2O) r -, or -(CH2CH2O) r -CH2- selected, r is an integer from 1 to 10, and the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, carbocyclo, heterocyclo, and arylene radicals may be unsubstituted or substituted as a part of another group. In some embodiments, the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, carbocyclo, heterocyclo, and arylene radicals are unsubstituted or not part of another group. In some embodiments, R 17 is -C1-C 10 Alkylene-, -carbocyclo-, -O-(C1-C8 alkylene)-, -arylene-, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10Alkylene-(carbocyclo)-, -(carbocyclo)-C1-C 10 Alkylene-, -C3-C8 heterocyclo-, -C1-C 10 Alkylene-(heterocyclo)-, -(heterocyclo)-C1-C 10 Alkylene-, -(CH2CH2O) r -, and -(CH2CH2O) r -CH2-, r is an integer from 1 to 10, the alkylene group is unsubstituted, and the remaining groups may be substituted.

[0176] Even if not clearly shown, it should be understood from all exemplary embodiments that 1 to 20 drug moieties (p = 1 to 20) can be linked to the antibody. TIFF2025113404000006.tif60128

[0177] An exemplary spacer unit is R 17 is a spacer unit of formula IIIa where R is -(CH2)5-. TIFF2025113404000007.tif28128

[0178] Another exemplary spacer unit is R 17 is -(CH2CH2O) r -CH2-, and r is 2, which is a spacer unit of formula IIIa. TIFF2025113404000008.tif28128

[0179] An exemplary spacer unit is R 17 is arylene- or arylene-C1-C 10 alkylene-, which is a spacer unit of formula IIIa. In some embodiments, the aryl group is an unsubstituted phenyl group.

[0180] Yet another exemplary spacer unit is R 17 is a spacer unit of formula IIIb where R is -(CH2)5-. TIFF2025113404000009.tif26128

[0181] In certain embodiments, the stretcher unit is linked to the antibody unit via a disulfide bond between a sulfur atom of the antibody unit and a sulfur atom of the stretcher unit. A representative stretcher unit of this embodiment is depicted within the brackets of Formula IV. Wherein, R 17 , L-, -W-, -Y-, -D, w, and y are as defined above. TIFF2025113404000010.tif24128

[0182] It should be noted that throughout this application, the S portion in the following formulas refers to the sulfur atom of the antibody unit, unless otherwise specifically defined by the context. TIFF2025113404000011.tif15128

[0183] In yet other embodiments, the stretcher contains a reactive site capable of forming a bond with a primary or secondary amino group of the antibody. Examples of these reactive sites include, but are not limited to, activated esters such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. A representative stretcher unit of this embodiment is depicted within the brackets of Formulas Va and Vb. Wherein, -R 17 -, L-, -W-, -Y-, -D, w, and y are as defined above. TIFF2025113404000012.tif57143

[0184] In one embodiment, the stretcher contains a reactive site that reacts with modified carbohydrate (-CHO) groups that may be present on the antibody. For example, the carbohydrate can be gently oxidized using a reagent such as sodium periodate, and the resulting (-CHO) units of the oxidized carbohydrate can be condensed with a stretcher containing functional groups such as hydrazide, oxime, primary or secondary amines, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide, such as the arylhydrazide described in Kaneko et al., 1991, Bioconjugate Chem. 2:133-41. Representative stretcher units of this embodiment are depicted within the square brackets of Formulas VIa, VIb, and VIc. In the formula, -R 17 -, L-, -W-, -Y-, -D, w, and y are as defined above. TIFF2025113404000013.tif83143

[0185] VII.) Amino acid unit When an amino acid unit (-W-) is present, if a spacer unit is present, the stretcher unit is linked to the spacer unit, if no spacer unit is present, the stretcher unit is linked to the drug moiety, and if neither a stretcher unit nor a spacer unit is present, the antibody unit is linked to the drug unit.

[0186] W w - may be, for example, a monopeptide, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide unit. Each -W- unit independently has the formula shown within the following square brackets, and w is an integer from 0 to 12. TIFF2025113404000014.tif29128

[0187] In the formula, R 19is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl, is TIFF2025113404000015.tif107148.

[0188] In some embodiments, the amino acid unit can be enzymatically cleaved by one or more enzymes including proteases associated with cancer or tumor in order to release the drug unit (-D). In one embodiment, the drug unit (-D) is protonated in vivo when released to become the drug (D).

[0189] In certain embodiments, the amino acid unit may include natural amino acids. In other embodiments, the amino acid unit may include non-natural amino acids. Exemplary W w units are represented by formulas (VII) - (IX). TIFF2025113404000016.tif25137 wherein R 20 and R 21 are as follows: TIFF2025113404000017.tif93128TIFF2025113404000018.tif28128 wherein R 20 , R 21 and R 22 are as follows: TIFF2025113404000019.tif27128TIFF2025113404000020.tif27128 wherein R20 , R 21 , R 22 , and R 23 are as follows: TIFF2025113404000021.tif20128

[0190] Exemplary amino acid units include, but are not limited to, the units of formula VII. In the formula, R 20 is benzyl, R 21 is -(CH2)4NH2, or R 20 is isopropyl, R 21 is -(CH2)4NH2, or R 20 is isopropyl, R 21 is -(CH2)3NHCONH2. Another exemplary amino acid unit is the unit of formula VIII. In the formula, R 20 is benzyl, R 21 is benzyl, R 22 is -(CH2)4NH2.

[0191] For the enzymatic cleavage by certain enzymes, such as tumor-related proteases, useful -W w - units can be designed and the selectivity of the -W w - units can be optimized. In one aspect, the -W w - unit is a unit that is cleaved catalytically by cathepsin B, C, and D, or plasmin protease.

[0192] In one aspect, -W w - is a dipeptide, tripeptide, tetrapeptide, or pentapeptide. When R 19 , R 20 , R 21 , R 22 , or R 23 is other than hydrogen, the carbon atom to which R 19 , R 20 , R 21 , R 22 , or R 23 is attached is chiral.

[0193] R 19 、R 20 、R 21 、R 22 、 or R 23 The carbon atoms to which R 19 、R 20 、R 21 、R 22 、 or R 23 is attached are each independently in the (S) configuration or the (R) configuration.

[0194] In one aspect of the amino acid unit, the amino acid unit is valine-citrulline (vc or Val-Cit). In another aspect, the amino acid unit is phenylalanine-lysine (i.e., fk). In yet another aspect of the amino acid unit, the amino acid unit is N-methylvaline-citrulline. In still another aspect, the amino acid unit is 5-aminovaleric acid, homophenylalanine lysine, tetraisocquinoline carboxylate lysine, cyclohexylalanine lysine, isonepecotic acid lysine, β-alanine lysine, glycine serine valine glutamine, and isonepecotic acid.

[0195] VIII.) Spacer unit A spacer unit (-Y-) is present, and when an amino acid unit is present, the spacer unit links the amino acid unit to the drug unit. Or, when no amino acid unit is present, the spacer unit links the stretcher unit to the drug unit. When neither an amino acid unit nor a stretcher unit is present, the spacer unit also links the drug unit to the antibody unit.

[0196] The spacer unit is of two broad types: a non-self-sacrificing spacer unit or a self-sacrificing spacer unit. A non-self-sacrificing spacer unit is a spacer unit in which, after an amino acid unit is cleaved from the antibody-drug conjugate, particularly after cleavage by an enzyme, some or all of the spacer unit remains bound to the drug moiety. Examples of non-self-sacrificing spacer units include, but are not limited to, (glycine-glycine) spacer units and glycine spacer units (both illustrated in Scheme 1) (below). When a conjugate containing a glycine-glycine spacer unit or a glycine spacer unit undergoes enzymatic cleavage by an enzyme (e.g., a tumor cell-related protease, a cancer cell-related protease, or a lymphocyte-related protease), the glycine-glycine-drug moiety or glycine-drug moiety is cleaved from L-A a -W w - to. In one embodiment, an independent hydrolysis reaction occurs within the target cell, cleaving the glycine-drug moiety bond and releasing the drug. Scheme 1 TIFF2025113404000022.tif47128

[0197] In some embodiments, the non-self-sacrificing spacer unit (-Y-) is -Gly-. In some embodiments, the non-self-sacrificing spacer unit (-Y-) is -Gly-Gly-.

[0198] In one embodiment, where no spacer unit is present (-Y y - where y = 0), a drug-linker conjugate, or a pharmaceutically acceptable salt or solvate thereof is provided.

[0199] Alternatively, a conjugate containing a self-sacrificing spacer unit can release -D. As used herein, the term "self-sacrificing spacer" refers to a bifunctional chemical moiety that can join two spaced chemical moieties together and covalently bond to a stable tripartite molecule. This spontaneously separates from the second chemical moiety if the bond between the first moiety and the second chemical moiety is cleaved.

[0200] In some embodiments, -Y y - is a p-aminobenzyl alcohol (PAB) unit (see Schemes 2 and 3) in which the phenylene moiety is substituted with Q m . In the formula, 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 m is an integer from 0 to 4. The alkyl, alkenyl, and alkynyl groups may be unsubstituted or substituted, either alone or as part of another group.

[0201] In some embodiments, -Y- is a PAB group that is linked to -W w - via the amino nitrogen atom of the PAB group and is directly connected to -D via a carbonate group, a carbamate group, or an ether group. Without being bound to a particular theory or mechanism, Scheme 2 illustrates a possible drug release mechanism for a PAB group directly attached to -D via a carbamate group or a carbonate group, as described in Toki et al., 2002, J. Org. Chem. 67:1866-1872. Scheme 2 TIFF2025113404000023.tif98128

[0202] In Scheme 2, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro, or -cyano; m is an integer from 0 to 4; and p is from 1 to about 20. The alkyl, alkenyl, and alkynyl groups may be unsubstituted or substituted, either alone or as part of another group.

[0203] Without being bound by a particular theory or mechanism, Scheme 3 illustrates a possible drug release mechanism of the PAB group directly attached to -D via an ether bond or an amine bond. In the formula, D contains an oxygen or nitrogen group that is part of the drug unit. Scheme 3 TIFF2025113404000024.tif121128

[0204] In Scheme 3, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro, or -cyano; m is an integer from 0 to 4; and p is from 1 to about 20. The alkyl, alkenyl, and alkynyl groups may be unsubstituted or substituted, either alone or as part of another group.

[0205] Other examples of self-sacrificing spacers include, but are not limited to, aromatic compounds electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho or para-aminobenzyl acetals. Spacers that cyclize upon hydrolysis of the amide bond, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring structures (Storm et al., 1972, J. Amer. Chem. Soc. 94:5815), and 2-aminophenylpropionic acid amide (Amsberry et al., 1990, J. Org. Chem. 55:5867) can be used. The elimination of amine-containing drugs substituted at the α position of glycine (Kingsbury et al., 1984, J. Med. Chem. 27:1447) is also an example of a self-sacrificing spacer.

[0206] In one aspect, the spacer unit is a branched bis(hydroxymethyl)-styrene (BHMS) unit as shown in Scheme 4. This can be used to incorporate and release multiple drugs. Scheme 4 TIFF2025113404000025.tif37128

[0207] In Scheme 4, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro, or -cyano; m is an integer from 0 to 4; n is 0 or 1; and p is from 1 to about 20. The alkyl, alkenyl, and alkynyl groups may be unsubstituted or substituted, either alone or as part of another group.

[0208] In some embodiments, the -D moiety is the same. In yet another embodiment, the -D moiety is different.

[0209] In one aspect, the spacer unit (-Y y -) is represented by Formulae (X) - (XII): TIFF2025113404000026.tif32128Wherein 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 m is an integer from 0 to 4. The alkyl, alkenyl, and alkynyl groups may be unsubstituted or substituted, either alone or as part of another group. TIFF2025113404000027.tif34128

[0210] Embodiments of Formulae I and II comprising an antibody-drug conjugate compound may include: TIFF2025113404000028.tif24128Wherein w and y are each 0, 1, or 2, and TIFF2025113404000029.tif23128and wherein w and y are each 0, TIFF2025113404000030.tif184140respectively.

[0211] IX.) Drug Unit The drug moiety (D) may be any cytotoxic agent, cell division inhibitor, or immunomodulatory agent (e.g., immunosuppressive agent). D is a drug unit (moiety) having an atom capable of forming a bond with a spacer unit, an amino acid unit, a stretcher unit, or an antibody unit. In some embodiments, the drug unit D has a nitrogen atom capable of forming a bond with a spacer unit. As used herein, the terms "drug unit" and "drug moiety" are synonyms and are used interchangeably.

[0212] Useful classes of cytotoxin, cell division inhibitor, or immunomodulator include, for example, antitubulin agents, DNA minor groove binders, DNA replication inhibitors, and alkylating agents.

[0213] In some embodiments, the drug is an auristatin, for example, auristatin E (also known in the art as a derivative of dolastatin-10), or a derivative thereof. The auristatin may be, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can react with para-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other representative auristatins include AFP, MMAF, and MMAE. Exemplary auristatin syntheses and structures are described in U.S. Patent Application Publication No. 2003-0083263; International Publication Nos. 04 / 010957, 02 / 088172, and U.S. Patent Nos. 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 these is hereby incorporated by reference in its entirety and for all purposes.

[0214] Auristatin has been shown to interfere with microtubule dynamics and mitosis and cell division and to have anti-cancer activity. Auristatin binds to tubulin and can exert a cytotoxic or cell division inhibitory effect on 191P4D12-expressing cells. There are many different assays known in the art that can be used to determine whether auristatin or the resulting antibody-drug conjugate exerts a cell division inhibitory or cytotoxic effect on a desired cell line.

[0215] Methods for determining whether a compound binds to tubulin are known in the art. See, for example, Muller et al., Anal. Chem 2006, 78, 4390-4397; Hamel et al., Molecular Pharmacology, 1995 47: 965-976; and Hamel et al., The Journal of Biological Chemistry, 1990 265:28, 17141-17149. For the purposes of the present invention, the relative affinity of a compound for tubulin can be determined. Among the preferred auristatins of the present invention, there are those that bind to tubulin from 1 / 10 (weak affinity) to 10-fold, 20-fold, and even 100-fold (strong affinity) of the binding affinity of MMAE for tubulin.

