EA040898B1 - ANTIBODY DRUG CONJUGATES (ADC) THAT BIND TO 158P1D7 PROTEINS - Google Patents

Description

Cross-reference to related applications

This application claims priority under US Provisional Application No. 61/692,448, filed August 23, 2012, the contents of which are incorporated by reference in their entirety.

Representing a sequence listing as an ASCII text file

The following contents are incorporated herein as an ASCII text file by reference in their entirety: Computer Form (CRF) of Sequence Listing (File Name: 511582005088SeqList.txt, Date of Record: August 21, 2013, Size: 40154 bytes).

The field of technology to which the invention belongs

The invention described herein concerns antibodies, their binding fragments, and antibody drug conjugates (ADCs) that bind proteins referred to as 158P1D7. The invention also relates to prognostic, prophylactic and therapeutic methods and compositions useful in the treatment of cancers in which 158P1D7 is expressed.

State of the art

Cancer is the second leading cause of death in humans after coronary heart disease. Worldwide, millions of people die of cancer every year. In the United States alone, according to the American Cancer Society, cancer causes more than half a million deaths a year, with more than 1.2 million new cases diagnosed each year. While deaths from heart disease are falling substantially, deaths from cancer in general are on the rise. Early in the next century, cancer is predicted to become the leading cause of death.

Worldwide, several types of cancer stand out as leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, ovaries, and bladder represent the leading causes of cancer death. These and virtually all other carcinomas have one common lethal feature. With very rare exceptions, metastasis is fatal in carcinoma. Moreover, even for those cancer patients who initially survived the primary cancer, the general experience showed that their life changed dramatically. Many cancer patients experience strong fears due to the possibility of recurrence or treatment failure. Many cancer patients experience physical disability after treatment. In addition, many cancer patients experience relapses.

Prostate cancer is the fourth most common cancer in men worldwide. In North America and Northern Europe, it is the most common form of cancer in men and is the second leading cause of death in men. In the United States alone, more than 30,000 men die every year from this disease - second only to lung cancer. Despite the magnitude of these numbers, there is as yet no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormonal ablation, surgical castration, and chemotherapy remain the main treatments. Unfortunately, for many, these treatments are ineffective and often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker capable of accurately detecting localized tumors at an early stage remains a significant limitation in the diagnosis and treatment of this disease. Although the serum prostate specific antigen (PSA) assay has proven to be a very useful tool, its specificity and overall usefulness is widely regarded as deficient in some important respects.

Progress in identifying additional specific markers of prostate cancer has been made by generating prostate cancer xenografts that can replicate various stages of the disease in mice. LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that survived passage in severe combined immunodeficiency (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). More recently established prostate cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate specific membrane antigen (PSMA) (Pinto et al., Clin. Cancer Res. 1996 Sep 2 (9): 1445-51), STEAP (Hubert et al., Proc. Natl. Acad. Sci. USA 1999, 96(25): 14523-8) and prostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

Although previously established markers such as PSA have contributed to efforts in the diagnosis and treatment of prostate cancer, there is a need to identify additional markers and therapeutic targets for prostate cancer and related cancers in order to further improve diagnosis and therapy.

In 2000, there were 130,200 cases of colorectal cancer in the United States, including 93,800 cases of colon cancer and 36,400 cases of rectal cancer. Colorectal cancer is the third most common in men and women. Its occurrence decreased significantly during 1992-1996 (-2.1% per year). Studies show that this decrease was due to increased screening and removal of polyps, which prevents them from progressing into invasive cancer. In 2000, there were 56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer), which is about 11% of all cancer deaths in the United States.

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Currently, the most common form of treatment for colorectal cancer is surgery, which is often curative for cancers that do not spread. Chemotherapy or chemotherapy plus radiation is given before or after surgery for most patients whose cancer has deeply perforated the intestinal wall or has spread to the lymph nodes. Colon cancer sometimes requires a permanent colostomy (creating an opening in the abdomen to remove body waste), which is rarely needed for rectal cancer. There is still a need for effective methods for the diagnosis and treatment of colorectal cancer.

Of all new cancers in the United States, bladder cancer accounts for approximately 5% in men (fifth most common) and 3% in women (eighth most common). Its occurrence is slowly increasing, along with the aging of the population. In 1998, there were 54,500 cases, including 39,500 in men and 15,000 in women. Adjusted for age, its incidence in the US is 32 per 100,000 males and 8 per 100,000 females. The historical 3:1 ratio between men and women may be declining due to the increase in female smoking. In 1998, there were 11,000 deaths from bladder cancer (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality increase strongly with age and will become an increasing problem as the population ages.

In most cases, bladder cancer recurs in the bladder. Bladder cancer is treated with a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer indicates the limitation of TUR. Most cases of muscle-invasive cancer are not cured by TUR alone. The most effective treatment for cancer is radical cystectomy with urinary diversion, but it has an undeniable effect on urinary and sexual function. There continues to be a significant need for treatments that will be beneficial for bladder cancer patients.

In 2000, there were 164,100 new cases of lung and bronchial cancer, accounting for 14% of all cancer diagnoses in the United States. The incidence of lung and bronchial cancers is declining significantly in men, from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s, the growth rate among women began to slow down. In 1996, its incidence in women was 42.3 per 100,000.

In 2000, lung and bronchial cancer caused approximately 156,900 deaths, accounting for 28% of all cancer deaths. In 1992-1996, lung cancer mortality decreased significantly among men (-1.7% per year), while among women it is still increasing significantly (0.9% per year). Since 1987, more women have died each year from lung cancer than from breast cancer, which has been the leading cause of cancer death in women for over 40 years. The decline in lung cancer incidence and mortality is most likely due to the decline in smoking over the past 30 years; however, the decline in smoking among women lags behind men. It is of concern that, despite the decline in adult tobacco use, tobacco use among young people is on the rise again.

Treatment options for lung and bronchial cancer depend on the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the best option. Since the disease is usually already spreading by the time it is diagnosed, radiotherapy and chemotherapy are often required in combination with surgery. Chemotherapy, alone or in combination with radiation therapy, is the best treatment for small cell lung cancer; with a large percentage of patients experiencing remission, which in some cases is long-term. However, there is still a need for effective methods for the treatment and diagnosis of lung and bronchial cancer.

In 2000, there were 182,800 new cases of invasive breast cancer in women in the United States. In addition, in 2000 there were about 1400 new cases of breast cancer in men. After increasing by about 4% per year in the 1980s, the incidence of breast cancer in women plateaued in the 1990s, at about 110.6 cases per 100,000.

In 2000, there were 41,200 deaths in the US alone (40,800 women, 400 men) due to breast cancer. Breast cancer is the second leading cause of cancer deaths in women. According to the most recent data, the death rate dropped significantly between 1992 and 1996, with the largest declines in young women, both white and black. It is likely that this decrease was the result of early detection and improved treatment.

Based on medical circumstances and patient preferences, treatment for breast cancer may include lumpectomy (local removal of the tumor) and removal of underarm lymph nodes; mastectomy (surgical removal of the breast) and removal of lymph nodes under the armpits; radiation therapy; chemotherapy; or hormone therapy. Often two or more methods are used in combination. Numerous studies have shown that in the early stages of the disease, long-term survival after lumpectomy plus radiation therapy is similar to that after modified radical mastectomy. Significant advances in reconstruction techniques

- 2 040898 provide several options for breast reconstruction after mastectomy. Recently, such a reconstruction is carried out simultaneously with a mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with sufficient surrounding normal breast tissue may prevent local recurrences of DCIS. Breast radiation and/or tamoxifen may reduce the chance of DCIS occurring in the remaining breast tissue. This is important because DCIS, if left untreated, can develop into invasive breast cancer. However, there are serious side effects or complications from these procedures. Therefore, there is a need for effective methods for the treatment of breast cancer.

In 2000, there were 23,100 new cases of ovarian cancer in the United States. It accounted for 4% of all cancers among women and ranked second among gynecological cancers. In 1992-1996, the incidence of ovarian cancer declined significantly. In 2000, there were 14,000 deaths from ovarian cancer. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.

Treatment options for ovarian cancer are surgery, radiation therapy, and chemotherapy. Surgery usually involves the removal of one or both of the ovaries, fallopian tubes (fallopian tubes), and uterus (hysterectomy). In some very early stages of tumors, only the affected ovary is removed, especially in young women who want to have children. In the later stages of the disease, all affected intra-abdominal parts should be removed to enhance the effect of chemotherapy. There continues to be an important need for effective treatments for ovarian cancer.

In 2000, there were 28,300 new cases of pancreatic cancer. Over the past 20 years, the incidence of pancreatic cancer has decreased in men. The incidence among women remains approximately constant, but may begin to decline. In the US, pancreatic cancer caused approximately 28,200 deaths in 2000. Over the past 20 years, there has been a small but significant decrease in mortality among men (about -0.9% per year), while among women it has increased slightly.

Treatment options for pancreatic cancer are surgery, radiation therapy, and chemotherapy. These treatments may improve survival and/or eliminate symptoms in many patients, but are unlikely to provide a cure in most.

There is a significant need for additional methods of treating and diagnosing cancer. These include the use of antibodies, vaccines, and small molecules as treatments. In addition, there is a need to use these tools as research tools for diagnosis, detection, monitoring and development of the state of the art in all areas of cancer treatment and research.

The therapeutic use of monoclonal antibodies (mAbs) is being implemented (G. Kohler and C. Milstein, Nature 256:495-497 (1975)). Monoclonal antibodies have already been approved as therapeutic agents in transplantation, cancer, infectious diseases, cardiovascular disease, and inflammation. Different isotypes have different effector functions. Such differences in function are reflected in different 3-dimensional structures for different immunoglobulin isotypes (P.M. Alzari et al., Annual Rev. Immunol., 6:555-580 (1988)).

Because mice are convenient for immunization and recognize most human antigens as foreign, mAbs against human targets with therapeutic potential have typically been derived from mice. However, mouse monoclonal antibodies have inherent disadvantages as a human therapy. They require more frequent dosing because mAbs have a shorter circulating half-life in humans than human antibodies. More importantly, reintroduction of mouse antibodies into the human immune system induces a human immune system response to recognize the mouse protein as foreign and a human anti-mouse antibody (HAMA) type response. Such a HAMA reaction can lead to an allergic reaction and rapid elimination of mouse antibodies from the system, thereby making the use of mouse antibodies useless. To avoid such effects, attempts were made to create a human immune system in mice.

At first, attempts were made to create transgenic mice capable of responding to antigens with antibodies containing human sequences (see Bruggemann et al., Proc. Natl. Acad. Sci. USA 86:6709-6713 (1989)), but they were limited by the amount of DNA that can be stably contained in available cloning vectors. The use of cloning vectors such as the yeast artificial chromosome (YAC) has paved the way for the introduction of large gamete fragments of the human Ig locus into transgenic mammals. Virtually most of the human V, D and J region genes located at the same intervals as in the human genome and human constant regions were introduced into mice with YACs. One such transgenic mouse line is known as XenoMouse® and is commercially available from Amgen Fremont, Inc. (Fremont, CA).

The essence of the invention

The invention provides antibodies, their binding fragments, and antibody drug conjugates (ADCs) that bind to the 158P1D7 protein and polypeptide fragments of the 158P1D7 protein. In some embodiments, the invention includes fully human antibodies,

- 3 040898 conjugated with therapeutic agents. In some embodiments, there is a condition that not all of the nucleic acid sequence of FIG. 3 and/or not the entire amino acid sequence from FIG. 2. In some embodiments, the entire nucleic acid sequence of FIG. 3 and/or the entire amino acid sequence from FIG. 2, any of which are in their respective human dosage unit forms.

The invention also provides various immunogenic or therapeutic compositions, such as antibody-drug conjugates, and methods for treating cancers in which 158P1D7 is expressed, such as those of the tissues listed in Table 1. I, especially bladder cancer.

Brief description of the figures

Fig. 1. In FIG. 1 shows the cDNA and amino acid sequence of 158P1D7. The parent methionine is underlined. The open reading frame extends from nucleotide 23 to 2548, including the stop codon.

Fig. 2. Nucleotide and amino acid sequence of antibodies to 158P1D7. In FIG. 2A shows the cDNA and amino acid sequence of the Ha15-10ac12 heavy chain. The heavy chain variable region is double underlined and the human IgG2 heavy chain constant region is underlined. In FIG. 2B shows the cDNA and light chain amino acid sequence of Ha15-10ac12. The light chain variable region is double underlined and the human kappa constant region is underlined.

Fig. 3. Amino acid sequence of antibodies to 158P1D7. In FIG. 3A shows the amino acid sequence of the Ha15-10ac12 heavy chain. The heavy chain variable region is double underlined and the human IgG2 heavy chain constant region is underlined. In FIG. 3B shows the amino acid sequence of the Ha15-10ac12 light chain. The light chain variable region is double underlined and the human kappa constant region is underlined.

Fig. 4. Alignment of Ha15-10ac12 antibodies with gametic human Ig. In FIG. 4A shows the heavy chain alignment of Ha15-10ac12 (SEQ ID NO: 3, positions 1-360; SEQ ID NO: 4, positions 1-120) with gametic human Ig. In FIG. 4B shows the light chain alignment of Ha15-10ac12 (SEQ ID NO: 5, positions 1-240; SEQ ID NO: 6, positions 1-80) with human gametic Ig IGKV2D-28*01 (SEQ ID NO: 10).

Fig. 5. Efficacy of Ha15-10ac12vcMMAE in a SCID human bladder cancer xenograft AG-B7 xenograft subcutaneous model in SCID mice.

Fig. 6. Efficacy of Ha15-10ac12vcMMAE in the RT-4-XCL subcutaneously grafted human bladder cancer xenograft model in SCID mice.

Fig. 7. Efficacy of Ha15-10ac12vcMMAE in NCI-H322M-XCL subcutaneously grafted human lung cancer xenograft model in SCID mice.

Fig. 8. Efficacy of Ha15-10ac12vcMMAE in the SCID mouse SCID human bladder cancer xenograft model AG-B7.

Fig. 9. Detection of the 158P1D7 protein in samples of cancer patients by the IHC method. In FIG. 9A and 9B are bladder cancer specimens. In FIG. 9C and 9D are breast cancer samples. In FIG. 9E and 9F are lung cancer samples. In FIG. 9G and 9H show glioblastoma samples.

Fig. 10. Efficacy of Ha15-10ac12vcMMAE in SW780 subcutaneously grafted human bladder cancer xenograft model in SCID mice.

Fig. 11. In vitro performance of Ha15-10ac12vcMMAE in CHP-212 cytotoxicity assay versus mAb M15-68(2)18 (aka 68(18)1.1).

Fig. 12. Efficacy of Ha15-10ac12vcMMAE in a model of grafted subcutaneously derived human bladder cancer AG-B8 xenografts in SCID mice.

Fig. 13. Saturation curve for Ha15-10ac12vcMMAE.

Fig. 14. Histogram showing the mean fluorescence intensity (MFI) of Ha1510ac12vcMMAE.

Fig. 15. Ha15-10ac12vcMMAE cytotoxicity assessment in vitro. Fig. 15(A) - on CHP-212 cells, FIG. 15(B) - on IGROV-1 cells.

Disclosure of the essence of the invention

Title by section.

I. Definitions.

II. Antibodies 158P1D7.

III. Antibody drug conjugates in general.

III(A). Maytansinoids.

III(B). Auristatins and dolastatins.

W(S). Calicheamicin.

III(D). Other cytotoxic substances.

IV. Antibody-drug conjugates that bind 158P1D7.

V. Linker unit.

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VI. Extension link.

VII. Amino acid link.

VIII. Spacer link.

IX. Drug unit.

X. Drug loading.

XI. Methods for determining the cytotoxic effect of ADCs.

XII. Treatment of cancer expressing 158P1D7.

XIII. 158P1D7 as a target for antibody-based therapy.

XIV. Cocktails with ADC to 158P1D7.

XV. Combined therapy.

XVI. Kits/industrial products.

I. Definitions.

Unless otherwise indicated, all professional terms, designations and other scientific terms or terminology used here have the generally accepted meanings understood by specialists in the field to which the present invention relates. In some cases, terms with generally accepted meanings are defined here for clarity and/or reference, and the inclusion here of such definitions should not necessarily be construed as representing a significant departure from what is generally accepted in the art. Many of the methods and procedures described or cited here are well known and widely used in standard methodology by those skilled in the art, such as the widely used molecular cloning techniques described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. When necessary, procedures involving the use of commercially available kits and reagents are generally performed in accordance with the manufacturer's established procedures and/or parameters, unless otherwise specified.

When trademarks are used in this document, reference to a trademark also refers to the formulation, generic drugs, and active pharmaceutical ingredients of medicinal products bearing that brand name, unless the context dictates otherwise.

The terms advanced cancer, locally advanced cancer, advanced disease, and locally advanced disease refer to cancer that has broken through the capsule of the appropriate tissue and covers American Urological Association (AUA) stage C disease, Whitmore-Jewett stages C1-C2, and stages TZ-T4 and N+ of the disease according to the TNM system (tumor, node, metastases). In general, patients with locally advanced disease are not recommended for surgery, and these patients have significantly worse outcomes than patients with clinically localized (within a single organ) cancer.

The abbreviation AFP stands for dimethylvaline-valine-dolaisolein-dolaproin-phenylalanine-pphenylenediamine (see formula XVI below).

The abbreviation MMAE stands for monomethylauristatin E (see formula XI below).

The abbreviation AEB stands for the ester obtained from the reaction of auristatin E with para-acetylbenzoic acid (see Formula XX below).

The abbreviation AEVB stands for the ester obtained from the reaction of auristatin E with benzoylvaleric acid (see formula XXI below).

The abbreviation MMAF stands for dovalin-valine-dolaisolein-dolaproin-phenylalanine (see formula XVIV below).

Unless otherwise indicated, the term alkyl refers to saturated linear or branched hydrocarbons containing from 1 to 20 carbon atoms (and to all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), preferably from 1 to 8 carbon atoms. 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-2pentyl, 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.

Alkyl groups, alone or as part of another group, may optionally be substituted with one or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogens), including, but not limited to, halo, -O-( C1- _C8 alkyl), -O-( _C2 - _C8 alkenyl), -O-(C2- _C8 alkynyl), -aryl, -C(O)R', -OC(O)R' , -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SO3R ', -S(O)2R', -S(O)R', -OH, =O, -N3, -NH2, -NH(R'), -N(R')2 and -CN, where each R' is independently selected from -H, -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl and -aryl, and the groups -O-(C1C ₈ -alkyl), -O-(C ₂ -C ₈ -alkenyl), -O-(C ₂ -C ₈ -alkynyl), -aryl, -C ₁ -C ₈ -alkyl, C ₂ -C ₈ -alkenyl and -C ₂ - C ₈ -alkynyl may optionally be further substituted with one or more groups, including, without limitation, -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl, -halogen, - O-(C1-C ₈ alkyl), -O-(C ₂ -C ₈ -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,

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-NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R ''), -N(R'')2 and -CN, where each R is independently selected from -H, -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ - alkynyl and -aryl.

Unless otherwise indicated, the terms alkenyl and alkynyl refer to straight or branched carbon chains containing from 2 to 20 carbon atoms (and to all combinations and subcombinations of intervals and specific numbers of carbon atoms therein), preferably from 2 to 8 carbon atoms. An alkenyl chain contains at least one double bond per chain, and an alkynyl chain contains at least one triple bond per chain. Examples of alkenyl groups include, without limitation, ethylene or vinyl, allyl, butenyl -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, 2-methyl -2-butenyl and -2,3-dimethyl-2-butenyl. Examples of alkynyl groups include, without limitation, acetylenyl, propargyl, acetylenyl, propynyl, -1-butynyl, -2-butynyl, -1pentynyl, -2-pentynyl, and -3-methyl-1-butynyl.

Alkenyl and alkynyl groups, alone or as part of another group, may optionally be substituted with one or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogens), including, without limitation, -halogen, -O -(C1-C ₈ -alkyl), -O-(C ₂ -C ₈ -alkenyl), -O-(C ₂ -C ₈ -alkynyl), -aryl, -C (O) R ', -OC ( O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C(O)N(R')2, -NHC(O)R', - SR', -SO3R', -S(O)2R', -S(O)R', -OH, =O, -N3, -NH2, -NH(R'), -N(R')2 and -CN, where each R' is independently selected from -H, -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -quinyl and -aryl, and the groups -O-(C1 -C ₈ -alkyl), -O- (C ₂ -C ₈ -alkenyl), -O- (C ₂ -C ₈ -alkynyl), -aryl, -C1-C ₈ -alkyl, -C ₂ -C ₈ -alkenyl and -C ₂ -C ₈ -alkynyl may optionally be further substituted with one or more groups, including, without limitation, -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl, -halogen, -O-(C1-C ₈ -alkyl), -O-(C ₂ -C ₈ -alkenyl), -O-(C ₂ -C ₈ -alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O )N(R'')2, -NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R'', -OH, - N3, -NH2, -NH(R''), -N(R'')2 and -CN, where each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8-alkynyl and -aryl.

Unless otherwise indicated, the term alkylene refers to branched or straight chain saturated hydrocarbon radicals containing from 1 to 20 carbon atoms (and to all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), preferably from 1 to 8 carbon atoms, which contain two univalent radical centers arising from the removal of two hydrogen atoms from the same or two different carbon atoms from the original alkane. Exemplary alkylenes include, without limitation, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decalene, 1,4-cyclohexylene, and the like. Alkylene groups, alone or as part of another group, may optionally be substituted with one or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogens), including, without limitation, -halo, -O-(C1-C ₈ -alkyl), -O-(C ₂ - C ₈ -alkenyl), -O-(C ₂ -C ₈ -alkynyl), -aryl, -C (O) R ', -OC (O) R ', -C (O) OR ', -C (O )NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S( O)R', -OH, =O, -N3, -NH2, -NH(R'), -N(R')2 and -CN, where each R' is independently selected from -H, -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl and -aryl, and the groups -O-(C1-C ₈ -alkyl), -O-(C ₂ -C ₈ alkenyl) , -O-(C ₂ -C ₈ -alkynyl), -aryl, -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl and -C ₂ -C ₈ -alkynyl may optionally be further substituted with one or several groups, including, without o restrictions, -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl, -halogen, -O-(C1-C ₈ -alkyl), -O-(C ₂ -C ₈ -alkenyl), -O-(C2-C8-alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C (O)NH2, -C(O)NHR'', -C(O)N(R'')2, -NHC(O)R'', -SR'', -SO3R'', -S(O )2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2 and -CN, where each R'' is independently selected from -H, -C1-C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl and -aryl.

Unless otherwise indicated, the term alkenylene refers to optionally substituted alkylene groups containing at least one carbon-carbon double bond. Examples of alkenylene groups include, for example, ethenylene (-CH=CH-) and propenylene (-CH=CHCH2-).

Unless otherwise indicated, the term alkynylene refers to optionally substituted alkylene groups containing at least one carbon-carbon triple bond. Examples of alkynylene groups include, for example, acetylene (-C^C-), propargyl (-CH ₂ C=C-) and 4-pentynyl (-CH ₂ CH ₂ CH ₂ C=CH-).

Unless otherwise noted, the term aryl refers to monovalent aromatic hydrocarbon radicals containing from 6 to 20 carbon atoms (and all combinations and subcombinations of intervals and specific numbers of carbon atoms therein) resulting from the removal of one hydrogen atom from one carbon atom in the original aromatic ring system. Some aryl groups in typical structures are represented as Ar. Representative aryl groups include, without limitation, radicals derived from benzene, substituted benzene, phenyl, naphthalene, anthracene, biphenyl, and others.

The aryl group, alone or as part of another group, may optionally be substituted with one or more, preferably 1 to 5 or even 1 to 2 groups, including, but not limited to, halo, -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl, -O- (C1-C ₈ -alkyl), -O- (C ₂ -C ₈ -alkenyl), -O- (C2-C8 -alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O )N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, =O, -N3, - NH2, -NH(R'), -N(R')2 and -CN, where each R' is independently selected from -H, -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl and -aryl, and groups -C ₁ -C ₈ -alkyl,

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-C2-C ₈ -alkenyl, -C2-C ₈ -alkynyl, -O- (C ₁ -C ₈ -alkyl), -O- (C2-C ₈ -alkenyl), -O- (C2-C ₈ - alkynyl) and -aryl may optionally be further substituted with one or more groups, including, without limitation, -C1- _C8 alkyl, -C2- _C8 alkenyl, -C2- _C8 alkynyl, -halogen, -O- (C ₁ -C ₈ -alkyl), -O- (C 2 -C ₈ -alkenyl), -O- (C ₂ -C ₈ -alkynyl), -aryl, -C (O) R '', -OC ( O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)N(R'') ₂ , -NHC(O)R '', -SR'', -SO3R'', -S(O) ₂ R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), - N(R'') ₂ and -CN, where each R is independently selected from -H, -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl and -aryl.

Unless otherwise indicated, the term arylene refers to those optionally substituted aryl groups that are divalent (i.e., formed by the removal of two hydrogen atoms from the same or two different carbon atoms on the parent aromatic ring system) and may be in ortho -, meta or para configuration as shown in the following structures with phenyl as an example of an aryl group:

while the aryl group (eg, phenyl group) may be unsubstituted or substituted. In some embodiments, arylene is a substituted arylene that is substituted with up to four groups, including, but not limited to, -C ₁ -C ₈ -alkyl, -O-(C ₁ -C ₈ -alkyl), -aryl, -C(O) R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O )R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R') ₂ and -CN, where each R' is independently selected from H, -C ₁ -C ₈ -alkyl and -aryl.

Unless otherwise indicated, the term heterocycle refers to monocyclic, bicyclic or polycyclic ring systems containing from 3 to 14 ring atoms (also referred to as ring), wherein at least one ring atom in at least one ring is a heteroatom selected from N, O, P and S (and to all combinations and subcombinations of intervals and specific amounts of carbon atoms and heteroatoms therein). The heterocycle may contain from 1 to 4 ring heteroatoms independently selected from N, O, P and S. One or more N, C or S atoms in the heterocycle may be oxidized. Monocyclic heterocycles preferably contain 3 to 7 ring atoms (e.g., 2 to 6 carbon atoms and 1 to 3 heteroatoms independently selected from N, O, P, and S), and bicyclic heterocycles preferably contain 5 to 10 ring atoms ( eg 4 to 9 carbon atoms and 1 to 3 heteroatoms independently selected from N, O, P and S). The ring that includes the heteroatom may be aromatic or non-aromatic. Unless otherwise indicated, a heterocycle is attached to its pendant group at any heteroatom or carbon atom resulting in the formation of a stable structure.