[0216] In some embodiments, -D is of formula D E or D F : Auristatin of TIFF2025113404000031.tif73128, or a pharmaceutically acceptable salt or solvate form thereof, wherein, independently, at each location, The wavy line indicates a bond; R 2 is -C1-C 20 alkyl, -C2-C 20 alkenyl, or -C2-C 20 alkynyl; R3 is -H, -C1-C 20 alkyl, -C2-C 20 alkenyl, -C2-C 20 alkynyl, -carbocycle, -C1-C 20 alkylene(carbocycle), -C2-C 20 alkenylene(carbocycle), -C2-C 20 alkynylene(carbocycle), -aryl, -C1-C 20 alkylene(aryl), -C2-C 20 alkenylene(aryl), -C2-C 20 alkynylene(aryl), heterocycle, -C1-C 20 alkylene(heterocycle), -C2-C 20 alkenylene(heterocycle), or -C2-C 20 alkynylene(heterocycle); R 4 is -H, -C1-C 20 alkyl, -C2-C 20 alkenyl, -C2-C 20 alkynyl, carbocycle, -C1-C 20 alkylene(carbocycle), -C2-C 20 alkenylene(carbocycle), -C2-C 20 alkynylene(carbocycle), aryl, -C1-C 20 alkylene(aryl), -C2-C 20 alkenylene(aryl), -C2-C 20 alkynylene(aryl), -heterocycle, -C1-C 20 alkylene(heterocycle), -C2-C 20 alkenylene(heterocycle), or -C2-C 20 alkynylene(heterocycle); R 5 is -H or -C1-C8 alkyl; or R 4 and R 5 together form a carbocycle ring, having the formula -(CR a R b ) s ; where in the formula, R aand R b is independently -H, -C1-C 20 alkyl, -C2-C 20 alkenyl, -C2-C 20 alkynyl, or -carbocycle, and s is 2, 3, 4, 5, or 6; R 6 is -H, -C1-C 20 alkyl, -C2-C 20 alkenyl, or -C2-C 20 alkynyl; R 7 is -H, -C1-C 20 alkyl, -C2-C 20 alkenyl, -C2-C 20 alkynyl, carbocycle, -C1-C 20 alkylene(carbocycle), -C2-C 20 alkenylene(carbocycle), -C2-C 20 alkynylene(carbocycle), -aryl, -C1-C 20 alkylene(aryl), -C2-C 20 alkenylene(aryl), -C2-C 20 alkynylene(aryl), heterocycle, -C1-C 20 alkylene(heterocycle), -C2-C 20 alkenylene(heterocycle), or -C2-C 20 alkynylene(heterocycle); Each R 8 is independently -H, -OH, -C1-C 20 alkyl, -C2-C 20 alkenyl, -C2-C 20 alkynyl, -O-(C1-C 20 alkyl), -O-(C2-C 20 alkenyl), -O-(C1-C 20 alkynyl), or -carbocycle; R 9 is -H, -C1-C 20 alkyl, -C2-C 20 alkenyl, or -C2-C 20 alkynyl; R 24is - aryl, - heterocycle, or - carbocycle; R 25 is - H, C1 - C 20 alkyl, - C2 - C 20 alkenyl, - C2 - C 20 alkynyl, - carbocycle, - O - (C1 - C 20 alkyl), - O - (C2 - C 20 alkenyl), - O - (C2 - C 20 alkynyl), or OR 18 wherein, R 18 is - H, a hydroxyl - protecting group, or OR 18 when = O, is a direct bond; R 26 is - H, - C1 - C 20 alkyl, - C2 - C 20 alkenyl, or - C2 - C 20 alkynyl, - aryl, - heterocycle, or - carbocycle; R 10 is - aryl or - heterocycle; Z is - O, - S, - NH, or - NR 12 wherein, R 12 is - C1 - C 20 alkyl, - C2 - C 20 alkenyl, or - C2 - C 20 alkynyl; R 11 is - H, - C1 - C 20 alkyl, - C2 - C 20 alkenyl, - C2 - C 20 alkynyl, - aryl, - heterocycle, - (R 13 O) m - R 14 or - (R 13 O) m - CH(R 15 )2; m is an integer from 1 to 1000, or m = 1 to 1000; R 13 is - C2 - C 20 alkylene, - C2 - C 20 alkenylene, or - C2 - C 20is an alkynylene; R 14 is -H, -C1-C 20 alkyl, -C2-C 20 alkenyl, or -C2-C 20 alkynyl; R 15 each occurrence of is independently -H, -COOH, -(CH2) n -N(R 16 )2, -(CH2) n -SO3H, -(CH2) n -SO3-C1-C 20 alkyl, -(CH2) n -SO3-C2-C 20 alkenyl, or -(CH2) n -SO3-C2-C 20 alkynyl; R 16 each occurrence of is independently -H, -C1-C 20 alkyl, -C2-C 20 alkenyl, -C2-C 20 alkynyl, or -(CH2) n -COOH; n is an integer from 0 to 6, the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, and heterocyclic radicals may be unsubstituted or substituted, either alone or as part of another group.

[0217] The auristatin of formula D E includes auristatin in which the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, and heterocyclic radicals are unsubstituted.

[0218] The auristatin of formula D E wherein the groups of R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are unsubstituted, and R19 , R 20 , and R 21 groups may be substituted as described herein and includes auristatin.

[0219] Formula D E of auristatin is R 2 is C1-C8 alkyl; R 3 , R 4 , and R 7 are independently -H, -C1-C 20 alkyl, -C2-C 20 alkenyl, -C2-C 20 alkynyl, monocyclic C3-C6 carbocycle, -C1-C 20 alkylene (monocyclic C3-C6 carbocycle), -C2-C 20 alkenylene (monocyclic C3-C6 carbocycle), -C2-C 20 alkynylene (monocyclic C3-C6 carbocycle), C6-C 10 aryl, -C1-C 20 alkylene (C6-C 10 aryl), -C2-C 20 alkenylene (C6-C 10 aryl), -C2-C 20 alkynylene (C6-C 10 aryl), heterocycle, -C1-C 20 alkylene (heterocycle), -C2-C 20 alkenylene (heterocycle), or -C2-C 20 alkynylene (heterocycle) and are selected, said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, carbocycle, aryl, and heterocyclic radicals may be substituted; R 5 is -H; R 6 is C1-C8 alkyl; Each R 8 is independently -OH, -O-(C1-C 20 alkyl), -O-(C2-C 20 alkenyl), or -O-(C2-C 20selected from (alkynyl), and said alkyl, alkenyl, and alkynyl radicals may be substituted; R 9 is -H or -C1-C8 alkyl; R 24 is optionally substituted -phenyl; R 25 is -OR 18 wherein R 18 is H, a hydroxyl protecting group, or OR 18 when =O, is a direct bond; R 26 is -H, -C1-C 20 alkyl, -C2-C 20 alkenyl, -C2-C 20 alkynyl, or -carbocycle, selected from the group consisting of said alkyl, alkenyl, alkynyl, and carbocycle radicals may be substituted, auristatin or a pharmaceutically acceptable salt form or solvate form thereof.

[0220] The auristatin of formula D E is such that R 2 is methyl; R 3 is -H, -C1-C8 alkyl, -C2-C8 alkenyl, or C2-C8 alkynyl, and said alkyl, alkenyl, and alkynyl radicals may be substituted; R 4 is -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, monocyclic C3-C6 carbocycle, -C6-C 10 aryl, -C1-C8 alkylene(C6-C 10 aryl), -C2-C8 alkenylene(C6-C 10 aryl), -C2-C8 alkynylene(C6-C 10is -C1 - C8 alkylene (monocyclic C3 - C6 carbocycle), -C2 - C8 alkenylene (monocyclic C3 - C6 carbocycle), -C2 - C8 alkynylene (monocyclic C3 - C6 carbocycle), and the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, and carbocycle radicals may be alone or part of another group and may be substituted; R 5 is -H; R 6 is methyl; R 7 is -C1 - C8 alkyl, -C2 - C8 alkenyl, or -C2 - C8 alkynyl, and each R 8 is methoxy; R 9 is -H or -C1 - C8 alkyl; R 24 is phenyl; R 25 is -OR 18 and R 18 is H, a hydroxyl protecting group, or when OR 18 is =O, is a direct bond; R 26 [[ID=�6]]is methyl, auristatin or a pharmaceutically acceptable salt form thereof.

[0221] Formula D E auristatin of which R 2 is methyl; R 3 is -H or -C1 - C3 alkyl; R 4 is -C1 - C5 alkyl; R 5 is -H; R 6 is methyl; R 7 is isopropyl or sec - butyl; R 8 is methoxy; R 9 is -H or -C1 - C8 alkyl; R 24 is phenyl; R 25 is -OR 18 and R 18 is -H, a hydroxyl protecting group, or OR18 When =O, it is a direct bond; R 26 includes auristatin where R is methyl, or a pharmaceutically acceptable salt or solvate form thereof.

[0222] Formula D E The auristatin of R 2 is methyl or C1-C3 alkyl; R 3 is -H or -C1-C3 alkyl; R 4 is C1-C5 alkyl; R 5 is H; R 6 is C1-C3 alkyl; R 7 is -C1-C5 alkyl; R 8 is -C1-C3 alkoxy; R 9 is -H or -C1-C8 alkyl; R 24 is phenyl; R 25 is -OR 18 and R 18 is -H, a hydroxyl protecting group, or OR 18 When =O, it is a direct bond; R 26 is C1-C3 alkyl, auristatin, or a pharmaceutically acceptable salt form thereof.

[0223] Formula D F The auristatin of R 2 is methyl; R 3 , R 4 , and R 7 are independently -H, -C1-C 20 alkyl, -C2-C 20 alkenyl, -C2-C 20 alkynyl, monocyclic C3-C6 carbocycle, -C1-C 20Alkylene (monocyclic C3-C6 carbocycle), -C2-C 20 Alkenylene (monocyclic C3-C6 carbocycle), -C2-C 20 Alkynylene (monocyclic C3-C6 carbocycle), -C6-C 10 Aryl, -C1-C 20 Alkylene (C6-C 10 Aryl), -C2-C 20 Alkenylene (C6-C 10 Aryl), -C2-C 20 Alkynylene (C6-C 10 Aryl), heterocycle, -C1-C 20 Alkylene (heterocycle), -C2-C 20 Alkenylene (heterocycle), or -C2-C 20 Alkynylene (heterocycle) is selected from; the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, carbocycle, aryl, and heterocyclic radicals may be alone or part of another group and may be substituted; R 5 is -H; R 6 is methyl; Each R 8 is methoxy; R 9 is -H, -C1-C 20 alkyl, -C2-C 20 alkenyl, or -C2-C 20 alkynyl; the alkyl, alkenyl, and alkynyl radicals may be substituted; R 10 is optionally substituted aryl or optionally substituted heterocycle; Z is -O-, -S-, -NH-, or -NR 12 where R 12 is -C1-C 20 alkyl, -C2-C 20 alkenyl, or -C2-C 20 alkynyl, and these may each be substituted; R 11 is -H, -C1-C 20 alkyl, -C2-C20 alkenyl, -C2-C 20 alkynyl, -aryl, -heterocycle, -(R 13 O) m -R 14 、 or -(R 13 O) m -CH(R 15 )2, and the alkyl, alkenyl, alkynyl, aryl, and heterocyclic radicals may be substituted; m is an integer from 1 to 1000 or m = 0; R 13 is -C2-C 20 alkylene, -C2-C 20 alkenylene, or -C2-C 20 alkynylene, and these may each be substituted; R 14 is -H, -C1-C 20 alkyl, -C2-C 20 alkenyl, or -C2-C 20 alkynyl, and the alkyl, alkenyl, and alkynyl radicals may be substituted, R 15 each occurrence of is independently -H, -COOH, -(CH2) n -N(R 16 )2, -(CH2) n -SO3H, -(CH2) n -SO3-C1-C 20 alkyl, -(CH2) n -SO3-C2-C 20 alkenyl, or -(CH2) n -SO3-C2-C 20 alkynyl, and the alkyl, alkenyl, and alkynyl radicals may be substituted; R 16 each occurrence of is independently -H, -C1-C 20 alkyl, -C2-C 20 alkenyl, -C2-C 20 alkynyl, or -(CH2) n -COOH, and the alkyl, alkenyl, and alkynyl radicals may be substituted; auristatin, where n is an integer from 0 to 6, or a pharmaceutically acceptable salt thereof.

[0224] In some of these embodiments, R 10 is optionally substituted phenyl.

[0225] For auristatin of formula D F , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 groups are unsubstituted, and R 10 and R 11 groups are as described herein.

[0226] For auristatin of formula D F , it includes auristatin in which the above alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, and heterocyclic radicals are unsubstituted.

[0227] For auristatin of formula D F , R 2 is -C1-C3 alkyl; R 3 is -H or -C1-C3 alkyl; R 4 is -C1-C5 alkyl; R 5 is -H; R 6 is -C1-C3 alkyl; R 7 is -C1-C5 alkyl; R 8 is -C1-C3 alkoxy; R 9 is -H or -C1-C8 alkyl; R 10 is optionally substituted phenyl; Z is -O-, -S-, or -NH-; R 11 is as defined herein, or a pharmaceutically acceptable salt thereof.

[0228] Formula D F The auristatin of 2 wherein R is methyl; R 3 is -H or -C1-C3 alkyl; R 4 is -C1-C5 alkyl; R 5 is -H; R 6 is methyl; R 7 is isopropyl or sec-butyl; R 8 is methoxy; R 9 is -H or -C1-C8 alkyl; R 10 is optionally substituted phenyl; Z is -O-, -S-, or -NH-; R 11 is auristatin as defined herein, or a pharmaceutically acceptable salt thereof.

[0229] Formula D F The auristatin of 2 wherein R is methyl; R 3 is -H or -C1-C3 alkyl; R 4 is -C1-C5 alkyl; R 5 is -H; R 6 is methyl; R 7 is isopropyl or sec-butyl; R 8 is methoxy; R 9 is -H or C1-C8 alkyl; R 10 is phenyl; Z is -O- or -NH-; R 11 is auristatin as defined herein, preferably hydrogen, or a pharmaceutically acceptable salt form thereof.

[0230] Formula D F The auristatin of 2 wherein R is -C1-C3 alkyl; R 3 is -H or -C1-C3 alkyl; R 4 is -C1-C5 alkyl; R 5 is -H; R 6 is -C1-C3 alkyl; R 7 is -C1-C5 alkyl; R8 is -C1 to C3 alkoxy; R 9 is -H or -C1 to C8 alkyl; R 10 is phenyl; Z is -O- or -NH-; R 11 is auristatin as defined herein, preferably hydrogen, or a pharmaceutically acceptable salt form thereof.

[0231] Formula D E or D F auristatin of has R 3 R 4 and R 7 are each independently isopropyl or sec - butyl; R 5 is -H; auristatin containing. In an exemplary embodiment, R 3 and R 4 are each isopropyl; R 5 is H; R 7 is sec - butyl. The remaining substituents are as defined herein.

[0232] Formula D E or D F auristatin of has R 2 and R 6 are each methyl; R 9 is H; auristatin containing. The remaining substituents are as defined herein.

[0233] Formula D E or D F auristatin of has R 8 each occurrence of which is -OCH3; auristatin containing. The remaining substituents are as defined herein.

[0234] Formula D E or D F auristatin of has R 3 and R 4 are each isopropyl; R 2 and R 6 are each methyl; R 5is H and R 7 is sec-butyl and R 8 each occurrence of is -OCH3 and R 9 contains an auristatin where is H. The remaining substituents are as defined herein.

[0235] Formula D F auristatins of contain auristatins where Z is -O- or -NH-. The remaining substituents are as defined herein.

[0236] Formula D F auristatins of contain auristatins where R 10 is aryl. The remaining substituents are as defined herein.

[0237] Formula D F auristatins of contain auristatins where R 10 is -phenyl. The remaining substituents are as defined herein.

[0238] Formula D F auristatins of contain auristatins where Z is -O- and R 11 is H, methyl, or t-butyl. The remaining substituents are as defined herein.

[0239] Formula D F auristatins of contain auristatins where Z is -NH- and R 11 is -(R 13 O) m -CH(R 15 )2 and R 15 is -(CH2) n -N(R 16 )2 and R 16 is -C1-C8 alkyl or -(CH2) n -COOH. The remaining substituents are as defined herein.

[0240] Formula D F auristatins of contain auristatins where Z is -NH- and R11 is -(R 13 O) m -CH(R 15 )2, and R 15 is -(CH2) n -SO3H, and contains an auristatin. The remaining substituents are as defined herein.

[0241] In a preferred embodiment, when D is an auristatin of formula D E , w is an integer from 1 to 12, preferably from 2 to 12, y is 1 or 2, and a is preferably 1.

[0242] In some embodiments, when D is an auristatin of formula D F , a is 1, and w and y are 0.

[0243] Exemplary drug units (-D) include a drug unit having the following structure: TIFF2025113404000032.tif94128TIFF2025113404000033.tif202142TIFF2025113404000034.tif164128, or a pharmaceutically acceptable salt or solvate thereof.

[0244] In one aspect, a hydrophilic group such as, but not limited to, triethylene glycol ester (TEG) can be attached to R 11 of the drug unit. Without being bound by theory, the hydrophilic group aids in the internal migration and non-aggregation of the drug unit.

[0245] 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.

[0246] An exemplary antibody-drug conjugate compound has the following structure where "L" or "mAb-s-" represents the 191P4D12 MAb named Ha22-2(2,4)6.1 as shown herein: having TIFF2025113404000035.tif174170, or a pharmaceutically acceptable salt thereof.

[0247] In some embodiments, the drug unit is calicheamicin, camptothecin, maytansinoid, or anthracycline. In some embodiments, the drug is a taxane, topoisomerase inhibitor, vinca alkaloid, etc.

[0248] In some representative embodiments, suitable cytotoxic agents include, for example, DNA minor groove binders (e.g., enediynes and lexitropsin, CBI compounds; see also U.S. Patent No. 6,130,237), duocarmycin, taxanes (e.g., paclitaxel and docetaxel), puromycin, and vinca alkaloids. Other cytotoxic agents include, for example, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, epothilones A and B, estramustine, cryptophysin, cemadotin, maytansinoid, discodermolide, erythrovidins, and mitoxantrone.

[0249] In some embodiments, the drug is an anti-tubulin agent. Examples of anti-tubulin agents include auristatin, taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik), and vinca alkaloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine). Other anti-tubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilones A and B), nocodazole, colchicine and colcimid, estramustine, cryptophysin, cemadotin, maytansinoid, combretastatin, discodermolide, and erythrovidins.