Heterocycles are described in Paquette, Principles of Modern Heterocyclic Chemistry (W.A. Benjamin, New York, 1968), in particular Chapters 1, 3, 4, 6, 7 and 9; The Chemistry of Heterocyclic Compounds, A Series of Monographs (John Wiley & Sons, New York, 1950 to present), in particular Vol. 13, 14, 16, 19 and 28; and J. Am. Chem. soc. 82:5566 (1960).

Examples of heterocyclic groups include, by way of example, but not limitation, pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 5H-1,2-thiazinyl, 2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxatinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolisinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4H-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, f enanthrolinyl, phenazinyl, phenothiazinyl, furazanil, phenoxazinil, isochromanil, chromanil, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, isatanoxazolyl, and benzoxazolinyl. Preferred heterocyclic groups include, without limitation, benzofuranyl, benzothiophenyl, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, and tetrazolyl.

Heterocyclic groups, alone or as part of another group, may optionally be substituted with one or more groups, preferably 1-2 groups, including, but not limited to, -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl, -halogen, -O- (C ₁ -C ₈ -alkyl), -O- (C ₂ -C ₈ -alkenyl), -O- (C2-C8 -alkynyl), - aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R' ) ₂ , -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, -N3, -NH2, -NH(R') , -N(R') ₂ and -CN, where each R' is independently selected from -H, -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl and - aryl, and groups -O-(C1-C ₈ -alkyl),

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-O-(C ₂ -C ₈ -alkenyl), -O-(C ₂ -C8-alkynyl), -C 1-C 8 -alkyl, -C ₂ -C 8 -alkenyl, -C ₂ -C 8 -alkynyl and -aryl may optionally be further substituted with one or more substituents including, but not limited to, -C1- _C8 alkyl, _-C2 - _C8 alkenyl, _-C2 - _C8 alkynyl, -halogen, -O-( _C1 -C ₈ -alkyl), -O-(C ₂ -C ₈ -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'', -SO ₃ R, -S(O) ₂ R'', -S(O)R'', -OH, -N ₃ , -NH ₂ , -NH(R''), -N(R'') ₂ and -CN, where each R'' is independently selected from -H, -C1-C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl, and -aryl.

By way of example, but not limitation, carbon-linked heterocycles can link at the following positions: the 2, 3, 4, 5, or 6 position of pyridine; position 3, 4, 5 or 6 of pyridazine; position 2, 4, 5 or 6 of the pyrimidine; position 2, 3, 5 or 6 of pyrazine; the 2, 3, 4 or 5 position of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole; position 2, 4 or 5 for oxazole, imidazole or thiazole; position 3, 4 or 5 for isoxazole, pyrazole or isothiazole; position 2 or 3 in aziridine; position 2, 3 or 4 of azetidine; position 2, 3, 4, 5, 6, 7 or 8 of quinoline; or position 1, 3, 4, 5, 6, 7 or 8 of isoquinoline. The most typical carbon-linked heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4 -pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl and 5-thiazolyl.

By way of example, but not limitation, nitrogen-linked heterocycles can link at the following positions: position 1 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; position 2 of isoindole or isoindoline; position 4 in morpholine; and position 9 for carbazole or β-carboline. The most typical nitrogen-linked heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

Unless otherwise indicated, the term carbocycle refers to saturated or unsaturated non-aromatic monocyclic, bicyclic or polycyclic ring systems containing from 3 to 14 ring atoms (and all combinations and subcombinations of intervals and specific numbers of carbon atoms therein), all atoms in rings are represented by carbon atoms. Monocyclic carbocycles preferably contain 3 to 6 ring atoms, even more preferably 5 or 6 ring atoms. Bicyclic carbocycles preferably contain from 7 to 12 ring atoms, e.g. arranged in the form of a bicyclosystem [5,6] or [6,6]. The term carbocycle includes, for example, a monocyclic carbocyclic ring fused to an aryl ring (eg, a monocyclic carbocyclic ring fused to a benzene ring). Carbocycles preferably contain from 3 to 8 carbon atoms in the ring.

Carbocyclic groups, alone or as part of another group, may optionally be substituted, for example, with one or more groups, preferably 1-2 groups (and any additional substituents selected from halogens), including, without limitation, -halogen, - C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl, -O- (C1-C ₈ -alkyl), -O- (C ₂ -C ₈ -alkenyl) , -O-(C ₂ -C ₈ -alkynyl), -aryl, -C (O) R ', -OC (O) R ', -C (O) OR ', -C (O) NH 2, -C (O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, =O, -N ₃ , -NH2, -NH(R'), -N(R') ₂ and -CN, where each R' is independently selected from -H, -C1-C ₈ -alkyl, - C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl and -aryl, and groups -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl, - O-(C1- _C8 alkyl), -O-( _C2 - _C8 alkenyl), -O-( _C2 - _C8 alkynyl), and -aryl may optionally be further substituted with one or more substituents, including , without limitation, -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl, -halogen, -O-(C1-C ₈ -alkyl), -O-(C ₂ -C ₈ -alkenyl), -O-(C2-C8-alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)N(R'')2, -NHC(O)R'', -SR'',-SO3R'',-S(O)2R'',-S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2 and -CN, where each R'' is independently selected from -H, -C1-C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl, and -aryl.

Examples of monocyclic carbocyclic substituents include -cyclopropyl, -cyclobutyl, -cyclopentyl, -1-cyclopent-1-enyl, -1-cyclopent-2-enyl, -1-cyclopent-3-enyl, cyclohexyl, -1-cyclohexene-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.

Carbocyclo, alone or as part of another group, refers to those optionally substituted carbocyclic groups, as defined above, which are divalent (i.e., they are obtained by removing two hydrogen atoms from the same or two different carbon atoms from the original carbocyclic ring system).

Unless otherwise indicated by context, a hyphen (-) indicates the point of attachment to the main molecule. Accordingly, the term -(C 1 -C ₈ -alkylene)aryl or -C ₁ -C ₈ -alkylene (aryl) refers to such C 1 -C ₈ -alkylene radicals, as defined herein, in which the alkylene radical is attached to the main molecule at any of carbon atoms at the alkylene radical, and one of the hydrogen atoms associated with the carbon atom of the alkylene radical is replaced by an aryl radical, as defined here. Typical groups

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-(C1- _C8 alkylene)aryl, -( _C2 - _C8 alkenylene)aryl, and -( _C2 - _C8 alkynylene)aryl include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethane-1-yl, 2-naphthylethen-1-yl, naphtobenzyl, 2-naphthophenylethan-1-yl, etc.

If a particular group is substituted, then that group may have one or more substituents, preferably 1 to 5 substituents, more preferably 1 to 3 substituents, most preferably 1 to 2 substituents, independently selected from a list of substituents. However, this group may generally have any number of substituents selected from halogens. In this case, the groups that are replaced are indicated.

It is assumed that the definition of any substituents or variables at a particular position in the molecule is independent of their definitions at other positions in that molecule. It is contemplated that the substituents and substitution profiles of the compounds of the invention may be chosen by one of ordinary skill in the art such that compounds are obtained that are chemically stable and can be readily synthesized by known methods as well as those set forth herein.

Protecting groups as used herein are those groups which selectively block, whether temporarily or permanently, one reactive site on a multifunctional compound. Suitable hydroxyl protecting groups for the present invention must be pharmaceutically acceptable and need not be cleaved from the parent compound upon administration to a subject in order for the compound to be active. Cleavage occurs through normal metabolic processes in the body. Hydroxyl protecting groups are well known in the art, see Protective Groups in Organic Synthesis by TW Greene and PGM Wuts (John Wiley & Sons, 3rd Ed.), incorporated herein by reference in its entirety to all occurrences, which include, to for example, protecting groups such as ethers (eg, alkyl ethers and silyl ethers, including, for example, dialkylsilyl ethers, trialkylsilyl ethers, dialkylalkoxysilyl ethers), esters, carbonates, carbamates, sulfonates, and phosphates. Examples of protecting groups for hydroxyl include, without limitation, methyl ester; methoxymethyl ether, methylthiomethyl ether, (phenyldimethylsilyl)methoxymethyl ether, benzyloxymethyl ether, p-methoxybenzyloxymethyl ether, p-nitrobenzyloxymethyl ether, o-nitrobenzyloxymethyl ether, (4-methoxyphenoxy)methyl ether, guaiacol methyl ether, tert-butoxymethyl ether, 4-pentenyloxymethyl ether, siloxymethyl ether, 2-methoxyethoxymethyl ether, 2,2,2-trichloroethoxymethyl ether, bis-(2-chloroethoxy)methyl ether, 2-(trimethylsilyl)ethoxymethyl ether, mentoxymethyl 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; substituted ethyl ethers such as 1-ethoxyethyl ether, 1-(2-chloroethoxy)ethyl ether, 1-[2-(trimethylsilyl)ethoxy]ethyl ether, 1-methyl-1-methoxyethyl ether, 1-methyl-1- benzyloxyethyl ether, 1-methyl-1-benzyloxy-2fluoroethyl ether, 1-methyl-1-phenoxyethyl ether, 2-trimethylsilyl ether, tert-butyl ether, allyl ether, propargyl ethers, p-chlorophenyl ether, p-methoxyphenyl ether, benzyl ether, p-methoxybenzyl ether, 3,4-dimethoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, tripropylsilyl ether, dimethylisopropylsilyl ether, diethylisopropylsilyl ether, dimethylhexylsilyl ether, tert-butyldimethylsilyl ether, diphenylmethylsilyl ether, benzoyl formate, acetate, chloroacetate, dichloroacetate esters, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenylacetate, benzoate, alkyl methyl carbonates, alkyl 9-fluorophenyl methyl carbonates, alkyl ethyl carbonates, alkyl 2,2,2 trichloroethyl carbonates, 1,1-dimethyl-2,2,2-trichloroethyl carbonates, alkylsulfonates, methanesulfonates, benzylsulfonates, tosylates, methylene acetal, ethylidene acetal and tert-butylmethylidenketal. Preferred protecting groups are represented by the formulas -Ra, -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) and -P(O)(OH)OR ^a where Ra is C1-C20 alkyl, C2-C20 alkenyl, C2-C2 ₀ -alkynyl, -C ₁ -C2 ₀ -alkylene (carbocycle), -C2-C2 ₀ -alkenylene (carbocycle), -C ₂ -C ₂₀ alkynylene (carbocycle), -C ₆ -C ₁₀ -aryl, -C ₁ -C ₂₀ -alkylene (aryl), -C ₂ -C ₂₀ -alkenylene (aryl), -C ₂ -C ₂₀ alkynylene (aryl), -C ₁ -C ₂₀ -alkylene (heterocycle), -C ₂ -C ₂₀ -alkenylene (heterocycle) or -C ₂ -C ₂₀ alkynylene (heterocycle), wherein the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocyclic and heterocyclic radicals, alone or as part of another group, are optionally substituted .

Altering the native glycosylation pattern in the present invention means removing one or more carbohydrate moieties that are present in the native 158P1D7 sequence (by removing the original glycosylation site or eliminating glycosylation by chemical and/or enzymatic means) and/or adding one or more glycosylation sites that are not present in the native sequence 158P1D7. In addition, this expression includes qualitative changes in the glycosylation of native proteins, including changes in the nature and content of various carbohydrate groups.

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The term analog refers to those molecules that are similar in structure or have similar or matching attributes to another molecule (eg, a 158P1D7 related protein). For example, a 158P1D7 protein analog can specifically bind to antibodies or T cells that specifically bind to 158P1D7.

The term antibody is used in its broadest sense, unless explicitly stated otherwise. In this case, the antibody can be natural or artificial, such as monoclonal antibodies obtained using standard hybridoma technology. Anti-158P1D7 antibodies include monoclonal and polyclonal antibodies, as well as fragments containing an antigen-binding domain and/or one or more complementarity-determining regions of these antibodies. In the present invention, the term antibody refers to any form of antibody or fragment thereof that specifically binds to 158P1D7 and/or exhibits the desired biological activity, and specifically it covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (for example , bispecific antibodies) and antibody fragments, as long as they specifically bind to 158P1D7 and/or exhibit the desired biological activity. Any specific antibody can be used in the methods and compositions provided herein. Thus, in one embodiment, the term antibody encompasses molecules comprising at least one light chain variable region of an immunoglobulin molecule and at least one heavy chain variable region, which in combination form a specific binding site for a target antigen. In one embodiment, the antibody is an IgG type antibody. For example, the antibody is an IgG1, IgG2, IgG3 or IgG4 type antibody. Antibodies useful in the methods and compositions of the present invention may be produced in cell culture, in phages, or in a variety of animals including, but not limited to, bovine, rabbit, goat, mouse, rat, hamster, guinea pig, sheep, dog, cat, apes, chimpanzees and apes. Thus, in one embodiment, the antibodies of the present invention are mammalian antibodies. Phage techniques can be used to isolate the parent antibody or generate variants with altered specificity or avidity. Such techniques are standard and well known in the art. In one embodiment, the antibodies are produced by recombinant methods known in the art. For example, a recombinant antibody can be obtained by transfecting host cells with a vector containing the DNA sequence encoding the antibody. One or more vectors can be used to transfect a DNA sequence expressing at least one V _L region and one V H region in host cells. Exemplary descriptions of recombinant antibody production and production methods include: Delves, Antibody Production: Essential Techniques (Wiley, 1997); Shephard et al., Monoclonal Antibodies (Oxford University Press, 2000); Goding, Monoclonal Antibodies: Principles and Practice (Academic Press, 1993); and Current Protocols in Immunology (John Wiley & Sons, latest editions). The antibodies of the present invention can be modified by recombinant methods to improve the effectiveness of antibodies in performing the desired function. Thus, it is within the scope of the invention that antibodies can be modified in recombinant ways by substitution. As a general rule, substitutions should be conservative. For example, you can replace at least one amino acid in the constant region of the antibody with another residue. For example, see US Patent No. 5,624,821, US Patent No. 6,194,551, Application No. W09958572; and Angal et al., Mol. Immunol. 30:105-08 (1993). Amino acid modifications include deletions, insertions, and amino acid substitutions. In some cases, such changes are made to reduce undesirable activity, such as complement-dependent cytotoxicity. Often, antibodies are labeled by attaching, covalently or non-covalently, a substance that emits a detectable signal. There are a large number of labels and methods of conjugation, which are widely represented in the scientific and patent literature. These antibodies can be screened for binding to normal or defective 158P1D7. For example, see Antibody Engineering: A Practical Approach (Oxford University Press, 1996). Suitable antibodies with the desired biological activity can be identified by the following in vitro assays, including but not limited to: proliferation, migration, adhesion, growth on soft agar, angiogenesis, cell-to-cell communication, apoptosis, transport, signaling, and in vivo assays such as tumor growth inhibition. The antibodies provided herein may also be useful for diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for the ability to bind to an antigen without inhibiting receptor binding or biological activity of the antigen. As neutralizing antibodies, they can be useful in competitive binding assays. They can also be used to quantify 158P1D7 or its receptor.

The term antigen-binding portion or antibody fragment (or simply antibody portion) as used herein refers to one or more anti-158P1D7 antibody fragments retaining the ability to specifically bind to an antigen (e.g., 158P1D7 and variants thereof; FIG. 1). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full length antibody. Examples of binding fragments encompassed by the term antigen-binding portion of an antibody include: (i) Fab fragment - a monovalent fragment consisting of the V _L , VH, _CL and C _H1 domains; (ii) F(ab') ₂ -fragment - a bivalent fragment, including two Fab fragments,

- 10 040898 connected by a disulfide bridge in the hinged area; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the V _L and VH domains of one antibody arm, (v) a dAb fragment (Ward et al. (1989) Nature 341:544-546) which consists of a VH domain; and (vi) complementarity determined regions (CDRs). In addition, although the two domains of the Fv fragment, V _L and VH, are encoded by separate genes, they can be connected recombinantly using a synthetic linker, allowing them to be obtained as a single protein chain, in which the V _L and VH regions are combined to form a monovalent molecule. (known as single chain Fv (scFv); for example, see Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) . Such single chain antibodies are also encompassed by the term antigen-binding antibody fragment. Such antibody fragments are prepared by standard methods known to those skilled in the art and the fragments are screened for utility in the same manner as intact antibodies.

In the present invention, any form of antigen can be used to generate antibodies specific for 158P1D7. Thus, the inducing antigen can be a single epitope, a multiple epitope, or the entire protein by itself or in combination with one or more immunogenicity enhancing agents known in the art. The inducing antigen can be a full-length protein, a cell surface protein (eg, when immunized with cells transfected with at least a portion of the antigen), or a soluble protein (eg, when immunized with only a portion of the protein's extracellular domain). The antigen can be obtained in genetically modified cells. Antigen-encoding DNA may be genomic or non-genomic (eg, cDNA) and encode at least a portion of the extracellular domain. In the present invention, the term moiety refers to the minimum number of amino acids or nucleic acids, as appropriate, constituting the immunogenic epitope of a given antigen. Any suitable genetic vectors can be used to transform the desired cells, including, but not limited to, adenovirus vectors, plasmids, and non-viral vectors, such as cationic lipids. In one embodiment, the antibody in the methods and compositions provided herein specifically binds to at least a portion of the extracellular domain of a given 158P1D7.

The antibodies presented herein or their antigen-binding fragments can be conjugated to a biologically active substance. In the present invention, the term bioactive refers to any synthetic or natural compounds that bind to an antigen and/or enhance or mediate the desired biological effect to enhance cell killing by toxins. In one embodiment, binding fragments useful in the present invention are biologically active fragments. In the present invention, the term biologically active refers to such antibodies or antibody fragments that are able to bind to the desired antigenic epitope and directly or indirectly exert a biological effect. Direct effects include, without limitation, modulating, stimulating and/or inhibiting the growth signal, modulating, stimulating and/or inhibiting the anti-apoptotic signal, modulating, stimulating and/or inhibiting the apoptotic or necrotic signal, modulating, stimulating and/or inhibiting the ADCC cascade, and modulating, stimulating and/or inhibiting the CDC cascade.

Bispecific antibodies can also be used in the methods and compositions of the present invention. As used herein, the term bispecific antibody refers to such antibodies, typically monoclonal antibodies, that have binding specificities for at least two different antigenic epitopes. In one embodiment, the epitopes refer to the same antigen. In another embodiment, the epitopes refer to two different antigens. Methods for producing bispecific antibodies are known in the art. For example, bispecific antibodies can be generated recombinantly by coexpressing two immunoglobulin heavy chain/light chain pairs. For example, see Milstein et al., Nature 305:537-39 (1983). In addition, bispecific antibodies can be obtained by chemical linkage. For example, see Brennan et al., Science 229:81 (1985). Bispecific antibodies also include fragments of bispecific antibodies. For example, see Hollinger et al., Proc. Natl. Acad. sci. USA 90:6444-48 (1993); Gruber et al., J. Immunol. 152:5368 (1994).

The monoclonal antibodies described herein specifically include chimeric antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, as long as they specifically bind to the target antigen and/or exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc Natl Acad Sci USA 81: 6851-6855 (1984)).

The term chemotherapeutic agent refers to all chemical compounds that effectively inhibit tumor growth. Non-limiting examples of chemotherapeutic agents include alkylating agents such as nitrogen mustard derivatives, ethyleneimine compounds, and alkylsulfonates; antimetabolites, for example folic acid, purine or pyrimidine antagonists;

- 11 040898 inhibitors of mitosis, for example antitubulin agents such as vinca alkaloids, auristatins and podophyllotoxin derivatives; cytotoxic antibiotics; compounds that damage or disrupt the expression or replication of DNA, such as those that bind to the minor groove of DNA; and growth factor receptor antagonists. In addition, chemotherapeutic agents include cytotoxic agents (as defined herein), antibodies, biological molecules, and small molecules.

The term compound means and encompasses the chemical compound itself and, whether expressly stated or not and unless it is clear from the context that it is to be excluded, the following: amorphous and crystalline forms of the compound, including polymorphic forms, which forms may be part of mixtures or individually; compounds in the form of a free acid and free base, which are usually presented in the structures shown here; isomers of a compound, which include optical isomers and tautomeric isomers, and optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and optical isomers include individual optical isomers, as well as mixtures of optical isomers, including racemic and non-racemic mixtures, in which the isomer can be isolated or in admixture with one or more other isomers; isotopes of the compound, including compounds containing deuterium and tritium, as well as compounds containing radioisotopes, including therapeutically and diagnostically effective radioisotopes; multimeric forms of the compound, including dimeric, trimeric, and the like; salts of the compound, preferably pharmaceutically acceptable salts, including salts with acids and salts with bases, including salts with organic counterions and inorganic counterions, including zwitterionic forms; however, if the compound is associated with two or more counterions, then these two or more counterions may be the same or different; and compound solvates, including hemisolvates, monosolvates, disolvates, etc., including organic solvates and inorganic solvates, the inorganic solvates including hydrates; moreover, if the compound is associated with molecules of two or more solvents, then these molecules of two or more solvents may be the same or different. In some instances, references herein to compounds of the invention include direct references to one or more of the foregoing forms, such as salts and/or solvates, but such references are only to highlight certain forms and should not be taken as excluding others from the above forms.

The terms complementarity determining region and CDR are known in the art and refer to non-contiguous amino acid sequences in the variable regions of antibodies that confer antigen specificity and binding affinity. In general, there are three (3) CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three (3) CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR -L3).

The exact boundaries of the amino acid sequence of a given CDR can be easily determined by any of a number of well known schemes, including those described in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (Kabat numbering system), Al-Lazikani et al. (1997) JMB 273, 927-948 (Chothia numbering system), MacCallum et al., J. Mol. Biol. 262:732-745 (1996) Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol. 262, 732-745 (Contact numbering system), Lefranc MP et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, Dev. Comp. Immunol., 2003, 27(1):55-77 (IMGT numbering system), and Honegger A. and Pluckthun A. Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool, J. Mol. Biol., 2001, 309(3):657-70 (ANO numbering system).

The boundaries of a given CDR may vary depending on the scheme used for identification. For example, the Kabat schema is based on structural alignments while the Chothia schema is based on structural information. Numbering in both Kabat and Chothia schemes is based on the most common lengths of antibody region sequences, with some antibodies appearing insertions, which are denoted by letters, for example, 30a, and deletions. In these two schemes, certain insertions and deletions (indels) are located in different positions, which leads to different numbering. The Contact scheme is based on the analysis of complex crystal structures and is in many ways similar to the Chothia numbering scheme. Below in table. V lists those provisions in CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3, which are defined by the scheme Kabat, Chothia and Contact, respectively. For CDR-H1, the numbering of residues is given according to the numbering system of both Kabat and Chothia.

Thus, unless otherwise indicated, the terms CDR and complementarity-determining region of a given antibody or variable region type region thereof, as well as individual CDRs (e.g., CDR-H1, CDR-H2) of an antibody or region thereof, shall encompass the complementarity-determining regions defined by any of the known schemes described above. In some cases, a scheme is given to identify certain CDRs, such as CDRs determined by the Kabat, Chothia or Contact method. In other cases, the amino acid sequence of a given CDR is given.

In the present invention, the term conservative substitution refers to those amino acid substitutions known to those skilled in the art that generally occur without altering the biological activity of the resulting molecule. It is known to those skilled in the art that single amino acid substitutions at non-essential regions of a polypeptide usually do not substantially alter biological activity (e.g., see Watson et al., Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Edition 1987) ). Such typical substitutions are preferably made in accordance with those given in table. II and tab. W(a-b). For example, such substitutions include substitutions of any of isoleucine (I), valine (V), and leucine (L) with any other of these hydrophobic amino acids; aspartic acid (D) to glutamic acid (E) and vice versa; glutamine (Q) to asparagine (N) and vice versa; and serine (S) to threonine (T) and vice versa. Other substitutions may also be considered conservative, depending on the environment of the amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) are often used interchangeably, as are alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, is often interchangeable with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are often interchangeable in those places where an important characteristic of an amino acid residue is its charge, and the differences in pK's of these two amino acid residues are insignificant. Also, other changes can be considered conservative in a certain environment (for example, see Table III(a) here; pp. 13-15 in Biochemistry 2nd ed., Lubert Stryer, ed. (Stanford University); Henikoff et al., PNAS 1992, 89: 10915-10919; Lei et al., J Biol Chem 1995, 270(20): 11882-6). Other substitutions are also acceptable and can be determined empirically or according to known conservative substitutions.

The term cytotoxic agent refers to those substances that inhibit or prevent cell expression activity, cell function, and/or cause cell destruction. The term includes radioactive isotopes, chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, without limitation, auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), auromycins, maytansinoids, ricin, ricin A chain, combrestatin, duocarmycins, dolastatins, doxorubicin, daunorubicin, taxols, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthricindione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE), PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, fenomycin , enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitor, glucocorticoids and other chemotherapeutic agents, as well as radioactive isotopes 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 radioactive isotopes including Lu ¹⁷⁷ . Antibodies can also be conjugated to an anticancer prodrug activating enzyme capable of converting the prodrug to its active form.

In the present invention, the term diabody refers to small fragments of antibodies with two antigen-binding sites, these fragments containing a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL) . With a linker too short to pair between two domains on the same strand, the domains will be forced to pair with complementary domains on the other strand and create two antigen-binding sites. Diabodies are described in more detail, for example, in EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. sci. USA 90:6444-48 (1993).

The term "deplete" in the context of the effect of a 158P1D7-binding agent on 158P1D7-expressing cells means to reduce or eliminate 158P1D7-expressing cells.

The term gene product in the present invention means a peptide/protein or mRNA. For example, the gene product of the invention is sometimes referred to as cancer amino acid sequence, cancer protein, cancer protein, shown in table. I, cancer mRNA, mRNA of the cancer shown in table. I etc. In one embodiment, the cancer protein is encoded by the nucleic acid of FIG. 1. The cancer protein may be a fragment or a full-length protein encoded by the nucleic acids of FIG. 1. In one embodiment, the cancer amino acid sequence is used to determine sequence identity or similarity. In another embodiment, these sequences are natural allelic variants of the protein encoded by the nucleic acid of FIG. 1. In another embodiment, these sequences are variants of the sequences described below.

Heteroconjugate antibodies may be used in the methods and compositions of the present invention. In the present invention, the term heteroconjugate antibody refers to two covalently linked antibodies. Such antibodies can be obtained by synthesis methods known in protein chemistry, including the use of cross-linking reagents. For example, see U.S. Patent No. 4,676,980.

The term homologue refers to molecules that show homology to another molecule, for example, by having the same or similar chemical residues in the sequence at the appropriate positions.

In one embodiment, the antibodies provided herein are human antibodies. In the present invention, the term human antibody refers to those antibodies that have almost

All light chain and heavy chain sequences, including complementarity determining regions (CDRs), are derived from human genes. In one embodiment, human monoclonal antibodies are generated by the trioma method, human B cell method (e.g., see Kozbor et al., Immunol. Today 4: 72 (1983), EBV transformation method (e.g., see Cole et al., Monoclonal Antibodies and Cancer Therapy 77-96 (1985)) or by phage display (e.g., see Marks et al., J. Mol. Biol. 222:581 (1991)).In a preferred embodiment, human antibodies are produced in transgenic mice. such partially or fully human antibodies are known in the art, and any such methods can be used.In accordance with one particularly preferred embodiment, fully human antibody sequences are obtained from transgenic mice engineered to express human antibody heavy and light chain genes. transgenic mice that produce human antibodies and their progeny are described in WO 02/43478 and United States Patent 6,657,103 (Abgenix). Then, B cells from transgenic mice producing the desired antibody are fused to obtain hybridoma cell lines for continuous antibody production. For example, see U.S. Patent Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; and Jacobovits, Adv. drugdel. Rev. 31:33-42 (1998); Green et al., J. Exp. Med. 188:483-95 (1998).

As used herein, the term humanized antibody refers to those forms of antibodies that contain sequences from non-human (eg, mouse) antibodies as well as from human antibodies. Such antibodies are chimeric antibodies that contain a minimal sequence derived from a non-human immunoglobulin. In general, a humanized antibody should contain substantially all of at least one, usually two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions (FRs) are derived from a human immunoglobulin sequence. . The humanized antibody optionally also contains at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. For example, see Cabilly, U.S. Patent No. 4,816,567; Queen et al. (1989) Proc. Natl. Acad. sci. USA 86:10029-10033; and Antibody Engineering: A Practical Approach (Oxford University Press, 1996).

The terms "inhibit" or "inhibition" as used herein means a reduction by a measurable amount or complete prevention.

The terms isolated or biologically pure refer to materials that are substantially or substantially free of the components normally associated with that material in its natural state. Thus, the isolated peptides of the invention preferably do not contain materials normally associated with peptides in their in situ environment. For example, a polynucleotide is said to be isolated if it is substantially separated from contaminating polynucleotides corresponding to or complementary to genes other than 158P1D7, or encoding polypeptides other than the products of the 158P1D7 gene or fragments thereof. Those skilled in the art will readily be able to use nucleic acid isolation procedures to obtain an isolated 158P1D7 polynucleotide. A protein is said to be isolated, for example, if physical, mechanical, or chemical methods are used to separate the 158P1D7 protein from the cellular components normally associated with it. Those skilled in the art can easily use standard purification methods to obtain isolated 158P1D7 protein. Alternatively, the isolated protein may be obtained chemically.

Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent molecules, chemiluminescent molecules, magnetic particles, and the like. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Additionally, the antibodies provided herein can be used as an antigen-binding component of a fluorotel. For example, see Zeytun et al., Nat. Biotechnol. 21:1473-79 (2003).

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

The terms metastatic cancer and metastatic disease refer to cancers that have spread to regional lymph nodes or to distant sites and cover AUA stage D disease and TNM stage TxNxM+.

The terms modulator or test compound or drug candidate, or grammatical equivalents used herein, describe any molecules, such as proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and the like, that are to be tested for their ability to directly or indirectly alter the cancer phenotype or expression cancer sequences, such as nucleic acid or protein sequences, or the effects of cancer sequences (eg, signaling, gene expression, protein interactions, etc.). In one aspect, the modulator neutralizes the effect of the cancer protein of the invention. By neutralization is meant the inhibition or blocking of the activity of a protein, along with a subsequent effect on cells. In another aspect, the modulator neutralizes the effect of a gene and its corresponding protein of the invention.

- 14 040898 tination by normalizing the level of this protein. In preferred embodiments, modulators alter the expression profiles of the nucleic acids or proteins provided herein, or the underlying effector pathways. In one embodiment, the modulator suppresses the cancerous phenotype, for example, to a normal tissue imprint. In another embodiment, the modulator induces a cancerous phenotype. As a rule, several mixtures are analyzed in parallel with different concentrations of a substance in order to obtain differential responses at different concentrations. Typically, one of these concentrations serves as a negative control, ie. at zero concentration or below the detection level.

The modulators, drug candidates or test compounds cover various chemical classes, although they are generally organic molecules, preferably small organic compounds with a molecular weight greater than 100 and less than 2500 daltons. Small molecules with a molecular weight less than 2000 or less than 1500 or less than 1000 or less than 500 D are preferred. a hydroxyl or carboxyl group, preferably at least two functional chemical groups. Candidate drugs often contain cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Modulators also include biomolecules such as peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof. Peptides are especially preferred. One class of modulators are peptides, for example, 5 to 35 amino acids in length, preferably 5 to 20 amino acids, and particularly preferably 7 to 15 amino acids. , which promotes solubility. In one embodiment, the moiety has the C-terminus as a free acid and the N-terminus as a free amine to facilitate attachment to, for example, cysteine. In one embodiment, the cancer protein of the invention is conjugated to an immunogenic agent as set forth herein. In one embodiment, the cancer protein is conjugated to BSA. The peptides of the invention, for example of a preferred length, can be combined with each other or with other amino acids to form a longer peptide/protein. Modulatory peptides can be cleaved natural proteins as described above, random peptides, or displaced random peptides. In a preferred embodiment, peptide/protein based modulators are antibodies and fragments thereof as defined herein.

The term monoclonal antibody in the present invention refers to antibodies derived from a population of substantially homogeneous antibodies, ie. the individual antibodies that make up the population are identical except for possible natural mutations, which may be present in small numbers. Monoclonal antibodies are highly specific because they are directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody formulations typically include a plurality of antibodies directed against (or specific to) different epitopes. In one embodiment, the polyclonal antibodies comprise a plurality of monoclonal antibodies with different epitope specificity, affinity, or avidity within a single antigen containing multiple antigenic epitopes. The definition of monoclonal indicates that the antibody is derived from a substantially homogeneous population of antibodies and should not be taken as if any particular method is required to produce the antibody. For example, monoclonal antibodies for use in the present invention may be prepared by the hybridoma method first described by Kohler et al., Nature 256: 495 (1975), or by recombinant DNA methods (eg, see U.S. Pat. No. 4,816,567). Monoclonal antibodies can also be isolated from antibody phage libraries using the methods described, for example, in Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991). Such monoclonal antibodies typically bind to a Kd of at least 1 μM, more often at least 300 nM, typically at least 30 nM, preferably at least 10 nM, more preferably at least 3 nM or better, usually as determined by the method ELISA.

Pharmaceutical excipients include materials such as adjuvants, carriers, pH adjusters and buffers, isotonic agents, wetting agents, preservatives, and others.

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

The term polynucleotide means the polymeric form of nucleotides at least 10 bases or base pairs long, whether ribonucleotides or deoxyribonucleotides or modified forms of any type of nucleotide, and encompasses single and double stranded forms of DNA and/or RNA. In the art, the term is often used interchangeably with oligonucleotide. The polynucleotide may include the nucleotide sequence shown here, which may include thymidine (T), as shown, for example, in FIG. 1, and uracil (U); this definition refers to the differences between

- 15 040898 chemical structures of DNA and RNA, in particular, to the fact that one of the four main bases in

RNA is uracil (U) instead of thymidine (T).

The term polypeptide means a polymer of at least 4, 5, 6, 7 or 8 amino acids. Throughout the description, standard three-letter (see Table III) or single-letter designations for amino acids are used. In the art, the term is often used interchangeably with peptide or protein.

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

In the present invention, the term single chain Fv or scFv or single chain antibody refers to antibody fragments containing the VH and V _L domains of an antibody, these domains being in the same polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the sFv to form the desired structure for antigen binding. See review on sFv: Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds. Springer-Verlag, New York, pp. 269-315 (1994).

As used herein, the terms specific, specific binding, and specifically binds refer to the selective binding of an antibody to an epitope of the antigen of interest. Antibodies can be tested for binding specificity by comparing binding to an appropriate antigen to binding to a foreign antigen or mixture of antigens under a given set of conditions. If an antibody binds to the corresponding antigen at least 2, 5, 7 or preferably 10 times stronger than to a foreign antigen or mixture of antigens, then it is considered specific. In one embodiment, a specific antibody is one that only binds to the 158P1D7 antigen, but does not bind to a foreign antigen. In another embodiment, specific is an antibody that binds to the human 158P1D7 antigen, but does not bind to the non-human 158P1D7 antigen, wherein the amino acid homology to the 158P1D7 antigen is 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more. In another embodiment, an antibody is specific that binds to the human 158P1D7 antigen and to the mouse 158P1D7 antigen, but binds more to the human antigen. In another embodiment, an antibody is specific that binds to the human 158P1D7 antigen and to the primate 158P1D7 antigen, but binds more to the human antigen. In another embodiment, the specific antibody binds to the human 158P1D7 antigen and to any non-human 158P1D7 antigen, but it binds to a greater extent to the human antigen or any combination thereof.

In the present invention, the terms treat or treatment and grammatically related terms refer to any improvement in any of the consequences of a disease, such as increased survival, reduced morbidity, and/or reduced side effects that are by-products of an alternative treatment; as is well known in the art, complete eradication of the disease is a preferred though not a requirement for the treatment itself.

The term variant refers to those molecules that exhibit deviations from the type or norm described, such as proteins containing one or more different amino acid residues at the appropriate positions of a specifically described protein (for example, the 158P1D7 protein shown in Fig. 1). An example of a protein variant is analog. Other examples of variants are spliceisoforms and single nucleotide polymorphisms (SNPs).

The 158P1D7 and/or 158P1D7 related proteins of the invention include the proteins specifically defined herein (see FIG. 1) as well as allelic variants, conservative substitution variants, analogs and homologues that can be isolated/generated and characterized without undue experimentation. , following the methods described here, or readily available in the art. Also included are fusion proteins that combine parts of different 158P1D7 proteins or fragments thereof, as well as fusion proteins from the 158P1D7 protein and heterologous polypeptides. Such 158P1D7 proteins are collectively referred to as 158P1D7 related proteins, proteins of the invention, or 158P1D7. The term 158P1D7 related protein refers to polypeptide fragments or sequences of the 158P1D7 protein 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more than 25 amino acids; or at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 155 160 165 170 175 180 185 190 195 200 225 250 275 300 325 330 335 345 355 365 375 385 395 405 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 830, 835, 840, 841 or more amino acids.

II. Antibodies to 158P1D7.

In another aspect of the invention, antibodies are provided that bind to related 158P1D7 proteins (see FIG. 1). In one embodiment, an antibody that binds to 158P1D7 related proteins is an antibody that specifically binds to the 158P1D7 protein containing the amino acid sequence of SEQ ID NO: 2. Antibodies that specifically bind to the 158P1D7 protein containing the amino acid sequence of SEQ ID NO: 2, include antibodies that can bind to other related 158P1D7 proteins. For example, antibodies that bind to the 158P1D7 protein containing the amino acid sequence SEQ

- 16 040898

ID NO: 2 can bind to related 158P1D7 proteins such as 158P1D7 variants and their homologues or analogs.

Anti-158P1D7 antibodies of the invention are particularly useful in cancer prognosis (eg see Table I), intrascopy, diagnostic and therapeutic. In addition, such antibodies are useful in the treatment and/or prognosis of bladder cancer and other cancers, insofar as 158P1D7 is also expressed or overexpressed in these other cancers. In addition, the anti-158P1D7 antibodies of the invention are therapeutically useful for the treatment of those cancers in which 158P1D7 expression is involved, especially bladder cancer of the advanced or metastatic bladder cancer type, when the antibodies are conjugated to monomethylauristatin E (MMAE) as described herein.

Various methods for producing antibodies, in particular monoclonal antibodies, are well known. For example, antibodies can be prepared by immunizing a suitable mammalian host with a related 158P1D7 protein, peptide or fragment, isolated or immunoconjugated (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies , Cold Spring Harbor Press, NY (1989)). In addition, 158P1D7 fusion proteins such as the 158P1D7 GST fusion protein can also be used. In a preferred embodiment, a GST fusion protein is produced comprising all or most of the amino acid sequence from FIG. 1, and then use it as an immunogen to produce the corresponding antibodies. In another embodiment, a 158P1D7 related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art (with or without purified 158P1D7 related protein or 158P1D7 expressing cells) are used to elicit an immune response to the encoded immunogen (for a review, see Donnelly et al., 1997, Ann. Rev. Immunol 15:617-648).

To select specific regions of the 158P1D7 protein for antibody production, the amino acid sequence of the 158P1D7 protein shown in FIG. 1. For example, hydrophobicity and hydrophilicity analysis of the 158P1D7 amino acid sequence is used to identify hydrophilic regions in the 158P1D7 structure. Regions of the 158P1D7 protein showing an immunogenic structure, as well as other regions and domains, can be easily identified using various other methods known in the art, such as the Chou-Fasman, Garnier-Robson, KyteDoolittle, Eisenberg, Karplus-Schultz, or Jameson assay. -Wolf. Hydrophilicity profiles can be obtained using the Knorr T.P. and Woods K.R., 1981, Proc. Natl. Acad. sci. USA 78: 3824-3828. Hydrophobicity profiles can be obtained by the method of Kyte J. and Doolittle R.F., 1982, J. Mol. Biol. 157:105-132. Residual availability profiles in percent (%) can be obtained by the method of Janin J., 1979, Nature 277:491-492. Average flex profiles can be obtained by the method of Bhaskaran R., Ponnuswamy R.K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta fold profiles can be obtained by the method of Deleage G., Roux B., 1987, Protein Engineering 1:289-294. Moreover, each site identified by any of these programs or methods is included in the scope of the present invention. Preferred methods for producing antibodies to 158P1D7 are further disclosed in the examples provided here. Methods for obtaining a protein or polypeptide for use as an immunogen are well known in the art. Techniques for preparing immunogenic conjugates of a protein with a carrier such as BSA, KLH or another carrier protein are also well known. In some cases, direct conjugation is used, for example, using carbodiimide reagents; in other cases, binding reagents such as those available from Pierce Chemical Co., Rockford, IL are effective. Administration of the 158P1D7 immunogen is often by injection over an appropriate period of time and using a suitable adjuvant, as provided in the art. The adequacy of antibody formation during immunization can be determined by antibody titers.

Monoclonal antibodies to 158P1D7 can be obtained by various methods well known in this field. For example, a line of immortalized cells secreting the desired monoclonal antibodies is prepared according to the standard hybridoma technology of Kohler and Milstein, or modifications that produce immortalized B cells that produce antibodies, as is generally known.

Cells of immortalized lines that secrete the desired antibodies are screened using an enzyme immunoassay in which the 158P1D7 related protein serves as the antigen. Once an appropriate culture of immortalized cells has been identified, the cells can be expanded and antibodies can be obtained from in vitro cultures or from ascitic fluid.

Antibodies or fragments of the invention can also be produced by recombinant methods. Regions that specifically bind to the desired regions of the 158P1D7 protein can also be generated in the context of chimeric or CDR grafted antibodies from other species. It is also possible to obtain humanized or human antibodies to 158P1D7, which are preferred for use in a therapeutic context. Methods are well known for humanizing mouse and other non-human antibodies by inserting one or more CDRs from the non-human antibody in place of the corresponding human antibody sequences.

- 17 040898 (e.g., see 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.

In a preferred embodiment, the antibodies of the present invention comprise fully human anti-158P1D7 antibodies (anti-158P1D7 mAbs). There are various ways to obtain fully human mAbs to 158P1D7. For example, in a preferred embodiment, methods are provided using antibody-inactivated transgenic mice inserted with human heavy and light chain loci, referred to as XenoMouse (Amgen Fremont, Inc.). A typical description of the production of transgenic mice that produce human antibodies is given in U.S. 6,657,103. See also U.S. Patent Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; and Mendez et al., Nature Genetics, 15: 146-156 (1998); Kellerman S.A. & Green L.L., Curr. Opin. Biotechnol. 13, 593-597 (2002).

In addition, human antibodies of the invention can be generated using HuMAb mice (Medarex, Inc.) that contain human immunoglobulin gene miniloci encoding unrearranged human immunoglobulin heavy (mu and gamma) and kappa light chain sequences, together with targeted mutations, inactivating endogenous mu and kappa chain loci (eg, see Lonberg et al. (1994) Nature 368(6474): 856-859).

In another embodiment, fully human antibodies of the invention can be generated using mice carrying human immunoglobulin sequences on transgenes and transchromosomes, such as mice carrying a human heavy chain transgene and a human light chain transchromosome. Such mice are referred to here as KM mice, which are described in Tomizuka et al. (2000) Proc. Natl. Acad. sci. USA 97:722-727 and PCT Publication WO 02/43478 to Tomizuka et al.

The human monoclonal antibodies of the invention can also be generated by phage display techniques for screening human immunoglobulin gene libraries. Such phage display techniques for isolating human antibodies are recognized in the art. For example, see U.S. Pat. nos. 5,223,409; 5,403,484; and 5,571,698 by Ladner et al.; U.S. Pat. nos. 5,427,908 and 5,580,717 on Dower et al.; U.S. Pat. nos. 5,969,108 and 6,172,197 by McCafferty et al.; and U.S. Pat. nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 on Griffiths et al.

The human monoclonal antibodies of the invention can also be generated using SCID mice in which human immune cells have been reconstituted so that human antibodies can be produced after immunization. Such mice are described, for example, in U.S. Pat. nos. 5,476,996 and 5,698,767 on Wilson et al.

Human monoclonal antibodies of the invention can also be generated using mice in which genomic sequences carrying endogenous mouse variable segments at immunoglobulin heavy chain loci (VH, D _H , J _H ) and/or kappa light chain (VK and J _K ) loci have been replaced, in whole or in part, with human genomic sequences carrying unrearranged gamete variable segments of the human immunoglobulin heavy chain (VH, DH and JH) and/or kappa light chain ( _VK and _JK ) loci (Regeneron, Tarrytown, NY) . For example, see US. Patent Nos. 6,586,251, 6,596,541, 7,105,348, 6,528,313, 6,638,768 and 6,528,314.

In one embodiment, the anti-158P1D7 mAbs of the invention comprise the heavy and light chain variable regions of an antibody designated Ha15-10ac12, which is produced in Chinese Hamster Ovary (CHO) cells deposited with the American Type Culture Collection (ATCC) under accession number PTA-13102 (see Fig. 3), or heavy and light chain variable regions, comprising amino acid sequences homologous to the amino acid sequences of the heavy and light chain variable regions Ha15-10ac12, such as functional fragments thereof, and these antibodies retain the required functional properties of mAbs to 158P1D7 according to the invention. The heavy chain variable region Ha15-10ac12 has an amino acid sequence ranging from the 1st residue Q to the 120th residue S in SEQ ID NO: 7, and the light chain variable region Ha15-10ac12 has an amino acid sequence ranging from the 1st residue D up to the 113th R residue in SEQ ID NO: 8.

In one embodiment, the anti-158P1D7 antibody comprises a heavy chain CDR from the HA15-10ac12 heavy chain variable region, such as heavy chain CDRs 1, 2, and/or 3 from the HA15-10ac12 heavy chain variable region, such as CDR1, CDR2, and/or CDR3 from the amino acid sequence shown in SEQ ID NO: 7, when determined by any known numbering scheme for identifying CDRs, such as any of those described here. In one embodiment, the anti-158P1D7 antibody comprises a light chain CDR from the HA15-10ac12 light chain variable region, such as light chain CDRs 1, 2 and/or 3 from the HA15-10ac12 light chain variable region, such as CDR1, CDR2 and/or CDR3 from the amino acid sequence shown in SEQ ID NO: 7, when determined by any known numbering scheme for identifying CDRs, such as any of those described here. In one aspect, the CDRs 1-3 of the heavy chain variable region Ha15-10ac12 comprise amino acid sequences within residues 31-35, residues 50-66, and residues 99-109, respectively, of SEQ ID NO: 7. In another aspect, the CDRs 1-3 from the light chain variable region Ha15-10ac12 contain amino acid sequences within residues 24-39, residues 55-61 and residues 94-102, respectively, from SEQ ID

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NO: 8. Thus, in some aspects, the anti-158P1D7 antibody comprises a heavy chain CDR1 containing residues 31-35 of SEQ ID NO: 7, a heavy chain CDR2 containing residues 50-66 of SEQ ID NO: 7, and/or a CDR3 a heavy chain containing residues 99-109 of SEQ ID NO: 7 and/or a light chain CDR1 containing residues 24-39 of SEQ ID NO: 8, a light chain CDR2 containing residues 55-61 of SEQ ID NO: 8 and /or a light chain CDR3 containing residues 94-102 of SEQ ID NO: 8. Also among the present embodiments are antibodies competing for antigen binding with antibodies having such variable region and/or CDR sequences. In some embodiments, the presented antibody includes a constant region. A constant region can be from any subclass of a constant region. In one embodiment, the heavy chain constant region is a human IgG2 constant region and the light chain constant region is an Ig kappa chain constant region.

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

(a) the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence of the heavy chain variable region shown in FIG. 3; and (b) the light chain variable region comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence of the light chain variable region shown in FIG. 3.

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

In another embodiment, 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 one, two, or three complementarity determining regions (CDRs) having the amino acid sequences of CDR regions 1, 2, and/or 3 from the heavy chain variable region shown in FIG. 3 when defined by any known numbering scheme, such as any of those described herein;

(b) the light chain variable region comprises one, two, or three CDRs having the amino acid sequences of CDRs 1, 2, and/or 3 from the light chain variable region shown in FIG. 3 when defined by any known numbering scheme, such as any of those described here.

In another embodiment, the antibody, or antigen-binding portion thereof, competes for binding with antibodies having such heavy and/or light chain CDRs.

Engineered antibodies of the invention include those in which changes have been made to the V _H and/or V _L framework residues (eg, to improve the properties of the antibodies). Typically, such framework modifications are made to reduce the immunogenicity of antibodies. For example, one approach is to back-mutate one or more backbone residues to the corresponding gamete sequence. In particular, if an antibody has undergone somatic mutations, then it may contain framework residues that differ from the gametic sequence from which it is derived. Such residues can be identified by comparing the framework sequences of the antibody with those gametic sequences from which the antibody is derived. In order to return the framework region sequences to their gametic configuration, somatic mutations can be back-mutated to a gamete sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis (eg, backmutation from leucine to methionine). Such reverse mutated antibodies are also within the scope of the present invention.

Another type of framework modifications include mutations of one or more residues in the framework region or even in one or more regions of the CDR to remove T-cell epitopes and thereby reduce the potential immunogenicity of antibodies. This approach is also referred to as deimmunization and is described in more detail in the U.S. Patent Publication No. 2003/0153043 by Carr et al.

In addition to or alternatively to changes made in the framework or CDR regions, the antibodies of the invention can be engineered to include modifications within the Fc region, typically to change one or more functional properties of the antibody, such as the half-life of serum, complement fixation, Fc receptor binding and/or antigen-dependent cellular cytotoxicity. In addition, the 158P1D7 mAbs of the invention may be chemically modified (eg, one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation, again to alter one or more of the functional properties of the mAb. Each of such embodiments is described in more detail below.

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In one embodiment, the hinge region CH1 is modified in order to change the amount of cysteine residues in the hinge region, for example, increase or decrease. This approach is detailed in the U.S. Pat. no. 5,677,425 by Bodmer et al. The number of cysteine residues in the CH1 hinge is changed, for example, to facilitate the assembly of light and heavy chains, or to increase or decrease the stability of the mAb to 158P1D7.

In another embodiment, the hinge region of the Fc region of the antibody is mutated so as to decrease the biological half-life of the anti-158P1D7 mAb. In particular, one or more amino acid mutations are introduced at the CH2-CH3 domain junction of the hinge region of the Fc fragment in order to disrupt the binding of the antibody to staphylococcal protein A (SPA) compared to binding to SpA of the Fc fragment with a native hinge region. This approach is described in more detail in U.S. Pat. no. 6,165,745 by Ward et al.

In another embodiment, the anti-158P1D7 mAb is modified to increase the biological half-life. Various approaches are possible. For example, mutations can be introduced as described in US Pat. no. 6,277,375 on Ward. On the other hand, to increase the biological half-life, the antibody can be altered within the C _H 1 or C _L region so that it contains a reserve receptor binding epitope taken from two loops of the CH2 domain of the IgG Fc region, as described in US Pat. nos. 5,869,046 and 6,121,022 by Presta et al.

In still other embodiments, the Fc region is altered by replacing at least one amino acid residue with another amino acid residue so as to change the effector function of the mAb to 158P1D7. For example, one or more amino acids from among certain amino acid residues can be replaced with other amino acid residues so that the antibody changes affinity for the effector ligand, but retains the ability to bind the antigen from the original antibody. The effector ligand for which the affinity is being changed may be, for example, the Fc receptor or the C1 complement component. This approach is described in more detail in U.S. Pat. nos. 5,624,821 and 5,648,260, both by Winter et al.

The reactivity of 158P1D7 antibodies to 158P1D7 related proteins can be determined using a number of well known methods, including Western blotting, immunoprecipitation, ELISA and FACS methods using, as appropriate, 158P1D7 related proteins expressing 158P1D7 cells or extracts thereof. Anti-158P1D7 antibodies, or fragments thereof, can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, without limitation, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators, or enzymes. In addition, bispecific antibodies specific to two or more epitopes of 158P1D7 are obtained using well-known methods. It is also possible to obtain homodimeric antibodies by cross-linking methods known in the art (eg, Wolff et al., Cancer Res. 53: 2560-2565).