[0250] In certain embodiments, the cytotoxic agent is a maytansinoid, another group of anti-tubulin agents. For example, in certain embodiments, the maytansinoid is maytansine or DM-1 (see also ImmunoGen, Inc.; Chari et al., 1992, Cancer Res. 52:127-131).

[0251] In certain embodiments, the cytotoxic or cytostatic agent is dolastatin. In certain embodiments, the cytotoxic or cytostatic agent is a cytotoxic or cytostatic agent of the auristatin class. Thus, in certain embodiments, the cytotoxic or cytostatic agent is MMAE (Formula XI). In another particular embodiment, the cytotoxic or cytostatic agent is AFP (Formula XVI). TIFF2025113404000036.tif37128

[0252] In certain embodiments, the cytotoxic or cytostatic agent is a compound of Formulas XII-XXI or a pharmaceutically acceptable salt thereof. TIFF2025113404000037.tif255140TIFF2025113404000038.tif160148

[0253] X.) DRUG LOADING Drug loading is represented by p and is the average number of drug moieties per antibody molecule within the molecule. The drug loading may be from 1 to 20 drug moieties (D) per antibody. The ADCs of the present invention include those in which antibodies conjugated with from 1 to 20 drug moieties are aggregated. The average number of drug moieties per antibody in the ADC preparation from the conjugation reaction can be characterized by conventional means such as mass spectrometry and ELISA assays. The quantitative distribution of the ADCs with respect to p can also be determined. In some cases, homogeneous ADCs with a particular value of p can be separated, purified, and characterized from ADCs with other drug loadings by means such as electrophoresis.

[0254] In some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, when the attachment is to a cysteine thiol, as in the exemplary embodiments above, the antibody may have only one cysteine thiol group or several cysteine thiol groups, and may have only one sufficiently reactive thiol group or several sufficiently reactive thiol groups to which a linker can be attached. In certain embodiments, as the drug loading increases, e.g., when p > 5, aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates may occur. In certain embodiments, the drug loading of the ADCs of the present invention is from 1 to about 8; about 2 to about 6; about 3 to about 5; about 3 to about 4; about 3.1 to about 3.9; about 3.2 to about 3.8; about 3.2 to about 3.7; about 3.2 to about 3.6; about 3.3 to about 3.8; or about 3.3 to about 3.7. In practice, for certain ADCs, it has been shown that the optimal ratio of drug moieties per antibody may be less than 8 and may be about 2 to about 5. See U.S. Patent No. 7,498,298, which is hereby incorporated by reference in its entirety.

[0255] In certain embodiments, during the conjugation reaction, fewer drug moieties than the maximum theoretical amount of the drug moiety are conjugated to the antibody. The antibody may contain, for example, lysine residues that do not react with the drug-linker intermediate or the linker reagent, as discussed below. Generally, the antibody does not contain free and reactive cysteine thiol groups that can be linked to the drug moiety. In practice, most cysteine thiol residues in the antibody exist as disulfide bridges. In certain embodiments, to create reactive cysteine thiol groups, the antibody can be reduced by a reducing agent such as dithiothreitol (DTT) or tricarbonyl ethylphosphine (TCEP) under partial or complete reducing conditions. In certain embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine.

[0256] The loading (drug / antibody ratio) of the ADC can be controlled in various ways, for example, (i) by limiting the drug-linker intermediate or linker reagent in molar excess to the antibody, (ii) by limiting the reaction time or temperature of conjugation, (iii) by partial or limited reduction conditions for cysteine thiol modification, (iv) by controlling the number and / or position of linker-drug attachment such that the number and position of cysteine residues are modified, by manipulating the amino acid sequence of the antibody by recombinant methods (e.g., thioMab or thioFab are prepared as disclosed in this specification and WO2006 / 034488, which is incorporated herein by reference in its entirety).

[0257] When multiple nucleophilic groups react with a drug-linker intermediate or react with a linker reagent and then with a drug moiety reagent, it will be understood that the resulting product is a mixture of ADC compounds in which one or more drug moieties attached to the antibody are distributed. The average number of drugs per antibody is specific to the antibody and can be calculated from the mixture by a dual ELISA antibody assay specific to the antibody and the drug. Individual ADC molecules can be identified in the mixture by mass spectrometry and separated by HPLC, for example, by hydrophobic interaction chromatography (see, e.g., Hamblett, KJ., et al. 「Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate」, Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, S.C., et al. 「Controlling the location of drug attachment in antibody-drug conjugates」, Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certain embodiments, homogeneous ADCs having a single loading value can be isolated from the conjugated mixture by electrophoresis or chromatography.

[0258] XI.) Method for Confirming the Cytotoxic Effect of ADC Methods for determining whether a drug or antibody-drug conjugate exerts a cell division inhibitory effect and / or a cytotoxic effect on cells are known. Generally, the cytotoxic activity or cell division inhibitory activity of an antibody-drug conjugate can be measured by exposing mammalian cells expressing the target protein of the antibody-drug conjugate in cell culture medium; culturing the cells for about 6 hours to about 5 days; and measuring cell viability. Cell-based in vitro assays can be used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of antibody-drug conjugates.

[0259] To determine whether an antibody-drug conjugate exerts a cell division inhibitory effect, a thymidine incorporation assay can be used. For example, cancer cells expressing the target antigen can be cultured at a density of 5,000 cells per well of a 96-well plate for 72 hours and exposed to 0.5 μCi of 3 3H-thymidine during the last 8 hours of the 72-hour period. Incorporation of 3 3H-thymidine into the cultured cells is measured in the presence and absence of the antibody-drug conjugate.

[0260] To confirm cytotoxicity, necrosis or apoptosis (programmed cell death) can be measured. Necrosis is typically achieved by increased permeability of the plasma membrane; swelling of the cells, and rupture of the plasma membrane. Apoptosis is typically characterized by membrane blebbing, cytoplasmic aggregation, and activation of endogenous endonucleases. Demonstration of any of these effects on cancer cells indicates that the antibody-drug conjugate is useful in cancer treatment.

[0261] Cell viability can be measured in cells by determining the uptake of neutral red, trypan blue, or ALAMAR™ Blue (see, for example, Page et al., 1993, Intl. J. Oncology 3:473-476). In such assays, cells are incubated in medium containing the dye, the cells are washed, and the remaining dye, which reflects the cellular uptake of the dye, is spectrophotometrically measured. To measure cytotoxicity, the protein-binding dye sulforhodamine B (SRB) can also be used (Skehan et al., 1990, J. Natl. Cancer Inst. 82:1107-12).

[0262] Alternatively, tetrazolium salts, such as MTT, are used in assays that quantitatively colorimetrically determine the survival and proliferation of mammalian cells by detecting live cells but not dead cells (see, for example, Mosmann, 1983, J. Immunol. Methods 65:55-63).

[0263] Apoptosis can be quantified, for example, by measuring DNA fragmentation. Commercial photometric methods can be utilized to quantitatively measure DNA fragmentation in vitro. Examples of such assays are described in Biochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals), including assays based on TUNEL (detection of incorporation of labeled nucleotides into fragmented DNA) and ELISA.

[0264] Apoptosis can also be confirmed by measuring morphological changes of cells. For example, similar to necrosis, loss of plasma membrane integrity can be confirmed by measuring the uptake of certain dyes (e.g., fluorescent dyes, such as acridine orange or ethidium bromide). Methods for measuring the number of apoptotic cells are described in 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 (e.g., acridine orange, ethidium bromide, or propidium iodide) and observed for chromatin condensation and marginalization along the inner nuclear membrane. Other morphological changes that can be measured to confirm apoptosis include, for example, cytoplasmic condensation, increased membrane vesiculation, and cell shrinkage.

[0265] The presence of apoptotic cells can be measured in both the adherent and "floating" compartments of the culture. For example, in both compartments, the supernatant is removed, the adherent cells are trypsinized, the preparations are combined after a washing step by centrifugation (e.g., 10 minutes at 2000 rpm), and collected by detecting apoptosis (e.g., by measuring DNA fragmentation). (See, for example, Piazza et al., 1995, Cancer Research 55:3110-16).

[0266] The effects of the 191P4D12 therapeutic composition can be evaluated in vivo in a suitable animal model. For example, a xenograft cancer model can be used. In a xenograft cancer model, cancer explants or passaged xenograft tissues are introduced into immunodeficient animals, such as nude mice or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-408). For example, PCT patent application WO98 / 16628 and US Patent No. 6,107,540 describe various human prostate cancer xenograft models that can reproduce the development of primary tumors, micrometastases, and the formation of osteoblastic metastases characteristic of advanced disease. Assays can be used to measure inhibition of tumor formation, tumor regression, or metastasis, etc., to predict efficacy.

[0267] In vivo assays for evaluating the promotion of apoptosis are useful in the evaluation of therapeutic compositions. In one aspect, xenografts derived from tumor-bearing mice treated with the therapeutic composition can be tested for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The therapeutic efficacy of the composition can be determined from the degree of apoptotic foci seen in the tumors of the treated mice.

[0268] The therapeutic compositions used in the practice of the methods described above can be formulated into pharmaceutical compositions containing a carrier suitable for the desired delivery method. Suitable carriers include any material that, when combined with the therapeutic composition, retains the anti-tumor 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 aqueous solutions, bacteriostatic water, etc. (generally, see Remington's Pharmaceutical Sciences 16th Edition, edited by A. Osal, 1980).

[0269] The therapeutic agent can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially efficient routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intraorgan, in situ, etc. Formulations preferred for intravenous injection include bacteriostatic water for injection preserved, solutions of sterile water for injection not preserved, and / or therapeutic compositions diluted in polyvinyl chloride bags or polyethylene bags containing 0.9% sterile sodium chloride injection, USP. The therapeutic protein preparation may be lyophilized, as a sterile powder, preferably stored under vacuum, and then may be dissolved and reconstituted in bacteriostatic water (e.g., containing benzyl alcohol preservative) or sterile water prior to injection.

[0270] The dosage and administration protocol for treating cancer using the methods described above will vary depending on the method and the target cancer and will generally be influenced by many other factors recognized in the art.

[0271] XII.) Treatment of cancers expressing 191P4D12 The identification of 191P4D12 as a protein that is normally expressed in a limited set of tissues but is also expressed in cancers such as those listed in Table I has opened up many therapeutic approaches for the treatment of such cancers.

[0272] Of note is that the targeted anti-tumor treatment was useful even when the targeted protein was expressed in normal tissues and even in vital normal organ tissues necessary for life support. Organs necessary for life support are organs necessary to maintain life, such as the heart or colon. Organs not necessary for life support are organs that can be removed and the individual can still survive. Examples of organs not necessary for life support are the ovaries, breast, and prostate.

[0273] The expression of a target protein in normal tissues, and in particular in normal tissues necessary for life support, does not invalidate the usefulness of a targeting agent against the target protein as a therapeutic agent for a specific tumor in which the target protein is overexpressed. For example, expression in an organ necessary for life support is not in itself and per se harmful. Furthermore, organs considered less important, such as the prostate and ovaries, can be removed without affecting mortality. Finally, some organs necessary for life support are not affected by normal organ expression due to immunoprivilege. Immunoprivileged organs are organs protected from the blood by the blood-organ barrier and are thus inaccessible to immunotherapy. Examples of immunoprivileged organs are the brain and the testis.

[0274] Accordingly, therapeutic approaches that inhibit the activity of the 191P4D12 protein are useful for patients suffering from cancers that express 191P4D12. These therapeutic approaches generally fall into three classes. The first class modulates 191P4D12 functions related to tumor cell proliferation, resulting in inhibition or delay of tumor cell proliferation, or induction of tumor cell death. The second class includes various methods for inhibiting the binding or association of the 191P4D12 protein with a binding partner of the 191P4D12 protein or with other proteins. The third class includes various methods for inhibiting the transcription of the 191P4D12 gene or the translation of 191P4D12 mRNA.

[0275] Accordingly, cancer patients can be evaluated for the presence and level of 191P4D12 expression, preferably using immunohistochemical evaluation of tumor tissue, quantitative 191P4D12 imaging, or other techniques that reliably demonstrate the presence and extent of 191P4D12 expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissue are well known in the art.

[0276] XIII.) 191P4D12 as a Target for Antibody-Based Therapy 191P4D12 is an attractive target for antibody-based therapeutic strategies. Many antibody strategies for both extracellular and intracellular molecules are known in the art (see, e.g., complement and ADCC-mediated killing and the use of intrabodies). Since 191P4D12 is expressed in various lines of cancer cells relative to corresponding normal cells, systemic administration of a 191P4D12 immunoreactive composition is provided that exhibits excellent sensitivity without causing non-specific and / or off-target effects of toxicity caused by binding of the immunoreactive composition to non-target organs and non-target tissues. Antibodies that specifically react with the domain of 191P4D12 are useful, preferably as antibody-drug conjugates (i.e., ADCs) with toxins or therapeutic agents, for systemic treatment of 191P4D12-expressing cancers.

[0277] One of ordinary skill in the art will understand that antibodies can be used to specifically target and bind to an immunogenic molecule (e.g., the immunogenic region of the 191P4D12 sequence shown in FIG. 1). Further, one of ordinary skill in the art will understand that conjugating an antibody to a cytotoxic agent is routine (see, e.g., Slevers et al., Blood 93:11 3678-3684 (June 1, 1999)). When a cytotoxic agent and / or therapeutic agent is delivered directly to the cell, for example, by conjugating these agents to an antibody specific for a molecule expressed by the cell (e.g., 191P4D12), the cytotoxic agent will exert a known biological effect (i.e., cytotoxicity) on that cell.

[0278] A wide variety of compositions and methods using antibody-cytotoxic agent conjugates to kill cells are known in the art. In the context of cancer, a representative method involves administering to a mammal having a tumor a biologically effective amount of a conjugate comprising a selected cytotoxic agent and / or therapeutic agent linked to a targeting agent (e.g., 191P4D12 MAb, preferably Ha22-2(2,4)6.1) that binds to an antigen (e.g., 191P4D12) expressed on the cell surface or locally available. A representative embodiment is a method of delivering a cytotoxic agent and / or therapeutic agent to cells expressing 191P4D12, the method comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to the 191P4D12 epitope and exposing the cells to the antibody-drug conjugate (ADC). Another exemplary embodiment is a method of treating an individual suspected of having metastatic cancer, the method comprising parenterally administering to the individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic agent and / or therapeutic agent.

[0279] Cancer immunotherapy using the 191P4D12 antibody can be carried out according to various approaches that have been successfully used in the treatment of other types of cancer (including, but not limited to, colorectal cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133 - 138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179 - 3186, Tsunenari et al., 1997, Blood 90:2437 - 2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res. 52:2771 - 2776), B - cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol. 19:93 - 101), leukemia (Zhong et al., 1996, Leuk. Res. 20:581 - 589), 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)). Among the treatment approaches, there are those involving the conjugation of naked antibodies to toxins or radioisotopes, for example, the conjugation of Y 91 or I 131 to anti - CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals), and there are also those involving the co - administration of antibodies and other therapeutic agents, for example, Herceptin™ (trastuzu MAb) and paclitaxel (Genentech, Inc.). In a preferred embodiment, the antibody can be conjugated to a cytotoxic agent (as described above), preferably an auristatin derivative named MMAE (Seattle Genetics, Inc.).

[0280] The 191P4D12 antibody therapy is useful for all stages of cancer, but antibody therapy may be particularly appropriate in advanced or metastatic cancer. Treatment with the antibody therapy of the present invention is applicable to patients who have received chemotherapy one or more times. Alternatively, the antibody therapy of the present invention is combined with a chemotherapy regimen or a radiation regimen for patients who have not received treatment with chemotherapy. Furthermore, antibody therapy may enable the use of low-dose combination chemotherapy, particularly for patients who are not very tolerant to the toxicity of chemotherapeutic agents. 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) have described the use of various antibodies together with chemotherapeutic agents.

[0281] The 191P4D12 monoclonal antibody that treats the cancers listed in Table I includes antibodies that elicit a strong immune response against tumors or antibodies that are directly cytotoxic. In this regard, the 191P4D12 monoclonal antibody (MAb) can induce tumor cell lysis by either the complement-mediated cytotoxic mechanism or the antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. Both of these mechanisms require an intact Fc portion of the immunoglobulin molecule due to the interaction with the effector cell Fc receptor site on the complement protein. Furthermore, the 191P4D12 MAb that exerts a direct biological effect on tumor growth is useful for treating cancers that express 191P4D12. The mechanisms by which cytotoxic MAb acts directly include the following: inhibition of cell proliferation, regulation of cell differentiation, regulation of the tumor neovascularization factor profile, and induction of apoptosis. The mechanism by which a specific 191P4D12 MAb exerts an antitumor effect is evaluated using any number of in vitro assays generally known in the art for assessing cell death, such as ADCC, complement-mediated cell lysis, and the like.