In a further preferred embodiment, the anti-158P1D7 mAb of the invention is an antibody comprising the heavy and light chain of an antibody designated Ha15-10ac12. The heavy chain of Ha15-10ac12 consists of an amino acid sequence ranging from 1st residue Q to 446th residue K in SEQ ID NO: 7, and the light chain of Ha15-10ac12 consists of an amino acid sequence ranging from 1st residue D to 219 th residue C in SEQ ID NO: 8. The sequence data is shown in FIG. 2 and 3. In a preferred embodiment, Ha15-10ac12 is conjugated to a cytotoxic agent.

Chinese hamster ovary (CHO) cells producing an antibody called Ha1510ac12 were shipped July 25, 2012 (via Federal Express) to the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, and received access number PTA-13102.

III. Antibody drug conjugates in general.

In a further aspect of the invention, antibody-drug conjugates (ADCs) are provided comprising an antibody conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a growth-inhibiting drug, an agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin). or a fragment thereof) or a radioactive isotope (i.e. it is a radioconjugate). In another aspect of the invention, methods for using ADCs are also provided. In one aspect, the ADC comprises any of the above anti-158P1D7 mAbs covalently linked to a cytotoxic agent or detectable substance.

The use of antibody-drug conjugates for local delivery of cytotoxic or cytostatic agents, i.e. drugs for killing or suppressing tumor cells in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; NiculescuDuvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; U.S. Patent 4,975,278), allows targeted delivery of drug molecules to tumors and intracellular accumulation in them, if the systemic administration of such unconjugated drugs can lead to unacceptable levels of toxicity to normal cells along with those tumor cells that need to be eliminated (Baldwin et al. (1986) Lancet pp. (Mar. 15, 1986):603-05 Thorpe (1985) Antibody Carriers of Cyto-20 040898 toxic Agents in Cancer Therapy: A Review, in Monoclonal Antibodies '84: Biological and Clinical Applications, A. Pinchera et al.( eds), pp. 475-506). At the same time, they strive to achieve maximum efficiency with minimal toxicity. Both polyclonal and monoclonal antibodies have proven useful in these strategies (Rowland et al. (1986) Cancer Immunol. Immunother., 21:183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al. (1986), supra). Toxins used in antibody-toxin conjugates include bacterial diphtheria toxin-type toxins, ricin-type plant toxins, geldanamycin-type small molecule toxins (Mandler et al. (2000) J. Nat. Cancer Inst. 92(19): 1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13:786791), 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). Toxins may exert their cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic agents tend to be inactive or less active when conjugated to large antibodies or receptor protein ligands.

Examples of antibody-drug conjugates include Zevalin® (ibritumomab tiuxetan, Biogen/Idec), which is an antibody-radioisotope conjugate consisting of a mouse IgG1 kappa monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes, and a ¹ⁿ In or ⁹⁰ Y radioisotope linked via a thiourea-chelator linker (Wiseman et al (2000) Eur. J. 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) .

In addition, Mylotarg™ (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate consisting of an anti-human CD33 antibody coupled to calicheamicin, was approved in 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future (2000) 25 (7):686; US Patent Nos. 4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001).

In addition, cantuzumab mertansin (Immunogen, Inc.), an antibody-drug conjugate consisting of the huC242 antibody coupled via an SPP disulfide linker to a DM1 maytansinoid drug molecule, is advancing into phase II trials for the treatment of CanAg-expressing cancers, somehow cancer of the colon, pancreas, stomach, etc.

In addition, MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), an antibody drug conjugate consisting of an anti-prostate specific membrane antigen (PSMA) monoclonal antibody coupled to a DM1 maytansinoid drug molecule, is under development for a potential treatment. prostate tumors.

Finally, auristatin peptides of the monomethylauristatin E (MMAE) type, synthetic analogues of dolastatin, were conjugated to the chimeric monoclonal antibodies cBR96 (it is specific for Lewis Y on carcinomas) and cAC10 (it is specific for CD30 on hematological cancers) (Doronina et al. (2003) ) Nature Biotechnology 21(7):778-784). cAC10 is under therapeutic development.

Further, chemotherapeutic agents suitable for the production of ADCs are described herein. Enzymatically active toxins and their fragments that can be used include diphtheria toxin A chain, non-binding diphtheria toxin active fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites proteins fordii, dianthine proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, fenomycin, enomycin and trichothecenes. For example, see WO 93/21232, published Oct. 28, 1993. Many radionuclides are available for making radioconjugated antibodies. Their examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re. Conjugates of antibodies with cytotoxic agents are prepared using a variety of bifunctional protein-crosslinking reagents such as N-succinimidyl-3-(2pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate-HCl), active esters ( disuccinimidyl suberate type), aldehydes (glutaraldehyde type), bis-azido compounds (bis-(para-azidobenzoyl)hexanediamine type), bis-diazonium derivatives (bis-(p-diazoniumbenzoyl)-ethylenediamine type), diisocyanates (toluene2,6- diisocyanate) and active bis-fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. (1987) Science 238:1098. A typical chelator for conjugating radionuclides to antibodies (WO 94/11026) is ^14C labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA).

The present invention also provides conjugates of antibodies with one or more small molecular weight toxins such as calicheamicin, maytansinoids, dolastatins, auristatins, trichothecenes and CC1065, as well as derivatives of these toxins having toxin activity.

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III(A). Maytansinoids.

Maytansin compounds suitable for use as maytansinoid drug molecules are well known in the art and can be isolated from natural sources according to known methods, obtained using genetic engineering methods (see Yu et al. (2002) PNAS 99:7968 -7973), or maytansinol and maytansinol analogs can be prepared synthetically according to known methods.

Examples of maytansinoid drug molecules include those containing a modified aromatic ring such as: C-19-dechlor (US 4,256,746) (prepared by reduction of ansamitocin P2 with lithium aluminum hydride); C-20-hydroxy (or C-20-demethyl) ± C-19-dechlor (US Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation with Streptomyces or Actinomyces or dechlorination with LAH); and C-20-demethoxy, C-20-acyloxy (-OCOR) ± dechlor (U.S. Pat. No. 4,294,757) (prepared by acylation with acyl chlorides); and those with modifications in other positions.

Examples of maytansinoid drug molecules also include molecules containing such modifications as: C-9-SH (US 4,424,219) (obtained by reacting maytansinol with H2S or P ₂ S ₅ ); C-14alkoxymethyl(demethoxy/CH ₂ OR) (US 4331598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or _CH2OAc ) (US 4,450,254) (obtained from Nocardia); C-15-hydroxy/acyloxy (US 4,364,866) (obtained from the conversion of Streptomyces maytansinol); C-15-methoxy (US Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudiflora); C-18-N-demethyl (US Pat. Nos. 4,362,663 and 4,322,348) (prepared by demethylation of Streptomyces maytansinol); and 4,5-deoxy (US 4,371,533) (prepared by reduction of maytansinol with titanium trichloride/LAH).

ADCs containing maytansinoids, methods for their preparation and their therapeutic use are set forth, for example, in US Patent Nos. 5,208,020; 5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, the contents of which are expressly incorporated here by reference. Liu et al., Proc. Natl. Acad. sci. USA 93:8618-8623 (1996) describes ADCs containing a maytansinoid called DM1 linked to the C242 monoclonal antibody against human colorectal cancer. The conjugate was found to be highly cytotoxic to cultured colon cancer cells and exhibited antitumor activity in an in vivo tumor growth assay. Chari et al., Cancer Research 52:127-131 (1992) describe ADCs in which the maytansinoid is conjugated via a disulfide linker to a mouse A7 antibody that binds to an antigen on human colon cancer cells, or to another mouse monoclonal antibody, TA. .1 binding to the HER-2/neu oncogene. Cytotoxicity of the TA.1-maytansinoid conjugate was tested in vitro on human breast cancer cell line SK-BR-3, which express 3×10 ⁵ HER-2 surface antigens per cell. The drug conjugate achieves a degree of cytotoxicity similar to the free maytansinoid drug, which can increase with an increase in the number of maytansinoid molecules per antibody molecule. The A7-maytansinoid conjugate exhibited low systemic cytotoxicity in mice.

W(H). Auristatins and dolastatins.

In some embodiments, the ADC conjugate comprises an antibody of the invention conjugated to dolastatin or a peptide analog or derivative of dolastatin, auristatin (US Patent Nos. 5,635,483; 5,780,588). Dolastatins and auristatins have been shown to disrupt microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents Chemother. 45(12):3580-3584) and have anticancer (US 5,663,149) and antifungal activity. ((Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). Dolastatin or auristatin molecules in the drug can be attached to the antibody at the N-terminus or C-terminus of the peptide drug molecule (WO 02/088172).

Exemplary auristatin embodiments include the N-terminally linked monomethylauristatin drug molecules DE and DF described in Senter et al., Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, presented March 28, 2004 and described in United States Patent Publication No. 2005/0238649, the contents of which are expressly incorporated herein by reference in their entirety.

A typical embodiment of auristatin is MMAE (with the wavy line representing covalent attachment of the antibody-drug conjugate to the linker (L)):

MMAE

Another exemplary embodiment of auristatin is MMAF, with the wavy line representing the covalent attachment of the antibody-drug conjugate to the linker (L) (US 2005/0238649):

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Additional exemplary embodiments involving MMAE or MMAF and various linker components (described in detail below) have the following structures and abbreviations (with Ab being an antibody and p being 1 to 8):

Ab-MC-MMAF

Typically, peptide-based drug molecules can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, by the liquid phase synthesis method (see E. Schroder and K. Lubke, The Peptides, volume 1, pp. 76-136, 1965, Academic Press), which is well known in the field of chemistry. peptides. Auristatin/dolastatin drug molecules can be prepared according to the methods in: US 5635483; US 5780588; Pettit et al. (1989) J. Am. Chem. soc. 111: 5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277; Pettit G.R. et al. (1996) Synthesis, 719-725; Pettit et al. (1996) J. Chem. soc. Perkin Trans. 15:859-863; and Doronina (2003) Nat. Biotechnol. 21(7): 778-784.

III(C). Calicheamicin.

In other embodiments, the ADC comprises an antibody of the invention conjugated to one or more molecules of calicheamicin. The calicheamicin family of antibiotics are capable of causing double-stranded DNA breaks at subpicomolar concentrations. For the preparation of calicheamicin conjugates, see US Patents 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296 (all from the American Cyanamid Company). Structural analogs of calicheamicin that can be used include, without limitation, γ1, a ₂ I, a ₃ ^I , X-acetyl-y/, PSAG, and θ\ (Hinman et al., Cancer Research 53:3336-3342 (1993) ; Lode et al., Cancer Research 58:2925-2928 (1998); and American Cyanamid US Patents cited above). Another anticancer drug with which an antibody can be conjugated is QFA, which is an antifolate. Both calicheamicin and QFA act intracellularly and hardly cross the plasma membrane. Therefore, uptake of these agents by cells through antibody-mediated internalization greatly enhances their cytotoxic effects.

III(D). Other cytotoxic agents.

Other antineoplastic agents that can be conjugated to the antibodies of the invention include BCNU, streptozocin, vincristine, and 5-fluorouracil, a family of substances collectively known as the LL-E33288 complex described in U.S. Patents 5,053,394, 5,770,710 and esperamycins (U.S. Patent 5,877,296).

Enzymatically active toxins and their fragments that can be used include diphtheria toxin A chain, non-binding diphtheria toxin active fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites proteins fordii, dianthine proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, fenomycin, enomycin and trichothecenes. For example, see WO 93/21232 published October 28, 1993.

The present invention also contemplates ADC conjugates formed between an antibody and a compound with nucleolytic activity (eg, a ribonuclease or a deoxyribonuclease-type DNA endonuclease; DNase).

To selectively destroy tumors, the antibody may contain a highly radioactive atom. Many radionuclides are available for the production of radioconjugated antibodies. Their examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu radioactive isotopes. When used for detection, conjugates may contain a radioactive atom for scintigraphy, such as Tc ^99m or I ¹²³ , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), once again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Radioactive or other labels can be included in the conjugate by known methods. For example, a peptide can be biosynthesized or synthesized by chemical synthesis from amino acids using suitable amino acid precursors, for example, containing fluoro-19 instead of hydrogen. Labels such as Tc ^99m or I ¹²³ , Re ¹⁸⁶ , Re ¹⁸⁸ and In ¹¹¹ can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached through a lysine residue. The Iodogen method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123. Other methods are detailed in Monoclonal Antibodies in Immunoscintigraphy (Chatal, CRC Press 1989).

IV. Antibody-drug conjugates that bind 158P1D7.

The present invention provides, inter alia, antibody-drug conjugates for targeted drug delivery. The inventors have discovered that antibody-drug conjugates have potent cytotoxic and/or cytostatic activity against cells expressing 158P1D7. Antibody-drug conjugates include an antibody unit covalently linked to at least one drug unit. The drug unit can be covalently attached directly or via a linker unit (-LU-).

In some embodiments, the antibody-drug conjugate has the following formula

L-(LU-D) _P (I) or a pharmaceutically acceptable salt or solvate thereof;

where L means an antibody link, for example mAb to 158P1D7 of the present invention; a (LU-D) means a linker unit-drug unit grouping, while:

LU- means linker unit,

- D means a drug unit having cytostatic or cytotoxic activity against target cells; and p is an integer from 1 to 20.

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

In some embodiments, the antibody-drug conjugate has the formula L—(Aa-Ww-Y y ^- D) ρ(II) or a pharmaceutically acceptable salt or solvate thereof;

where L means an antibody link, for example mAb to 158P1D7; A

- A _a -W _w -Y _y - means linker unit (LU), while:

- A- means an extension unit, a is 0 or 1, each -W- independently means an amino acid link, w is an integer from 0 to 12,

- Y- means a self-destructing spacer link, y is equal to 0, 1 or 2;

- D means a drug unit having cytostatic or cytotoxic activity against target cells; and p is an integer from 1 to 20.

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 between 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, if w is not 0, then y is 1 or 2. In some embodiments, if w is 1 to 12, then y is 1 or 2. In some embodiments, w is 2 to 12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0.

For formulations containing multiple antibodies, drug loading is represented by p, i. e. the average number of drug molecules per antibody. The drug loading can be from 1 to 20 drug molecules (D) per antibody. The average number of drug molecules per antibody during conjugation reactions can be determined by standard methods such as mass spectroscopy, ELISA and HPLC. It is also possible to quantify the distribution of antibody-drug conjugates by p-values. In some cases, it is possible to separate, purify, and characterize homogeneous antibody drug conjugates with a specific p-value from antibody-drug conjugates with different drug loading by methods such as reverse phase HPLC or electrophoresis. In typical embodiments, p is from 2 to 8.

- 24 040898

The preparation of antibody-drug conjugates may be carried out by any method known to those skilled in the art. Briefly, antibody-drug conjugates include an anti-158P1D7 mAb as the antibody unit, a drug, and optionally a linker that connects the drug and the binding agent. In a preferred embodiment, the antibody is an anti-158P1D7 mAb comprising the heavy and light chain variable regions of an antibody designated Ha15-10ac12 as described above. In a more preferred embodiment, the antibody is an anti-158P1D7 mAb comprising the heavy and light chain of the antibody designated Ha15-10ac12 as described above. There are several different reactions for the covalent attachment of drugs and/or linkers to binding agents. This is often done by reacting amino acid residues of a binding agent, for example an antibody molecule, including lysine amino groups, free carboxyl groups of glutamic and aspartic acid, cysteine sulfhydryl groups, and various aromatic amino acid moieties.

One of the most widely used non-specific methods of covalent attachment is the carbodiimide reaction between the carboxy group (or amino group) of a compound and the amino group (or carboxy group) of an antibody. In addition, bifunctional reagents such as dialdehydes or imidoesters were also used to link the amino groups of the compound with the amino groups of the antibody molecule. Reactions with Schiff bases are also available for the attachment of drugs to binders. This method involves periodate oxidation of a drug containing glycol or hydroxyl groups to form an aldehyde, which then reacts with a binder. The attachment proceeds via the formation of a Schiff base with the amino groups of the binder. Also, isothiocyanates can be used as condensing reagents for the covalent attachment of drugs to binding agents. Specialists are aware of other methods within the scope of the present invention.

In some embodiments, the linker precursor intermediate is reacted with the drug under appropriate conditions. In some embodiments, reactive groups on the drug itself and/or on an intermediate are used. Thereafter, the reaction product between the drug and the intermediate or the derivatized drug is reacted with the anti-158P1D7 mAb under appropriate conditions.

Further, each of the individual units of the antibody-drug conjugates is described in more detail below. The synthesis and structure of exemplary linker units, extender units, amino acid units, self-destructing spacer units, and drug units are also described in U.S. Patent Application Publication Nos. 2003-0083263, 2005-0238649 and 2005-0009751, each of which is incorporated here by reference in its entirety and in all cases.

V. Linker unit.

Typically, antibody-drug conjugates include a linker unit between the drug unit and the antibody unit. In some embodiments, the linker may be cleaved under intracellular conditions such that upon cleavage of the linker in the intracellular environment, the drug moiety is separated from the antibody. In other embodiments, the linker unit cannot be cleaved off and the drug is released, for example, upon degradation of the antibody.

In some embodiments, the linker is cleaved off by a cleaving agent present in the intracellular environment (eg, in lysosomes or endosomes or caveolae). The linker can be, for example, a peptidyl linker that is cleaved off by an intracellular enzyme, a peptidase or a protease, including, but not limited to, lysosomal or endosomal proteases. In some embodiments, the peptidyl linker is at least 2 amino acids long or 3 amino acids long. Degrading agents may include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in release of the active drug within the target cells (e.g., see Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67 -123). Most typical are peptidyl linkers, which are cleaved by enzymes found in 158P1D7 expressing cells. For example, a peptidyl linker that is cleaved by the thiol-dependent protease cathepsin B, which is highly expressed in cancer tissues, can be used (eg Phe-Leu or Gly-Phe-Leu-Gly linker (SEQ ID NO: 9)). Other examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, incorporated herein by reference in its entirety and in all instances. In a specific embodiment, the peptidyl linker cleavable by intracellular proteases is a Val-Cit linker or a Phe-Lys linker (for example, see U.S. Patent 6,214,345 which describes the synthesis of doxorubicin with a Val-Cit linker). One of the advantages of using intracellular proteolytic release of therapeutic agents is that the agents are generally attenuated upon conjugation and the serum stability of the conjugates is generally high.

In other embodiments, the cleavable linker is pH sensitive, ie. sensitive to hydrolysis at certain pH values. As a rule, pH-sensitive linkers are hydrolyzed under acidic conditions. For example, an acid labile linker can be used that will expose

- 25 040898 hydrolysis in lysosomes (e.g. hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, etc.) (e.g. see 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 at neutral pH, as in blood, but unstable below pH 5, 5 or 5.0, i.e. about the same as the pH in lysosomes. In some embodiments, the hydrolysable linker is a thioether linker (such as, for example, a thioether attached to a therapeutic agent through an acetylhydrazone bond (eg, see U.S. Patent No. 5,622,929).

In still other embodiments, the linker is cleaved under reducing conditions (eg, a disulfide linker). Several disulfide linkers are known, including, for example, linkers that are prepared using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3 -(2-pyridyldithio)butyrate) and SMPT (N-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene), SPDB and SMPT (e.g. see 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.

And in 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).

In other embodiments, the linker unit is not cleaved off and the drug is released upon degradation of the antibody (eg, see U.S. Publication No. 2005/0238649, incorporated herein by reference in its entirety and in all cases).

As a rule, the linker is practically insensitive to the extracellular environment. In the present description, practically insensitive to the extracellular environment in the context of a linker means that when the antibody-drug conjugate is found in the extracellular environment (for example, in plasma), no more than 20% is cleaved, usually no more than 15%, more often no more than 10% and more often, not more than 5%, not more than 3%, or not more than 1% of the linker in the sample of the antibody-drug conjugate. Whether the linker will be substantially insensitive to the extracellular environment can be determined, for example, by incubating the antibody-drug conjugate with plasma for a predetermined time (eg, 2, 4, 8, 16, or 24 hours) and then determining the amount of free drug. agent present in plasma.

In other, non-exclusive embodiments, the linker promotes cellular internalization. In some embodiments, the linker promotes cellular internalization when conjugated to a therapeutic agent (ie, as part of a linker-therapeutic agent moiety in an antibody-drug conjugate, as described herein). In other embodiments, the linker promotes cellular internalization when conjugated simultaneously with the connection of auristatin and mAb to 158P1D7.

Various exemplary linkers that can be used with the compositions and methods of the present invention are described in WO 2004/010957, U.S. Publication no. 2006/0074008, U.S. Publication no. 2005/0238649 and U.S. Publication no. 2006/0024317 (each of which is incorporated herein by reference in its entirety and in all cases).

A linker unit (LU) is a bifunctional compound that can be used to join a drug unit and an antibody unit to form an antibody drug conjugate. In some embodiments, the linker unit corresponds to the formula -Aa-Ww-Yy- where -A- means the extension unit;

a is 0 or 1;

each W is independently an amino acid unit;

w is an integer from 0 to 12;

-Y - means a link of a self-liquidating spacer; and y is 0, 1, or 2.

In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1, or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, if w is 2 to 12, then y is 1 or 2. In some embodiments, w is 2 to 12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0.

VI. Extension link.

The extension unit (A), when present, can link the antibody unit to the amino acid unit (-W-), if present, to the spacer unit (-Y-), if present, or to the drug unit (-D). Useful functional groups that may be present on an anti-158P1D7 mAb (eg, Ha1510ac12), naturally or through chemical manipulation, include, without limitation, sulfhydryl, amino, hydroxyl, anomeric carbohydrate hydroxyl groups, and carboxy groups. Suitable functional groups are sulfhydryl and amino groups. In one embodiment, sulfhydryl groups can be obtained by reducing intramolecular

- 26 040898 disulfide bonds in mAb to 158P1D7. In another embodiment, sulfhydryl groups can be generated by reacting the mAb's lysine amino group to 158P1D7 with 2-iminothiolane (Traut's reagent) or other sulfhydryl-generating reagents. In some embodiments, the anti-158P1D7 mAb is a recombinant antibody and is engineered to carry one or more lysine residues. In some other embodiments, the recombinant anti-158P1D7 mAb is engineered to carry additional sulfhydryl groups, such as additional cysteine residues.

In one embodiment, the extension unit forms a bond with the sulfur atom of the antibody unit. The sulfur atom may be derived from the sulfhydryl group of the antibody. Representative extension units of this embodiment are shown in square brackets of formula IIIa and IIIb, where L-, -W-, -Y-, -D, w and y are defined above and R ¹⁷ is selected from -C ₁ -C ₁₀ -alkylene- , -C ₂ -C ₁₀ -alkenylene-, -C ₂ -C ₁₀ -alkynylene-, -carbocyclo-, -O- (C ₁ -C ₈ -alkylene) -, O- (C ₂ -C ₈ -alkenylene) -, -O- (C ₂ -C ₈ -alkynylene) -, -arylene-, -C ₁ -C ₁₀ -alkylenarilene-, -C ₂ -C ₁₀ -alkenylene-arylene, -C ₂ -C ₁₀ -alkynylene- arylene, -arylene-C ₁ -C ₁₀ -alkylene-,

-arylene-C ₂ -C ₁₀ -alkenylene-, -arylene-C ₂ -C ₁₀ -alkynylene-, -C ₁ -C ₁₀ -alkylene-(carbocyclo)-,

-C ₂ -C ₁₀ -alkenylene-(carbocyclo)-, -C ₂ -C ₁₀ -alkynylene-(carbocyclo)-, (carbocyclo)-C ₁ -C ₁₀ -alkylene-, -(carbocyclo)-C ₂ -C ₁ o-alkenylene-, -(carbocyclo)-C ₂ -C ₁ o-alkynylene, -heterocyclo-, -C ₁ -C ₁₀ -alkylene (heterocyclo) -, -C ₂ -C ₁₀ -alkenylene-(heterocyclo)- , -C ₂ -C ₁₀ -alkynylene-(heterocyclo)-, -(heterocyclo)-C ₁ C ₁₀ -alkylene-, (heterocyclo)-C ₂ -C ₁₀ -alkenylene-, -(heterocyclo)-C ₂ -C ₁₀ -alkynylene-, -(CH ₂ CH ₂ O) _r - and -(CH ₂ CH ₂ O) _g -CH ₂ -, where r is an integer from 1 to 10, and the radicals are alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, carbocyclo, heterocyclo and arylene, alone or as part of another group, are optionally substituted. In some embodiments, the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, carbocyclo, heterocyclo, and arylene radicals, alone or as part of another group, are unsubstituted. In some embodiments, R ¹⁷ is selected from -C ₁ -C ₁₀ -alkylene-, -carbocyclo-, -O-(C ₁ -C ₈ -alkylene)-, -arylene-, -C ₁ -C ₁₀ -alkylene-arylene- , -arylene-C ₁ -C ₁₀ -alkylene-, -C ₁ -C ₁₀ -alkylene-(carbocyclo)-, -(carbocyclo)-C ₁ -C ₁₀ -alkylene-,

-C ₃ -C ₈ -heterocyclo-, -C ₁ -C ₁₀ -alkylene-(heterocyclo)-, -(heterocyclo)-C ₁ -C ₁₀ -alkylene-, -(CH ₂ CH ₂ O) _r - and - (CH ₂ CH ₂ O) _r -CH ₂ -, where r is an integer from 1 to 10, and the alkylene radicals are not substituted, and the remaining groups are optionally substituted.

From all exemplary embodiments, it should be understood that, even where not expressly indicated, between 1 and 20 drug molecules (p=1-20) can bind to an antibody.

[^NR ¹⁷ -C(O)--W _w -Y _y -D

- ° 1 Sha b-CH ₂ -CONH-R ¹⁷ -C (O) - W _w -Y _y -D ^J IIIb

A typical extension unit is represented by formula IIIa, in which R ¹⁷ means -(CH ₂ ) ₅ -

Another exemplary extension unit is represented by formula IIIa in which R ¹⁷ is -(CH2CH2OX-CH2 and r is 2

Another exemplary extension unit is represented by formula IIIa, in which R ¹⁷ is arylene- or arylene-C ₁ -C ₁₀ -alkylene-. In some embodiments, the aryl group is an unsubstituted phenyl group.

Another typical extension unit is represented by formula IIIb, in which R ¹⁷ means -(CH ₂ ) ₅ -

In some embodiments, the extension unit is connected to the antibody unit through a disulfide bond between the sulfur atom of the antibody unit and the sulfur atom of the extension unit. A representative extension unit of this embodiment is shown in square brackets of formula IV, where R ¹⁷ , L, -W-, -Y-, -D, w and y are already defined above.

- 27 040898

LSS-R ¹⁷ -C(O)--W _w -Y _y -D

IV

It should be noted that throughout the application in the formula below, S denotes a sulfur atom at the link of the antibody, unless the context requires otherwise.

L-S-<

And in other embodiments, the extension contains a reactive site that can form a bond with the primary or secondary amino group of the antibody.

Examples of such reactive sites include, without limitation, activated esters such as succinimide esters, 4-nitrophenyl ethers, pentafluorophenyl ethers, tetrafluorophenyl ethers, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. Representative extension units of this embodiment are shown in square brackets of the formulas Va and Vb, where -R ¹⁷ -, L-, -W-, -Y-, -D, w and y are already defined above.

L--C(O)NH-R ¹⁷ -C(O)--W _w -Y _y -D

And 17 b - -CN HR ¹⁷ -C (O) - W _w - Yy-D

In some embodiments, the extender contains a reactive site that reacts with a modified carbohydrate (-CHO) group, which may be on the antibody. For example, a carbohydrate can be subjected to mild oxidation using a reagent such as sodium periodate, and the resulting unit (-CHO) of the oxidized carbohydrate can be condensed with an extension containing a functional group such as hydrazide, oxime, primary or secondary amine, hydrazine, thiosemicarbazone, hydrazine carboxylate or an aryl hydrazide such as those described in Kaneko et al., 1991, Bioconjugate Chem. 2:133-41. Representative extension units of this embodiment are shown in square brackets of formulas VIa, VIb and VIc, where -R ¹⁷ -, L-, -W-, -Y-, -D, w and y are already defined above:

\u003d \u003d Ν-Ν H - R ¹⁷ -C (O) - -W _w -Y _y -D

Via \u003d \u003d NO-R ¹⁷ -C (O) W _w -Yy-D

VIb :N-NH-I ΰ-r ¹⁷ -c(O)-w _v

-Yy-D

VII. Amino acid link.

The amino acid unit (-W-), when present, connects the extension unit to the spacer unit if there is a spacer unit, connects the extender unit to the drug unit if there is no spacer unit, and connects the antibody unit to the drug unit if there is no extension unit and spacer link.

The W _w - unit can be, for example, a monopeptide, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide. Each -W- unit independently corresponds to the formula below in square brackets, aw is an integer from 0 to 12 where R ¹⁹ is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl,

-CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -( CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2,

-CH ₂ CH ₂ CH(OH)CH ₂ NH ₂ , 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,

- 28 040898

In some embodiments, the amino acid unit can be enzymatically cleaved by one or more enzymes, including proteases associated with cancer or tumors, releasing the drug (-D) unit, which in one embodiment, upon release, is protonated in vivo to form the drug (D).

In some embodiments, the amino acid unit may contain naturally occurring amino acids. In other embodiments, the amino acid unit may contain non-naturally occurring amino acids. Typical units W _w are represented by formulas (VII)-(IX) where R ²⁰ and R ²¹ mean:

^R20

R ²¹ benzyl methyl isopropyl isopropyl benzyl isobutyl emo/2-butyl

benzyl benzyl (CH ₂ ) ₄ NH ₂ , (CH ₂ ) ₄ NH ₂ , (CH ₂ ) ₄ NH ₂ , (CH ₂ ) ₃ NHCONH ₂ , (CH ₂ ) ₃ NHCONH ₂ , (CH ₂ ) ₃ NHCONH ₂ , (CH ₂ ) ₃ NHCONH ₂ , (CH ₂ ) ₃ NHCONH ₂ , methyl, (CH ₂ ) ₃ NHC(=NH)NH ₂ ;

where R ²⁰ , R ²¹ and R ²² mean:

where R ²⁰ , R ²¹ , R ²² and R ²³ mean:

^R20

H methyl

R ²¹ R ²² R ²³ benzyl isobutyl H, isobutyl methyl isobutyl

Exemplary amino acid units include, without limitation, those of Formula VII,

- 29 040898 where R ²⁰ means benzyl, and R ²¹ means -(CH ₂ ) ₄ NH ₂ ; or R ²⁰ is isopropyl and R ²¹ is

-(CH ₂ ) ₄ NH ₂ ; or R ²⁰ is isopropyl and R ²¹ is -(CH ₂ )3NHCONH ₂ . Another exemplary amino acid unit is a unit according to formula VIII, where R ²⁰ means benzyl, R ²¹ means benzyl, and R ²² means

-(CH2)4NH2.

Useful -W _w - units can be designed and optimized for selectivity for enzymatic cleavage by certain enzymes, such as tumor-associated proteases. In one embodiment, the -W _w - unit is such that its cleavage is catalyzed by cathepsin B, C, or D, or a plasmin protease.

In one embodiment, -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, then the carbon atom to which R ¹⁹ , R ²⁰ , R ²¹ , R ²² or R ²³ is attached is chiral.

Each carbon atom to which R ¹⁹ , R ²⁰ , R ²¹ , R ²² or R ²³ is attached independently has the (S) or (R) configuration.

In one aspect of the amino acid link, they are valine-citrulline (VC or Val-Cit). In another aspect, the amino acid unit is phenylalanine-lysine (ie FK). In the next aspect of the amino acid link, they are N-methylvaline-citrulline. In a further aspect, the amino acid unit is 5-aminovaleric acid, homophenylalanine-lysine, tetraisoquinolinecarboxylate-lysine, cyclohexylalanine-lysine, isonipecotic acid-lysine, beta-alanine-lysine, or glycine-serine-valinglutamine-isonipecotic acid.

VIII. Spacer link.

The spacer unit (-Y-), when present, connects the amino acid unit to the drug unit, if there is an amino acid unit. On the other hand, the spacer unit connects the extension unit to the drug unit if there is no amino acid unit. The spacer unit also connects the drug unit to the antibody unit if neither an amino acid unit nor an extension unit is present.

Spacer links are of two main types: non-self-destructive (non self-immolative) and self-destructive (self-immolative). Non-self-destructing spacer units are such that part or all of the spacer unit remains associated with the drug unit after cleavage, especially the enzymatic amino acid unit, from the antibody-drug conjugate. Examples of non-self-destructing spacer units include, but are not limited to, a (glycine-glycine) spacer unit and a glycine single spacer unit (both shown in Scheme 1 below). When a conjugate containing a glycine-glycine spacer unit or a single glycine spacer unit is enzymatically cleaved by an enzyme (e.g., a tumor cell-associated protease, a cancer cell-associated protease, or a lymphocyte-associated protease), LA _a -W _w is cleaved there is a glycine-glycine-drug moiety or a glycine-drug moiety. In one embodiment, an independent hydrolysis reaction occurs in the target cells, cleaving the glycine-drug moiety bond and releasing the drug.

Scheme 1

ENZYMATIVE

SPLIT

Gly~ϋ|

ENZYMATIVE I

SPLITTING | —Gly—

Gly-D HYDROLYSIS | MEDICINE

Gly-Gly-D

HYDROLYSIS |

MEDICINE

In some embodiments, the non-self-destructing spacer unit (-Y-) is represented by -Gly-. In some embodiments, the non-self-destructing spacer unit (-Y-) is represented by -Gly-Gly-.

In one embodiment, drug-linker conjugates lacking a spacer unit (y=0), or pharmaceutically acceptable salts or solvates thereof, are provided.

On the other hand, conjugates containing a self-destructing spacer unit can release -D. As used herein, the term self-destructing spacer refers to a bifunctional chemical moiety that is capable of covalently bonding two separated chemical moieties to each other to form a stable triple molecule. It should spontaneously separate from the second chemical moiety when its bond with the first moiety is cleaved.

In some embodiments, -Y _y - is a p-aminobenzyl alcohol (PAB) unit (see Schemes 2 and 3) in which the phenylene moiety is replaced by Q _m , where Q is -C ₁ -C ₈ -alkyl, -C ₂ - C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl, -O- (C ₁ -C ₈ -alkyl), -O- (C ₂ -C ₈ -alkenyl), -O- (C ₂ -C ₈ - alkynyl), -halogen, -nitro or -cyano; and m is an integer from 0 to 4. The alkyl, alkenyl and alkynyl radicals, alone or as part of another group, may optionally be substituted.

In some embodiments, -Y- is a PAB group that is coupled to -Ww- via the nitrogen atom of the PAB amino group and is directly linked to -D via a carbonate, carbamate, or simple radical.

- 30 040898 ether. Without adhering to any particular theory or mechanism, Scheme 2 shows a possible mechanism for the detachment of a drug from a PAB group that couples directly to -D via a carbamate or carbonate radical, as described in Toki et al., 2002, J. Org. Chem.

67:1866-1872.

Scheme 2

ENZYMATIVE Cleavage

1.6 ELIMINATION

MEDICINE

In scheme 2, Q means -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl, -O-(C ₁ -C ₈ -alkyl), -O-(C ₂ -C ₈ -alkenyl), -O- (C ₂ -C ₈ -alkynyl), -halogen, -nitro or -cyano; m is an integer from 0 to 4; and p is from 1 to 20. The alkyl, alkenyl and alkynyl radicals, alone or as part of another group, may optionally be substituted.

Without adhering to any particular theory or mechanism, Scheme 3 shows a possible mechanism for detaching the drug from the PAB group, which is directly connected to -D through an ether or amine bond, and D includes an oxygen or nitrogen atom that is part of the drug unit.

Scheme 3

ENZYMATIVE

SPLIT

In scheme 3, Q means -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl, -O-(C ₁ -C ₈ -alkyl), -O-(C ₂ -C ₈ -alkenyl), -O- (C ₂ -C ₈ -alkynyl), -halogen, -nitro or -cyano; m is an integer from 0 to 4; and p is from 1 to 20. The alkyl, alkenyl and alkynyl radicals, alone or as part of another group, may optionally be substituted.

Other examples of self-destructing spacers include, but are not limited to, aromatic compounds that are electronically similar to PABs, such as 2-aminoimidazole-5-methanol derivatives (Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho- or para-aminobenzylacetals. Spacers that undergo cyclization after amide bond hydrolysis can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2:223), suitably substituted with bicyclo[2.2.1]- and bicyclo [2.2.2]-ring systems (Storm et al., 1972, J. Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acid amides (Amsberry et al.,

- 31 040898

1990, J. Org. Chem. 55:5867). Elimination of amine-containing drugs substituted at the α-position of glycine (Kingsbury et al., 1984, J. Med. Chem. 27:1447) also provides examples of self-destructing spacers.

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

-W _w -NH a _g

CH ₂ (0SS (0))) _n -0 \ / tn ₂ (0 (C (0Sh-0

ENZYMATIVE Cleavage

MEDICINES

In scheme 4, Q means -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl, -O-(C ₁ -C ₈ -αkyl), -O-(C ₂ -C ₈ -alkenyl), -O- (C ₂ -C ₈ -alkynyl), -halogen, -nitro or -cyano; m is an integer from 0 to 4; n is 0 or 1; and p is an integer from 1 to 20. The alkyl, alkenyl and alkynyl radicals, alone or as part of another group, may optionally be substituted.

In some embodiments, the -D groupings are the same. In another embodiment, the -D groupings are different.

In one aspect, the spacer unit (-Y _y -) is represented by formulas (X)-(XII)

hn-ch ₂ -coH _χι

NHCH ₂ C(O)-NHCH ₂ C(O)-5 ^? ? XII where Q means -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl, -O-(C ₁ -C ₈ -alkyl), -O-(C ₂ -C ₈ -alkenyl), -O-(C ₂ -C ₈ -alkynyl), -halogen, -nitro or -cyano; and m is an integer from 0 to 4. The alkyl, alkenyl and alkynyl radicals, alone or as part of another group, may optionally be substituted.

Embodiments of formulas I and II containing antibody-drug conjugates may include

where w and y are 0, 1 or 2,

where w and y are 0,

- 32 040898

IX. Drug unit.

The drug unit (D) can be any cytotoxic, cytostatic, or immunomodulatory (eg, immunosuppressive) agent or drug. D is a drug unit (molecule) containing an atom that can form a bond with a spacer unit, with an amino acid unit, with an extension unit, or with an antibody unit. In some embodiments, the D drug unit contains a nitrogen atom that can form a bond with the spacer unit. In the present specification, the terms drug unit and drug molecule are synonymous and are used interchangeably.

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

In some embodiments, the drug is an auristatin of the auristatin E type (also known as a dolastatin-10 derivative) or derivatives thereof. Auristatin can be, for example, an ester between auristatin E and a keto acid. For example, auristatin E can be reacted with para-acetylbenzoic acid or benzoylvaleric acid to give AEB and AEVB, respectively. Other exemplary auristatins include AFP, MMAF and MMAE. The synthesis and structure of typical auristatins are described in U.S. Patent Application Publication Nos. 20030083263, 2005-0238649 and 2005-0009751; International Patent Publication No. WO 04/010957, International Patent Publication No. WO 02/088172, and U.S. Patent Nos. 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, which are all incorporated herein by reference in their entirety and in all instances.

It has been shown that auristatins disrupt microtubule dynamics and nuclear and cell division and exhibit anticancer activity. Auristatins bind tubulin and may have cytotoxic or cytostatic effects on 158P1D7-expressing cells. There are several different assays known in the art that can be used to determine whether auristatin or the resulting antibody-drug conjugate will have a cytostatic or cytotoxic effect on the cell line of interest.

Methods for determining whether a compound will bind to tubulin are known in the art. For example, see Muller et al., Anal. Chem. 2006, 78, 4390-4397; Hamel et al., Molecular Pharmacology, 1995, 47: 965-976; and Hamel et al., Journal of Biological Chemistry, 1990, 265(28): 17141-17149. For the purposes of the present invention, the relative affinity of a compound for tubulin can be determined. Some preferred auristatins of the present invention bind tubulin with an affinity that is 10 times less (weaker affinity) than that of MMAE binding to tubulin, up to 10 times, times, or even 100 times higher (higher affinity) than MMAE binding affinity for tubulin.

In some embodiments, -D means auristatin according to the formula DE or DF:

De

Df or a pharmaceutically acceptable salt or solvate thereof; where, independently at each position: a squiggly line indicates a connection;

R ² means -C ₁ -C ₂ o-alkyl, -C ₂ -C ₂ o-alkenyl or -C ₂ -C ₂₀ alkynyl;

R ³ means -H, -C ₁ -C ₂₀ -alkyl, -C ₂ -C ₂₀ -alkenyl, -C ₂ -C ₂₀ -alkynyl, carbocycle,

- C ₁ -C ₂ o-alkylene (carbocycle), -C ₂ -C ₂₀ -alkenylene (carbocycle), -C ₂ -C ₂₀ -alkynylene (carbocycle), -aryl, -C ₁ -C ₂₀ -alkylene (aryl ), -C ₂ -C ₂₀ -alkenylene (aryl), -C ₂ -C ₂₀ -alkynylene (aryl), -heterocycle,

- C ₁ -C ₂ o-alkylene (heterocycle), -C ₂ -C ₂₀ -alkenylene (heterocycle) or -C ₂ -C ₂₀ -alkynylene (heterocycle);

R ⁴ means -H, -C ₁ -C ₂₀ -alkyl, -C ₂ -C ₂₀ -alkenyl, -C ₂ -C ₂₀ -alkynyl, carbocycle,

- C ₁ -C ₂ o-alkylene (carbocycle), -C ₂ -C ₂₀ -alkenylene (carbocycle), -C ₂ -C ₂₀ -alkynylene (carbocycle), -aryl, -C ₁ -C ₂₀ -alkylene (aryl ), -C ₂ -C ₂₀ -alkenylene (aryl), -C ₂ -C ₂₀ -alkynylene (aryl), -heterocycle,

- C ₁ -C ₂₀ -alkylene (heterocycle), -C ₂ -C ₂₀ -alkenylene (heterocycle) or -C ₂ -C ₂₀ -alkynylene (heterocycle);

R ⁵ means -H or -C ₁ -C ₈ -alkyl; or

R ⁴ and R ⁵ together form a carbocyclic ring according to the formula -(CR ^a R ^b )s-, where R ^a and R ^b independently mean -H, -C ₁ -C ₂₀ -alkyl, -C ₂ -C ₂₀ -alkenyl, -C ₂ -C ₂₀ alkynyl or -carbocycle, as is 2, 3, 4, 5 or 6;

R ⁶ is -H, -C ₁ -C ₂₀ alkyl, -C ₂ -C ₂₀ alkenyl or -C ₂ -C ₂₀ alkynyl;

R ⁷ means -H, -C ₁ -C ₂₀ -alkyl, -C ₂ -C ₂₀ -alkenyl, -C ₂ -C ₂₀ -alkynyl, -carbocycle,

- C ₁ -C ₂ o-alkylene (carbocycle), -C ₂ -C ₂₀ -alkenylene (carbocycle), -C ₂ -C ₂₀ -alkynylene (carbocycle), -aryl, -C ₁ -C ₂₀ -alkylene (aryl ), -C ₂ -C ₂₀ -alkenylene (aryl), -C ₂ -C ₂₀ -alkynylene (aryl), heterocycle,

- C ₁ -C ₂ o-alkylene (heterocycle), -C ₂ -C ₂₀ -alkenylene (heterocycle) or -C ₂ -C ₂₀ -alkynylene (heterocycle);

each R ⁸ is independently -H, -OH, -C ₁ -C ₂₀ alkyl, -C ₂ -C ₂₀ alkenyl, -C ₂ -C ₂₀ alkynyl, -O-(C ₁ -C ₂ o-alkyl ), -O-(C ₂ -C ₂₀ alkenyl), -O-(C ₂ -C ₂₀ alkynyl) or -carbocycle;

R ⁹ is -H, -C ₁ -C ₂₀ alkyl, -C ₂ -C ₂₀ alkenyl or -C ₂ -C ₂₀ alkynyl;

R ²⁴ is -aryl, -heterocycle or -carbocycle;

R ²⁵ means -H, -C ₁ -C ₂₀ -alkyl, -C ₂ -C ₂₀ -alkenyl, -C ₂ -C ₂₀ -alkynyl, -carbocycle, -O-(C ₁ -C ₂₀ -alkyl), - O-(C ₂ -C ₂₀ -alkenyl), -O-(C ₂ -C ₂₀ -alkynyl) or OR ¹⁸ where R ¹⁸ means -H, a hydroxyl protecting group or direct bond, where OR ¹⁸ means =O;

R ²⁶ is -H, -C ₁ -C ₂₀ alkyl, -C ₂ -C ₂₀ alkenyl or -C ₂ -C ₂₀ alkynyl, -aryl, -heterocycle or -carbocycle;

R ¹⁰ is -aryl or -heterocycle;

Z means -O-, -S-, -NH- or -NR ¹² -, where R ¹² means -C ₁ -C ₂₀ -alkyl, -C ₂ -C ₂₀ -alkenyl or -C ₂ -C ₂₀ -alkynyl;

R ¹¹ is -H, -C ₁ -C ₂₀ alkyl, -C ₂ -C ₂₀ alkenyl, -C ₂ -C ₂₀ alkynyl, -aryl, -heterocycle, -(R ¹³ O)mR ¹⁴ or -( R ¹³ O)m-CH(R ¹⁵ )2;

m is an integer from 1 to 1000;

R ¹³ is -C ₂ -C ₂₀ alkylene, -C ₂ -C ₂₀ alkenylene or -C ₂ -C ₂₀ alkynylene;

R ¹⁴ is -H, -C ₁ -C ₂₀ alkyl, -C ₂ -C ₂₀ alkenyl or -C ₂ -C ₂₀ alkynyl;

R ¹⁵ is in each case independently -H, -COOH, -(CH2)nN(R ¹⁶ )2, -(CH ₂ ) _n -SO ₃ H, -(CH ₂ ) _n -SO ₃ -C ₁ C ₂₀ - alkyl, -(CH ₂ ) _n -SO ₃ -C ₂ -C ₂₀ -alkenyl or -(CH ₂ ) _n -SO ₃ -C ₂ -C ₂₀ alkynyl;

R ¹⁶ is at each occurrence independently -H, -C ₁ -C ₂₀ alkyl, -C ₂ -C ₂₀ alkenyl, -C ₂ -C ₂₀ alkynyl, or -(CH2)p-COOH; and n is an integer from 0 to 6;

wherein the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, and heterocycle radicals, alone or as part of another group, are optionally substituted.

Auristatins of formula D _E include those in which the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, and heterocycle radicals are unsubstituted.

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Auristatins of formula DE include those in which the groups R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ are unsubstituted and the groups R ¹⁹ , R ²⁰ and R ²¹ are optionally substituted, as described here.

The DE formula auristatins include those for which:

R ² means -C ₁ -C ₈ -alkyl;

R ³ , R ⁴ and R ⁷ are independently selected from -H, -C ₁ -C ₂₀ alkyl, -C ₂ -C ₂₀ alkenyl, -C ₂ -C ₂₀ alkynyl, monocyclic C ₃ -C ₆ carbocycle, -C ₁ -C ₂₀ -alkylene (monocyclic C ₃ -C ₆ carbocycle),

-C ₂ -C ₂₀ -alkenylene (monocyclic C ₃ -C ₆ carbocycle), -C ₂ -C ₂₀ -alkynylene (monocyclic

C ₃ -C ₆ -carbocycle), -C ₆ -C ₁₀ -aryl, -C ₁ -C ₂₀ -alkylene (C ₆ -C ₁₀ -aryl), -C ₂ -C ₂₀ -αlkenylene (C ₆ -C ₁₀ -aryl), -C ₂ -C ₂₀ -alkynylene (C ₆ -C ₁₀ -aryl), -heterocycle, -C ₁ -C ₂₀ -alkylene (heterocycle), -C ₂ -C ₂₀ alkenylene (heterocycle) and -C ₂ -C ₂₀ -alkynylene (heterocycle); wherein the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, carbocycle, aryl, and heterocycle radicals are optionally substituted;

R ⁵ means -H;

R ⁶ means -C ₁ -C ₈ -alkyl;

each R ⁸ is independently selected from -OH, -O-(C ₁ -C ₂₀ -alkyl), -O-(C ₂ -C ₂₀ -alkenyl) and -O-(C ₂ -C ₂₀ -alkynyl), and the radicals alkyl, alkenyl and alkynyl are optionally substituted;

R ⁹ means -H or -C ₁ -C ₈ -alkyl;

R ²⁴ means optionally substituted -phenyl;

R ²⁵ means -OR ¹⁸ where R ¹⁸ means H, a hydroxyl protecting group or a direct bond, where OR ¹⁸ means =O;

R ²⁶ is selected from -H, -C ₁ -C ₂₀ alkyl, -C ₂ -C ₂₀ alkenyl, -C ₂ -C ₂₀ alkynyl and -carbocycle; wherein the alkyl, alkenyl, alkynyl and carbocycle radicals are optionally substituted;

or their pharmaceutically acceptable salts.

The DE formula auristatins include those for which:

R ² means methyl;

R ³ means -H, -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl or -C ₂ -C ₈ -alkynyl, and the radicals alkyl, alkenyl and alkynyl are optionally substituted;

R ⁴ means -H, -C ₁ -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl, -C ₂ -C ₈ -alkynyl, monocyclic C ₃ -C ₆ carbocycle, -C ₆ -C ₁₀ -aryl , -C ₁ -C ₈ -alkylene (C ₆ -C ₁₀ -aryl), -C ₂ -C ₈ -alkenylene (C ₆ -C ₁₀ -aryl), -C ₂ -C ₈ -alkynylene (C ₆ -C ₁₀ -aryl), -C ₁ -C ₈ -alkylene (monocyclic C ₃ -C ₆ carbocycle), -C ₂ -C ₈ -alkenylene (monocyclic C ₃ -C ₆ carbocycle), -C ₂ -C ₈ -alkynylene (monocyclic C ₃ -C ₆ carbocycle); wherein the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, and carbocycle radicals, alone or as part of another group, are optionally substituted;

R ⁵ means -H;

R ⁶ means methyl;

R ⁷ means -C 1 -C ₈ -alkyl, -C ₂ -C ₈ -alkenyl or -C ₂ -C ₈ -alkynyl;

each R ⁸ is methoxy;

R ⁹ means -H or -C ₁ -C ₈ -alkyl;

R ²⁴ means -phenyl;

R ²⁵ is -OR ¹⁸ where R ¹⁸ is -H, a hydroxyl protecting group or a direct bond, where OR ¹⁸ is ═O;

R ²⁶ means methyl;

or their pharmaceutically acceptable salts.

The DE formula auristatins include those for which:

R ² means methyl; R ³ is -H or C1-C3 alkyl; R ⁴ means C1-C ₅ -alkyl; R ⁵ means -H; R ⁶ means methyl; R ⁷ is isopropyl or sec-butyl; R ⁸ is methoxy; R ⁹ means -H or C ₁ -C ₈ -alkyl; R ²⁴ means phenyl; R ²⁵ is OR ¹⁸ where R ¹⁸ is H, a hydroxyl protecting group or a direct bond, where OR ¹⁸ is ═O; and R ²⁶ is methyl; or their pharmaceutically acceptable salts or solvates.

Auristatins according to the formula D _E include those in which:

R ² is methyl or C1-C3 alkyl; R ³ means -H or C1-C ₃ -alkyl; R ⁴ is C1-C5 alkyl; R ⁵ means -H; R ⁶ means C1-C ₃ -alkyl; R ⁷ is C1-C5 alkyl; R ⁸ means C1-C ₃ -alkoxy; R ⁹ is -H or C1-C8 alkyl; R ²⁴ means phenyl; R ²⁵ is OR ¹⁸ where R ¹⁸ is H, a hydroxyl protecting group or a direct bond, where OR ¹⁸ is ═O; and R ²⁶ is C1- _C3 alkyl; or their pharmaceutically acceptable salts.