[0282] Accordingly, the preferred monoclonal antibodies used in the treatment methods of the present invention are fully human antibodies that specifically bind to the target 191P4D12 antigen with high affinity.

[0283] XIV.) 191P4D12 ADC Cocktail The treatment methods of the present invention contemplate the administration of a single 191P4D12 ADC, and combinations of different MAbs (i.e., 191P4D12 MAb or MAbs that bind to other proteins), i.e., cocktails. Such MAb cocktails can have certain advantages as long as these cocktails include MAbs that target different epitopes, utilize different effector mechanisms, or directly combine a MAb that depends on immune effector functions with a cytotoxic MAb. Such combined MAbs can exhibit a synergistic therapeutic effect. Furthermore, the 191P4D12 MAb can be administered concurrently with other treatment modalities including, but not limited to, various chemotherapeutic and biological agents, androgen blockers, immunomodulators (e.g., IL-2, GM-CSF), surgery or radiation. In a preferred embodiment, the 191P4D12 MAb is administered in a conjugated form.

[0284] The 191P4D12 ADC formulation is administered via any route capable of delivering the antibody to tumor cells. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumoral, intradermal, etc. Generally, the treatment typically involves repeated administration of the 191P4D12 ADC preparation at a dosage in the range of about 0.1 mg / kg body weight, 0.2 mg / kg body weight, 0.3 mg / kg body weight, 0.4 mg / kg body weight, 0.5 mg / kg body weight, 0.6 mg / kg body weight, 0.7 mg / kg body weight, 0.8 mg / kg body weight, 0.9 mg / kg body weight, 1 mg / kg body weight, 2 mg / kg body weight, 3 mg / kg body weight, 4 mg / kg body weight, 5 mg / kg body weight, 6 mg / kg body weight, 7 mg / kg body weight, 8 mg / kg body weight, 9 mg / kg body weight, 10 mg / kg body weight, 15 mg / kg body weight, 20 mg / kg body weight, or 25 mg / kg body weight via an acceptable route of administration, such as intravenous injection (IV). Generally, dosages in the range of 10 - 1000 mg MAb per week are effective and well tolerated.

[0285] Based on clinical experience with Herceptin® (Trastuzumab) in the treatment of metastatic breast cancer, an initial loading dose of IV of about 4 mg / kg patient body weight, followed by a weekly dose of about 2 mg / kg IV of the MAb preparation, represents an acceptable dosing regimen. Preferably, this initial loading dose is administered as an infusion over 90 minutes or more. Regular maintenance doses are administered as an infusion over 30 minutes or more, provided the initial dose has been well tolerated. As will be appreciated by those skilled in the art, various factors can affect the ideal dosing regimen in a particular case. Such factors include, for example, the binding affinity and half-life of the MAb used, the degree of 191P4D12 expression in the patient, the degree of free 191P4D12 antigen in circulation, the desired steady-state antigen concentration level, the treatment frequency, as well as the influence of chemotherapeutic agents or other drugs used in combination with the methods of the invention, and the health status of the particular patient.

[0286] Optionally, in order to assist in determining the most effective dosing regimen, etc., patients should be evaluated for 191P4D12 levels in a given sample (e.g., the level of circulating 191P4D12 antigen and / or 191P4D12-expressing cells). Such evaluations are also used for monitoring purposes over the course of treatment and are useful in combination with the evaluation of other parameters (e.g., urine cytology and / or ImmunoCyt levels in the treatment of bladder cancer, or serum PSA levels in the treatment of prostate cancer, or the like) to measure treatment success.

[0287] It is an object of the present invention to provide a 191P4D12 ADC that inhibits or delays the growth of tumor cells expressing 191P4D12. A further object of the present invention is to provide a method of inhibiting neovascularization and other biological functions, whereby in a mammal, preferably a human, such a 191P4D12 ADC is used, in particular in combination with other drugs or immunologically active treatments and such a 191P4D12 ADC, to reduce tumor growth.

[0288] XV.) Combination Therapy In one aspect, there is a synergistic effect when a tumor, including a human tumor, is treated with a 191P4D12 ADC together with a chemotherapeutic agent or radiation or a combination thereof. In other words, the inhibition of tumor growth by the 191P4D12 ADC is stronger than expected when combined with a chemotherapeutic agent or radiation or a combination thereof. The synergistic effect can be demonstrated, for example, by inhibition of tumor growth by combination treatment that is greater than expected from the additive effect of treatment with the 191P4D12 ADC alone or treatment with the 191P4D12 ADC and a chemotherapeutic agent or radiation. Preferably, the synergistic effect is demonstrated by remission of cancer when the remission is not expected from treatment with the 191P4D12 ADC or additional combinations of the 191P4D12 ADC and a chemotherapeutic agent or radiation.

[0289] A method of inhibiting the growth of tumor cells using 191P4D12 ADC and chemotherapy and / or radiation, or both, comprises administering 191P4D12 ADC before, during, or after (i.e., before and during, before and after, during and after, or before, during, and after starting chemotherapy and / or radiation therapy) starting chemotherapy and / or radiation therapy. For example, 191P4D12 ADC is typically administered 1 to 60 days, preferably 3 to 40 days, more preferably 5 to 12 days before starting radiation therapy and / or chemotherapy. However, depending on the treatment protocol and the specific patient's requirements, this method is carried out in a manner that provides the most effective treatment and ultimately extends the patient's lifespan.

[0290] Administration of chemotherapeutic agents can be achieved in various ways, including systemic administration by parenteral and enteral routes. In one aspect, 191P4D12 ADC and chemotherapeutic agents are administered as separate molecules. Specific examples of chemotherapeutic agents or chemotherapy include cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozotocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon α, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozotocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, gemcitabine, chlorambucil, taxol, and combinations thereof.

[0291] The radiation source used in combination with the 191P4D12 ADC can be either external or internal to the patient being treated. When this radiation source is external to the patient, this therapy is known as external beam radiation therapy (EBRT). When the radiation source is internal to the patient, this treatment is referred to as brachytherapy (BT).

[0292] The treatment regimen may further be combined with additional cancer therapeutics and / or regimens, such as additional chemotherapy, cancer vaccines, signal transduction inhibitors, agents useful for treating abnormal cell growth or cancer, antibodies that inhibit tumor growth by binding to IGF-1R (e.g., anti-CTLA-4 antibodies as described in WO / 2005 / 092380 (Pfizer)) or other ligands, and cytokines.

[0293] If the mammal is to be subjected to additional chemotherapy, the chemotherapy agents described above can be used. Additionally, growth factor inhibitors, biological response modifiers, antihormonal therapy, selective estrogen receptor modulators (SERMs), neovascularization inhibitors, and antiandrogens can be used. For example, antihormones such as antiestrogens such as Nolvadex (tamoxifen) or antiandrogens such as Casodex (4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3'-(trifluoromethyl)propionanilide) can be used.

[0294] The treatment approach can be used in combination with any one of a wide variety of surgical, chemotherapy, or radiotherapy regimens. The treatment approach of the present invention may enable the use of low-dose chemotherapy (or other treatments) and / or less frequent administrations. This is advantageous for all patients, particularly those who are not sufficiently tolerant to the toxicity of chemotherapy agents.

[0295] XVI.) Kits / Manufactured Articles For use in the research, prognostic, prophylactic, diagnostic, and therapeutic applications described herein, kits are included within the scope of the present invention. Such kits may comprise a carrier, package, or container compartmentalized to hold one or more containers, such as vials, tubes, etc. Each container contains one of the distinct elements used in the methods, along with a label or insert containing instructions for use, e.g., for the uses described herein. For example, the container may contain an antibody that is detectably labeled or an antibody that can be detectably labeled. The kit may comprise a container containing a drug unit. The kit may contain all or a portion of the amino acid sequences or their analogs in FIG. 2 or FIG. 3, or a nucleic acid molecule encoding such amino acid sequences.

[0296] Kits of the present invention typically comprise the aforementioned container and one or more other containers associated therewith. These other containers contain substances that are desirable from a commercial or user perspective. Such substances include, for example, buffers, diluents, filters, needles, syringes, carriers, packages, containers, vials, and / or tube labels listing the contents and / or instructions for use, and package inserts with instructions for use.

[0297] The label may be present on or with the container to indicate that the composition is for use for a particular therapeutic or non-therapeutic use, such as a prognostic use, a prophylactic use, a diagnostic use, or a research use, and may indicate instructions for either in vivo or in vitro use, as described herein. The instructions and / or other information may also be included with the kit or on an insert or label on the kit. The label may also be on or attached to the container. If the characters, numbers or other words forming the label are molded or etched on the container itself, the label may be present on the container. If the label is present, for example, as a package insert, in a receptacle or carrier holding the container, it may be attached to the container. The label may indicate that the composition is for use for the diagnosis, treatment, prophylaxis or prognosis of a condition, such as cancer of the tissue shown in Table I.

[0298] The terms "kit" and "manufactured article" can be used synonymously.

[0299] In another aspect of the invention, there is provided a manufactured article comprising a composition, such as an antibody or an antibody-drug conjugate (ADC), which is useful for the diagnosis, prognosis, prevention and / or treatment of cancer of tissues, such as those shown in Table I. The manufactured article typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The container can be formed from various materials such as glass, metal or plastic. The container may hold an amino acid sequence, a small molecule, a nucleic acid sequence, a cell population and / or an antibody. In another aspect, the container contains an antibody, a binding fragment thereof or a specific binding protein for use in the evaluation of protein expression of 191P4D12 in cells and tissues, or for related research purposes, prognostic purposes, diagnostic purposes, preventive purposes and therapeutic purposes. Instructions and / or directions for such use may be provided on or with such container, and the same may be true for reagents and other compositions or tools used for these purposes.

[0300] Alternatively, the container may also hold a composition effective for treating, diagnosing, prognosticating or preventing a condition and may have a sterile access port (for example, the container can be an intravenous fluid bag or vial having a stopper penetrable by a hypodermic needle). The active agent in the composition can be an antibody that specifically binds to 191P4D12 or an antibody-drug conjugate that specifically binds to 191P4D12.

[0301] The manufactured article may further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution and / or dextrose solution. The manufactured article may further contain other substances desirable from a commercial and user perspective, including other buffers, diluents, filters, stirrers, needles, syringes and / or package inserts with instructions and / or directions for use.

Examples

[0302] Various aspects of the present invention are further described and illustrated by the following several examples. None of these are intended to limit the scope of the present invention.

[0303] Example 1 191P4D12 antigen The 191P4D12 gene sequence was discovered using the suppression subtractive hybridization (SSH) method known in the art. Using standard methods, a 223-bp 191P4D12 SSH sequence was identified from bladder tumor minus cDNA derived from a pool of nine normal tissues. A full-length 191P4D12 cDNA clone was isolated from a bladder cancer cDNA library. This cDNA is 3464 bp in length and encodes a 510-amino acid ORF (see Figure 1). The 191P4D12 gene shows homology to the nectin-4 gene. For further reference, see US2004 / 0083497 (Agensys, Inc., Santa Monica, CA) and PCT publication WO2004 / 016799 (Agensys, Inc., Santa Monica, CA). For exemplary embodiments of the 191P4D12 antigen, see Figure 1.

[0304] Example 2 Preparation of 191P4D12 Monoclonal Antibody (MAb) In one aspect, therapeutic monoclonal antibodies ("MAbs") against 191P4D12 and 191P4D12 variants bind to 191P4D12 or 191P4D12 variants, internalize, and disrupt or modulate the biological function of 191P4D12 or 191P4D12 variants, and react with epitopes specific to each protein or specific to sequences common among variants, for example, antibodies that disrupt the interaction with ligands, substrates, and binding partners. Immunogens for generating such MAbs include immunogens designed to encode or contain the extracellular domain or the entire 191P4D12 protein sequence, regions predicted to contain functional motifs from computer analysis of amino acid sequences, and regions of 191P4D12 protein variants predicted to have antigenicity. Immunogens include peptides and recombinant proteins, such as tag5-191P4D12, His-tagged proteins derived from purified mammalian cells. Furthermore, cells engineered to express high levels of 191P4D12, such as rat 1-191P4D12 or 300.19-191P4D12, are used to immunize mice.

[0305] MAbs against 191P4D12 were generated using XenoMouse technology (registered trademark) (Amgem Fremont), in which the mouse heavy and κ light chain loci are inactivated and most of the human heavy and κ light chain immunoglobulin loci are inserted. Ha22 - 2(2,4)6.1 The MAb named was generated from the immunization of human γ1-producing XenoMice with pTag5 / mychis-191P4D12 (amino acids 23-351).

[0306] 191P4D12 MAb Ha22 - 2(2,4)6.1 specifically binds to the pTag5 / mychis-191P4D12 protein by ELISA and also specifically binds to recombinant 191P4D12-expressing cells and multiple 191P4D12-expressing cancer cell lines.

[0307] Ha22 - 2(2,4)6.1 The hybridomas that produce the antibody namedAugust 18, 2010 It was sent (via Federal Express) to the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, and accession number PTA - 11267 was assigned.

[0308] After isolating mRNA from each hybridoma cell using TRIzol reagent (Life Technologies, Gibco BRL), the DNA coding sequence of 191P4D12 MAb Ha22 - 2(2,4)6.1 was determined.

[0309] Using the following protocol, the variable heavy nucleic acid sequence and variable light nucleic acid sequence of anti-191P4D12 Ha22-2(2,4)6.1 were sequenced from hybridoma cells. The Ha22-2(2,4)6.1 secreting hybridoma cells were lysed with TRIzol reagent (Life Technologies, Gibco BRL). Total RNA was purified and quantified. First-strand cDNA was prepared from total RNA using oligo(dT)12-18 priming and the Gibco-BRL Superscript Preamplification system. The first-strand cDNA was amplified using human immunoglobulin heavy chain variable primers and human immunoglobulin light chain variable primers. The PCR products were sequenced to determine the variable heavy region and variable light region.

[0310] The nucleic acid sequences and amino acid sequences of the variable heavy region and variable light region are listed in FIGS. 2 and 3. The alignment of Ha22-2(2,4)6.1 MAb with the human Ig germline is shown in FIGS. 4A-4B.

[0311] Example 3 Expression of Ha22 - 2(2,4)6.1 Using Recombinant DNA Methods To recombinantly express the Ha22-2(2,4)6.1 MAb in transfected cells, the Ha22-2(2,4)6.1 MAb heavy chain variable sequence and light chain variable sequence were each cloned upstream of the human heavy chain IgG1 constant region and the human light chain Igκ constant region. The complete Ha22-2(2,4)6.1 MAb human heavy chain cassette and light chain cassette were cloned downstream of the CMV promoter / enhancer in a cloning vector. A polyadenylation site was included downstream of the MAb coding sequence. The recombinant Ha22-2(2,4)6.1 MAb expression construct was transfected into CHO cells. The binding of the Ha22-2(2,4)6.1 MAb secreted from the recombinant cells to cell surface 191P4D12 was evaluated by flow cytometry (Figure 5A). Rat-control cells and rat-191P4D12 cells were stained with the Ha22-2(2,4)6.1 MAb from either the hybridoma or CHO cells transfected with the Ha22-2(2,4)6.1 heavy chain vector construct and light chain vector construct. Binding was detected by flow cytometry.

[0312] From the results, it can be seen that the recombinantly expressed Ha22-2(2,4)6.1 expressed in CHO cells binds to 191P4D12 in the same manner as the Ha22-2(2,4)6.1 purified from the hybridoma. The Ha22-2(2,4)6.1 MAb secreted from the recombinant cells was also evaluated by ELISA for binding to the 191P4D12 recombinant protein. As shown in Figure 5B, the binding between Ha22-2(2,4)6.1 and the 191P4D12 protein was identical between the CHO-derived MAb material and the hybridoma cell-derived MAb material.

[0313] Example 4 Antibody - Drug Conjugation of Ha22 - 2(2,4)6.1 MAb Using the following protocol, the antibody-drug conjugate (ADC) of the present invention, named Ha22-2(2,4)6.1vcMMAE, was created by conjugating Ha22-2(2,4)6.1 Mab (Figure 2) with an auristatin derivative named MMAE (Formula XI) using the vc(Val-Cit) linker described herein. To create cytotoxic vcMMAE, conjugation of the vc(Val-Cit) linker with MMAE (Seattle Genetics, Inc., Seattle, WA) was completed using the general method shown in Table IV (see U.S. Patent No. 7,659,241).