Auristatins according to the formula D _F include those in which:

R ² means methyl;

R ³ , R ⁴ and R ⁷ are independently selected from -H, -C ₁ -C ₂₀ -alkyl, -C ₂ -C ₂₀ -alkenyl, -C ₂ -C ₂₀ alkynyl, monocyclic C ₃ -C ₆ carbocycle, -C ₁ -C ₂₀ -alkylene (monocyclic C ₃ -C ₆ carbocycle),

-C ₂ -C ₂₀ -alkenylene (monocyclic C ₃ -C ₆ carbocycle), -C ₂ -C ₂₀ -alkynylene (monocyclic

C ₃ -C ₆ -carbocycle), -C ₆ -C ₁₀ -aryl, -C ₁ -C ₂₀ -alkylene (C ₆ -C ₁₀ -aryl), -C ₂ -C ₂₀ -αlkenylene (C ₆ -C ₁₀ -aryl), -C ₂ -C ₂₀ -alkynylene (C ₆ -C ₁₀ -aryl), -heterocycle, -C ₁ -C ₂₀ -alkylene (heterocycle),

-C ₂ -C ₂₀ -alkenylene (heterocycle) and -C ₂ -C ₂₀ -alkynylene (heterocycle); moreover, the radicals alkyl, alkenyl, al- 35 040898 kinyl, alkylene, alkenylene, alkynylene, carbocycle, aryl and heterocycle, individually or as part of another group, are optionally substituted;

R ⁵ means -H;

R ⁶ means methyl;

each R ⁸ is methoxy;

R ⁹ is -H, -C ₁ -C ₂₀ alkyl, -C ₂ -C ₂₀ alkenyl or -C ₂ -C ₂₀ alkynyl; wherein the alkyl, alkenyl and alkynyl radicals are optionally substituted;

R ¹⁰ means optionally substituted aryl or optionally substituted heterocycle;

Z means -O-, -S-, -NH- or -NR ¹² -, where R ¹² means -C ₁ -C ₂₀ -alkyl, -C ₂ -C ₂₀ -alkenyl or -C ₂ -C ₂₀ -alkynyl, each of which is optionally substituted;

R ¹¹ means -H, -C1-C20-alkyl, -C2-C20-alkenyl, -C2-C20-alkynyl, -aryl, -heterocycle, -(R ¹³ O)mR ¹⁴ or -(R ¹³ O)m- CH(R ¹⁵ )2, wherein the alkyl, alkenyl, alkynyl, aryl and heterocycle radicals are optionally substituted;

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

R ¹³ is -C ₂ -C ₂₀ alkylene, -C ₂ -C ₂₀ alkenylene, or -C ₂ -C ₂₀ alkynylene, each of which is optionally substituted;

R ¹⁴ is -H, -C ₁ -C ₂₀ alkyl, -C ₂ -C ₂₀ alkenyl or -C ₂ -C ₂₀ alkynyl, the alkyl, alkenyl and alkynyl radicals being optionally substituted;

R ¹⁵ is in each case independently -H, -COOH, -(CH2)nN(R ¹⁶ )2, -(CH2) _n -SO ₃ H, -(CH ₂ ) _n -SO ₃ -C ₁ C ₂₀ -alkyl , -(CH ₂ ) _n -SO ₃ -C ₂ -C ₂₀ -alkenyl or -(CH ₂ ) _n -SO ₃ -C ₂ -C ₂₀ alkynyl, the alkyl, alkenyl and alkynyl radicals being optionally substituted;

R ¹⁶ in each case independently means -H, -C ₁ -C ₂₀ -alkyl, -C ₂ -C ₂₀ -alkenyl, -C ₂ -C ₂₀ -alkynyl or -(CH ₂ ) _p -COOH, and the radicals are alkyl, alkenyl and alkynyl are optionally substituted;

n is an integer from 0 to 6;

or their pharmaceutically acceptable salts.

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

Auristatins of formula DF include those in which the R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ groups are unsubstituted and the R ¹⁰ and R ¹¹ groups are as described herein.

Auristatins of formula D _F include those in which the alkyl, alkenyl, alkynyl, alkylene, alkenylene alkynylene, aryl, carbocycle, and heterocycle radicals are unsubstituted.

Auristatins according to the formula D _F include those in which:

R ² means -C1-C3-alkyl; R ³ means -H or -C1-C ₃ -alkyl; R ⁴ is -C1-C5 alkyl; R ⁵ means -H; R ⁶ means -C1-C ₃ -alkyl; R ⁷ is -C1-C5 alkyl; R ⁸ means -C1-C ₃ -alkoxy; R ⁹ means -H or -C ₁ -C ₈ -alkyl; R ¹⁰ means optionally substituted phenyl; Z means O, S or NH; a R ¹¹ is already defined here; or their pharmaceutically acceptable salts.

Auristatins in the DF formula include those for which:

R ² means methyl; R ³ is -H or -C1-C3 alkyl; R ⁴ means -C1-C ₅ -alkyl; R ⁵ means -H; R ⁶ means methyl; R ⁷ is isopropyl or sec-butyl; R ⁸ is methoxy; R ⁹ means -H or -C ₁ -C ₈ -alkyl; R ¹⁰ means optionally substituted phenyl; Z is -O-, -S- or -NH-; a R ¹¹ is already defined here; or their pharmaceutically acceptable salts.

Auristatins according to the formula D _F include those in which:

R ² means methyl; R ³ is -H or -C1-C3 alkyl; R ⁴ means -C1-C ₅ -alkyl; R ⁵ means -H; R ⁶ means methyl; R ⁷ is isopropyl or sec-butyl; R ⁸ is methoxy; R ⁹ is -H or -Ci-C ₈ alkyl; R ¹⁰ means phenyl; Z is -O- or -NH-; and R ¹¹ is already defined here, preferably hydrogen; or their pharmaceutically acceptable salts.

Auristatins in the DF formula include those for which:

R ² means -C1-C3-alkyl; R ³ means -H or -C1-C ₃ -alkyl; R ⁴ is -C1-C5 alkyl; R ⁵ means -H; R ⁶ means -C1-C ₃ -alkyl; R ⁷ is -C1-C5 alkyl; R ⁸ means -C1-C ₃ -alkoxy; R ⁹ means -H or -C ₁ -C ₈ -alkyl; R ¹⁰ means phenyl; Z is -O- or -NH-; a R ¹¹ is already defined here, preferably hydrogen; or their pharmaceutically acceptable salts.

Auristatins of formula DE or D _F include those in which R ³ , R ⁴ and R ⁷ are independently isopropyl or sec-butyl; a R ⁵ means -H. In a typical embodiment, R ³ and R ⁴ are each isopropyl, R ⁵ is H, and R ⁷ is sec-butyl. The rest of the substituents are already defined here.

Auristatins of the formula D _E or D _F include those in which R ² and R ⁶ are each methyl and R ⁹ is H. The remaining substituents are already defined here.

Auristatins of the formula DE or DF include those in which R ⁸ in each occurrence is -OCH3. The rest of the substituents are already defined here.

Auristatins of formula DE or DF include those wherein R ³ and R ⁴ are each isopropyl, R ² and R ⁶ are each methyl, R ⁵ are H, R ⁷ are sec-butyl, R ⁸ are each -OCH3, a R ⁹ is H. The rest of the substituents are already defined here.

Auristatins of the formula D _F include those in which Z is -O- or -NH-. The rest of the substituents are already defined here.

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Auristatins of formula DF include those in which R ¹⁰ is aryl. The rest of the substituents are already defined here.

Auristatins of the formula DF include those in which R ¹⁰ is -phenyl. The rest of the substituents are already defined here.

Auristatins of formula DF include those in which Z is -O- and R ¹¹ is H, methyl or mpem-butyl. The rest of the substituents are already defined here.

Auristatins of the formula DF include those in which if Z is -NH then R ¹¹ is -(R ¹³ O)m-CH(R ¹⁵ )2, where R ¹⁵ is -(CH2)nN(R ¹⁶ )2, a R ¹⁶ means -C1-C8-alkyl or -(CH2)P-COOH. The remaining substituents have already been defined above.

Auristatins of formula D _F include those in which if Z is -NH then R ¹¹ is -(R ¹³ O)m-CH(R ¹⁵ )2 where R ¹⁵ is -(CH ₂ ) _n -SO ₃ H The remaining substituents have already been defined above.

In preferred embodiments, if D is auristatin of the formula DE, then w is an integer from 1 to 12, preferably from 2 to 12, y is 1 or 2, and a is preferably 1.

In some embodiments, if D is auristatin of formula DF, then a is 1 and w and y are 0.

Exemplary drug units (-D) include drug units having the following structures:

- 37 040898

or their pharmaceutically acceptable salts or solvates.

In one aspect, hydrophilic groups, such as but not limited to triethylene glycol (TEG) esters, can be attached to the R ¹¹ drug unit. Without being bound by any theory, hydrophilic groups promote internalization and prevent agglomeration of the drug unit.

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.

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Exemplary antibody-drug conjugates have the following structures, wherein L or mAb-S- is the anti-158P1D7 mAb shown here, designated Ha15-10ac12:

L-MC-vc-PAB-MMAE:

L-MC-vc-PAB-MMAF:

or

°=<

hh ₂

L-MC-MMAF:

or their pharmaceutically acceptable salts.

In some embodiments, the drug moiety is calicheamicin, camptothecin, maytansinoid, or anthracycline. In some embodiments, the drug is a taxane, a topoisomerase inhibitor, a vinca alkaloid, and the like.

In some exemplary embodiments, suitable cytotoxic agents include, for example, DNA minor groove binding agents (e.g., enediins and lexitropsins, CBI compounds; see also U.S. Patent No. 6,130,237), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins and vinca alkaloids. Other cytotoxic agents include, for example, CC1065, CH-38, topotecan, morpholino-doxorubicin, risoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide, eleutherobin, and mitoxantrone. .

In some embodiments, the drug is an antitubulin agent. Examples of antitubulin agents include auristatins, taxanes (eg Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik) and vinca alkaloids (eg vincristine, vinblastine, vindesine and vinorelbine). Other antitubulin agents include, for example, baccatin derivatives, taxane analogs (eg, epothilone A and B), nocodazole, colchicine and colcimide, estramustine, cryptophycins, cemadotin, maytansinoids, combretastatins, discodermolide, and eleutherobin.

In some embodiments, the cytotoxic agent is a maytansinoid, from a distinct group of antitubulin agents. For example, in certain embodiments, the maytansinoid is maytansin or DM-1 (ImmunoGen, Inc.; also see Chari et al., 1992, Cancer Res. 52:127-131).

In some embodiments, the cytotoxic or cytostatic agent is dolastatin. In some embodiments, the cytotoxic or cytostatic agent belongs to the auristatin class. Thus, in one particular embodiment, the cytotoxic or cytostatic agent is MMAE (Formula XI). In another specific embodiment, the cytotoxic or cytostatic agent is AFP (Formula XVI).

- 39 040898

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

- 40 040898

X. Drug loading.

Drug loading is expressed as p and represents the average number of drug molecules per antibody per molecule. The drug loading can be from 1 to 20 drug molecules (D) per antibody. The ADC conjugates of the invention comprise a pool of antibodies conjugated to a range of drug molecules from 1 to 20. The average number of drug molecules per antibody in ADC preparations in a conjugation reaction can be determined by standard methods such as mass spectroscopy and ELISA. It is also possible to determine the quantitative distribution of ADC conjugates by p-values. In some cases it is possible to separate, purify and characterize homogeneous ADC conjugates with a certain p value from ADC conjugates with different drug loading by methods such as electrophoresis.

For some antibody-drug conjugates, the p-value may be limited by the number of attachment sites on the antibody. For example, if the site of attachment is a cysteine thiol, as in the above exemplary embodiments, then the antibody may contain only one or a few cysteine thiol groups, or it may contain only one or a few sufficiently reactive cysteine thiol groups at which the linker can be attached. In some embodiments, higher drug loading, eg p>5, may cause aggregation, insolubility, toxicity, or loss of cell permeability in some antibody-drug conjugates. In some embodiments, the drug loading of an ADC of the invention is from 1 to 8; from 2 to 6; from 3 to 5; 3 to 4; from 3.1 to 3.9; from 3.2 to 3.8; from 3.2 to 3.7; from 3.2 to 3.6; from 3.3 to 3.8; or from 3.3 to 3.7. Thus, it has been shown that in some ADCs the optimal number of drug molecules per antibody may be less than 8 or may be from 2 to 5. See US 2005-0238649 A1 (incorporated here by reference in its entirety).

In some embodiments, the antibody conjugation reaction conjugates fewer drug molecules than the theoretical maximum. For example, the antibody may contain lysine residues that are not reactive with the drug-linker intermediate or linker reagent, as described below. In general, antibodies contain few free and reactive cysteine thiol groups that can bind to the drug molecule; and most cysteine thiol groups in antibodies exist as disulfide bridges. In some embodiments, to obtain reactive cysteine thiol groups, an antibody can be reduced using a reducing reagent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partially or fully reducing conditions. In some

- 41 040898 embodiments, the antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.

The loading (drug/antibody ratio) of the ADC can be controlled in various ways, for example by: (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the time or temperature of the conjugation reaction, (iii) partial limiting reductive conditions to modify cysteine thiol groups, (iv) modifying by recombinant methods the amino acid sequence of the antibody so as to modify the number and location of cysteine residues to control the amount and/or location of attachment of the linker-drug (such as obtaining a thio-Mab or thio- Fab as set forth here and in WO 2006/034488 (incorporated here by reference in its entirety)).

It should be borne in mind that if more than one nucleophilic group reacts with the drug-linker intermediate or linker reagent and then with the drug molecule reagent, the resulting product will be a mixture of ADC conjugates distributed over one or more drug molecules. attached to the antibody. The average number of drug molecules per antibody in a mixture can be calculated by a dual antibody-specific and drug-specific ELISA method. In a mixture, individual ADC molecules can be identified by mass spectroscopy and separated by HPLC, such as hydrophobic chromatography (e.g., see Hamblett, K.J. 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 some embodiments, homogeneous ADCs with the same loading value can be isolated from the conjugation mixture by electrophoresis or chromatography.

XI. Methods for determining the cytotoxic effect of ADC conjugates.

Methods for determining whether a drug or antibody-drug conjugate will have a cytostatic and/or cytotoxic effect on cells are known. In general, the cytotoxic or cytostatic activity of an antibody-drug conjugate can be measured as follows: mammalian cells expressing a given protein are exposed to the antibody-drug conjugate in a cell culture medium; cells are cultured for 6 hours to 5 days; and cell viability is measured. In vitro cellular assay methods can be used to measure the viability (proliferation), cytotoxicity, and apoptosis (caspase activation) induction of an antibody-drug conjugate.

To determine whether the antibody-drug conjugate will have a cytostatic effect, the thymidine inclusion method can be used. For example, cancer cells expressing the target antigen can be cultured at a density of 5000 cells per well in a 96-well plate for 72 hours and exposed to 0.5 μCi ³ H-thymidine for the last 8 hours of this 72 hour period. Incorporation of ³ H-thymidine into culture cells is measured in the presence and absence of the antibody-drug conjugate.

Necrosis or apoptosis (programmed cell death) can be measured to determine cytotoxicity. Necrosis is usually accompanied by increased plasma membrane permeability, cell swelling, and rupture of the plasma membrane. Apoptosis is usually characterized by the formation of membrane vesicles (blebbing), condensation of the cytoplasm, and activation of endogenous endonucleases. Determination of any of these effects on cancer cells means that the antibody drug conjugate is useful in the treatment of cancer.

Cell viability can be measured by determining the uptake of a dye such as neutral red, trypan blue, or Alamar™ Blue on cells (eg, see Page et al., 1993, Int. J. Oncology 3:473-476). In this assay, the cells are incubated in a medium containing the dye, then the cells are washed and the remaining dye is measured spectrophotometrically, which reflects the uptake of the dye by the cells. The protein-binding dye sulforodamine B (SRB) can also be used to measure cytotoxicity (Skehan et al., 1990, J. Natl. Cancer Inst. 82:1107-12).

On the other hand, in the quantitative colorimetric determination of the viability and proliferation of mammalian cells, tetrazolium salts of the MTT type are used, which detect living, but not dead cells (for example, see Mosmann, 1983, J. Immunol. Methods 65:55-63).

Apoptosis can be quantified, for example, by DNA fragmentation. There are commercial photometric methods for the in vitro quantitative determination of DNA fragmentation. Examples of such methods, including TUNEL (which detects the incorporation of labeled nucleotides into fragmented DNA) and ELISA-based methods, are given in Biochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

- 42 040898

Apoptosis can also be determined by morphological changes in cells.

For example, as in necrosis, it is possible to determine the integrity of the plasma membrane by the absorption of certain dyes (for example, fluorescent dyes, such as, for example, acridine orange or ethidium bromide). A method for measuring the number of apoptotic cells is described in Duke and Cohen, Current Protocols in Immunology (Coligan et al., eds., 1992, pp. 3.17.1-3.17.16). It is also possible to label the cells with a DNA dye (eg, acridine orange, ethidium bromide, or propidium iodide) and note the condensation of chromatin in the cells and its accumulation along the inner nuclear membrane. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane blebbing, and cell shrinkage.

The presence of apoptotic cells can be measured in both fixed and free-floating culture compartments. For example, both compartments can be harvested by removing the supernatant, trypsinizing adherent cells, pooling preparations after washing them by centrifugation (eg, 10 min at 2000 rpm), and detecting apoptosis (eg, by DNA fragmentation) (eg, see Piazza et al. , 1995, Cancer Research 55:3110-16).

The effect of therapeutic compositions against 158P1D7 can be tested in vivo in suitable animal models. For example, xenogenic cancer models can be used in which explants or passaged xenografts of cancer tissues are administered to immunocompromised animals such as nude or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-408). For example, in PCT Patent Application WO 98/16628 and U.S. Patent 6,107,540 describes various models of human prostate cancer xenografts capable of reproducing the development of primary tumors, micrometastases, and the formation of osteoblastic metastases characteristic of the late stage of the disease. Efficacy can be predicted by methods that measure tumor formation inhibition, tumor regression or metastasis, etc.

When evaluating therapeutic compositions, in vivo methods are used in which an increase in apoptosis is determined. In one embodiment, xenografts from tumor-bearing mice treated with the therapeutic composition can be examined for apoptotic foci and compared to untreated xenograft control mice. The degree to which foci of apoptosis are found in tumors in treated mice is indicative of the therapeutic efficacy of the composition.

Therapeutic compositions used in the practice of the above methods may be formulated as pharmaceutical compositions containing suitable carriers for the desired mode of delivery.

Suitable carriers include any materials which, when combined with the therapeutic composition, retain the antitumor function of the therapeutic composition and generally do not react with the patient's immune system. Examples of these include, without limitation, any of a variety of standard pharmaceutical carriers such as sterile phosphate buffered saline, bacteriostatic water, and the like. (generally see Remington's Pharmaceutical Sciences, 16th Edition, A. Osal., Ed., 1980).

Dosage forms can be solubilized and administered by any method suitable for delivering the therapeutic composition to the site of the tumor. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intraorganic, orthotopic, and others. in polyvinyl chloride or polyethylene bags containing 0.9% sterile sodium chloride for injection, USP. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing, for example, benzyl alcohol as a preservative) or sterile water prior to administration.

Dosages and routes of administration in the treatment of cancers using the above methods will depend on the method and the cancer and generally depend on a number of other factors considered in practice.

XII. Treatment of cancer expressing 158P1D7.

Identification of 158P1D7 as a protein that is normally expressed in a limited set of tissues but is also expressed in the cancers listed in Table 1. I, opens up a range of therapeutic approaches for the treatment of such cancers.

It should be noted that targeted antitumor therapy is useful even when the desired protein is expressed in normal tissues, even in normal tissues of vital organs. Vital organs are those organs that are necessary to sustain life, such as the heart or large intestine. Non-vital organs - such organs, after the removal of which the individual is still able to survive. Examples of non-vital organs are the ovaries, the mammary gland, and the prostate gland.

Expression of the protein of interest in normal tissue, even in vital normal tissue, does not detract from the usefulness of a protein targeting agent as a therapeutic agent for some

- 43 040898 tumors in which this protein is overexpressed. For example, expression in vital organs does not in itself cause harm. In addition, organs considered optional, such as the prostate and ovaries, can be removed without causing mortality. Finally, some vital organs do not depend on normal expression in the organ due to its immune privilege. Immunoprivileged organs are those organs that are protected from blood by the blood-organ barrier and therefore are not available for immunotherapy. Examples of immune-privileged organs are the brain and testicles.

Accordingly, therapeutic approaches that inhibit the activity of the 158P1D7 protein are beneficial for patients suffering from cancer expressing 158P1D7. These therapeutic approaches generally fall into three classes. The first class modulates the function of 158P1D7, as it is associated with the growth of tumor cells, which leads to inhibition or slowdown in the growth of tumor cells or causes their death. The second class includes various ways of inhibiting the binding or association of the 158P1D7 protein with its binding partner or with other proteins. The third class includes various methods for inhibiting 158P1D7 gene transcription or 158P1D7 mRNA translation.

Accordingly, cancer patients can be examined for the presence and level of expression of 158P1D7, preferably by immunohistochemical assessment of tumor tissue, quantitative imaging of 158P1D7, or other methods that reliably indicate the presence and level of expression of 158P1D7. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for the immunohistochemical analysis of tumor tissues are well known in the art.

XIII. 158P1D7 as a target for antibody-based therapy.

158P1D7 is an attractive target for antibody-based therapeutic strategies. Various antibody-based strategies are known for targeting both extracellular and intracellular molecules (eg complement and ADCC-mediated killing as well as intrabodies). Since 158P1D7 is expressed in cancer cells of various lines relative to the corresponding normal cells, systemic administration of such 158P1D7 immunoreactive compositions is used, which exhibit high sensitivity without toxic, non-specific and / or off-target effects caused by binding of the immunoreactive composition to non-target organs and tissues. Antibodies specifically reactive with 158P1D7 domains are useful for the systemic treatment of 158P1D7-expressing cancers, preferably in the form of antibody-drug conjugates (ie ADCs), namely toxin or therapeutic agent conjugates.

Those skilled in the art will recognize that antibodies can be used to specifically target and bind immunogenic molecules such as the immunogenic region in the 158P1D7 sequence shown in FIG. 1. In addition, it is known to those skilled in the art that conjugation of antibodies to cytotoxic agents is common (eg, see Slevers et al., Blood 93(11): 36783684 (1999)). By delivering cytotoxic and/or therapeutic agents directly to cells, such as by conjugating them with antibodies specific to a molecule expressed in those cells (e.g. 158P1D7), the cytotoxic agent will exert its known biological effect (i.e. cytotoxicity) to these cells.

A wide variety of compositions and methods of using conjugates of antibodies with cytotoxic agents for cell killing are known. In the context of cancer, exemplary methods include administering to a mammal with a tumor a biologically effective amount of a conjugate containing a selected cytotoxic and/or therapeutic agent coupled to a targeted agent (e.g., an anti-158P1D7 mAb, preferably Ha15-10ac12) that binds to an antigen (e.g., 158P1D7) , which is expressed, available for binding, or localized on the cell surface. A typical embodiment is a method of delivering a cytotoxic and/or therapeutic agent to cells expressing 158P1D7, which comprises conjugating the cytotoxic agent to an antibody that binds immunospecifically to the 158P1D7 epitope and exposing the cells to an antibody-drug conjugate (ADC). Another typical embodiment is a method of treating individuals with suspected metastatic cancer, which includes the step of parenteral administration to this individual of a pharmaceutical composition containing a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.

Immunotherapy of cancer with anti-158P1D7 antibodies can be carried out in accordance with various approaches that have been successfully used in the treatment of other types of cancer, including, but not limited to, colon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18 :133-138), multiple myeloma (Ozaki et al., 1997, Blood 90: 3179-3186, Tsunenari et al., 1997, Blood 90: 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). Some therapeutic approaches include conjugation of the naked antibody to a toxin or radioactive isotope, such as conjugation of Y ⁹¹ or I ¹³¹ with anti-CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals

- 44 040898

Corp., or Bexxar™, Coulter Pharmaceuticals), respectively, while others include the co-administration of antibodies and other therapeutics such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). In a preferred embodiment, the antibodies must be conjugated to a cytotoxic agent, see above, preferably an auristatin derivative called MMAE (Seattle Genetics).

While anti-158P1D7 antibody therapy is applicable to all stages of cancer, it may be particularly appropriate for advanced or metastatic cancer. Treatment with antibody therapy of the invention is indicated for those patients who have already received one or more courses of chemotherapy. On the other hand, antibody therapy of the invention is combined with chemotherapy or radiation therapy for those patients who have not received chemotherapy. In addition, antibody therapy may help to reduce the dose of concomitant chemotherapy, especially for those patients who very poorly tolerate the toxicity of the chemotherapeutic agent. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al. (Int. J. Oncol. 9:217-224, 1996) and Hancock et al. (Cancer Res. 51:4575-4580, 1991) describes the use of various antibodies in conjunction with chemotherapeutic agents.

Accordingly, preferred monoclonal antibodies for use in the therapeutic methods of the invention are those that are fully human or that specifically bind to the 158P1D7 antigen of interest with high affinity. Although the antibody-drug conjugates of the invention are useful in the treatment of cancers in which 158P1D7 is expressed, the antibody-drug conjugates of the invention may be of particular therapeutic utility in the treatment of bladder cancer.

XIV. Cocktails with ADC to 158P1D7.

Therapeutic methods of the invention involve the administration of single anti-158P1D7 ADCs, as well as combinations or cocktails of different mAbs (ie, anti-158P1D7 mAbs or other protein-binding mAbs). Such mAb cocktails may be advantageous as they contain mAbs that target different epitopes, use different effector mechanisms, or combine directly cytotoxic mAbs with mAbs that depend on immunoeffector functionality. Such combinations of mAbs may have a synergistic therapeutic effect. In addition, anti-158P1D7 mAbs can be administered simultaneously with other therapeutic agents, including, but not limited to, various chemotherapeutic and biological agents, androgen blockers, immunomodulators (eg, IL-2, GM-CSF), surgery, or radiation. In a preferred embodiment, anti-158P1D7 mAbs are administered in conjugated form.

Dosage forms with ADC to 158P1D7 are administered by any method suitable for delivery of antibodies to tumor cells. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intradermal, intratumoral, intradermal, etc. Treatment generally includes repeated administration of the anti-158P1D7 ADC drug by an acceptable route of administration such as intravenous injection (IV), typically in a dose range, without limits, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 mg/kg body weight. In general, doses in the range of 10-1000 mg mAb per week are effective and well tolerated.

Based on clinical experience with Herceptin® (trastuzumab) in the treatment of metastatic breast cancer, an acceptable dosage regimen is an initial loading dose of approximately 4 mg/kg of the patient's body weight IV followed by a weekly dose of the mAb preparation of approximately 2 mg/kg IV. Preferably, the initial loading dose is administered as a 90 minute infusion or longer. The intermittent maintenance dose is administered as a 30-minute infusion or longer, provided that the initial dose is well tolerated. As will be known to those skilled in the art, various factors may influence the ideal dosage regimen in any given case. Such factors include, for example, the binding affinity and half-life of the mAbs used, the degree of expression of 158P1D7 in the patient, the level of circulating 158P1D7 detached antigen, the desired steady-state level of antibody concentration, the frequency of treatment, and the effect of chemotherapeutic or other agents used in combination with the method of treatment. according to the invention, as well as the state of health of the patient.

Optionally, patients should be screened for the level of 158P1D7 in a given sample (eg, levels of circulating 158P1D7 antigen and/or 158P1D7 expressing cells) to help determine the most effective dosing regimen, etc. Such examinations are also used for monitoring purposes throughout therapy and are useful for assessing therapeutic success in combination with the assessment of other parameters (for example, urine cytology and/or immunocyte levels in bladder cancer therapy, or, by analogy, serum PSA levels). in the treatment of prostate cancer).

The subject of the present invention is to provide such ADCs to 158P1D7, which inhibit or slow down the growth of tumor cells expressing 158P1D7. Another object of the invention is to provide methods for inhibiting angiogenesis and other biological functions and thereby

- 45 040898 attenuation of tumor growth in mammals, preferably humans, using such ADCs to

158P1D7, in particular, by such ADCs to 158P1D7 in combination with other drugs or immunologically active procedures.

XV. Combined therapy.

In one embodiment, there is synergy in the treatment of tumors, including tumors in humans, using ADCs to 158P1D7 in combination with chemotherapeutic agents or radiation, or a combination of both. In other words, tumor growth inhibition by ADCs to 158P1D7 is enhanced more than expected when combined with chemotherapeutic agents or radiation, or a combination thereof. Synergism can manifest, for example, greater inhibition of tumor growth with combination therapy than would be expected with treatment with 158P1D7 ADCs alone, or an additive effect of treatment with 158P1D7 ADCs and a chemotherapeutic agent or radiation. Preferably synergism is manifested by cancer remission when remission is not expected when treated with 158P1D7 ADCs alone or when treated with an additive combination of 158P1D7 ADCs and a chemotherapeutic agent or radiation.

A method for inhibiting tumor cell growth with an anti-158P1D7 ADC in combination with chemotherapy or radiation, or both, comprises administering an anti-158P1D7 ADC before, during, or after the initiation of chemotherapy or radiotherapy, as well as any combination thereof (i.e., before and during, before and after, during and after, or before, during and after the start of chemotherapy and/or radiotherapy). For example, anti-158P1D7 ADCs are typically administered 1 to 60 days, preferably 3 to 40 days, more preferably 5 to 12 days prior to the start of radiotherapy and/or chemotherapy. However, depending on the method of treatment and the specific needs of the patient, the method is performed in such a way as to provide the most effective treatment and ultimately prolong the life of the patient.

The introduction of chemotherapeutic agents can be carried out in various ways, including systemic administration by parenteral and enteral route. In one embodiment, the anti-158P1D7 ADCs and the chemotherapeutic agent are administered as separate molecules. Specific examples of chemotherapeutic agents or chemotherapy include cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide , methotrexate, 5fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, alpha interferon, leuprolide, megestrol, melphalan , mercaptopurine, plicamycin, mitotane, pegaspargas, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, gemcitabine, chlorambucil, taxol, and combinations thereof.

The radiation source, when used in combination with ADC to 158P1D7, may be external or internal to the patient being treated. When the source is external to the patient, the treatment is known as external beam radiation therapy (EBRT). If the radiation source is internal to the patient, then the treatment is called brachytherapy (BT).

The above treatment regimens may also be combined with additional cancer treatments and/or regimens, e.g., additional chemotherapy, cancer vaccines, signaling inhibitors, agents in the treatment of abnormal cell growth or cancer, antibodies (e.g., anti-CTLA-4 antibodies described in WO/2005/092380 (Pfizer)), or with other ligands that inhibit tumor growth by binding to IGF-1R, and cytokines.

When mammals are subjected to additional chemotherapy, the chemotherapeutic agents described above can be used. In addition, growth factor inhibitors, biological response modifiers, antihormonal therapy, selective estrogen receptor modulators (SERMs), angiogenesis inhibitors, and antiandrogens can be used. For example, antihormones such as antiestrogen such as Nolvadex (tamoxifen) or antiandrogens such as Casodex (4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3'(trifluoromethyl )propionanilide).

The above therapeutic approaches can be combined with any of a variety of surgical, chemotherapeutic, or radiotherapy regimens. Therapeutic approaches of the invention may facilitate dose reduction in chemotherapy (or other therapy) and/or less frequent administration, which is an advantage for all patients and especially for those who do not tolerate the toxicity of chemotherapeutic agents.

XVI. Kits/industrial products.

Kits for use in the laboratory, prognostic, prophylactic, diagnostic, and therapeutic applications described herein are within the scope of the invention. Such kits may include a carrier, package or container that is divided so that it contains one or more containers such as vials, test tubes, etc., and each container contains one of the individual elements used in the method, along with a label or insert. , containing instructions for using the type of application described here. For example, the container may contain an antibody into which

- 46 040898 a detectable label has been entered or can be entered. Kits may include a container containing a drug unit. The set may include all or part of the amino acid sequences from FIG. 2 or 3 or their analogues or nucleic acid molecules encoding such amino acid sequences.

Typically, the kit according to the invention includes the container described above and one or more associated other containers (containers) containing materials desired from a commercial and consumer point of view, including buffers, diluents, filters, needles, syringes; media, packaging, containers, vials and / or tubes with labels showing their contents and / or instructions for use, and packaging inserts with instructions for use.

The label may be on or with the container and indicate that the composition is for specific therapy or non-therapeutic use, such as prognostic, prophylactic, diagnostic or laboratory use, and may also contain instructions for in vivo or in vitro use such as those that are described here. Instructions and/or other information may also be included on inserts or labels included with or on the kit itself. The label may be on or associated with the container. A label is on a container if the letters, numbers or other symbols that make up the label are molded or engraved on the container itself; a label is associated with a container if it is in a receptacle or carrier that also holds the container, for example in the form of a package insert. The label may indicate that the composition is used for the diagnosis, treatment, prevention or prognosis of a disease of the type of tissue cancer shown in Table I.

The terms kit and manufactured product may be used interchangeably.

In another embodiment of the invention, articles of manufacture are provided containing compositions such as antibodies or antibody-drug conjugates (ADCs), such as materials useful in the diagnosis, prognosis, prevention and/or treatment of tissue cancers of the type shown in Table 1. I. Typically, an article of manufacture includes at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes and test tubes. Containers can be made from various materials such as glass, metal or plastic. The container may contain amino acid sequences, small molecules, nucleic acid sequences, cell populations, and/or antibodies. In another embodiment, the container contains an antibody, its binding fragment or a specific binding protein for use in assessing the expression of the 158P1D7 protein in cells and tissues, or for appropriate laboratory, prognostic, diagnostic, prophylactic and therapeutic purposes; indications and/or instructions for such applications, as well as reagents and other compositions or means used for these purposes, may be on or with the container.

On the other hand, the container may contain a composition that is effective in treating, diagnosing, predicting or preventing a disease and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that is pierced by a hypodermic needle) . The active substances in the composition can be antibodies capable of specifically binding to 158P1D7, or antibody drug conjugates that specifically bind to 158P1D7.

The article of manufacture may also include a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution and/or dextrose solution. It may also include other materials desired from a commercial and consumer point of view, including other buffers, diluents, filters, stirrers, needles, syringes and/or package inserts with directions and/or instructions for use.

Examples

Various aspects of the invention are described in more detail and illustrated in the following few examples, none of which should limit the scope of the invention.

Example 1 Antigen 158P1D7.

The 158P1D7 gene sequence was discovered by suppressive subtractive hybridization (SSH) techniques known in the art. SSH sequence 158P1D7 in 223 bp was identified by subtracting from the bladder cancer pool minus normal bladder cDNA. A full-length cDNA clone for 158P1D7 was isolated from a pool of bladder cancer tissues. The length of the cDNA is 2555 bp. and it encodes an 841 amino acid ORF (see FIG. 1). The 158P1D7 gene shows homology with the SLITRK6 gene. For further references see US 6,863,892 (Agensys, Inc., Santa Monica, CA), US 7,358,353 (Agensys, Inc., Santa Monica, CA), and EP 1,311,675 (Agensys, Inc., Santa Monica, CA). Representative variants of the 158P1D7 antigen are shown in FIG. 1.

Example 2. Obtaining monoclonal antibodies (mAbs) to 158P1D7.

In one embodiment, the therapeutic monoclonal antibodies (mAbs) to 158P1D7 and 158P1D7 variants include antibodies that react with epitopes specific to each protein or specific to sequences shared between variants that are to bind, internalize, disrupt, or modulate the biological function of 158P1D7 or 158P1D7 variants. ,

- 47 040898 for example antibodies, which should disrupt the interaction with ligands, substrates and binding partners. Immunogens for making such mAbs include those designed to encode or contain extracellular domains or the entire 158P1D7 protein sequence, regions suspected of containing functional motifs, and regions of 158P1D7 protein variants suspected to be antigenic by amino acid sequence computer analysis. The immunogens include peptides and recombinant proteins of the tag5-158PlD7 type, a mammalian-derived purified His-tagged protein. In addition, cells engineered for high expression of 158P1D7, such as UMUC-158P1D7 or 3T3158P1D7, are used to immunize mice.

MAbs to 158P1D7 were generated by XenoMouse® technology (Amgem Fremont) in which the mouse kappa heavy and light chain loci were inactivated and most of the human immunoglobulin kappa heavy and light chain loci were inserted. The MAb designated Ha15-10ac12 was generated by immunizing human γ2 producing xeno mice with recombinant 3T3 cells expressing 158P1D7.

The anti-158P1D7 MAb Ha15-10ac12 binds specifically to 158P1D7 expressing cells (recombinant and endogenous) as well as to the recombinant 158P1D7 protein by ELISA.

The DNA coding sequences for the anti-158P1D7 mAb Ha15-10ac12 were determined after mRNA isolation from the respective hybridoma cells using the Trizol reagent (Life Technologies, Gibco BRL).

The variable heavy and light chain nucleic acid sequences of the anti-158P1D7 antibody Ha1510ac12 from hybridoma cells were sequenced by the following method. Ha15-10ac12 secreting hybridoma cells were lysed with Trizol reagent (Life Technologies, Gibco BRL). Total RNA was isolated and quantified. From the total RNA, the first strand of cDNA was prepared with primers oligo(dT)12-18 using the Gibco-BRL Superscript Preamplification system. The single strand cDNA was amplified with primers for the heavy chain variable region and primers for the human immunoglobulin variable light chain. The PCR products were sequenced and the heavy and light chain variable regions were determined.

The nucleotide and amino acid sequences of the heavy and light chain variable regions are shown in FIG. 2 and 3. The alignment of mAb Ha15-10ac12 with gametic human Ig is shown in FIG. 4A-4B.

Example 3 Ha15-10ac12 expression by recombinant DNA methods.

For recombinant expression of mAb Ha15-10ac12 in transfected cells, mAb Ha15-10ac12 heavy and light chain variable region sequences were cloned forward of the human IgG2 heavy chain and human IgK light chain constant regions, respectively. The complete heavy chain and light chain cassettes of the human mAb Ha15-10ac12 were cloned behind the CMV promoter/enhancer in the cloning vector. A polyadenylation site was inserted behind the mAb coding sequence. Constructs expressing the recombinant Ha15-10ac12 mAb were transfected into CHO-K1SV cells. Related Ha15-10ac12 mAbs secreted from recombinant CHO cells were evaluated for cell surface binding to 158P1D7 by flow cytometry. Control UMUC cells and UMUC-158P1D7 cells were stained with Ha15-10ac12 mAb from hybridoma cells or from CHO cells transfected with Ha15-10ac12 heavy and light chain vector constructs. Binding was detected by flow cytometry.

The results show that the recombinantly expressed Ha15-10ac12 mAb, when expressed in CHO cells, binds to 158P1D7 in a manner similar to Ha15-10ac12 isolated from hybridoma. MAb Ha1510ac12 secreted from recombinant cells was also evaluated for binding to the recombinant 158P1D7 protein by ELISA. The binding of Ha15-10ac12 to the 158P1D7 protein was identical between the mAb material derived from CHO cells and from hybridoma cells.

Chinese hamster ovary (CHO) cells producing an antibody called Ha1510ac12 were shipped July 25, 2012 (via Federal Express) to the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, and received access number PTA-13102.

Example 4 Drug Conjugation of mAb Ha15-10ac12.

Mab Ha15-10ac12 (FIG. 2) was conjugated to an auristatin derivative named MMAE (Formula XI) using the vc linker (Val-Cit) described here to give an antibody-drug conjugate (ADC) of the invention, designated Ha15-10ac12vcMMAE, according to the following methodology. Conjugation of the vc linker (Val-Cit) with MMAE (Seattle Genetics, Seattle, WA) was performed by the General method shown in table. IV, receiving cytotoxic vcMMAE (see US/2006/0074008).

An antibody-drug conjugate (ADC) of the invention, named Ha15-10ac12vcMMAE, was then prepared according to the following procedure.

Briefly, mAb Ha15-10ac12 in composition buffer (10 mM acetate pH 5.0 with 5% sorbitol) was buffer exchanged with reconstitution buffer (25 mM sodium borate, 300 mM sodium chloride, pH 9.0 ± 0.1). mAb Ha15-10ac12 was then partially reduced by adding 5 mM EDTA and

- 48 040898

2.65 TCEP molar equivalents (relative to moles of mAb Ha15-10ac12). This mixture was then stirred at 37° C. for 3 (three) hours. After reduction of the disulfides, the mixture was cooled to the desired temperature of 15-17° C. and five (5) equivalents of vcMMAE drug per 1 mol as 4% (vol. /v) solution in DMSO. After 60-75 minutes, excess unreacted vc linker was quenched by adding 1 mole of N-acetyl-L-cysteine per mole of vcMMAE added at the start of conjugation. After 15 minutes, Ha15-10ac12vcMMAE was adjusted to pH 6.0-6.4 with concentrated histidine buffer pH 5.2 and filtered through a 0.5/0.2 μm PES membrane to remove the aggregated antibody-drug conjugate. Immediately after filtration, tangential filtration was performed to remove DMSO and the quenched linker-drug conjugate and to buffer the antibody-drug conjugate with 20 mM histidine (pH 6.0±0.1) containing 5.5% anhydrous trehalose. After tangential filtration, Ha15-10ac12vcMMAE was diluted to a final concentration of 6±1 mg/mL and polysorbate 20 was added to a final concentration of 0.01%.

The resulting antibody drug conjugate (ADC) is designated Ha15-10ac12vcMMAE and has the following formula in this example, is from 3.5 to 3.7.

Example 5 Characterization of Ha15-10ac12vcMMAE.

Antibody-drug conjugates that bind to 158P1D7 were prepared using the procedure outlined in the example titled Drug Conjugation of mAb Ha15-10ac12, screened, identified, and characterized using a combination of methods known in the art.

A. Affinity determination by FACS method.

Ha15-10ac12vcMMAE was tested for binding affinity to 158P1D7 expressed on the surface of SW780 cells. Briefly, eleven (11) dilutions of Ha15-10ac12vcMMAE were incubated with SW780 cells (50,000 cells per well) overnight at 4° C. at a final concentration of 6.67 to 0.0001 nM. At the end of the incubation, the cells were washed and incubated with detecting antibody against hIgG-PE for 45 min at 4°C. After washing off the unbound detection antibodies, the cells were analyzed by FACS. Received the values of the average fluorescence intensity (MFI), shown in table. VI. The MFI values were entered into the Graphpad Prism program and analyzed by the single site (hyperbole) equation Y=B _max xX/(K _d +X) to give the Ha1510ac12vcMMAE saturation curve shown in FIG. 13. B _max - MFI value at maximum binding of Ha1510ac12vcMMAE to 158P1D7; Kd is the binding affinity of Ha15-10ac12vcMMAE, which is the concentration of Ha15-10ac12vcMMAE required to achieve half-maximal binding.

The calculated affinity (Kd) for binding of Ha15-10ac12vcMMAE to 158P1D7 expressed on the surface of SW780 cells is 0.005 nM.

B. Binding by FACS data.

Ha15-10ac12vcMMAE was tested by FACS for binding to 158P1D7 expressed on the surface of UMUC, SW780 and TEC-212 cells. Briefly, cells were harvested and seeded at a concentration of 50,000 cells per well. Ha15-10ac12vcMMAE was diluted to 3 μg/ml (for UMUC and SW780 cells) or 10 μg/ml (for CHP-212 cells) and incubated with cells (1 h at 4°C). At the end of the incubation, the cells were washed and incubated with detecting antibody against hIgG-PE for 1 h at 4°C. After washing off the unbound detection antibodies, the cells were analyzed by FACS. The obtained mean fluorescence intensity (MFI) values (Table VII) and histograms (FIG. 14) are shown.

Example 6 Ha15-10ac12vcMMAE mediated cellular cytotoxicity in vitro.

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

Briefly, CHP-212 and IGROV-1 cells were plated in 50 μl of complete medium at a density of 1000 cells per well in 96-well plates and placed in a tissue culture incubator at 37° C., 5% CO ₂ . The following day, a stock solution of 2x Ha15-10ac12vcMMAE and isotype control antibody conjugated to vcMMAE in complete medium was prepared and 50 μl serial dilutions of ADCs were added to the appropriate wells. Cells were treated with Ha15-10ac12vcMMAE and isotype control antibody conjugated with vcMMAE for six (6) days in a tissue culture incubator at 37°C, 5%

CO2. At the end of the incubation period, 12 µl of Alamar Blue or Presto Blue was added to each well and incubated for four (4) hours. The plates were read on a BioTek Synergy H4 reader at 540 nm excitation and 590 nm emission.

These data show that the ADC named Ha15-10ac12vcMMAE can selectively induce cytotoxicity in the SLITRK-6 expressing CHP-212 cell line, but is unable to induce cytotoxicity in the IGROV-1 cell line that does not express SLITRK-6. Thus, these results indicate that Ha15-10ac12vcMMAE can selectively deliver a cytotoxic drug to cells expressing 158P1D7, resulting in their death (Fig. 15).

In another experiment, human neuroblastoma cells, CHP-212, which express SLITRK6 (target for mAb Ha15-10ac12 and mAb M15-68(2)18) (aka 68(18)1.1, see WO 2004/072263)), were incubated with investigational devices, Ha15-10ac12vcMMAE and M15-68(2)18, to demonstrate their in vitro cell death-inducing activity (cytotoxicity). Briefly, 4000 CHP-212 cells/mL were plated in 96-well plates and test antibodies were added at serial dilutions from 10,000 ng/mL to 0.006 ng/mL using 6-fold dilutions at 9 points plus 0.0 ng/mL in the last hole. The wells were placed in three repetitions. In addition, control antibodies and control cells (IGROV-1) were set up in the same way for comparison with test products. Incubation was carried out for 4 days, after which Presto Blue (a pigment that stains living cells) was added to the wells to obtain a colorimetric index of cell viability. Cell viability in each well was then calculated and the data analyzed using a non-linear sigmoid dose-response fit. IC ₅₀ values were calculated using the Graphpad Prism program and the cytotoxic effects of Ha15-10ac12vcMMAE were compared with M15-68(2)18 and other controls used in this experiment.

The results show that Ha15-10ac12vcMMAE has greater in vitro cytotoxicity compared to the M15-68(2)18 antibody and other controls (FIG. 11). The IC ₅₀ value for Ha1510ac12vcMMAE was 0.9076 and the IC ₅₀ for M15-68(2)18 could not be calculated.

Example 7 Ha15-10ac12vcMMAE inhibits tumor growth in vivo.

Significant expression of 158P1D7 on the cell surface of tumor tissues, together with its limited expression in normal tissues, makes 158P1D7 a good target for antibody therapy as well as ADC therapy. Therefore, the therapeutic efficacy of Ha1510ac12vcMMAE was evaluated in xenograft models of human bladder, lung, breast, and glioblastoma cancers in mice.

The effect of the antibody-drug conjugate on tumor growth and metastasis formation was studied in mouse cancer xenograft models (eg, subcutaneous and orthotopic).

Subcutaneous (sc) tumors were obtained by injecting 5×10 ⁴ -10 ⁶ cancer cells mixed 1:1 with Matrigel (Collaborative Research) into the right flank of male SCID mice. To test the effect of ADC on tumor formation, ADC injections were started on the same day as tumor cell injections. As controls, mice were injected with either purified human IgG or PBS, or a purified mAb that recognizes a foreign antigen not expressed in human cells. In preliminary experiments, no difference was found between the effect of control IgG or PBS on tumor growth. Tumor sizes were determined using calipers and tumor volume was calculated as width ² χ length/2, with width being the smallest size and length being the largest. Mice with subcutaneous tumors larger than 1.5 cm in diameter were sacrificed.

The advantage of cancer xenograft models is the ability to study neovascularization and angiogenesis. The growth of tumors depends in part on the formation of new blood vessels. Although the capillary system and the resulting circulatory network originate from the host organism, the initiation and architecture of the new vasculature is regulated by the tumor xenograft (Davidoff et al., Clin. Cancer Res. (2001) 7:2870; Solesvik et al., Eur. J. Cancer Clin Oncol (1984) 20:1295). The effect of antibodies and small molecules on neovascularization was investigated according to known methods such as IHC analysis of tumor tissues and their microenvironment.

ADCs to 158P1D7.

Monoclonal antibodies against 158P1D7 were prepared as described in the example entitled Preparation of monoclonal antibodies (mAbs) against 158P1D7. The mAbs were further conjugated to the toxin as described in the example titled Drug Conjugation of the mAb Ha15-10ac12 Antibody, to give Ha15-10ac12vcMMAE. Ha15-10ac12vcMMAE was characterized by FACS and other known methods to determine its ability to bind 158P1D7.

Cell lines and xenografts.

Cells were maintained in DMEM supplemented with L-glutamine and 10% FBS, as is known in the art. AG-B7, RT-4-XCL and NCI-H322M-XCL xenografts were maintained by serial propagation in SCID mice. The SW780 and RT-4-XCL cells are derived from a urinary cancer cell line.

- 50 040898 bubbles obtained from ATCC (Manassas, VA). AG-B7 and AG-B8 xenografts are derived from human bladder cancer specimens obtained from patients. Those skilled in the art will appreciate that many in vivo experiments have been performed on the ADCs of the invention. This is partly due to the fact that each in vivo model, even if it refers to the same disease, exhibits a certain degree of unpredictability for specialists. Therefore, those skilled in the art will appreciate that the use of several types of in vivo models will allow those skilled in the art to better evaluate the ADCs of the invention.

Evaluation of Ha15-10ac12vcMMAE in a SCID xenograft AG-B7 xenograft model of human bladder cancer in SCID mice.

In this experiment, human bladder cancer AG-B7 cells (5×10 ⁶ cells/mouse) were injected into the flank of individual SCID mice and the tumors were allowed to grow without treatment until they reached a volume of approximately 250 mm ³ . At this point, animals were divided into groups based on tumor volume at the start of treatment to ensure the same average tumor size and variability within each group using the Study Director software (v. 1.7; Studylog Systems, Inc., South San Francisco, CA ). All ADC-treated groups received a single dose of 10 mg/kg by intravenous bolus injection. Tumor growth in each group was monitored twice a week by caliper measurements until the end of the study. Statistical analysis of tumor volume was performed at the last time point, when data were collected from all groups, by non-parametric analysis of variance (ANOVA) on ranked data.

The results show that Ha15-10ac12vcMMAE exhibits a strong inhibitory effect compared to the untreated control (p<0.0001) (FIG. 5). RT-4-XCL.

Evaluation of Ha15-10ac12vcMMAE in a SCID xenograft RT-4-XCL xenograft model of human bladder cancer in SCID mice.

In another experiment, parental tumors were harvested from AG-87 human bladder cancer xenografts under sterile conditions and minced into small pieces (approximately 1 mm ³ ). 6 pieces were implanted subcutaneously in the flank of individual SCID mice. When the mean tumor volume reached a target size of 100 mm ³ , animals were randomly divided into ADC-treated groups and an untreated control group (see Tumor Volume Graph) with similar mean tumor size and variability within each group using Study Director (v. 1.7; Studylog Systems, Inc., South San Francisco, CA). All ADC-treated groups, including two control ADCs, received a single dose of 5 mg/kg by intravenous bolus injection. Tumor growth in each group was monitored twice a week by caliper measurements until the end of the study. Statistical analysis of tumor volume was performed at the last time point, when data were collected from all groups, by non-parametric analysis of variance (ANOVA) on ranked data.

The results show that Ha15-10ac12vcMMAE exhibited a strong tumor inhibitory effect compared to the untreated control or the corresponding control ADC - H31.4.1.2vcMMAE (both p<0.0001) (FIG. 6).