[0314] Next, the antibody-drug conjugate (ADC) of the present invention, named Ha22-2(2,4)6.1vcMMAE, was prepared using the following protocol.

[0315] Briefly, a 15 mg / mL solution of Ha22-2(2,4)6.1 MAb dissolved in 10 mM acetate pH 5.0, 1% sorbitol, 3% L-agrinine was added with 20% volume of 0.1 M TrisCl, pH 8.4, 25 mM EDTA, and 750 mM NaCl, 5 mM EDTA, and 150 mM sodium chloride to adjust the pH of this solution to 7.5. The MAb was then partially reduced by adding 2.3 molar equivalents of TCEP (based on the moles of MAb), and then stirred at 37 °C for 2 hours. The partially reduced MAb solution was then cooled to 5 °C, and 4.4 molar equivalents of vcMMAE (based on the moles of antibody) was added as a 6% (v / v) DMSO solution. The mixture was stirred at 5 °C for 60 minutes, and then stirred for an additional 15 minutes after adding 1 molar equivalent of N-acetylcysteine based on vcMMAE. Excess quenched vcMMAE and other reaction components were removed by ultrafiltration / diafiltration of the antibody-drug conjugate (ADC) using 10 volumes of 20 mM histidine, pH 6.0.

[0316] The resulting antibody-drug conjugate (ADC) was named Ha22-2(2,4)6.1vcMMAE and has the following formula: It has TIFF2025113404000039.tif36162. In the formula, MAb is Ha22-2(2,4)6.1 (Figures 2 and 3), and p is from 1 to 8. The p value of the antibody-drug conjugate shown in this example was approximately 3.8.

[0317] Example 5 Characterization of Ha22 - 2(2,4)6.1vcMMAE Using the procedure shown in the example titled "Antibody-drug conjugation of Ha22-2(2,4)6.1 MAb", an antibody-drug conjugate that binds to 191P4D12 was prepared and screened, identified, and characterized using a combination of assays known in the art.

[0318] A. Determination of Affinity by FACS The binding affinity of Ha22-2(2,4)6.1vcMMAE for 191P4D12 expressed on the surface of PC3-human-191P4D12 cells, the binding affinity of Ha22-2(2,4)6.1vcMMAE for 191P4D12 expressed on the surface of PC3-cynomolgus monkey-191P4D12 cells, and the binding affinity of Ha22-2(2,4)6.1vcMMAE for 191P4D12 expressed on the surface of PC3-rat-191P4D12 cells were tested. Briefly, 11 (eleven) types of Ha22-2(2,4)6.1vcMMAE dilution solutions with final concentrations ranging from 160 nM to 0.011 nM were each incubated with the respective cell type (50,000 cells / well) at 4 °C overnight. At the end of the incubation, the cells were washed and incubated with an anti-hIgG-PE detection antibody at 4 °C for 45 minutes. After washing away the unbound detection antibody, the cells were analyzed by FACS. Mean fluorescence intensity (MFI) values were obtained as listed in Figures 6 - 8. The MFI values were input into Graphpad Prisim software, Y = Bmax *The saturation curves of Ha22-2(2,4)6.1vcMMAE shown in FIGS. 6 to 8 were generated by analyzing using the one-site binding (hyperbola) equation of X / (Kd+X). Bmax is the MFI value at the maximum binding of Ha22-2(2,4)6.1vcMMAE and 191P4D12, and 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 reach the maximum half-binding.

[0319] The calculated values of the affinity (Kd) of Ha22-2(2,4)6.1vcMMAE for 191P4D12 expressed on the surface of PC3-human-191P4D12 cells, the affinity (Kd) of Ha22-2(2,4)6.1vcMMAE for 191P4D12 expressed on the surface of PC3-cynomolgus monkey-191P4D12 cells, and the affinity (Kd) of Ha22-2(2,4)6.1vcMMAE for 191P4D12 expressed on the surface of PC3-rat-191P4D12 cells are 0.69 nM (FIG. 6), 0.34 nM (FIG. 7), and 1.6 nM (FIG. 8), respectively.

[0320] B. Determination of Affinity by SPR The affinity of Ha22-2(2,4)6.1 MAb and Ha22-2(2,4)6.1vcMMAE for purified recombinant 191P4D12 (ECD amino acids 1-348) was determined by surface plasmon resonance (SPR) (BIAcore). Briefly, goat anti-human Fcγ polyclonal Ab (Jackson Immuno Research Labs, Inc.) was covalently immobilized on the surface of a CM5 sensor chip (Biacore). Subsequently, purified Ha22-2(2,4)6.1 MAb or Ha22-2(2,4)6.1vcMMAE was captured on the surface of the chip. On average, approximately 300 RU of test Ha22-2(2,4)6.1 MAb or Ha22-2(2,4)6.1vcMMAE was captured in each cycle. Thereafter, a series of 5-6 dilutions of recombinant 191P4D12 (ECD amino acids 1-348) ranging from 1 nM to 100 nM were injected onto such surface and processed using BIAevaluation 3.2 and CLAMP software (Myszka and Morton, 1998) to generate a binding curve (sensogram) globally fit to a 1:1 interaction model (Figure 22). Table V summarizes the association rate constants, dissociation rate constants, and affinities of Ha22-2(2,4)6.1 MAb and Ha22-2(2,4)6.1vcMMAE for recombinant 191P4D12 (ECD amino acids 1-348).

[0321] C. Domain Mapping of Ha22 - 2(2.4)6.1 MAb To map the binding site of the Ha22-2(2,4)6.1 MAb to specific domains of the 191P4D12 protein, several types of rat 1(E) recombinant cell lines expressing such domains (or combinations thereof) were generated (Table VI). Using standard protocols, the binding of Ha22-2(2,4)6.1 to the cell surface was evaluated by FACS. As shown in Figure 10, the Ha22-2(2,4)6.1 MAb binds to VC1 domain-expressing cells as well as wild-type 191P4D12, but does not bind to C1C2 domain-expressing cells. Furthermore, another 191P4D12 MAb named Ha22-8e6.1 recognizes the 191P4D12 C1C2 domain on the cell surface, but does not recognize the VC1 domain. This suggests that the binding site of the Ha22-2(2,4)6.1 MAb is located in the 1-147aa domain of 191P4D12, but not all MAbs that bind to 191P4D12 recognize this domain.

[0322] To further confirm the results shown in Figure 10, Western blot analysis was performed. Briefly, the entire extracellular portion (full-length) of 191P4D12 as well as the specific domains shown in Table VI were expressed as mouse Fc fusion proteins in 293T cells and purified. Goat anti-mouse-HRP was used as a control. As shown in Figure 11, when separated by SDS-PAGE (non-reducing) and probed with streptavidin-HRP after Ha22-2(2,4)6.1-biotin, bands corresponding to the full-length 191P4D12 (lane 1), V (lane 2), and VC1 (lane 3) fusion constructs were observed, but no band corresponding to the C1C2 fusion construct (lane 4) was observed. This further suggests that the binding epitope of the Ha22-2(2,4)6.1 MAb is located within the 1-147aa domain of 191P4D12.

[0323] Example 6 Cytotoxicity Mediated by Ha22 - 2(2,4)6.1vcMMAE In PC3 cells engineered to express human 191P4D12, cynomolgus monkey 191P4D12, and rat 191P4D12, the ability of Ha22-2(2,4)6.1vcMMAE to mediate 191P4D12-dependent cytotoxicity was evaluated. Briefly, on day 1, PC3-Neo, PC3-human-191P4D12 cells, PC3-cynomolgus monkey-191P4D12 cells, or PC3-rat-191P4D12 cells (1500 cells / well) were seeded in 96-well plates. The next day, equal volumes of medium containing the indicated concentrations of Ha22-2(2,4)6.1vcMMAE or control MAb conjugated to vcMMAE (i.e., control-vcMMAE) were added to each well. The cells were incubated at 37 °C for 4 days. At the end of the incubation period, Alamar Blue was added to each well and incubation was continued for an additional 4 hours. The resulting fluorescence was detected using a Biotek plate reader at an excitation wavelength of 620 nm and an emission wavelength of 540 nm.

[0324] From the results in Figures 9A-9D, it can be seen that Ha22-2(2,4)6.1vcMMAE mediated cytotoxicity in PC3-human-191P4D12 (Figure 9A), PC3-cynomolgus monkey-191P4D12 (Figure 9B), and PC3-rat-191P4D12 cells (Figure 9C), whereas 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 to PC3-Neo cells that do not express 191P4D12 (Figure 9D). Thus, from these results, it can be seen that Ha22-2(2,4)6.1vcMMAE can selectively deliver a cytotoxic drug to 191P4D12-expressing cells and kill 191P4D12-expressing cells.

[0325] Example 7 Ha22 - 2(2,4)6.1vcMMAE Inhibits Tumor Growth In Vivo Due to the substantial 191P4D12 expression on the surface of tumor tissue cells and limited 191P4D12 expression in normal tissues, 191P4D12 is an excellent target for antibody therapy and also an excellent target for therapy via ADC. Therefore, evaluate the therapeutic efficacy of Ha22-2(2,4)6.1vcMMAE in human bladder cancer, lung cancer, breast cancer, and pancreatic cancer xenograft mouse models.

[0326] Study the efficacy of antibody-drug conjugates against tumor growth and metastasis formation in mouse cancer xenograft models (e.g., subcutaneous and orthotopic).

[0327] 5x10 4 ~10 6 Subcutaneous (s.c.) tumors are generated by injecting 5x10 2 ~10

[0328] Ovarian tumors often metastasize and grow intraperitoneally. Therefore, grow ovarian tumors in the mouse peritoneal cavity by directly injecting 2 million cells into the peritoneal cavity of female mice. Monitor the overall health, physical activity, and appearance of the mice until they are near death. At the time of sacrifice, examine the peritoneal cavity to confirm the tumor burden and collect the lungs to evaluate metastasis to distant sites. Alternatively, death can be used as an endpoint. Then, divide the mice into groups for appropriate treatment and inject 191P4D12 or control MAb i.p.

[0329] The advantage of the xenograft cancer model is that it enables the study of angiogenesis and vasculogenesis. Tumor growth depends to some extent on the development of new blood vessels. The capillary system and the developing blood network are derived from the host, but the initiation and construction of the new vascular structure are regulated by the xenograft tumor (Davidoff et al., Clin Cancer Res. (2001) 7:2870; Solesvik et al., Eur J Cancer Clin Oncol. (1984) 20:1295). The effects of antibodies and small molecules on angiogenesis are studied according to procedures known in the art, such as IHC analysis of tumor tissue and its surrounding microenvironment.

[0330] Ha22-2(2,4)6.1 ADC inhibits the formation of lung cancer xenografts, bladder cancer xenografts, breast cancer xenografts, and pancreatic cancer xenografts. These results indicate the usefulness of Ha22-2(2,4)6.1 ADC in the treatment of cancer, preferably the local and advanced stages of the cancers shown in Table I.

[0331] 191P4D12 ADC: As described in the example entitled "Production of Monoclonal Antibody (MAb) 191P4D12", monoclonal antibodies were produced against 191P4D12. Further, as described in the example entitled "Antibody-Drug Conjugation of Ha22-2(2,4)6.1 MAb", this MAb was conjugated with a toxin to form Ha22-2(2,4)6.1vcMMAE. To confirm the ability of Ha22-2(2,4)6.1vcMMAE to bind to 191P4D12, Ha22-2(2,4)6.1vcMMAE was characterized by FACS and other methods known in the art.

[0332] Cell Lines and Xenografts: BT-483 and HPAC cells were maintained in DMEM supplemented with L-glutamine and 10% FBS as is known in the art. The AG-B8, AG-Panc4, AG-Panc2, AG-B1, AG-L4, and AG-Panc3 xenografts were maintained by serial passage in SCID mice.

[0333] Evaluation of Ha22 - 2(2,4)6.1vcMMAE MAb in a Subcutaneous Tumor - Formation Model of Human Lung Cancer Xenograft AG - L4 in SCID Mice In this experiment, the patient-derived lung cancer xenograft AG-L4 was maintained by serial passage in SCID mice. Stock tumors were aseptically harvested and enzymatically digested to a single cell suspension. Two million cells were transplanted into the flanks of individual SCID mice. The animals were then randomly assigned to seven groups: six 191P4D12 antibody treatment groups and a control antibody H3-1.10.1.2 group (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 3-4 days. Tumor volume was calculated as width 2 x length / 2. Width was the shortest dimension and length was the longest dimension.

[0334] The results showed that the 191P4D12 MAb did not significantly inhibit tumor growth in the human lung cancer xenograft AG-L4 in SCID mice. Furthermore, other 191P4D12 MAbs were used in this study. The results are not shown (Figure 12).

[0335] Evaluation of Ha22 - 2(2,4)6.1 MAb in a Subcutaneous Tumor - Formation Model of Human Pancreatic Cancer Xenograft HPAC in SCID Mice In another experiment, human pancreatic cancer HPAC cells (2 million cells / mouse) were injected into the flanks of individual SCID mice. The animals were then randomly assigned to eight groups: seven 191P4D12 antibody treatment groups and a control antibody H3-1.4.1.2 group (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 3-4 days. Tumor volume was calculated as width 2 x length / 2. Width was the shortest dimension and length was the longest dimension.

[0336] From the results, it can be seen that compared with the control antibody, the 191P4D12 MAb did not inhibit tumor growth in human pancreatic xenografts in SCID mice. Furthermore, other 191P4D12 MAbs were used in this study. The results are not shown (Figure 13).

[0337] Evaluation of Ha22-2(2,4)6.1 MAb in a subcutaneous tumor formation model of human pancreatic cancer xenograft AG-Panc3 in SCID mice In another experiment, patient-derived pancreatic cancer xenograft AG-Panc3 was maintained by serial passage in SCID mice. The stock tumor was aseptically collected and minced into 1 mm 3 fragments. Six fragments were transplanted into the flanks of individual SCID mice. The animals were then randomly assigned to the following cohorts (n = 10): two 191P4D12 MAb treatment groups and a control antibody H3-1.4.1.2 group. 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 - 4 days. Tumor volume was calculated as width 2 × length / 2. The width is the shortest dimension and the length is the longest dimension.

[0338] From the results, it can be seen that compared with the control antibody, the 191P4D12 MAb did not inhibit tumor growth in human pancreatic xenografts in SCID mice. Furthermore, other 191P4D12 MAbs were used in this study. The results are not shown (Figure 14).

[0339] Efficacy of Ha22 - 2(2,4)6.1 - vcMMAE in Subcutaneously Established Human Lung Cancer Xenograft AG - L4 in SCID Mice In another experiment, patient-derived lung cancer xenograft AG-L13 was maintained by serial passage in SCID mice. The stock tumor was aseptically collected and minced into 1 mm 3 fragments. Six fragments were transplanted into the flanks of individual SCID mice. When the tumor reached 200 mm 3The tumor was allowed to grow untreated until it reached an approximate volume. 10 mg / kg of Ha22-2(2,4)6.1vcMMAE and control ADC were administered by intravenous bolus injection for two doses every 7 days. The amount of ADC administered was based on the individual body weight of each animal obtained immediately before dosing. Tumor growth was monitored using caliper measurements every 3 - 4 days. Tumor volume was calculated as width 2 x length / 2. The width was the shortest dimension and the length was the longest dimension.

[0340] The results showed that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of AG-L4 lung cancer xenografts subcutaneously implanted in nude mice compared to control ADC. Additionally, another 191P4D12 MAb was used in this study. The results are not shown (Figure 15).

[0341] Efficacy of Ha22-2(2,4)6.1-vcMMAE in the human breast cancer xenograft BT-483 established subcutaneously in SCID mice In this experiment, stock xenografts were generated using human breast cancer BT-483 cells. The stock xenografts were maintained by serial passage in SCID mice. Stock tumors were aseptically harvested and minced into 1 mm 3 fragments. Six fragments were implanted into the flanks of individual SCID mice. The tumors were allowed to grow untreated until they reached an approximate volume of 100 mm 3 . 5 mg / kg of Ha22-2(2,4)6.1vcMMAE and control ADC were administered by intravenous bolus injection for four doses every 4 days. The amount of ADC administered was based on the individual body weight of each animal obtained immediately before dosing. Tumor growth was monitored using caliper measurements every 3 - 4 days. Tumor volume was calculated as width 2 x length / 2. The width was the shortest dimension and the length was the longest dimension.