Evaluation of Ha15-10ac12vcMMAE in the NCI-H322MXCL subcutaneous xenograft xenograft model of human lung cancer in SCID mice

In another experiment, human lung cancer cells NCI-H322M-XCL (2.5×10 ⁶ cells per mouse) were injected flankally into individual SCID mice and the tumors were allowed to grow without treatment until they reached a volume of about 200 mm ³ . At this point, animals were divided into groups based on tumor volume at the start of treatment to ensure the same average tumor size and variability within each group using the Study Director software (v. 1.7; Studylog Systems, Inc., South San Francisco, CA ). All ADC treated groups, including two control ADCs, received doses of 3 mg/kg twice a week for a total of 5 doses via intravenous bolus injection. Tumor growth in each group was monitored twice a week by caliper measurements until the end of the study. Statistical analysis of tumor volume was performed at the last time point, when data were collected from all groups, by non-parametric analysis of variance (ANOVA) on ranked data.

The results show that Ha15-10ac12vcMMAE exhibited a strong inhibitory effect compared to the vehicle control (p<0.0001) or the corresponding control ADC - Ha312bc1.1vcMMAE (p=0.0041) (FIG. 7).

Efficacy of Ha15-10ac12vcMMAE in a SCID mouse AG-B7 xenograft xenograft model of human bladder cancer

In another experiment, parental tumors were harvested from AG-37 human bladder cancer xenografts under sterile conditions and minced into small pieces (approximately 1 mm ³ ). 5 pieces were implanted subcutaneously in the flank of individual SCID mice. The tumors were allowed to grow without treatment until they reached a volume of about 200 mm ³ . At this point, the animals were divided into groups depending on the volume of tumors at the time of treatment in order to ensure the same average size.

- 51 040898 tumors and variability within each group using the Study Director software (v.1.7; Studylog Systems, Inc., South San Francisco, CA). All ADC treated groups, including the two control ADCs, received doses of 0.5 mg/kg twice a week for a total of 7 doses via intravenous bolus injection. Tumor growth in each group was monitored twice a week by caliper measurements until the end of the study. Statistical analysis of tumor volume was performed at the last time point, when data were collected from all groups, by non-parametric analysis of variance (ANOVA) on ranked data.

The results show that Ha15-10ac12vcMMAE derived from both hybridoma (Ha1510ac12.1vcMMAE) and CHO cells (Ha15-10ac12vcMMAE) exhibited a strong tumor inhibitory effect compared to the vehicle control (p<0.0001) or the corresponding control ADC - Ha3-12abc1vcMMAE (p<0.0001) (Fig. 8).

Efficacy of Ha15-10ac12vcMMAE in SW780 subcutaneously grafted human bladder cancer xenograft model in SCID mice.

In another experiment, human bladder cancer xenograft SW780 cells were implanted in the flank of SCID mice and the tumors were allowed to grow until they reached a volume of approximately 200 mm ³ . At this point, the animals were divided into experimental groups depending on the volume of tumors at the time of treatment in order to ensure the same average tumor size and variability in each group. The division of mice into experimental groups was carried out using the Study Director software (v.1.7; Studylog Systems, Inc., South San Francisco, CA) so that the size of the tumors corresponded to mice. All ADC-treated groups, including two control ADCs, received doses of 1 mg/kg by intravenous bolus injection at baseline. Tumor growth in each group was monitored twice a week by caliper measurements until the end of the study. Statistical analysis of tumor volume was performed at the last time point, when data were collected from all groups, by non-parametric analysis of variance (ANOVA) on ranked data.

The results show that Ha15-10ac12vcMMAE has greater tumor inhibitory activity compared to the Ha15-10ac12 mAb. In addition, it can be concluded that mAb Ha15-10ac12 does not have an inhibitory effect on tumors, since the dynamics of tumor growth in this experimental group follows that of isotype control antibodies. In addition, after a single dose of 1 mg/kg, Ha1510ac12vcMMAE exhibited statistically significant growth inhibition compared to the isotype control ADC (p<0.001) (FIG. 10).

Efficacy of Ha15-10ac12vcMMAE in a grafted subcutaneous patient-derived xenograft AG-B8 human bladder cancer model in SCID mice.

In another experiment, patient-derived pieces of human bladder cancer AG-B8 tumors were implanted in the flank of SCID mice and the tumors were allowed to grow until they reached a volume of approximately 200 mm ³ . At this point, the animals were divided into experimental groups depending on the volume of tumors at the time of treatment in order to ensure the same average tumor size and variability in each group. The division of mice into experimental groups was carried out using the Study Director software (v.1.7; Studylog Systems, Inc., South San Francisco, CA) so that the size of the tumors corresponded to mice. All ADC-treated groups, including two control ADCs, received doses of 5 mg/kg by intravenous bolus injection at baseline (day 0). Tumor growth in each group was monitored twice a week by caliper measurements until the end of the study. Statistical analysis of tumor volume was performed at the last time point, when data were collected from all groups, by non-parametric analysis of variance (ANOVA) on ranked data.

The results show that Ha15-10ac12vcMMAE has greater tumor inhibitory activity than the untreated control (p<0.0001) or other controls corresponding to ADC gamma2 (p<0.0001). In addition, Ha15ac12vcMMAE showed a higher statistically significant effect compared to Ha15-10ac12mcMMAF (p<0.0458) (Fig. 12).

Conclusion.

So, from FIG. 5-8, 10 and 12 show that the ADC to 158P1D7 called Ha15-10ac12vcMMAE significantly inhibits the growth of tumor cells expressing 158P1D7 compared to control ADCs. Thus, Ha15-10ac12vcMMAE can be used therapeutically to treat and control the cancers shown in Table 1. I. In particular, these results indicate that Ha15-10ac12vcMMAE has an inhibitory effect on various types of bladder cancer models, suggesting that it may be of particular therapeutic utility in the treatment of bladder cancer.

Example 8 Human clinical trials for the treatment and diagnosis of human carcinoma using ADCs to 158P1D7.

In accordance with the present invention, ADCs to 158P1D7, which specifically bind to 158P1D7, can be used in the treatment of certain tumors, preferably those listed in table. I. For each of these indications, two clinical approaches have been successfully applied.

I. Adjuvant therapy. In adjuvant therapy, patients are treated with ADCs to 158P1D7 in concomitant

- 52 040898 in combination with a chemotherapeutic or antitumor agent and/or radiation therapy, or a combination thereof. Primary cancer targets such as those shown in Table. I, are treated according to standard protocol by adding ADCs to 158P1D7 to standard first and second line therapy. Methods are designed with an emphasis on efficacy, as evidenced by the following examples, including, but not limited to, weight reduction in primary or metastatic tumors, improved progression-free survival, overall survival, improved patient health, disease stabilization, and the possibility of reducing the usual doses of standard chemotherapy agents and other biological agents. Such dosage reductions allow additional and/or prolonged therapy by reducing the dose-related toxicity of the chemotherapeutic or biological agent. ADCs to 158P1D7 have been used in several ancillary clinical trials in combination with chemotherapeutic or anticancer agents.

II. Monotherapy. In connection with the use of ADCs to 158P1D7 in tumor monotherapy, they are administered to patients without chemotherapeutic or antitumor agents. In one embodiment, the monotherapy is administered in the clinic in end-stage cancer patients with extensive metastatic disease. Methods are designed with an emphasis on efficacy, as evidenced by the following examples, including, but not limited to, weight reduction in primary or metastatic tumors, improved progression-free survival, overall survival, improved patient health, disease stabilization, and the possibility of reducing the usual doses of standard chemotherapy agents and other biological agents.

Dosage.

Dosing regimens can be adjusted to provide the optimum desired response. For example, one may administer as a single bolus, several divided doses over time, or one may decrease or increase the dose as required by the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and dosage uniformity. Dosage unit form in the present invention means physically discrete units suitable as a unit of dose for mammalian subjects to be treated; each unit contains a predetermined amount of active compound calculated to provide the desired therapeutic effect in combination with the desired pharmaceutical carrier. Specifications for unit dosage forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the antibody and/or ADC and the particular therapeutic or prophylactic effect achieved, and (b) existing limitations in formulating such active compounds for the treatment of susceptibility in individuals.

A typical, without limitation, range of therapeutically effective amount of anti-158P1D7 ADC when administered in combinations of the invention is 0.5 to 10 mg/kg, 1 to 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 typical, non-limiting ranges are, for example, 0.5 to 5 mg/kg, or 0.8 to 5 mg/kg, or 1 to 7.5 mg/kg. High dose embodiments of the invention relate to dosages greater than 10 mg/kg. It should be noted that the dosage may vary depending on the type and severity of the disease being treated and may include single or multiple doses. It should also be appreciated that for any given subject, certain dosage regimens will need to be adjusted over time according to individual needs and the professional judgment of the prescriber or supervisor of administration of the compositions, and the dosage ranges given herein are only exemplary and are not intended to be limiting. scope or practical application of the claimed composition.

Clinical Development Plan (CDP).

CDP is following and developing treatments with ADCs to 158P1D7 in conjunction with adjuvant therapy or monotherapy. Trials first demonstrate safety and then confirm efficacy at repeated doses. The trials are open and compare standard chemotherapy with standard therapy plus anti-158P1D7 ADCs. As should be known, one of the non-restrictive criteria that can be applied in connection with enrollment of patients is the level of expression of 158P1D7 in tumors, which is determined by biopsy.

As with any protein or antibody infusion therapy, safety concerns are mainly associated with (i) cytokine release syndrome, i.e. hypotension, fever, trembling, chills; (ii) the occurrence of an immune response to the material (ie, the appearance in the patient of human antibodies to the therapeutic antibody, or a HAMA response); and (iii) toxicity to normal cells expressing 158P1D7. Standard tests and follow-up are used to monitor each of these security issues. ADCs to 158P1D7 have been shown to be safe when administered to humans.

Example 9 IHC Detection of 158P1D7 Protein in Cancer Patient Samples.

Expression of the 158P1D7 protein was tested by immunohistochemistry in patient tumor samples from (i) bladder, (ii) breast, (iii) lung, and (iv) glioblastoma cancer patients. Briefly, formalin-fixed and solid paraffin-embedded tissues were cut into sections 4

- 53 040898 microns and mounted on glass slides. Sections were deparaffinized, rehydrated and treated with Citra Antigen Recovery Solution (Biogenex, San Ramon, CA) in an EZRetriever (Biogenex, San Ramon, CA) microwave for 15 min at 95°C. The sections were then treated with 3% hydrogen peroxide to inactivate endogenous peroxidase activity. Serum-free blocking protein (Dako, Carpenteria, CA) was used to inhibit non-specific binding prior to incubation with mouse anti-158P1D7 monoclonal antibody or isotope control. Sections were then processed with the Super Sensitive™ Polymer horseradish peroxidase (HRP) detection system, which consists of incubation in Super Enhancer™ reagent followed by incubation with a polymer-HRP-secondary antibody conjugate (BioGenex, San Ramon, CA). The sections were then developed with the DAB kit (BioGenex, San Ramon, CA). The nuclei were stained with hematoxylin and analyzed by bright field microscopy. Specific staining in patient samples was detected with an immunoreactive antibody to 158P1D7, brown (see FIGS. 9A, 9C, 9E and 9G). In contrast, the control antibody did not stain any of the patient samples (see FIGS. 9B, 9D, 9F and 9H).

The results indicate the expression of 158P1D7 in tumor cells of patients with bladder, breast, lung, and glioblastoma cancers. These results indicate that 158P1D7 is expressed in human cancer tissues, and antibodies against this antigen and an antibody-drug conjugate called Ha15-10ac12vcMMAE are useful for diagnostic and therapeutic purposes (FIG. 9).

Links are provided throughout the application to data websites with various content, publications, patent applications and patents (websites are identified by Uniform Resource Locator addresses, ie URLs, on the World Wide Web). The contents of each of these references are incorporated herein by reference in their entirety.

The present invention should not be limited in scope to the embodiments disclosed herein, which serve merely as illustrations of certain aspects of the invention, and their functional equivalents are within the scope of the invention. Various modifications of the models and methods of the invention, in addition to those described here, should be obvious to experts in this field from the foregoing description and its provisions, and should also be included in the scope of the invention. Such modifications or other embodiments may be practiced without departing from the true scope and spirit of the invention.

Tables.

Table I. Tissues Expressing 158P1D7 in Glioblastoma Cancers

Lungs

Bladder

Breast

Table II. Abbreviations for amino acids

Single letter three-letter Full title F Phe phenylalanine L Leu leucine S Ser serine Y Tugh tyrosine With Cys cysteine W trp tryptophan R Pro proline n His histidine Q gin glutamine R Arg arginine I He isoleucine m Met methionine T Thr threonine N Asn asparagine TO Lys lysine V Vai valine A Ala alanine D asp aspartic acid E Glu glutamic acid G gly glycine

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Table III. Amino Acid Substitution Matrix (Adapted from Amino Acid Substitution Matrix (block substitution matrix) BLOSUM62 of GCG Software 9.0.

The higher the value, the more likely that this substitution occurs in related natural proteins)

Table IV. General method for synthesizing vcMMAE

- AAi-Dil-Dap-AA ₅

PINKER - -' -AΑί-DII -Dap -AAs where: AAl = amino acid 1

AA2 = amino acid 2

AA5 = amino acid 5

DIL - dolaisolein DAP = dolaproin Linker = Val-Cit (vc)

Table V. Positions of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as established by the Kabat, Chothia and Contact schemes, respectively. For CDR-H1, residue numbering is given according to the numbering system of both Kabat and Chothia

CDR Kabat Chothia contact CDR-L1 L24-L34 L24-L34 L30-L36 CDR-L2 L50-L56 L50-L56 L46-L55 CDR-L3 L89-L97 L89-L97 L89-L96 CDR-H1 (Kabat ¹ numbering) H31-H35B H26-H32..34 H30-H35B CDR-H1 (Chothia ² numbering) H31-H35 H26-H32 H30-H35 CDR-H2 H50-H65 H52-H56 H47-H58 CDR-H3 H95-H102 H95-H102 H93-H101

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Claims (31)

Table VI. Mean fluorescence intensity (MFI) values from FACS nM Hal5-10acl2vcMMAE 6.667 155.0 2.222 149.6 0.741 140.9 0.247 133.8 0.082 124.0 0.027 107.6 0.0091 87.0 0.0030 60.8 0.0010 25.7 0.0003 14.9 0.0001 4.3 Table VII. MFI values for Ha15-10ac12vcMMAE binding from FACS data UMUC-AGS15 SW780 SNR-212 MFI 115 159 142 CLAIM 1. An antibody-drug conjugate comprising an anti-158P1D7 antibody or antigen-binding fragment thereof, which is conjugated to monomethylauristatin E (MMAE), wherein the antibody or fragment thereof includes a heavy chain variable region containing an amino acid sequence of at least 80% homologous to the sequence heavy chain variable region set forth 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 sequence set forth in SEQ ID NO: 8, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing complementarity determining regions (CDRs) containing the amino acid sequences of CDRs from the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 7, and a light chain variable region containing CDRs containing amino acid sequences the CDR sequence from the light chain variable region amino acid sequence shown in SEQ ID NO: 8. 2. The antibody-drug conjugate of claim 1, wherein the antibody or fragment thereof comprises a heavy chain variable region containing an amino acid sequence of at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 , 95, 96, 97, 98, or 99% homologous to the heavy chain variable region sequence shown in SEQ ID NO: 7 and a light chain variable region containing an amino acid sequence of at least 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the light chain variable region sequence shown in SEQ ID NO: 8. 3. An antibody-drug conjugate comprising an anti-158P1D7 antibody or an antigen-binding fragment thereof, which is conjugated to monomethylauristatin E (MMAE), wherein the antibody or fragment thereof comprises a heavy chain variable region containing an amino acid sequence of at least 80% homology to the variable sequence. heavy chain region of an antibody produced by Chinese Hamster Ovary (CHO) cells deposited with the American Type Culture Collection (ATCC) under accession number PTA-13102, and a light chain variable region containing an amino acid sequence of at least 80% homologous to the light chain variable region sequence. chain of an antibody produced by Chinese hamster ovary (CHO) cells deposited with ATCC under accession number PTA-13102, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing complementarity-determining regions (CDRs) containing amino acidic CDR sequences from the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region containing CDRs containing CDR amino acid sequences from the light chain variable region amino acid sequence set forth in SEQ ID NO: 8. 4. The antibody-drug conjugate of claim 3, wherein the antibody or fragment thereof comprises a heavy chain variable region containing an amino acid sequence of at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 , 95, 96, 97, 98, or 99% homologous to the heavy chain variable region sequence of an antibody produced by Chinese hamster ovary cells - 56 040898 (CHO) deposited with ATCC under accession number PTA-13102, and a light chain variable region containing an amino acid sequence of at least 85, 86, 87, 88, 89, 90, 91, 92, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the light chain variable region sequence of an antibody produced by Chinese Hamster Ovary (CHO) cells deposited with ATCC under accession number PTA-13102. 5. An antibody-drug conjugate according to any one of claims 1 to 4, wherein the antibody or fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 from the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 from the amino acid sequence shown in SEQ ID NO: 8, wherein the CDRs are defined according to the Kabat, Chotia or Contact numbering system, preferably wherein the antibody or its antigen-binding fragment includes a heavy chain variable region containing a CDR-H1 consisting of an amino acid sequence ranging from 31 to 35 of SEQ ID NO: 7, a CDR-H2 consisting of an amino acid sequence ranging from 50 to 66 of SEQ ID NO: 7, and a CDR-H3 consisting of an amino acid sequence ranging from 99 to 109 of SEQ ID NO: 7 and a light chain variable region containing a CDR-L1 consisting of an amino acid sequence ranging from 24 d o 39 SEQ ID NO: 8, CDR-L2 consisting of amino acid sequences ranging from 55 to 61 of SEQ ID NO: 8, and CDR-L3 consisting of amino acid sequences ranging from 94 to 102 of SEQ ID NO: 8. 6. The antibody-drug conjugate of any one of claims 1 to 5, wherein the antibody further comprises a heavy chain constant region and a human IgG light chain constant region. 7. An antibody-drug conjugate according to any one of claims 1-6, wherein the antibody is: (i) a fully human antibody; or (ii) an antibody produced by a recombinant method. 8. An antibody-drug conjugate according to any one of claims 1 to 7, wherein the fragment is a Fab, F(ab')2, Fv or scFv fragment. 9. The antibody-drug conjugate of any one of claims 1 to 8, wherein the antibody or fragment thereof is conjugated to MMAE via a linker unit. 10. The antibody-drug conjugate of claim 9, wherein the linker unit is an enzyme-cleavable peptidyl linker, preferably wherein the linker unit forms a bond to a sulfur atom in the antibody or fragment thereof. 11. An antibody drug conjugate according to any one of claims 1 to 10, wherein the antibody drug conjugate has the following formula L-(LU-D) _P where L is an anti-158P1D7 antibody or fragment thereof as disclosed in any one of claims 1-10; (LU-D) is a linker unit-drug unit, where LU- is a linker unit, -D stands for MMAE; and p is an integer from 1 to 20, preferably where p is in the range from 1 to 10, or where p is in the range from 1 to 5. 12. The antibody-drug conjugate according to any one of claims 9-11, wherein the linker unit has the following formula Aa-Ww-Yywhere -A- means extension link, a is 0 or 1, W- means an amino acid unit, w is an integer from 0 to 12, -Y- means a self-destructing spacer link, y is 0, 1 or 2; preferably wherein the amino acid unit comprises valine-citrulline (Val-Cit). 13. The antibody-drug conjugate according to any one of claims 11 or 12, wherein the extension unit is represented by the structure of formula IIIa formula Xa and the amino acid unit is valine-citrulline (Val-Cit), and the spacer unit is represented by the structure of formula Xa - 57 040898 formula Xa, preferably wherein the extension unit is bonded to the sulfur atom of the antibody or fragment thereof, where the spacer unit is a p-aminobenzyl alcohol (PAB) unit, and where the spacer unit is linked to MMAE via a carbamate group. 14. An antibody drug conjugate according to any one of claims 1 to 13, wherein the antibody drug conjugate has the following structure: where p is in the range from 1 to 10, preferably where p is in the range from 2 to 5. 15. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 14 in a human dosage form, wherein the pharmaceutical composition further comprises a chemotherapeutic agent in a human unit dosage form. 16. Use of the antibody-drug conjugate of any one of claims 1 to 14 in a method of treating cancer in a subject, wherein the cancer is glioblastoma, lung cancer, bladder cancer, or breast cancer. 17. Use according to claim 16, wherein the subject is a human. 18. Use according to claim 16 or 17, wherein the method comprises administering to the subject an antibody-drug conjugate in an amount of 1.2, 3, 4, 5, or 6 mg/kg of body weight. 19. Use according to claim 16, wherein the method further comprises exposing the subject to radiation or a chemotherapeutic agent. 20. An Ahtu-158P1D7 antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence shown in SEQ ID NO: 7 and a light variable region. a chain containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence shown in SEQ ID NO: 8, where CDRs are defined according to the Kabat, Chotia or Contact numbering system. 21. The Ahtu-158P1D7 antibody or antigen-binding fragment thereof according to claim 20, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing a CDR-H1 consisting of an amino acid sequence ranging from 31 to 35 of SEQ ID NO: 7, CDR- H2, consisting of an amino acid sequence ranging from 50 to 66 of SEQ ID NO: 7, and CDR-H3, consisting of an amino acid sequence ranging from 99 to 109 of SEQ ID NO: 7, and a light chain variable region containing CDR-L1, consisting of an amino acid sequence ranging from 24 to 39 of SEQ ID NO: 8, a CDR-L2 consisting of an amino acid sequence ranging from 55 to 61 of SEQ ID NO: 8, and a CDR-L3 consisting of an amino acid sequence ranging from 94 to 102 SEQ ID NO: 8. 22. Ahtu-158P1D7 antibody or antigen-binding fragment thereof, wherein the antibody or fragment comprises a heavy chain variable region containing an amino acid sequence at least 80% homologous to the heavy chain variable region sequence shown in SEQ ID NO: 7 and a light chain variable region. a chain containing an amino acid sequence at least 80% homologous to the light chain variable region sequence shown in SEQ ID NO: 8, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing complementarity determining regions (CDRs) containing amino acid sequences of CDRs from the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 7 and the light chain variable region containing CDRs containing the amino acid sequences of the CDRs from the amino acid sequence of the light chain variable region shown in SEQ ID NO: 8. - 58 040898 23. Ahtu-158P1D7 antibody or fragment thereof according to claim 22, wherein the antibody or fragment thereof comprises a heavy chain variable region containing an amino acid sequence of at least 85, 86, 87, 88, 89, 90, 91, 92, 93 , 94, 95, 96, 97, 98 or 99% homologous to the sequence of the heavy chain variable region shown in SEQ ID NO: 7 and the light chain variable region containing the amino acid sequence at least 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the light chain variable region sequence shown in SEQ ID NO: 8. 24. An Ahtu-158P1D7 antibody or antigen-binding fragment thereof according to any one of claims 20-23, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region consisting of an amino acid sequence ranging from position 1 to position 120 in SEQ ID NO: 7, and a light chain variable region consisting of an amino acid sequence ranging from position 1 to position 113 in SEQ ID NO: 8. 25. The Ahtu-158P1D7 antibody or antigen-binding fragment thereof according to any one of claims 20-24, wherein the antibody comprises a heavy chain consisting of an amino acid sequence ranging from position 1 to position 446 in SEQ ID NO: 7 and a light chain consisting of amino acid sequence ranging from position 1 to position 219 in SEQ ID NO: 8. 26. Ahtu-158P1D7 antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 from the heavy chain variable region of a Chinese Hamster Ovary (CHO) antibody deposited with the American Type Culture Collection (ATCC) under accession number PTA-13102 and a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3 from the light chain variable region of a Chinese Hamster Ovary (CHO) antibody. ) deposited with ATCC under accession number PTA-13102, where CDRs are defined according to the Kabat, Chotia or Contact numbering system. 27. Ahtu-158P1D7 antibody or antigen-binding fragment thereof, wherein the antibody or fragment comprises a heavy chain variable region containing an amino acid sequence at least 80% homologous to the heavy chain variable region sequence of an antibody produced by Chinese hamster ovary (CHO) cells deposited with American Type Culture Collection (ATCC) accession number PTA-13102, and a light chain variable region containing an amino acid sequence at least 80% homologous to the light chain variable region sequence of an antibody produced by Chinese hamster ovary cells (CHO) deposited with ATCC number Access PTA-13102, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing complementarity determining regions (CDRs) containing CDR amino acid sequences from the amino acid sequence of the heavy chain variable region, represented by specified in SEQ ID NO: 7, and a light chain variable region containing CDRs containing the amino acid sequences of the CDRs from the amino acid sequence of the light chain variable region shown in SEQ ID NO: 8. 28. Ahtu-158P1D7 antibody or antigen-binding fragment thereof according to claim 27, wherein the antibody or fragment thereof comprises a heavy chain variable region containing an amino acid sequence of at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the heavy chain variable region sequence of the Chinese Hamster Ovary (CHO) antibody deposited with ATCC under accession number PTA-13102, and the light chain variable region containing the amino acid sequence at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the light chain variable region sequence of a Chinese Hamster Ovary (CHO) antibody deposited with ATCC under access number PTA-13102. 29. The Ahtu-158P1D7 antibody or antigen-binding fragment thereof according to any one of claims 26-28, wherein the antibody or fragment thereof comprises a heavy chain variable region consisting of the amino acid sequence of the heavy chain variable region of a Chinese Hamster Ovary (CHO) antibody, deposited with ATCC under accession number PTA-13102, and a light chain variable region consisting of the amino acid sequence of the light chain variable region of an antibody produced by Chinese hamster ovary (CHO) cells deposited with ATCC under accession number PTA-13102. 30. Ahtu-158P1D7 antibody or antigen-binding fragment thereof according to any one of claims 26-29, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of the heavy chain of an antibody produced by Chinese hamster ovary (CHO) cells deposited with ATCC under accession number PTA -13102, and a light chain consisting of the light chain amino acid sequence of an antibody produced by Chinese Hamster Ovary (CHO) cells deposited with ATCC under accession number PTA-13102. 31. An Ahtu-158P1D7 antibody or fragment thereof according to any one of claims 20-24 or 26-29, further comprising a heavy chain constant region and a human IgG light chain constant region. -