[0342] From the results, it can be seen that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of BT-483 breast tumor xenografts subcutaneously implanted in SCID mice compared with the control ADC. Furthermore, another 191P4D12 MAb was used in this study. The results are not shown (Figure 16).

[0343] Efficacy of Ha22-2(2,4)6.1-vcMMAE in the human bladder cancer xenograft AG-B1 established subcutaneously in SCID mice In another experiment, the patient-derived bladder cancer xenograft AG-B1 was maintained by serial passage in SCID mice. Stock tumors were collected aseptically and minced into 1 mm 3 fragments. Six fragments were implanted into the flanks of individual SCID mice. The tumors were allowed to grow untreated until they reached an approximate volume of 230 mm 3 . 4 mg / kg of Ha22-2(2,4)6.1vcMMAE and the control ADC were administered once by intravenous bolus injection. The amount of ADC administered was based on the individual body weight of each animal obtained immediately before administration. Tumor growth was monitored using caliper measurements every 3-4 days. Tumor volume was calculated as width 2 × length / 2. The width was the shortest dimension and the length was the longest dimension.

[0344] From the results, it can be seen that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of AG-B1 bladder cancer xenografts compared with the control ADC. Furthermore, another 191P4D12MAb was used in this study. The results are not shown (Figure 17).

[0345] Efficacy of Ha22-2(2,4)6.1-vcMMAE in the human pancreatic cancer xenograft AG-Panc2 in SCID mice In another experiment, the patient-derived pancreatic cancer xenograft AG-Panc2 was maintained by serial passage in SCID mice. Stock tumors were collected aseptically and minced into 1 mm 3 fragments. Five fragments were implanted into the flanks of individual SCID mice. The tumors were allowed to grow until they reached 100 mm 3The tumor was allowed to grow in an untreated state until it reached an approximate volume. 5 mg / kg of Ha22-2(2,4)6.1vcMMAE and control ADC were administered by intravenous bolus injection for 4 doses at 4-day intervals. The amount of ADC administered was based on the individual body weight of each animal obtained immediately prior to administration. Tumor growth was monitored using caliper measurements every 3 - 4 days. Tumor volume was calculated as width 2 x length / 2. The width was the shortest dimension and the length was the longest dimension.

[0346] From the results, it can be seen that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of AG-Panc2 pancreatic cancer xenografts compared to control ADC. Additionally, another 191P4D12 MAb was used in this study. The results are not shown (Figure 18).

[0347] Efficacy of Ha22-2(2,4)6.1-vcMMAE in the human pancreatic cancer xenograft AG-Panc4 established subcutaneously in SCID mice In another experiment, patient-derived pancreatic cancer xenograft AG-Panc4 was maintained by serial passage in SCID mice. Stock tumors were collected aseptically and minced into 1 mm 3 fragments. Six fragments were transplanted into the flanks of individual SCID mice. 5 mg / kg of Ha22-2(2,4)6.1vcMMAE and control ADC were administered by intravenous bolus injection for 3 doses at 7-day intervals. The amount of ADC administered was based on the individual body weight of each animal obtained immediately prior to administration. Tumor growth was monitored using caliper measurements every 3 - 4 days. Tumor volume was calculated as width 2 x length / 2. The width was the shortest dimension and the length was the longest dimension.

[0348] From the results, it can be seen that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of AG-Panc4 pancreatic cancer xenografts compared to control ADC. Additionally, another 191P4D12 MAb was used in this study. The results are not shown (Figure 19).

[0349] Efficacy of comparative doses of Ha22-2(2,4)6.1-vcMMAE in the human bladder cancer xenograft AG-B8 established subcutaneously in SCID mice In this experiment, the patient-derived bladder cancer xenograft AG-B8 was maintained by serial passage in SCID mice. Stock tumors were aseptically harvested and minced into 1 mm 3 fragments. Six fragments were implanted into the flanks of individual SCID mice. Tumors were allowed to grow untreated until they reached an approximate volume of 200 mm 3 . The animals were then randomly assigned to the following three cohorts (n = 6): two Ha22-2(2,4)6.1-vcMMAE treatment groups and the control ADC VCD37-5ce5p-vcMMAE group. Ha22-2(2,4)6.1-vcMMAE was administered at 5 mg / kg or 10 mg / kg, and the control ADC was given at 5 mg / kg. All ADCs were administered as a single dose by intravenous bolus injection. The amount of ADC administered was based on the individual body weight of each animal obtained immediately prior to dosing. Tumor growth was monitored using caliper measurements every 3 - 4 days. Tumor volume was calculated as width 2 x length / 2. The width was the shortest dimension and the length was the longest dimension.

[0350] From the results, it can be seen that treatment with 10 mg / kg Ha22-2(2,4)6.1vcMMAE inhibited the growth of AG-B8 bladder cancer xenografts compared to 5 mg / kg Ha22-2(2,4)6.1vcMMAE (Figure 20).

[0351] Conclusion In summary, Figures 12 - 20 show that the 191P4D12 ADC named Ha22-2(2,4)6.1vcMMAE significantly inhibited the proliferation of tumor cells expressing 191P4D12 compared to the control ADC. Therefore, Ha22-2(2,4)6.1vcMMAE can be used for therapeutic purposes to treat and manage the cancers shown in Table I.

[0352] Example 8 Human clinical trials for treating and diagnosing human carcinomas using 191P4D12 ADC The 191P4D12 ADC that specifically binds to 191P4D12 was used in accordance with the present invention and used in the treatment of specific tumors, preferably tumors listed in Table I. In connection with each of these indicators, two clinical approaches were successfully implemented.

[0353] I.) Adjuvant therapy: In adjuvant therapy, the patient is treated with a chemotherapeutic or anti-tumor agent and / or radiation therapy or a combination thereof in combination with the 191P4D12 ADC. The target of the primary cancer, for example, the cancers listed in Table I, is treated under a standard protocol by adding the 191P4D12 ADC to standard primary and secondary treatments. The design of the protocol addresses efficacy as evaluated by the following examples. Reduction in tumor mass of primary or metastatic lesions, prolongation of progression-free survival, overall survival, improvement in patient health, stabilization of the disease, and the ability to reduce the usual doses of standard chemotherapy and other biological agents, but is not limited thereto. These dose reductions enable additional therapy and / or long-term therapy by reducing the dose-related toxicity of the chemotherapeutic or biological agent. The 191P4D12 ADC is utilized in several adjuvant clinical trials in combination with a chemotherapeutic or anti-tumor agent.

[0354] II.) Monotherapy: In connection with the use of the 191P4D12 ADC in monotherapy of tumors, the 191P4D12 ADC is administered to the patient without a chemotherapeutic or anti-tumor agent. In one aspect, monotherapy is clinically performed in end-stage cancer patients with extensive metastatic disease. The design of the protocol addresses efficacy as evaluated by the following examples. Reduction in tumor mass of primary or metastatic lesions, prolongation of progression-free survival, overall survival, improvement in patient health, stabilization of the disease, and the ability to reduce the usual doses of standard chemotherapy and other biological agents, but is not limited thereto.

[0355] Dosage The dosing regimen can be adjusted to provide the optimal desired response. For example, a single bolus may be administered, several divided doses may be administered over a period of time, or the dose may be proportionally decreased or increased as indicated by the exigencies of the treatment situation. It is particularly advantageous to formulate the parenteral composition in unit dosage form for ease of administration and uniformity of dosing. As used herein, unit dosage form refers to physically discrete units suitable as a single dosage for the mammalian subject to be treated. Each unit containing a predetermined amount of the active compound is calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications for the unit dosage forms of the present invention are determined by and directly depend on (a) the particular 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 formulating such active compounds with respect to the susceptibility of the individual to treatment.

[0356] Exemplary, non-limiting ranges for the therapeutically effective amount of 191P4D12 ADC administered in combination according to the present invention are from about 0.5 to about 10 mg / kg, from 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, from about 0.5 to about 5 mg / kg, or for example, from about 0.8 to about 5 mg / kg, or for example, from about 1 to about 7.5 mg / kg. The high-dose aspect of the present invention relates to dosages greater than 10 mg / kg. It should be noted that the dosage values can vary depending on the type and severity of the condition to be alleviated and may include a single dose or multiple doses. For any particular subject, the specific dosing regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and it is further to be understood that the dosage ranges set forth herein are merely exemplary and are not intended to limit the scope or practice of the claimed composition.

[0357] Clinical Development Plan (CDP) CDP pursues and develops the treatment with 191P4D12 ADC related to adjuvant therapy or monotherapy. The trial first proves safety and then confirms the efficacy with repeated dosing. The trial is a non-blinded one that compares standard chemotherapy with the standard therapy plus 191P4D12 ADC. As is recognized, one non-limiting criterion available for patient enrollment is the expression level of 191P4D12 in the patient's tumor as determined by biopsy.

[0358] Similar to any treatment based on the injection of a protein or antibody, safety issues mainly include the following: (i) cytokine release syndrome, namely, low blood pressure, fever, tremors, chills; (ii) the occurrence of an immunogenic response to the substance (i.e., the generation of human antibodies by the patient against the antibody treatment, i.e., the HAMA reaction); and (iii) toxicity to normal cells expressing 191P4D12. Standard tests and follow-up investigations are used to monitor each of these safety issues. 191P4D12 ADC has been found to be safe in human administration.

[0359] Example 9 Detection of 191P4D12 protein in cancer patient specimens by IHC The expression of the 191P4D12 protein was tested by immunohistochemistry in patient tumor specimens derived from (i) bladder cancer patients, (ii) breast cancer patients, (iii) pancreatic cancer patients, (iv) lung cancer patients, (v) ovarian cancer patients, (vi) esophageal patients, and (vii) head and neck patients. Briefly, formalin-fixed, paraffin-embedded tissue was sectioned at 4 microns and placed on glass slides. The sections were deparaffinized, rehydrated, and placed in an EZ-Retriever microwave (Biogenex, San Ramon, CA) and treated with an EDTA antigen retrieval solution (Biogenex, San Ramon, CA) at 95°C for 30 minutes. The sections were then treated with a 3% hydrogen peroxide solution to inactivate endogenous peroxidase activity. After inhibiting non-specific binding using a serum-free protein block (Dako, Carpenteria, CA), the sections were incubated with a monoclonal mouse anti-191P4D12 antibody or an isotype control. The sections were then processed by a Super Sensitive™ Polymer-horseradish peroxidase (HRP) Detection System consisting of incubation in a Super Enhancer™ reagent followed by incubation with a polymer-HRP secondary antibody conjugate (BioGenex, San Ramon, CA). The sections were then developed using a DAB kit (BioGenex, San Ramon, CA). The nuclei were stained with hematoxylin and analyzed by bright-field microscopy. Specific staining was detected in patient specimens using the 191P4D12 immunoreactive antibody as shown by brown staining (see FIGS. 21(A), 21(C), 21(E), 21(G), 21(I), 21(K), and 21(M)). In contrast, the control antibody did not stain either patient specimen (see FIGS. 21(B), 21(D), 21(F), 21(H), 21(J), 21(L), and 21(N)).

[0360] From the results, it can be seen that 191P4D12 is expressed in tumor cells of patients' bladder cancer tissue, breast cancer tissue, pancreatic cancer tissue, lung cancer tissue, ovarian cancer tissue, esophageal cancer tissue, and head and neck cancer tissue. From these results, it can be seen that 191P4D12 is expressed in human cancer, and that antibodies against this antigen and antibody-drug conjugates named Ha22-2(2,4)6.1vcMMAE) are useful for diagnostic and therapeutic purposes (Figure 21).

[0361] Example 10 Determination of the binding epitope of the Ha22-2(2,4)6.1 MAb To confirm the cross-reactivity of Ha22-2(2,4)6.1 with 191P4D12 proteins derived from humans, cynomolgus monkeys, rats, and mice, these orthologs were recombinantly overexpressed in the PC3 cell line. Ha22-2(2,4)6.1 was shown to strongly cross-react with the cynomolgus monkey ortholog and rat ortholog of 191P4D12 (Figure 23). The EC50 binding values are shown in Table VII. The binding of Ha22-2(2,4)6.1 to the mouse ortholog shows a significant decrease in the binding EC50 value. From this, it can be seen that important amino acid substitutions in the V domain (compared to the human and rat sequences) affected the affinity of Ha22-2(2,4)6.1 for 191P4D12.

[0362] Table VIII shows the aa1-180 protein sequence alignment of 191P4D12 orthologs containing the V domain. Only two amino acids, Thr-75 and Ser-90, in the rat ortholog sequence are substituted in place of Ile and Asn, respectively, in the mouse ortholog sequence ( Indicated in bold)。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 are included in the binding epitope of Ha22-2(2,4)6.1, several mutant constructs of 191P4D12 and its mouse ortholog were prepared and expressed in PC3 cells (Table IX). Instead of standard alanine substitution mutagenesis, "mouse" amino acids were introduced into the human sequence, and conversely, "human" amino acids were introduced into the mouse sequence.

[0363] When Ser-91 of 191P4D12 was mutated to Asn, it was shown that the binding of Ha22-2(2,4)6.1 was significantly impaired. This supports that this amino acid Ser-91 is absolutely necessary for binding and must contain the epitope recognized by the Ha22-2(2,4)6.1 MAb. Further mutations of Ala at position 76 (A76I, S91N double mutant) were also introduced into 191P4D12. The binding of Ha22-2(2,4)6.1 to the double mutant A76I, S91N was shown to be very similar to the mouse ortholog binding (Figure 24). Conversely, when Asn-90 in the mouse sequence was mutated to Ser, the binding of Ha22-2(2,4)6.1 to the mouse mutant ortholog was dramatically improved. This further supports that the amino acid at this position is important for the binding of Ha22-2(2,4)6.1. The binding of Ha22-2(2,4)6.1 to the mouse ortholog double mutant A90S, I75A seems to be very similar to that of the 191P4D12 human ortholog.

[0364] In summary, from these data, it can be seen that Ser-91 and Ala-76 play important roles in the binding of Ha22-2(2,4)6.1 to the 191P4D12 protein on the cell surface and constitute part of the epitope recognized by Ha22-2(2,4)6.1 on the 191P4D12 surface.

[0365] To visualize this idea, the inventors generated a computer model of the 191P4D12 V domain using PyMOL based on the published crystal structure data of family members consisting of 191P4D12 and Ig domain-containing proteins (Figure 25). The positions of Ala-76 (stippled) and Ser-91 (hatched) are indicated.

[0366] Furthermore, to further refine the Ha22-2(2,4)6.1 binding site on the 191P4D12 molecule, the inventors designed and expressed a 191P4D12 fragment corresponding to the V domain on the surface of rat (1) E cells. The following constructs were generated in a retroviral vector. 191P4D12(aa1~150, 347~510)

[0367] Binding of the Ha22-2(2,4)6.1 MAb was evaluated by FACS. As shown in Figure 26, Ha22-2(2,4)6.1 binds to V domain-expressing cells (A) as well as wild-type 191P4D12 (B), but does not bind to previously generated C1C2 domain-expressing cells (C). This indicates that the binding site of this antibody is located in the 191P4D12 V domain within the first 150 amino acids.

[0368] The results indicate that the Ha22-2(2,4)6.1 MAb binds to the 191P4D12 protein v domain at positions aa1~150, and further that a specific epitope containing aaSer-91 and aaAla-76 is important for binding of the Ha22-2(2,4)6.1 MAb.

[0369] Throughout this application, data content, publications, patent applications, and patents of various websites are referenced (websites are referenced by their Uniform Resource Locators, i.e., URLs, which are addresses on the World Wide Web). The disclosure of each of these references is hereby incorporated by reference in its entirety into this specification.

[0370] The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any embodiments 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 disclosure and are also intended to be included within the scope of the invention. Such modifications or other embodiments can be made without departing from the true scope and spirit of the invention.

[0371] Table I. Tissues that express 191P4D12 when malignant colon pancreas ovaries breast lung bladder

[0372] Table II: Amino acid abbreviations TIFF2025113404000040.tif95158

[0373] Table III: Amino acid substitution matrix Compiled from GCG Software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the more likely the substitution will be found in related native proteins. TIFF2025113404000041.tif96146

[0374] Table IV: General method for synthesizing vcMMAE Where AA1 = amino acid 1 AA2 = amino acid 2 AA5 = amino acid 5 DIL = Dry Solo In DAP = Dolaproin Linker = Val-Cit(vc) TIFF2025113404000042.tif98160

[0375] (Table V) Calculation of Biacore association rate, dissociation rate, and resulting affinity TIFF2025113404000043.tif17128

[0376] (Table VI) 191P4D12 constructs used in the domain mapping assay TIFF2025113404000044.tif46128

[0377] (Table VII) TIFF2025113404000045.tif26151

[0378] (Table VIII) SEQ ID NO:11 - 13, in order of appearance TIFF2025113404000046.tif49161

[0379] (Table IX) TIFF2025113404000047.tif24169

[0380] Sequence information SEQUENCE LISTING <110> AGENSYS, INC. SEAGEN INC. <120> ANTIBODY DRUG CONJUGATES (ADC) THAT BIND TO 191P4D12 PROTEINS <150> US 61 / 387,933 <151> 2010 - 09 - 29 <160> 13 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 3464 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (264)...(1796) <220> <221> misc_feature <222> (1)...(3464) <223> 191P4D12 <400> 1 ggccgtcgtt gttggccaca gcgtgggaag cagctctggg ggagctcgga gctcccgatc 60 acggcttctt gggggtagct acggctgggt gtgtagaacg gggccggggc tggggctggg 120 tcccctagtg gagacccaag tgcgagaggc aagaactctg cagcttcctg ccttctgggt 180 cagttcctta ttcaagtctg cagccggctc ccagggagat ctcggtggaa cttcagaaac 240 gctgggcagt ctgcctttca acc atg ccc ctg tcc ctg gga gcc gag atg tgg 293 Met Pro Leu Ser Leu Gly Ala Glu Met Trp 1 5 10 ggg cct gag gcc tgg ctg ctg ctg ctg cta ctg ctg gca tca ttt aca 341 Gly Pro Glu Ala Trp Leu Leu Leu Leu Leu Leu Leu Ala Ser Phe Thr 15 20 25 ggc cgg tgc ccc gcg ggt gag ctg gag acc tca gac gtg gta act gtg 389 Gly Arg Cys Pro Ala Gly Glu Leu Glu Thr Ser Asp Val Val Thr Val 30 35 40 gtg ctg ggc cag gac gca aaa ctg ccc tgc ttc tac cga ggg gac tcc 437 Val Leu Gly Gln Asp Ala Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser 45 50 55 ggc gag caa gtg ggg caa gtg gca tgg gct cgg gtg gac gcg ggc gaa 485 Gly Glu Gln Val Gly Gln Val Ala Trp Ala Arg Val Asp Ala Gly Glu 60 65 70 ggc gcc cag gaa cta gcg cta ctg cac tcc aaa tac ggg ctt cat gtg 533 Gly Ala Gln Glu Leu Ala Leu Leu His Ser Lys Tyr Gly Leu His Val 75 80 85 90 agc ccg gct tac gag ggc cgc gtg gag cag ccg ccg ccc cca cgc aac 581 Ser Pro Ala Tyr Glu Gly Arg Val Glu Gln Pro Pro Pro Pro Arg Asn 95 100 105 ccc ctg gac ggc tca gtg ctc ctg cgc aac gca gtg cag gcg gat gag 629 Pro Leu Asp Gly Ser Val Leu Leu Arg Asn Ala Val Gln Ala Asp Glu 110 115 120 ggc gag tac gag tgc cgg gtc agc acc ttc ccc gcc ggc agc ttc cag 677 Gly Glu Tyr Glu Cys Arg Val Ser Thr Phe Pro Ala Gly Ser Phe Gln 125 130 135 gcg cgg ctg cgg ctc cga gtg ctg gtg cct ccc ctg ccc tca ctg aat 725 Ala Arg Leu Arg Leu Arg Val Leu Val Pro Pro Leu Pro Ser Leu Asn 140 145 150 cct ggt cca gca cta gaa gag ggc cag ggc ctg acc ctg gca gcc tcc 773 Pro Gly Pro Ala Leu Glu Glu Gly Gln Gly Leu Thr Leu Ala Ala Ser 155 160 165 170 tgc aca gct gag ggc agc cca gcc ccc agc gtg acc tgg gac acg gag 821 Cys Thr Ala Glu Gly Ser Pro Ala Pro Ser Val Thr Trp Asp Thr Glu 175 180 185 gtc aaa ggc aca acg tcc agc cgt tcc ttc aag cac tcc cgc tct gct 869 Val Lys Gly Thr Thr Ser Ser Arg Ser Phe Lys His Ser Arg Ser Ala 190 195 200 gcc gtc acc tca gag ttc cac ttg gtg cct agc cgc agc atg aat ggg 917 Ala Val Thr Ser Glu Phe His Leu Val Pro Ser Arg Ser Met Asn Gly 205 210 215 cag cca ctg act tgt gtg gtg tcc cat cct ggc ctg ctc cag gac caa 965 Gln Pro Leu Thr Cys Val Val Ser His Pro Gly Leu Leu Gln Asp Gln 220 225 230 agg atc acc cac atc ctc cac gtg tcc ttc ctt gct gag gcc tct gtg 1013 Arg Ile Thr His Ile Leu His Val Ser Phe Leu Ala Glu Ala Ser Val 235 240 245 250 agg ggc ctt gaa gac caa aat ctg tgg cac att ggc aga gaa gga gct 1061 Arg Gly Leu Glu Asp Gln Asn Leu Trp His Ile Gly Arg Glu Gly Ala 255 260 265 atg ctc aag tgc ctg agt gaa ggg cag ccc cct ccc tca tac aac tgg 1109 Met Leu Lys Cys Leu Ser Glu Gly Gln Pro Pro Pro Ser Tyr Asn Trp 270 275 280 aca cgg ctg gat ggg cct ctg ccc agt ggg gta cga gtg gat ggg gac 1157 Thr Arg Leu Asp Gly Pro Leu Pro Ser Gly Val Arg Val Asp Gly Asp 285 290 295 act ttg ggc ttt ccc cca ctg acc act gag cac agc ggc atc tac gtc 1205 Thr Leu Gly Phe Pro Pro Leu Thr Thr Glu His Ser Gly Ile Tyr Val 300 305 310 tgc cat gtc agc aat gag ttc tcc tca agg gat tct cag gtc act gtg 1253 Cys His Val Ser Asn Glu Phe Ser Ser Arg Asp Ser Gln Val Thr Val 315 320 325 330 gat gtt ctt gac ccc cag gaa gac tct ggg aag cag gtg gac cta gtg 1301 Asp Val Leu Asp Pro Gln Glu Asp Ser Gly Lys Gln Val Asp Leu Val 335 340 345 tca gcc tcg gtg gtg gtg gtg ggt gtg atc gcc gca ctc ttg ttc tgc 1349 Ser Ala Ser Val Val Val Val Gly Val Ile Ala Ala Leu Leu Phe Cys 350 355 360 ctt ctg gtg gtg gtg gtg gtg ctc atg tcc cga tac cat cgg cgc aag 1397 Leu Leu Val Val Val Val Val Leu Met Ser Arg Tyr His Arg Arg Lys 365 370 375 gcc cag cag atg acc cag aaa tat gag gag gag ctg acc ctg acc agg 1445 Ala Gln Gln Met Thr Gln Lys Tyr Glu Glu Glu Leu Thr Leu Thr Arg 380 385 390 gag aac tcc atc cgg agg ctg cat tcc cat cac acg gac ccc agg agc 1493 Glu Asn Ser Ile Arg Arg Leu His Ser His His Thr Asp Pro Arg Ser 395 400 405 410 cag ccg gag gag agt gta ggg ctg aga gcc gag ggc cac cct gat agt 1541 Gln Pro Glu Glu Ser Val Gly Leu Arg Ala Glu Gly His Pro Asp Ser 415 420 425 ctc aag gac aac agt agc tgc tct gtg atg agt gaa gag ccc gag ggc 1589 Leu Lys Asp Asn Ser Ser Cys Ser Val Met Ser Glu Glu Pro Glu Gly 430 435 440 cgc agt tac tcc acg ctg acc acg gtg agg gag ata gaa aca cag act 1637 Arg Ser Tyr Ser Thr Leu Thr Thr Val Arg Glu Ile Glu Thr Gln Thr 445 450 455 gaa ctg ctg tct cca ggc tct ggg cgg gcc gag gag gag gaa gat cag 1685 Glu Leu Leu Ser Pro Gly Ser Gly Arg Ala Glu Glu Glu Glu Asp Gln 460 465 470 gat gaa ggc atc aaa cag gcc atg aac cat ttt gtt cag gag aat ggg 1733 Asp Glu Gly Ile Lys Gln Ala Met Asn His Phe Val Gln Glu Asn Gly 475 480 485 490 acc cta cgg gcc aag ccc acg ggc aat ggc atc tac atc aat ggg cgg 1781 Thr Leu Arg Ala Lys Pro Thr Gly Asn Gly Ile Tyr Ile Asn Gly Arg 495 500 505 gga cac ctg gtc tga cccaggcctg cctcccttcc ctaggcctgg ctccttctgt 1836 Gly His Leu Val * 510 tgacatggga gattttagct catcttgggg gcctccttaa acacccccat ttcttgcgga 1896 agatgctccc catcccactg actgcttgac ctttacctcc aacccttctg ttcatcggga 1956 gggctccacc aattgagtct ctcccaccat gcatgcaggt cactgtgtgt gtgcatgtgt 2016 gcctgtgtga gtgttgactg actgtgtgtg tgtggagggg tgactgtccg tggaggggtg 2076 actgtgtccg tggtgtgtat tatgctgtca tatcagagtc aagtgaactg tggtgtatgt 2136 gccacgggat ttgagtggtt gcgtgggcaa cactgtcagg gtttggcgtg tgtgtcatgt 2196 ggctgtgtgt gacctctgcc tgaaaaagca ggtattttct cagaccccag agcagtatta 2256 atgatgcaga ggttggagga gagaggtgga gactgtggct cagacccagg tgtgcgggca 2316 tagctggagc tggaatctgc ctccggtgtg agggaacctg tctcctacca cttcggagcc 2376 atgggggcaa gtgtgaagca gccagtccct gggtcagcca gaggcttgaa ctgttacaga 2436 agccctctgc cctctggtgg cctctgggcc tgctgcatgt acatattttc tgtaaatata 2496 catgcgccgg gagcttcttg caggaatact gctccgaatc acttttaatt tttttctttt 2556 ttttttcttg ccctttccat tagttgtatt ttttatttat ttttattttt attttttttt 2616 agagatggag tctcactatg ttgctcaggc tggccttgaa ctcctgggct caagcaatcc 2676 tcctgcctca gcctccctag tagctgggac tttaagtgta caccactgtg cctgctttga 2736 atcctttacg aagagaaaaa aaaaattaaa gaaagccttt agatttatcc aatgtttact 2796 actgggattg cttaaagtga ggcccctcca acaccagggg gttaattcct gtgattgtga 2856 aaggggctac ttccaaggca tcttcatgca ggcagcccct tgggagggca cctgagagct 2916 ggtagagtct gaaattaggg atgtgagcct cgtggttact gagtaaggta aaattgcatc 2976 caccattgtt tgtgatacct tagggaattg cttggacctg gtgacaaggg ctcctgttca 3036 atagtggtgt tggggagaga gagagcagtg attatagacc gagagagtag gagttgaggt 3096 gaggtgaagg aggtgctggg ggtgagaatg tcgcctttcc ccctgggttt tggatcacta 3156 attcaaggct cttctggatg tttctctggg ttggggctgg agttcaatga ggtttatttt 3216 tagctggccc acccagatac actcagccag aatacctaga tttagtaccc aaactcttct 3276 tagtctgaaa tctgctggat ttctggccta agggagaggc tcccatcctt cgttccccag 3336 ccagcctagg acttcgaatg tggagcctga agatctaaga tcctaacatg tacattttat 3396 gtaaatatgt gcatatttgt acataaaatg atattctgtt tttaaataaa cagacaaaac 3456 ttgaaaaa 3464 <210> 2 <211> 510 <212> PRT <213> Homo sapiens <220> <221> misc_feature <222> (1)...(510) <223> 191P4D12 <400> 2 Met Pro Leu Ser Leu Gly Ala Glu Met Trp Gly Pro Glu Ala Trp Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu Ala Ser Phe Thr Gly Arg Cys Pro Ala Gly 20 25 30 Glu Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly Gln Asp Ala 35 40 45 Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser Gly Glu Gln Val Gly Gln 50 55 60 Val Ala Trp Ala Arg Val Asp Ala Gly Glu Gly Ala Gln Glu Leu Ala 65 70 75 80 Leu Leu His Ser Lys Tyr Gly Leu His Val Ser Pro Ala Tyr Glu Gly 85 90 95 Arg Val Glu Gln Pro Pro Pro Pro Arg Asn Pro Leu Asp Gly Ser Val 100 105 110 Leu Leu Arg Asn Ala Val Gln Ala Asp Glu Gly Glu Tyr Glu Cys Arg 115 120 125 Val Ser Thr Phe Pro Ala Gly Ser Phe Gln Ala Arg Leu Arg Leu Arg 130 135 140 Val Leu Val Pro Pro Leu Pro Ser Leu Asn Pro Gly Pro Ala Leu Glu 145 150 155 160 Glu Gly Gln Gly Leu Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly Ser 165 170 175 Pro Ala Pro Ser Val Thr Trp Asp Thr Glu Val Lys Gly Thr Thr Ser 180 185 190 Ser Arg Ser Phe Lys His Ser Arg Ser Ala Ala Val Thr Ser Glu Phe 195 200 205 His Leu Val Pro Ser Arg Ser Met Asn Gly Gln Pro Leu Thr Cys Val 210 215 220 Val Ser His Pro Gly Leu Leu Gln Asp Gln Arg Ile Thr His Ile Leu 225 230 235 240 His Val Ser Phe Leu Ala Glu Ala Ser Val Arg Gly Leu Glu Asp Gln 245 250 255 Asn Leu Trp His Ile Gly Arg Glu Gly Ala Met Leu Lys Cys Leu Ser 260 265 270 Glu Gly Gln Pro Pro Pro Ser Tyr Asn Trp Thr Arg Leu Asp Gly Pro 275 280 285 Leu Pro Ser Gly Val Arg Val Asp Gly Asp Thr Leu Gly Phe Pro Pro 290 295 300 Leu Thr Thr Glu His Ser Gly Ile Tyr Val Cys His Val Ser Asn Glu 305 310 315 320 Phe Ser Ser Arg Asp Ser Gln Val Thr Val Asp Val Leu Asp Pro Gln 325 330 335 Glu Asp Ser Gly Lys Gln Val Asp Leu Val Ser Ala Ser Val Val Val 340 345 350 Val Gly Val Ile Ala Ala Leu Leu Phe Cys Leu Leu Val Val Val Val 355 360 365 Val Leu Met Ser Arg Tyr His Arg Arg Lys Ala Gln Gln Met Thr Gln 370 375 380 Lys Tyr Glu Glu Glu Leu Thr Leu Thr Arg Glu Asn Ser Ile Arg Arg 385 390 395 400 Leu His Ser His His Thr Asp Pro Arg Ser Gln Pro Glu Glu Ser Val 405 410 415 Gly Leu Arg Ala Glu Gly His Pro Asp Ser Leu Lys Asp Asn Ser Ser 420 425 430 Cys Ser Val Met Ser Glu Glu Pro Glu Gly Arg Ser Tyr Ser Thr Leu 435 440 445 Thr Thr Val Arg Glu Ile Glu Thr Gln Thr Glu Leu Leu Ser Pro Gly 450 455 460 Ser Gly Arg Ala Glu Glu Glu Glu Asp Gln Asp Glu Gly Ile Lys Gln 465 470 475 480 Ala Met Asn His Phe Val Gln Glu Asn Gly Thr Leu Arg Ala Lys Pro 485 490 495 Thr Gly Asn Gly Ile Tyr Ile Asn Gly Arg Gly His Leu Val 500 505 510 <210> 3 <211> 1432 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (32)...(1432) <220> <221> misc_feature <222> (1)...(1432) <223> Ha22-2(2,4)6.1 heavy chain <400> 3 ggtgatcagc actgaacaca gaggactcac c atg gag ttg ggg ctg tgc tgg 52 Met Glu Leu Gly Leu Cys Trp 1 5 gtt ttc ctt gtt gct att tta gaa ggt gtc cag tgt gag gtg cag ctg 100 Val Phe Leu Val Ala Ile Leu Glu Gly Val Gln Cys Glu Val Gln Leu 10 15 20 gtg gag tct ggg gga ggc ttg gta cag cct ggg ggg tcc ctg aga ctc 148 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 25 30 35 tcc tgt gca gcc tct gga ttc acc ttc agt agc tat aac atg aac tgg 196 Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Asn Met Asn Trp 40 45 50 55 gtc cgc cag gct cca ggg aag ggg ctg gag tgg gtt tca tac att agt 244 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser<C 60 65 70 agt agt agt agt acc ata tac tac gca gac tct gtg aag ggc cga ttc 292 Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 75 80 85 acc atc tcc aga gac aat gcc aag aac tca ctg tct ctg caa atg aac 340 Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser Leu Gln Met Asn 90 95 100 agc ctg aga gac gag gac acg gct gtg tat tac tgt gcg aga gca tac 388 Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Tyr 105 110 115 tac tac ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tcc 436 Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 120 125 130 135 tca gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc 484 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 140 145 150 aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac 532 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 155 160 165 tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc 580 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 170 175 180 agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac 628 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 185 190 195 tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acc cag 676 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 200 205 210 215 acc tac atc tgc aac gtg aat cac aag ccc agc aac acc aag gtg gac 724 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 220 225 230 aag aga gtt gag ccc aaa tct tgt gac aaa act cac aca tgc cca ccg 772 Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 235 240 245 tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc 820 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 250 255 260 cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca 868 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 265 270 275 tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac 916 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 280 285 290 295 tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg 964 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 300 305 310 gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc 1012 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 315 320 325 ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc 1060 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 330 335 340 aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa 1108 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 345 350 355 ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg gag 1156 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 360 365 370 375 gag atg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc 1204 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 380 385 390 tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag 1252 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 395 400 405 aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc 1300 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 410 415 420 ttc ctc tat agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg 1348 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 425 430 435 aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac 1396 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 440 445 450 455 acg cag aag agc ctc tcc ctg tcc ccg ggt aaa tga 1432 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys * 460 465 <210> 4 <211> 466 <212> PRT <213> Homo sapiens <220> <221> misc_feature <222> (1)...(466) <223> Ha22-2(2,4)6.1 heavy chain <400> 4 Met Glu Leu Gly Leu Cys Trp Val Phe Leu Val Ala Ile Leu Glu Gly 1 5 10 15 Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Ser Leu Ser Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln 115 120 125 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460 Gly Lys 465 <210> 5 <211> 735 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (25)...(735) <220> <221> misc_feature <222> (1)...(735) <223> Ha22-2(2,4)6.1 light chain <400> 5 agtcagaccc agtcaggaca cagc atg gac atg agg gtc ccc gct cag ctc 51 Met Asp Met Arg Val Pro Ala Gln Leu 1 5 ctg ggg ctc ctg ctg ctc tgg ttc cca ggt tcc aga tgc gac atc cag 99 Leu Gly Leu Leu Leu Leu Trp Phe Pro Gly Ser Arg Cys Asp Ile Gln 10 15 20 25 atg acc cag tct cca tct tcc gtg tct gca tct gtt gga gac aga gtc 147 Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val 30 35 40 acc atc act tgt cgg gcg agt cag ggt att agc ggc tgg tta gcc tgg 195 Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Gly Trp Leu Ala Trp 45 50 55 tat cag cag aaa cca ggg aaa gcc cct aag ttc ctg atc tat gct gca 243 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile Tyr Ala Ala 60 65 70 tcc act ttg caa agt ggg gtc cca tca agg ttc agc ggc agt gga tct 291 Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 75 80 85 ggg aca gat ttc act ctc acc atc agc agc ctg cag cct gaa gat ttt 339 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 90 95 100 105 gca act tac tat tgt caa cag gct aac agt ttc cct ccc act ttc ggc 387 Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro Thr Phe Gly 110 115 120 gga ggg acc aag gtg gag atc aaa cga act gtg gct gca cca tct gtc 435 Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val 125 130 135 ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga act gcc tct 483 Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 140 145 150 gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag 531 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 155 160 165 tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc 579 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 170 175 180 185 aca gag cag gac agc aag gac agc acc tac agc ctc agc agc acc ctg 627 Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 190 195 200 acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa 675 Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 205 210 215 gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg 723 Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 220 225 230 gga gag tgt tag 735 Gly Glu Cys * 235 <210> 6 <211> 236 <212> PRT <213> Homo sapiens <220> <221> misc_feature <222> (1)...(236) <223> Ha22-2(2,4)6.1 light chain <400> 6 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Ser Gly Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Phe Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Ala Asn Ser Phe Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 7 <211> 466 <212> PRT <213> Homo sapiens <220> <221> misc_feature <222> (1)...(466) <223> Ha22-2(2,4)6.1 heavy chain <400> 7 Met Glu Leu Gly Leu Cys Trp Val Phe Leu Val Ala Ile Leu Glu Gly 1 5 10 15 Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95 Ser Leu Ser Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln 115 120 125 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 165 170 175 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 180 185 190 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 195 200 205 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 210 215 220 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 225 230 235 240 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 275 280 285 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455 460 Gly Lys 465 <210> 8 <211> 236 <212> PRT <213> Homo sapiens <220> <221> misc_feature <222> (1)...(236) <223> Ha22-2(2,4)6.1 light chain <400> 8 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Ser Gly Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Phe Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Ala Asn Ser Phe Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 9 <211> 98 <212> PRT <213> Homo sapiens <220> <221> misc_feature <222> (1)...(98) <223> Human Ig germline VH3-48 <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val [[ID=3x]]50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr ]65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 10 <211> 96 <212> PRT <213> Homo sapiens <220> <221> misc_feature It seems there is a small error in the original text where "3x" should likely be "50". The above translation is based on the corrected assumption. If this is not the case, please let me know for a more accurate translation.<222> (1)...(96) <223> Human Ig germline L5 <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro 85 90 95 <210> 11 <211> 179 <212> PRT <213> Mus musculus <220> <221> misc_feature <222> (1)...(179) <223> 191P4D12 ortholog containing the V-domain <400> 11 Met Pro Leu Ser Leu Gly Ala Glu Met Trp Gly Pro Glu Ala Trp Leu 1 5 10 15 Arg Leu Leu Phe Leu Ala Ser Phe Thr Gly Gln Tyr Ser Ala Gly Glu 20 25 30 Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly Gln Asp Ala Lys 35 40 45 Leu Pro Cys Phe Tyr Arg Gly Asp Pro Asp Glu Gln Val Gly Gln Val 50 55 60 Ala Trp Ala Arg Val Asp Pro Asn Glu Gly Ile Arg Glu Leu Ala Leu 65 70 75 80 Leu His Ser Lys Tyr Gly Leu His Val Asn Pro Ala Tyr Glu Asp Arg 85 90 95 Val Glu Gln Pro Pro Pro Pro Arg Asp Pro Leu Asp Gly Ser Val Leu 100 105 110 Leu Arg Asn Ala Val Gln Ala Asp Glu Gly Glu Tyr Glu Cys Arg Val 115 120 125 Ser Thr Phe Pro Ala Gly Ser Phe Gln Ala Arg Met Arg Leu Arg Val 130 135 140 Leu Val Pro Pro Leu Pro Ser Leu Asn Pro Gly Pro Pro Leu Glu Glu 145 150 155 160 Gly Gln Gly Leu Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly Ser Pro 165 170 175 Ala Pro Ser <210> 12 <211> 179 <212> PRT <213> Rattus norvegicus <220> <221> misc_feature <222> (1)...(179) <223> 191P4D12 ortholog containing the V-domain <400> 12 Met Pro Leu Ser Leu Gly Ala Glu Met Trp Gly Pro Glu Ala Trp Leu 1 5 10 15 Leu Leu Leu Phe Leu Ala Ser Phe Thr Gly Arg Tyr Ser Ala Gly Glu 20 25 30 Leu Glu Thr Ser Asp Leu Val Thr Val Val Leu Gly Gln Asp Ala Lys 35 40 45 Leu Pro Cys Phe Tyr Arg Gly Asp Pro Asp Glu Gln Val Gly Gln Val 50 55 60 Ala Trp Ala Arg Val Asp Pro Asn Glu Gly Thr Arg Glu Leu Ala Leu 65 70 75 80 Leu His Ser Lys Tyr Gly Leu His Val Ser Pro Ala Tyr Glu Asp Arg 85 90 95 Val Glu Gln Pro Pro Pro Pro Arg Asp Pro Leu Asp Gly Ser Ile Leu 100 105 110 Leu Arg Asn Ala Val Gln Ala Asp Glu Gly Glu Tyr Glu Cys Arg Val 115 120 125 Ser Thr Phe Pro Ala Gly Ser Phe Gln Ala Arg Met Arg Leu Arg Val 130 135 140 Leu Val Pro Pro Leu Pro Ser Leu Asn Pro Gly Pro Pro Leu Glu Glu 145 150 155 160 Gly Gln Gly Leu Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly Ser Pro 165 170 175 Ala Pro Ser <210> 13 <211> 180 <212> PRT <213> Homo sapiens <220> <221> misc_feature <222> (1)...(180) <223> 191P4D12 ortholog containing the V-domain <400> 13 Met Pro Leu Ser Leu Gly Ala Glu Met Trp Gly Pro Glu Ala Trp Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu Ala Ser Phe Thr Gly Arg Cys Pro Ala Gly 20 25 30 Glu Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly Gln Asp Ala 35 40 45 Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser Gly Glu Gln Val Gly Gln 50 55 60 Val Ala Trp Ala Arg Val Asp Ala Gly Glu Gly Ala Gln Glu Leu Ala 65 70 75 80 Leu Leu His Ser Lys Tyr Gly Leu His Val Ser Pro Ala Tyr Glu Gly 85 90 95 Arg Val Glu Gln Pro Pro Pro Pro Arg Asn Pro Leu Asp Gly Ser Val 100 105 110 Leu Leu Arg Asn Ala Val Gln Ala Asp Glu Gly Glu Tyr Glu Cys Arg 115 120 125 Val Ser Thr Phe Pro Ala Gly Ser Phe Gln Ala Arg Leu Arg Leu Arg 130 135 140 Val Leu Val Pro Pro Leu Pro Ser Leu Asn Pro Gly Pro Ala Leu Glu 145 150 155 160 Glu Gly Gln Gly Leu Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly Ser 165 170 175 Pro Ala Pro Ser 180

Claims

1. An antibody-drug conjugate comprising an anti-191P4D12 antibody or its antigen-binding fragment, wherein the anti-191P4D12 antibody or its antigen-binding fragment binds to an epitope within the V domain of 191P4D12, the V domain comprising amino acids 1 to 150 of SEQ ID NO:2, and the antibody or its antigen-binding fragment binds to at least A76 and S91 of SEQ ID NO:

2.

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

8.

3. An antibody-drug conjugate as described in claim 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, or 90% homologous to the heavy chain variable region amino acid sequence set forth in SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, or 90% homologous to the light chain variable region amino acid sequence set forth in SEQ ID NO:

8.

4. The antigen-binding fragments are Fab, F(ab') 2 4. The antibody-drug conjugate of any one of claims 1 to 3, which is an Fv or scFv fragment.

5. 2. The antibody-drug conjugate of claim 1, wherein the antibody is a fully human antibody.

6. The antibody-drug conjugate of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is recombinantly produced.

7. 7. The antibody-drug conjugate of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is conjugated to monomethyl auristatin E (MMAE) via a linker.

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

9. 9. The antibody-drug conjugate of claim 7 or 8, wherein the linker is an enzyme-cleavable linker, and the linker forms a bond with a sulfur atom of the antibody or antigen-binding fragment thereof.

10. The linker unit is of formula -A a -W w -Y y -having; -A- is a stretcher unit, a is 0 or 1; -W- is an amino acid unit, w is an integer ranging from 0 to 12; and -Y- is a spacer unit, y is 0, 1, or 2; The stretcher unit has a structure of the following formula (1): wherein the amino acid unit is valine citrulline; and the spacer unit has the structure of formula (2): is a PAB group having the formula: the Stretcher unit forms a bond with a sulfur atom of an antibody or antigen-binding fragment thereof; and the spacer unit is attached to MMAE via a carbamate group; The antibody-drug conjugate of any one of claims 7 to 9.

11. 11. The antibody drug conjugate of any one of claims 7 to 10, comprising 1 unit to 10 units of MMAE per antibody or antigen-binding fragment.

12. 12. The antibody drug conjugate of any one of claims 7 to 11, comprising 2 to 5 units of MMAE per antibody or antigen-binding fragment.

13. 13. The antibody drug conjugate of any one of claims 7 to 12, comprising 3 to 5 units of MMAE per antibody or antigen-binding fragment.

14. The following structure: and 14. The antibody drug conjugate of any one of claims 1 to 13, wherein L- represents an anti-191P4D12 antibody or antigen-binding fragment, and p ranges from 1 to 10.

15. 15. The antibody-drug conjugate of claim 14, wherein p is 3 to 5.

16. The antibody-drug conjugate of claim 14, wherein p is 3.

8.

17. A composition comprising a plurality of antibody-drug conjugates described in any one of claims 14 to 16, wherein the average p-value of the antibody-drug conjugates in the composition is about 3.

8.

18. A pharmaceutical composition comprising a therapeutically effective amount of an antibody-drug conjugate described in any one of claims 1 to 16 and a pharmaceutically acceptable excipient.

19. A pharmaceutical composition comprising a therapeutically effective amount of an antibody-drug conjugate according to any one of claims 14 to 16 and a pharmaceutically acceptable excipient, wherein the average p-value of the antibody-drug conjugate in the pharmaceutical composition is about 3.

8.

20. An anti-191P4D12 antibody or antigen-binding fragment thereof, wherein the anti-191P4D12 antibody or antigen-binding fragment thereof binds to an epitope within the V domain of 191P4D12, the V domain comprising amino acids 1 to 150 of SEQ ID NO:2, and the antibody or antigen-binding fragment thereof binds to at least A76 and S91 of SEQ ID NO:

2.

21. An antibody or antigen-binding fragment thereof described in claim 20, comprising a heavy chain variable region comprising an amino acid sequence at least 80% homologous to the heavy chain variable region amino acid sequence shown in SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence at least 80% homologous to the light chain variable region amino acid sequence shown in SEQ ID NO:

8.

22. An antibody or antigen-binding fragment thereof described in claim 21, comprising a heavy chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, or 90% homologous to the heavy chain variable region amino acid sequence shown in SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, or 90% homologous to the light chain variable region amino acid sequence shown in SEQ ID NO:

8.

23. The antibody or antigen-binding fragment thereof according to claim 20, wherein the antigen-binding fragment is a Fab, F(ab') 2 , Fv, or scFv fragment.

24. The antibody or antigen-binding fragment thereof described in claim 20, wherein the antibody is a fully human antibody.

25. An antibody or antigen-binding fragment thereof described in any one of claims 20 to 24, which is recombinantly produced.

26. A pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in any one of claims 20 to 25 and a pharmaceutically acceptable excipient, for preventing or treating cancer in a subject.

27. 27. The pharmaceutical composition of claim 26, wherein the subject is a human subject.

28. The pharmaceutical composition of claim 26 or 27, wherein the cancer comprises tumor cells that express 191P4D12.

29. 29. The pharmaceutical composition of any one of claims 26 to 28, wherein the cancer is colon cancer, ovarian cancer, esophageal cancer, head and neck cancer, pancreatic cancer, lung cancer, bladder cancer, or breast cancer.

30. The pharmaceutical composition of claim 29, wherein the cancer is bladder cancer.

31. 30. The pharmaceutical composition of claim 29, wherein the cancer is pancreatic cancer.

32. 30. The pharmaceutical composition of claim 29, wherein the cancer is lung cancer.

33. 30. The pharmaceutical composition of claim 29, wherein the cancer is breast cancer.

34. 30. The pharmaceutical composition of claim 29, wherein the cancer is colon cancer.

35. 30. The pharmaceutical composition of claim 29, wherein the cancer is ovarian cancer.

36. 30. The pharmaceutical composition of claim 29, wherein the cancer is esophageal cancer.

37. 30. The pharmaceutical composition of claim 29, wherein the cancer is head and neck cancer.

38. 31. The pharmaceutical composition of claim 30, wherein the bladder cancer is advanced bladder cancer.

39. 31. The pharmaceutical composition of claim 30, wherein the bladder cancer is metastatic bladder cancer.

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